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WO2025097067A2 - Préparations cellulaires modifiées pour le traitement de l'hypophosphatasie - Google Patents

Préparations cellulaires modifiées pour le traitement de l'hypophosphatasie Download PDF

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Publication number
WO2025097067A2
WO2025097067A2 PCT/US2024/054285 US2024054285W WO2025097067A2 WO 2025097067 A2 WO2025097067 A2 WO 2025097067A2 US 2024054285 W US2024054285 W US 2024054285W WO 2025097067 A2 WO2025097067 A2 WO 2025097067A2
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seq
cell
alp
sequence
population
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WO2025097067A3 (fr
WO2025097067A9 (fr
Inventor
Hanlan Liu
Sarah Grace LEACH
JR. Wayne BAINTER
Madison Ashley CLEMENTS
Richard A. Morgan
Timothy James MULLEN
Anja Fides Hohmann
Shamael Rabia Dastagir
Sharvari GUJJA
Sean Patrick KEEGAN
Andre STANLIE
Toshiro Kendrick Ohsumi
Monika MUSIAL-SIWEK
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Be Biopharma Inc
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Be Biopharma Inc
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Publication of WO2025097067A9 publication Critical patent/WO2025097067A9/fr
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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/13—B-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0635—B lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/231—Interleukin-10 (IL-10)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2315—Interleukin-15 (IL-15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2321—Interleukin-21 (IL-21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/52—CD40, CD40-ligand (CD154)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03001—Alkaline phosphatase (3.1.3.1)

Definitions

  • Cell-based therapeutics are an emerging class of medicine that makes use of innate cellular machinery to combat disease. Unlike traditional treatment methods, cell therapies make use of cellular localization, migration, and proliferation within the body, which can translate to improved biodistribution and targeted delivery of therapeutics. Cell-based therapies have potential applications for a broad range of diseases, including those that have proved intractable or difficult to manage with traditional treatment options.
  • modified immune cells e.g., B lineage cells
  • modified immune cells comprise nucleic acid constructs comprising one or more nucleic acid sequences encoding an alkaline phosphatase (ALP) or fusion protein thereof. Methods of producing and using such modified immune cells are also provided.
  • ALP alkaline phosphatase
  • the present disclosure also encompasses, among other things, nucleic acid constructs comprising one or more nucleic acid sequences encoding an ALP or fusion protein thereof (e.g., a fusion protein comprising ALP and a human immunoglobulin G fragment crystallizable region (IgG Fc; “ALP-Fc fusion protein”) described herein) and methods of producing the same.
  • the present disclosure provides, inter alia, engineered B lineage cell populations expressing an ALP or fusion protein thereof (e.g., an ALP-Fc fusion protein).
  • the present disclosure provides methods of preparing an engineered B lineage cell population that express an alkaline phosphatase or fusion protein thereof.
  • methods comprise the steps of: (a) isolating primary B cells so that a primary B cell population is obtained, (b) activating the primary B cell population, and (c) during or after the activating step, engineering the primary B cell population by integrating a transgene encoding an alkaline phosphatase or fusion protein thereof, thereby generating an engineered B lineage cell population.
  • a fusion protein comprises an alkaline phosphatase and immunoglobulin fragment crystallizable region (ALP-Fc) fusion protein.
  • ALP-Fc fusion protein comprises a Fc derived from human immunoglobulin G1 (IgGl) or human immunoglobulin G4 (IgG4).
  • an ALP-Fc fusion protein further comprises either an octa- or deca-aspartate tag.
  • an ALP-Fc fusion protein further comprises one or more substitutions within an Fc region.
  • one or more substitutions within an Fc region comprise K392D, D399K, and K409D of IgGl (numbered according to full-length IgGl as shown, e.g., in Gunasekaran et al., J. Biol. Chem. 2010).
  • one or more substitutions within an Fc region comprise K392D, D399K, and R409D of IgG4 (numbered according to full-length IgG4).
  • one or more substitutions within an Fc region comprise S228P, F234A, and/or L235A.
  • an ALP-Fc fusion protein further comprises a linker.
  • a linker comprises a tetraglycine and mono-serine linker (GGGGS).
  • GGGGS mono-serine linker
  • a linker comprises three GGGS linkers continuously.
  • a linker comprises two GGGS linkers continuously followed by a mono-glycine.
  • a linker comprises a leucine and a lysine.
  • an ALP-Fc fusion protein further comprises one or more substitutions within the ALP.
  • a transgene is integrated at a site selected from a CCR5 site, a IgH site, CD19, B2M, and a JCHAIN site.
  • an ALP or ALP-Fc fusion protein comprises or has an amino acid sequence of any one of SEQ ID NOs: 62- 75, 91-94, 101, and 109-117.
  • an ALP or ALP-Fc fusion protein is encoded by a nucleic acid sequence comprising any one of SEQ ID NOs: 55-61, 87-90, 100, and 102-108.
  • methods further comprise a step of expanding engineered B lineage cell population described herein.
  • a differentiation step comprises contacting an engineered B lineage cell population with culture media comprising: (a) IL-2, (b) IL-6, (c) IL- 10, and/or (d) IL- 15.
  • a differentiation step comprises contacting a population of plasmablasts with culture media comprising: (a) IL-6, (b) IL-15, and/or (c) IFN-alpha-2-beta (IFNa-2P).
  • a method further comprises a step of transferring an engineered B lineage cell population to cell culture media without human or bovine serum for about 24 hours.
  • a step of engineering further comprises introducing a expression cassette comprising a transgene into a primary B cell population.
  • an expression cassette comprises: (a) a 5’ homology arm that is at least 95% identical to a sequence 5’ to a double-stranded break site, and (b) a 3’ homology arm that is at least 95% identical to a sequence 3’ to a double- stranded break site.
  • a 5’ homology arm comprises or is: (i) a length about 375-850 base pairs in size, (ii) a PAM site or absence of a PAM site, and/or (iii) symmetrical or asymmetrical in length with a 3’ homology arm.
  • a 3’ homology arm comprises or is: (i) a length about 375-850 base pairs in size, (ii) a PAM site or absence of a PAM site, and/or (iii) symmetrical or asymmetrical in length with a 5’ homology arm.
  • a 5’ homology arm has at least 95% identity with one or more of SEQ ID NOs: 39-43, 50, 52, 54, 118, 121, and 123.
  • a 3’ homology arm has at least 95% identity with one or more of SEQ ID NOs: 44-49, 51, 53, 119, 122, and 124.
  • an expression cassette comprises a sequence with at least 95% identity with SEQ ID NO: 77.
  • an expression cassette further comprises one or more of: (a) a promoter sequence selected from SEQ ID NOs: 76, and 125-128, (b) a nucleic acid sequence encoding for alkaline phosphatase or a fusion protein thereof selected from SEQ ID NOs: 55-61, 87-90, 100, and 102-108, (c) an enhancer sequence of SEQ ID NOs: 95-99, and (d) a polyA sequence of SEQ ID NO: 78.
  • an expression cassette is or comprises an adcno-associatcd viral (AAV) vector.
  • a step of engineering comprises contacting a primary B cell population with a targeted nuclease capable of introducing double- stranded breaks.
  • the targeted nuclease is or comprises a CRISPR-associated (Cas) protein, zinc finger nuclease (ZFN), transcription activator- like effector-based nuclease (TALEN), or meganuclease.
  • a Cas protein is or comprises Cas9, Casl2a, or Casl3a, or a variant thereof.
  • a Cas protein is complexed with a guide RNA (gRNA).
  • a gRNA is a single guide RNA (sgRNA). In some embodiments, a gRNA is or comprises a sequence with at least 95% sequence identity to any one of SEQ ID NOs: 79- 86, and 129-132.
  • a step of engineering further comprises electroporation of the primary B cell population; and/or transduction of a primary B cell population with an expression cassette.
  • electroporation is performed on a composition comprising: (a) a Cas protein complexed with a gRNA, and (b) a primary B cell population.
  • a JCHAIN site has at least 95% identity with one or more SEQ ID NOs: 80-82, and 129-130.
  • an IgH site has at least 95% identity with SEQ ID NO: 83.
  • an CD19 site has at least 95% identity with SEQ ID NO: 84-86.
  • a transgene has at least 95% identity with SEQ ID NO: 77.
  • the present disclosure provides populations of genetically modified B lineage cells.
  • a transgene is expressed from an endogenous CCR5, IgH, CD19, B2M, or JCHAIN locus.
  • cells express an ALP-Fc fusion protein and at least partially disrupt expression of the endogenous CCR5, IgH, CD19, B2M, or JCHAIN locus.
  • a population of genetically modified B lineage cells is or comprises a population of genetically modified plasma cells.
  • a population of genetically modified B lineage cells is or comprises a population of plasmablasts.
  • a population of genetically modified B lineage cells is or comprises a population of plasma cell precursors.
  • the present disclosure provides a pharmaceutical composition comprising one or more B lineage cells selected from a population of genetically modified B lineage cells.
  • a pharmaceutical composition comprises one or more B lineage cells selected from a population of genetically modified B lineage cells and further comprises one or more pharmaceutically acceptable excipients.
  • a method of administering a pharmaceutical composition wherein the pharmaceutical composition comprises: (a) one or more B lineage cells selected from the population of genetically modified B lineage cells of any aspect or embodiment described herein, and (b) one or more pharmaceutically acceptable excipients, wherein the pharmaceutical composition is administered to a subject.
  • the present disclosure provides methods of treating a disease, disorder, or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition, thereby treating the disease, disorder, or condition in the subject.
  • a pharmaceutical composition is administered intravenously or intraperitoneally.
  • a pharmaceutical composition is administered to an adult subject.
  • a pharmaceutical composition is administered to a pediatric subject.
  • a subject has hypophosphatasia (HPP).
  • an assay comprises or is one or more of: fluorescence-activated cell sorting (FAC- sort), Western Blot, flow cytometry, enzyme-linked immunosorbent spot assay (ELISpot), and enzyme-linked immunosorbent assay (ELISA).
  • FAC- sort fluorescence-activated cell sorting
  • ELISpot enzyme-linked immunosorbent spot assay
  • ELISA enzyme-linked immunosorbent assay
  • one or more B lineage cells selected from a population of genetically modified B lineage cells engraft within bone marrow of a subject.
  • a method of monitoring engraftment of genetically modified B lineage cells within a subject comprising one or more of: bioluminescence, ELISpot, flow cytometry, and enzyme-linked immunosorbent assay.
  • one or more B lineage cells selected from the population of genetically modified B lineage cells engraft within the bone marrow of the subject.
  • a polynucleotide sequence for use in engineering a population of B lineage cells comprises one or more of: (i) a 5’ homology arm that is or comprises SEQ ID NO: 39; (ii) a promoter that is or comprises SEQ ID NO: 76; (iii) a signal peptide encoded by a polynucleotide sequence that is or comprises SEQ ID NO: 143; (iv) a transgene that is or comprises SEQ ID NO: 139; (v) a terminator that is or comprises SEQ ID NO: 78; and/or (vi) a 3’ homology arm that is or comprises SEQ ID NO: 44.
  • a polynucleotide sequence for use in engineering a population of B lineage cells encoding a polypeptide that comprises one or more of: (i) a signal peptide encoded that is or comprises SEQ ID NO: 144; (ii) a transgene that is or comprises SEQ ID NO: 147; and/or (iii) a linker encoded that is or comprises SEQ ID NO: 141.
  • a polynucleotide sequence is or comprises SEQ ID NO: 100.
  • a polynucleotide sequence is or comprises SEQ ID NO: 149.
  • a polynucleotide sequence encodes a polypeptide sequence that is or comprises SEQ ID NO: 101.
  • Figure IB shows Western blots of histidine purified unengineered control, alkaline phosphatase and immunoglobulin fusion protein (ALP-IgGlFc-His), or alkaline phosphatase and immunoglobulin including K392D/D399K/K409D 3x substitutions (ALP-IgGlFc-3xsubst-His) using either an anti-alkaline phosphatase antibody (top blot) or anti-IgG-CHl antibody (bottom blot).
  • ALP-IgGlFc-His alkaline phosphatase and immunoglobulin including K392D/D399K/K409D 3x substitutions
  • Figure 3A-3B show assessment of ALP-Fc fusion proteins.
  • Figure 3A shows a schematic of an osteogenic stimulation protocol involving osteoblast precursor (MC3T3) cells. These cells are subjected to pyrophosphate (PPi) and engineered B lineage cell derived alkaline phosphatase human immunoglobulin fusion protein with a deca-aspartate tag (ALP-Fc-DlO) or alkaline phosphatase with a deca-aspartate tag (ALP-D10).
  • PPi pyrophosphate
  • ALP-Fc-DlO engineered B lineage cell derived alkaline phosphatase human immunoglobulin fusion protein with a deca-aspartate tag
  • ALP-D10 alkaline phosphatase with a deca-aspartate tag
  • Figure 3B shows ARS staining images (left) and a graph of calcium concentration (right) of osteogenic precursor cells treated with either 50, 250, or 500 ng/mL concentration of engineered B lineage cell derived ALP-D10 or ALP-Fc-DlO.
  • Figure 4 shows a graph of mean calcium concentration of osteogenic precursor cells under osteogenic conditions treated with or without pyrophosphate and engineered B lineage cell derived ALP-D10, ALP-Fc-DlO.
  • Figures 5A-5B shows assessment of engineered B lineage cell derived ALP-Fc.
  • Figure 5A shows a graph of calcium concentration of osteogenic precursor cells under osteogenic conditions treated with or without pyrophosphate and different concentrations of engineered B lineage cell derived ALP-Fc.
  • Figure 5B shows a graph of calcium concentration of osteogenic precursor cells under osteogenic conditions treated with or without pyrophosphate and different concentrations of recombinant human alkaline phosphatase (“rhALP”).
  • Figure 6 shows a graph of a 4-Methylumbelliferyl phosphate (4-MUP) hydrolysis activity assay of recombinant ALP or engineered B lineage cell derived ALP fusion proteins (ALP-Fc, ALP-D10, or ALP-Fc-DlO) as described herein.
  • 4-MUP 4-Methylumbelliferyl phosphate
  • Figure 7 shows a graph of enzymatic activity of ALP-IgGlFc or ALP and IgGl Fc with K392D, D399K, and K409D substitutions (ALP-IgGl-Fc-3xsubst).
  • Figure 8A-8B show assessment of engineered B lineage cells.
  • Figure 8A shows a graph of plasma measurements of ALP or ALP-Fc over time in the NOG-IL-6 model treated with engineered B lineage cells as described herein.
  • Figure 8B shows a graph of plasma human immunoglobulin G (huIgG) over time in the NOG-IL-6 model treated with engineered B lineage cells as described herein.
  • Figure 9C shows a graph of enzymatic activity of ALP-IgGlFc or ALP and IgGl Fc with K392D, D399K, and K409D substitutions (ALP-IgGl-Fc-3xsubst-His) comprising a linker comprising leucine and lysine (LK) or a linker comprising three continuous regions of tetra-glycine and mono-serine (GGGGS(x3)).
  • ALP-IgGl-Fc-3xsubst-His comprising a linker comprising leucine and lysine (LK) or a linker comprising three continuous regions of tetra-glycine and mono-serine (GGGGS(x3)).
  • Figure 9D shows a graph of enzymatic activity of ALP and IgG4 Fc with S288P, F234A, and L235A substitutions and deca-aspartate tag fusion protein (ALP-IgG4Fc-PAA-D10) or ALP, long linker (GGGGSx3) and IgG4 Fc S288P, F234A, L235A, K392D, D399K, and R409D substitutions and deca-aspartate tag fusion protein ( ALP- LL-IgG4Fc-3xsubst-PAA-D10).
  • Figures 10A-10B show assessment of ALP-Fc fusion proteins.
  • Figure 10A demonstrates secretion rate of ALP in B lineage cells engineered at either CCR5 or JCHAIN 31.
  • Figure 10B demonstrates activity of ALP produced by B lineage cells engineered at either CCR5 or JCHAIN 31.
  • Figure 11 is a schematic demonstrating integration of a payload (e.g., expression cassette) at a target locus.
  • a payload e.g., expression cassette
  • Figure 12 shows a schematic of: ALP-Fc fusion protein comprising human immunoglobulin G1 (hu-IgGl) Fc (left), soluble ALP (center), and ALP-Fc fusion protein comprising a human immunoglobulin G4 (hu-IgG4) and additional substitutions.
  • ALP-Fc fusion protein comprising human immunoglobulin G1 (hu-IgGl) Fc (left), soluble ALP (center), and ALP-Fc fusion protein comprising a human immunoglobulin G4 (hu-IgG4) and additional substitutions.
  • Figure 13 shows ALP-Fc fusion protein secretion levels from B lineage cells engineered to express ALP-Fc fusion proteins or variants thereof, such as codon-optimized variants (CO-1: GeneArt codon optimized valiant, CO-2: alternative codon-optimized valiant).
  • Figure 14 shows ALP-Fc fusion protein secretion levels from B lineage cells engineered to express ALP-Fc fusions proteins from a payload (c.g., expression cassette) comprising one or more signal peptides (SP-1: ALP signal peptide, SP-2: A1AT signal peptide).
  • SP-1 ALP signal peptide
  • SP-2 A1AT signal peptide
  • Figure 15 shows ALP-Fc fusion protein secretion levels from B lineage cells engineered to express ALP-Fc fusion proteins at either the CCR5 locus or JCHAIN1 locus (“Locus B”).
  • Figures 16A-16C shows assessment of ALP-Fc fusion proteins.
  • Figure 16A shows a schematic of ALP-Fc fusion proteins with linkers of various lengths between ALP and IgG-Fc components.
  • Figure 16B shows a crystal structure of ALP dimer, PDB#1HQA.
  • Figure 16C shows projected crystal structures for the following in order from left to right: ALP-Fc fusion protein, ALP-Short Linker (“SL”)-Fc fusion protein, ALP-Long Linker (“LL”)-Fc fusion protein, and overlay between ALP-SL-Fc and ALP-LL-Fc fusion proteins.
  • SL ALP-Short Linker
  • LL ALP-Long Linker
  • Figure 17 shows ALP-Fc fusion protein secretion levels from B lineage cells engineered to express ALP-Fc fusion proteins at either the CCR5 locus, JCHAIN173 locus (“Locus A”), or JCHAIN1 locus (“Locus B”) which were compared to control electroporated plasma cells without vector (“EPC”).
  • FIG 18 shows 4-Methylumbelliferyl phosphate (4-MUP) hydrolysis activity assay of B lineage cells engineered to express ALP fusion proteins at either the CCR5 locus, JCHAIN173 locus (“Locus A”), or JCHAIN1 locus (“Locus B”) which were compared to control electroporated plasma cells without vector (“EPC”).
  • 4-MUP 4-Methylumbelliferyl phosphate
  • FIG 19 shows 4-Methylumbelliferyl phosphate (4-MUP) specific hydrolysis activity of B lineage cells engineered to express ALP fusion proteins at either the CCR5 locus, JCHA1N 173 locus (“Locus A”), or JCHA1N 1 locus (“Locus B”) which were compared to a control electroporated plasma cells without vector (“EPC”).
  • 4-MUP 4-Methylumbelliferyl phosphate
  • Figure 22 shows ALP-Fc protein secretion levels from two different donor populations of B lineage cells (LKP23185 and LKP23187) engineered with constructs described herein comprising either an MND, Fu_l, Fu_2, Fu_3, B29 promoter with or without an Ep enhancer.
  • Figure 23 shows 4-Methylumbelliferyl phosphate (4-MUP) hydrolysis activity of two different donor populations of B lineage cells (LKP23185 and LKP23187) engineered with constructs described herein comprising either an MND, Fu_l, Fu_2, Fu_3, B29 promoter with or without an Ep enhancer.
  • 4-MUP 4-Methylumbelliferyl phosphate
  • Figures 24A-24C show assessment of engineered B lineage cells.
  • Figure 24A shows percentage of homology-directed repair (% HDR) for B lineage cells engineered with constructs described herein comprising an MND promoter with or without an Ep enhancer.
  • Figure 24B shows ALP-Fc protein secretion levels from B lineage cells engineered with constructs described herein comprising an MND promoter with or without an Ep enhancer.
  • Figure 24C shows 4-Methylumbelliferyl phosphate (4-MUP) hydrolysis activity of B lineage cells engineered with constructs described herein comprising an MND promoter with or without an Ep enhancer.
  • 4-MUP 4-Methylumbelliferyl phosphate
  • FIGs 25A-D depict assessment of B lineage cells engineered to express ALP- Fc (ALP-BCM). ALP-BCMs or control cells electroporated without vector were administered to immunodeficient mice.
  • Figure 25A shows levels of secreted ALP-Fc isolated from mice to which ALP-BCMs were administered.
  • Figure 25B shows assessment of ALP-Fc activity in serum isolated from mice to which ALP-BCMs were administered.
  • Figure 25C shows assessment of human IgG secretion in mice to which ALP-BCMs were administered.
  • Figure 25D shows weight assessment of mice to which ALP-BCMs were administered.
  • Figure 26 shows Western blots of histidine purified non-engineered control (Uneng), alkaline phosphatase and immunoglobulin fusion protein (ALP-IgG4Fc-His), or alkaline phosphatase and immunoglobulin including K392D/D399K/K409D 3 substitutions (ALP-IgG4Fc-3xsubst-His) using an anti- ALP antibody.
  • ALP-IgG4Fc-His alkaline phosphatase and immunoglobulin including K392D/D399K/K409D 3 substitutions
  • Figure 27 shows Alizarin Red S (ARS) staining images (left) and calcium levels (right) of osteoblast precursor cells (MC3T3) cultured under osteogenic conditions for 21 days as indicated with media alone, PPi, 50 ng/mL of supernatant from cultured B lineage cells engineered to express ALP-Fc (ALP-BCM), or 50 ng/mL of supernatant from cultured control B lineage cells electroporated without vector (EPC).
  • ARS Alizarin Red S
  • Figures 28A-B depict assessment of B lineage cells engineered to express ALP-Fc (ALP-BCM). ALP-BCMs or control cells electroporated without vector were administered to immunodeficient mice.
  • Figure 28A shows levels of secreted ALP-Fc isolated from mice to which ALP-BCMs were administered.
  • Figure 28B shows assessment of hALP activity in serum isolated from mice to which ALP-BCMs were administered.
  • Figures 29A-B depict assessment of B lineage cells engineered to express ALP-Fc (ALP-BCM). ALP-BCMs or control cells electroporated without vector were administered to immunodeficient mice.
  • Figure 29A shows levels of secreted ALP-Fc isolated from mice to which ALP-BCMs were administered.
  • Figure 29B shows assessment of hALP activity in serum isolated from mice to which ALP-BCMs were administered.
  • Figure 30 depicts assessment of B lineage cells engineered to express ALP-Fc (ALP-BCM) after cryopreservation and expansion. ALP-BCMs or control cells electroporated without vector were administered to immunodeficient mice. Figure 30 shows levels of secreted ALP-Fc isolated from mice to which ALP-BCMs were administered.
  • ALP-BCMs or control cells electroporated without vector were administered to immunodeficient mice.
  • Figure 30 shows levels of secreted ALP-Fc isolated from mice to which ALP-BCMs were administered.
  • Figures 31A-B depict assessment of B lineage cells engineered to express ALP-Fc (ALP-BCM) after cryoprcscrvation and expansion. ALP-BCMs or control cells electroporated without vector were administered to immunodeficient mice.
  • Figure 31 A shows levels of secreted ALP-Fc isolated from mice to which ALP-BCMs were administered.
  • Figure 3 IB shows assessment of hALP activity in serum isolated from mice to which ALP-BCMs were administered.
  • Figures 32A-B depict assessment of B lineage cells engineered to express ALP-Fc (ALP-BCM) through use of different expression cassettes. ALP-BCMs or control cells electroporated without vector were administered to immunodeficient mice.
  • Figure 32A shows levels of secreted ALP-Fc isolated from mice to which ALP-BCMs were administered.
  • Figure 32B shows assessment of hALP activity in serum isolated from mice to which ALP-BCMs were administered.
  • Figures 33A-C depict assessment of B lineage cells engineered to express ALP-Fc (ALP-BCM) through use of different expression cassettes (p00215 and p00983) and viral transduction protocols (protocols 1 and 2).
  • Figure 33 A depicts percent targeted integration of ALP constructs into B-lineage cells.
  • Figure 33B shows levels of ALP in supernatant of B lineage cells engineered to express ALP.
  • Figure 33C depicts ALP activity of B-lineage cells engineered to express ALP.
  • Figures 35A-C depict assessment of ALP constructs driven by Eu enhancers or variants thereof.
  • Figure 35A depicts percent targeted integration of ALP constructs into B- lineage cells.
  • Figure 35B shows levels of ALP in supernatant of B-lineage cells engineered to express ALP.
  • Figure 35C depicts ALP activity of B-lineage cells engineered to express ALP.
  • Figure 43 shows assessment of purified ALP-Fc protein binding to FcRn.
  • Figure 44 depicts a quantification of sialic acids detected on purified ALP-Fc proteins.
  • Figure 46 shows assessment of purified ALP-Fc protein binding to FcyRI.
  • Figure 47 shows assessment of purified ALP-Fc protein binding to FcRn.
  • Figures 49A-B depict assessment of ALP-Fc proteins purified from HEK cells after administration to mice.
  • Figure 49A shows levels of secreted ALP-Fc isolated from mice to which ALP-Fc proteins were administered.
  • Figure 49B shows assessment of ALP-Fc activity in serum isolated from mice to which ALP-Fc proteins were administered.
  • Figures 50A-B depict assessment of ALP-Fc proteins purified from HEK cells or B lineage cells engineered to express ALP-Fc.
  • Figure 50A shows levels of secreted ALP-Fc isolated from mice to which ALP-Fc proteins were administered.
  • Figure 50B shows assessment of ALP-Fc activity in serum isolated from mice to which ALP-Fc proteins were administered.
  • Activation refers to the state of a cell, for example a B cell that has been sufficiently stimulated to induce detectable cellular proliferation or has been stimulated to exert its effector function. Activation can also be associated with induced cytokine production, cell signaling, differentiation, and/or antigen processing and presentation.
  • Administration typically refers to the administration (e.g., of a composition or treatment) to a subject or system (e.g., that is or comprises one or more cells, tissues, organisms, etc), for example to achieve delivery of an agent that is, is included in, or is otherwise delivered or generated by, such composition or treatment.
  • a subject or system e.g., that is or comprises one or more cells, tissues, organisms, etc
  • administration may be ocular, oral, parenteral, topical, etc.
  • administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular’, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e. g. intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc.
  • bronchial e.g., by bronchial instillation
  • buccal which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc
  • enteral intra-arterial, intradermal, intragastric
  • administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.
  • agent refers to a molecule that may be expressed, released, secreted or delivered to a target by a modified cell described herein.
  • An agent includes, but is not limited to, a nucleic acid, an antibiotic, an anti-inflammatory agent, an antibody or fragments thereof, an antibody agent or fragments thereof, a growth factor, a cytokine, an enzyme, a protein (e.g., an RNAse inhibitor), a peptide, a fusion protein, a synthetic molecule, an organic molecule (e.g., a small molecule), a carbohydrate, a lipid, a hormone, a microsome, a derivative or a variation thereof, and any combinations thereof.
  • An agent may bind any cell moiety, such as a receptor, an antigenic determinant, or other binding site present on a target or target cell. An agent may diffuse or be transported into a cell, where it may act
  • Alloantigen refers to an antigen associated with allorecognition and/or graft rejection (e.g., an antigen against which a rejection immune response is directed).
  • alloantigens are agents that are present in or on tissue from one individual (e.g., a donor individual) of a particular species, but not in or on tissue from another individual (e.g., a recipient individual, for example who is genetically different from the donor individual) of the species, so that transfer of tissue from the donor individual to the recipient individual risks and/or results in a rejection immune response.
  • an antigen may be or include any chemical entity such as, for example, a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, etc.
  • an alloantigen is or comprises a polypeptide.
  • a variety of polypeptides are known in the art whose amino acid sequences can vary between and among individuals of the same species such that they might act as alloantigcns.
  • Allogeneic refers to any material (e.g., a population of cells) derived from a different animal of the same species.
  • Allorecognition typically refers to an immune response mounted by the immune system of an individual (i.e., a recipient) who receives a tissue graft from another individual (i.e., a donor, who for example is genetically distinct from the recipient individual) of the same species, which immune response involves recognition of an alloantigen on the grafted tissue.
  • allorecognition involves T cell recognition of the alloantigen.
  • T cells recognize an alloantigen peptide, for example, encoded by a polymorphic gene whose sequence differs between the donor and recipient individuals.
  • Amelioration' refers to the prevention, reduction or palliation of a state, or improvement of the state of a subject. Amelioration includes, but does not require complete recovery or complete prevention of a disease, disorder or condition (e.g., radiation injury).
  • a disease, disorder or condition e.g., radiation injury
  • Antigen' refers to a molecule that is capable of provoking an immune response. This immune response may involve either antibody production, activation of specific immunologically-competent cells, or both. A skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA that comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response encodes an “antigen” as that term is used herein.
  • an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.
  • antibody agent refers to a polypeptide that may be expressed, released, secreted, or delivered to a target by a modified cell described herein.
  • the polypeptide includes canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen.
  • an antibody agent comprises an antibody.
  • antibodies as produced in nature are approximately 150 kD tetrameric agents comprising two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about 25 kD each) that associate with each other into what is commonly referred to as a “Y-shaped” structure.
  • Each heavy chain comprises at least four domains (each about 110 amino acids long) - an amino-terminal variable (VH) domain (located at the tips of the Y structure), followed by three constant domains: CHI, CH2, and the carboxy-terminal CH3 (located at the base of the Y’s stem).
  • VH amino-terminal variable
  • CH2 amino-terminal variable
  • CH3 carboxy-terminal CH3
  • Each light chain comprises two domains - an aminoterminal variable (VL) domain, followed by a carboxy-terminal constant (CL) domain, separated from one another by another “switch”.
  • Intact antibody agent tetramers comprise two heavy chain-light chain dimers in which the heavy and light chains are linked to one another by a single disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and a tetramer is formed.
  • Antibody agents are also glycosylated, typically on the CH2 domain.
  • Each domain in a natural antibody has a structure characterized by an “immunoglobulin fold” formed from two beta sheets (e.g., 3-, 4-, or 5-stranded sheets) packed against each other in a compressed antiparallel beta barrel.
  • Each variable domain contains three hypervariable loops known as “complementarity determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4).
  • CDR1, CDR2, and CDR3 three hypervariable loops known as “complementarity determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4).
  • any polypeptide or complex of polypeptides that includes sufficient immunoglobulin domain sequences as found in natural antibodies can be referred to and/or used as an “antibody agent”, whether such polypeptide is naturally produced (e.g., generated by an organism reacting to an antigen), or produced by recombinant engineering, chemical synthesis, or other artificial system or methodology.
  • an antibody agent is polyclonal.
  • an antibody agent is monoclonal.
  • an antibody agent has constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies.
  • antibody agent sequence elements are humanized, primatized, chimeric, etc, as is known in the art.
  • an antibody agent can refer in appropriate embodiments (unless otherwise stated or clear' from context) to any of the art-known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation.
  • an antibody agent may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally.
  • an antibody agent may contain a covalent modification (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc], or other pendant group [e.g., poly-ethylene glycol, etc.].
  • Autologous refers to any material derived from an individual to which it is later to be re-introduced into the same individual.
  • Biomarker' refers to an observable biological effect or result achieved by an agent or entity of interest.
  • a specific binding interaction is a biological activity.
  • modulation (e.g., induction, enhancement, or inhibition) of a biological pathway or event is a biological activity.
  • presence or extent of a biological activity is assessed through detection of a direct or indirect product produced by a biological pathway or event of interest.
  • biomarker is used herein, consistent with its use in the art, to refer to a to an entity, event, or characteristic whose presence, level, degree, type, and/or form, correlates with a particular biological event or state of interest, so that it is considered to be a “marker” of that event or state.
  • a biomarker may be or comprise a marker for a particular disease state, or for likelihood that a particular disease, disorder or condition may develop, occur, or reoccur.
  • a biomarker may be or comprise a marker for a particular disease or therapeutic outcome, or likelihood thereof.
  • a biomarker is predictive, in some embodiments, a biomarker is prognostic, in some embodiments, a biomarker is diagnostic, of the relevant biological event or state of interest.
  • a biomarker may be or comprise an entity of any chemical class, and may be or comprise a combination of entities.
  • a biomarker may be or comprise a nucleic acid, a polypeptide, a lipid, a carbohydrate, a small molecule, an inorganic agent (e.g., a metal or ion), or a combination thereof.
  • a biomarker is a cell surface marker.
  • a biomarker is intracellular.
  • a biomarker is detected outside of cells (e.g., is secreted or is otherwise generated or present outside of cells, e.g., in a body fluid such as blood, urine, tears, saliva, cerebrospinal fluid, etc.
  • a biomarker may be or comprise a genetic or epigenetic signature.
  • a biomarker may be or comprise a gene expression signature.
  • Conservative sequence modifications refers to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody compatible with various embodiments by standard techniques known in the ail, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g., threonine, valine, isolcucinc
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine.
  • one or more amino acid residues within the CDR regions of an antibody can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for the ability to bind antigens using the
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, 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 gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • Engineered' refers to the aspect of having been manipulated by the hand of man.
  • a polynucleotide is considered to be “engineered” when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in an engineered polynucleotide and/or when a particular residue in a polynucleotide is non-naturally occurring and/or is caused through action of the hand of man to be linked with an entity or moiety with which it is not linked in nature.
  • a cell or organism is considered to be “engineered” if it has been subjected to a manipulation, so that its genetic, epigenetic, and/or phenotypic identity is altered relative to an appropriate reference cell such as otherwise identical cell that has not been so manipulated.
  • such manipulation is or comprises a genetic manipulation, so that its genetic information is altered (c.g., new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols).
  • an engineered cell is one that has been manipulated so that it contains and/or expresses a particular agent of interest (e.g., a protein, a nucleic acid, and/or a particular form thereof) in an altered amount and/or according to altered timing relative to such an appropriate reference cell.
  • a particular agent of interest e.g., a protein, a nucleic acid, and/or a particular form thereof
  • progeny of an engineered polynucleotide or cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.
  • Expand refers to increasing in number, as in an increase in the number of cells, for example, monocytes, macrophages, and/or dendritic cells.
  • monocytes, macrophages, or dendritic cells that are expanded ex vivo increase in number relative to the number originally present in a culture.
  • monocytes, macrophages, or dendritic cells that are expanded ex vivo increase in number relative to other cell types in a culture.
  • expansion may occur in vivo.
  • expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and/or 3’ end formation); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.
  • a nucleotide fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more monomeric units (e.g., nucleic acids) as found in the whole nucleotide.
  • monomeric units e.g., nucleic acids
  • a nucleotide fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the monomeric units (e.g., residues) found in the whole nucleotide.
  • the whole material or entity may in some embodiments be referred to as the “parent” of the whole.
  • Functional' As used herein, a “functional” biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.
  • Gene product or expression product generally refers to an RNA transcribed from a gene (pre-and/or postprocessing) or a polypeptide (pre- and/or post-modification) encoded by an RNA transcribed from a gene.
  • homology refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e. ., DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical.
  • polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions).
  • sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions).
  • a variety of algorithms are available that permit comparison of sequences in order to determine their degree of homology, including by permitting gaps of designated length in one sequence relative to another when considering which residues “correspond” to one another in different sequences.
  • Calculation of the percent homology between two nucleic acid sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-corresponding sequences can be disregarded for comparison purposes).
  • the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence.
  • the nucleotides at corresponding nucleotide positions are then compared.
  • the percent homology between the two sequences is a function of the number of identical and similar positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
  • Identity refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules. When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an Arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage.
  • the identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half (e.g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical.
  • Immune cell refers to a cell that is involved in an immune response, e.g., promotion of an immune response.
  • immune cells include, but are not limited to, plasma cells, plasmablasts, B-lymphocytes, macrophages, monocytes, dendritic cells, neutrophils, eosinophils, mast cells, platelets, large granular lymphocytes, Langerhans' cells, natural killer (NK) cells, or T-lymphocytes.
  • a source of immune cells can be obtained from a subject.
  • immune cells comprise or are B lineage cells.
  • Immune response refers to a cellular and/or systemic response to an antigen that occurs when lymphocytes identify antigenic molecules as foreign and induce the formation of antibodies and/or activate lymphocytes to remove the antigen.
  • Immunoglobulin refers to a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as a BCR (B cell receptor) or antigen receptor. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE.
  • IgA is the primary antibody that is present in body secretions, such as saliva, tears, breast milk, gastrointestinal secretions and mucus secretions of the respiratory and genitourinary tracts.
  • TgG is the most common circulating antibody.
  • IgM is the main immunoglobulin produced in the primary immune response in most subjects.
  • IgD is an immunoglobulin that has no known antibody function, but may serve as an antigen receptor.
  • IgE is an immunoglobulin that mediates immediate hypersensitivity by causing release of mediators from mast cells and basophils upon exposure to allergen.
  • Isolated refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting) and/or otherwise previously associated, and/or (2) designed, produced, prepared, and/or manufactured by the hand of man.
  • a substance may be considered to be “isolated” if it is (or has been caused to be) free of or separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of other components (e.g., components with which it was previously associated).
  • isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.
  • a substance is “pure” if it is substantially free of other components.
  • a substance may still be considered “isolated” or even “pure”, after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of a substance is calculated without including such carriers or excipients.
  • carriers or excipients e.g., buffer, solvent, water, etc.
  • a biological polymer such as a polypeptide or polynucleotide that occurs in nature is considered to be "isolated” when, a) by virtue of its origin or source of derivation is not associated with some or all of the components that accompany it in its native state in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species from the species that produces it in nature; c) is expressed by or is otherwise in association with components from a cell or other expression system that is not of the species that produces it in nature.
  • a polypeptide that is chemically synthesized or is synthesized in a cellular system different from that which produces it in nature is considered to be an "isolated” polypeptide.
  • a polypeptide that has been subjected to one or more purification techniques may be considered to be an "isolated" polypeptide to the extent that it has been separated from other components a) with which it is associated in nature; and/or b) with which it was associated when initially produced.
  • a marker refers to an entity or moiety whose presence or level is a characteristic of a particular state or event. In some embodiments, presence or level of a particular marker may be characteristic of presence or stage of a disease, disorder, or condition. To give but one example, in some embodiments, the term refers to a gene expression product that is characteristic of a particular tumor, tumor subclass, stage of tumor, etc.
  • a presence or level of a particular marker correlates with activity (or activity level) of a particular signaling pathway, for example that may be characteristic of a particular class of tumors.
  • the statistical significance of presence or absence of a marker may vary depending upon a particular' marker.
  • detection of a marker is highly specific in that it reflects a high probability that such tumor is of a particular subclass. Such specificity may come at the cost of sensitivity (i.e., a negative result may occur even if the tumor is a tumor that would be expected to express the marker).
  • markers with a high degree of sensitivity may be less specific that those with lower sensitivity.
  • a useful marker need not distinguish with 100% accuracy.
  • Modified refers to a changed state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids.
  • Modulating refers to mediating a detectable increase or decrease in the level of a response and/or a change in the nature of a response in a subject compared with the level and/or nature of a response in the subject in the absence of a treatment or compound, and/or compared with the level and/or nature of a response in an otherwise identical but untreated subject.
  • the term encompasses perturbing and/or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.
  • nucleic acid refers to a polymer of at least three nucleotides.
  • a nucleic acid comprises DNA.
  • a nucleic acid comprises RNA.
  • a nucleic acid is single stranded.
  • a nucleic acid is double stranded.
  • a nucleic acid comprises both single and double stranded portions.
  • a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages.
  • a nucleic acid comprises a backbone that comprises both phosphodiester and non- phosphodiester linkages.
  • a nucleic acid may comprise a backbone that comprises one or more phosphorothioate or 5'-N-phosphoramidite linkages and/or one or more peptide bonds, e.g., as in a “peptide nucleic acid”.
  • a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxy thymidine, guanine, thymine, uracil).
  • a nucleic acid comprises one or more, or all, non-natural residues.
  • a non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5 -fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2- aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)- methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof).
  • a non-natural residue comprises one or more modified sugars (e.g., 2'- fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared to those in natural residues.
  • a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide.
  • a nucleic acid has a nucleotide sequence that comprises one or more introns.
  • a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro, reproduction in a recombinant cell or system, or chemical synthesis.
  • enzymatic synthesis e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro, reproduction in a recombinant cell or system, or chemical synthesis.
  • a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long.
  • operably linked refers to functional linkage between, for example, a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • Payload' refers to an agent that may be delivered or transported by association with another entity.
  • association may be or include a covalent linkage; in some embodiments such association may be or include non-covalent interaction(s).
  • association may be direct; in some embodiments, association may be indirect.
  • a payload is not limited to a particular chemical identity or type; for example, in some embodiments, a payload may be or comprise, for example, an entity of any chemical class including, for example, a lipid, a metal, a nucleic acid (e.g., a transgene), a polypeptide, a saccharide (e.g., a polysaccharide), small molecule, or a combination or complex thereof.
  • a payload may be or comprise a biological modifier, a detectable agent (e.g., a dye, a fluorophore, a radiolabel, etc.), a detecting agent, a nutrient, a therapeutic agent, etc., or a combination thereof.
  • a payload may be or comprise a cell or organism, or a fraction, extract, or component thereof.
  • a payload may be or comprise a natural product in that it is found in and/or is obtained from nature; alternatively or additionally, in some embodiments, the term may be used to refer to one or more entities that is man-made in that it is designed, engineered, and/or produced through action of the hand of man and/or is not found in nature.
  • an payload may be or comprise an agent in isolated or pure form; in some embodiments, such agent may be in crude form.
  • composition refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers.
  • active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.
  • compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.
  • oral administration for example, drenches (aqueous or non-aqueous solutions or suspension
  • Polynucleotide refers to a chain of nucleotides.
  • nucleic acids are polymers of nucleotides.
  • nucleic acids and polynucleotides as used herein are interchangeable.
  • nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides.
  • polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCRTM, and the like, and by synthetic means.
  • recombinant means i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCRTM, and the like, and by synthetic means.
  • Polypeptide refers to any polymeric chain of residues (e.g., amino acids) that are typically linked by peptide bonds.
  • a polypeptide has an amino acid sequence that occurs in nature.
  • a polypeptide has an amino acid sequence that does not occur in nature.
  • a polypeptide has an amino acid sequence that is engineered in that it is designed and/or produced through action of the hand of man.
  • a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both.
  • a polypeptide may comprise or consist of only natural amino acids or only non- natural amino acids.
  • a polypeptide may comprise D-amino acids, L- amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof.
  • a polypeptide may be cyclic, and/or may comprise a cyclic portion. In some embodiments, a polypeptide is not cyclic and/or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides.
  • exemplary polypeptides within the class whose amino acid sequences and/or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family.
  • a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and/or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class).
  • a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%.
  • a conserved region that may in some embodiments be or comprise a characteristic sequence element
  • Such a conserved region usually encompasses at least 3-4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids.
  • a useful polypeptide may comprise or consist of a fragment of a parent polypeptide.
  • a useful polypeptide as may comprise or consist of a plurality of fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to one another than is found in the polypeptide of interest (e.g., fragments that are directly linked in the parent may be spatially separated in the polypeptide of interest or vice versa, and/or fragments may be present in a different order in the polypeptide of interest than in the parent), so that the polypeptide of interest is a derivative of its parent polypeptide.
  • Protein refers to a polypeptide (i.e. , a string of at least two amino acids linked to one another by peptide bonds). Proteins may include moieties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and/or may be otherwise processed or modified. Those of ordinary skill in the ail will appreciate that a “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means.
  • Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the ail. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc.
  • proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof.
  • the term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids.
  • proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.
  • Reference' As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the ait will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control.
  • a response to treatment may refer to a beneficial alteration in a subject’s condition that occurs as a result of or correlates with treatment.
  • alteration may be or comprise stabilization of a condition (e.g., prevention of deterioration that would have taken place in the absence of a treatment), amelioration of symptoms of a condition, and/or improvement in prospects for cure of a condition, etc.
  • the term “response” may refer to a response of a particular system or components thereof (e.g., of a particular cell, tissue, organism, or subject). Those skilled in the ail will be aware of technologies available to assess a response of interest.
  • sample typically refers to an aliquot of material obtained or derived from a source of interest, as described herein.
  • a source of interest is a biological or environmental source.
  • a source of interest may be or comprise a cell or an organism, such as a microbe, a plant, or an animal (e.g., a human).
  • a source of interest is or comprises biological tissue or fluid.
  • a biological tissue or fluid may be or comprise amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humor, vomit, and/or combinations or component(s) thereof.
  • a biological fluid may be or comprise an intracellular fluid, an extracellular fluid, an intravascular fluid (blood plasma), an interstitial fluid, a lymphatic fluid, and/or a transcellular fluid.
  • a biological fluid may be or comprise a plant exudate.
  • a biological tissue or sample may be obtained, for example, by aspirate, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing or lavage (e.g., brocheoalvealar, ductal, nasal, ocular, oral, uterine, vaginal, or other washing or lavage).
  • a biological sample is or comprises cells obtained from an individual.
  • a sample is a “primary sample” obtained directly from a source of interest by any appropriate means.
  • the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane.
  • a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques such as amplification or reverse transcription of nucleic acid, isolation and/or purification of certain components, etc.
  • a sample may be a “crude” sample in that it has been subjected to relatively little processing and/or is complex in that it includes components of relatively varied chemical classes.
  • Signal transduction pathway refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell.
  • cell surface receptor includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the plasma membrane of a cell.
  • the term “significant” typically refers to the context wherein the difference or relationship between two variables e.g., sequence identity, protein production, spatiotemporal conditions, etc.) are certain and exist. Significance can be statistically measured (e.g., statistically significant) by various mathematical formulas and models as understood by one skilled in the art. These methods include, but are not limited to, student t-test, two-tailed test, analysis of variance (ANOVA), etc. Furthermore, significance can impart differences within structure and chemistry between two different entities.
  • Source typically refers to a context in which an agent of interest (e.g., that may be or comprise a carbohydrate, a lipid, a nucleic acid, a metal, polypeptide, a small molecule, or a combination thereof) may be found in nature, or from which such agent can be or has been obtained (e.g., isolated).
  • a source may be or comprise a biological source (c.g., an organism, tissue, or cell, or sample thereof); in some embodiments, a source may be an environmental source.
  • a source may be or comprise a primary sample from an organism (e.g., which may be or comprise a tissue or fluid of such organism, and/or may be or comprise cell(s) of such organism).
  • an organism may be or comprise a prokaryotic organism (e.g., a bacterium) or a eukaryotic organism (e.g., a fungus or yeast, an insect, a mammal, a plant, a reptile, etc.).
  • an infectious agent such as a virus or phage may be considered an organism for purposes of this disclosure, and in particular with respect to being a source.
  • a source may be or comprise an engineered source, such as a cell line or culture, an in vitro system, etc.
  • Subject refers to an organism, for example, a mammal (e.g., a human, a non-human mammal, a non-human primate, a primate, a laboratory animal, a mouse, a rat, a hamster, a gerbil, a cat, or a dog).
  • a human subject is an adult, adolescent, or pediatric subject.
  • a subject is suffering from a disease, disorder or condition, e.g., a disease, disorder, or condition that can be treated as provided herein, e.g., a cancer or a tumor listed herein.
  • a subject is susceptible to a disease, disorder, or condition; in some embodiments, a susceptible subject is predisposed to and/or shows an increased risk (as compared to the average risk observed in a reference subject or population) of developing the disease, disorder, or condition.
  • a subject displays one or more symptoms of a disease, disorder, or condition.
  • a subject does not display a particular symptom (e.g., clinical manifestation of disease) or characteristic of a disease, disorder, or condition.
  • a subject does not display any symptom or characteristic of a disease, disorder, or condition.
  • a subject is a patient.
  • a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.
  • Substantial identity refers to a comparison between amino acid or nucleic acid sequences. As will be appreciated by those of ordinary skill in the art, two sequences are generally considered to be “substantially identical” if they contain identical residues in corresponding positions. As is well known in this art, amino acid or nucleic acid sequences may be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences.
  • two sequences are considered to be substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are identical over a relevant stretch of residues.
  • the relevant stretch is a complete sequence.
  • the relevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.
  • the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.
  • One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result.
  • the term “substantially” is therefore used herein to capture a potential lack of completeness inherent in many biological and chemical phenomena.
  • substantially purified refers to a cell that is essentially free of other cell types.
  • a substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state.
  • a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state.
  • the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.
  • Target refers to a cell, tissue, organ, or site within the body that is the subject of provided methods, systems, and/or compositions, for example, a cell, tissue, organ or site within a body that is in need of treatment or is preferentially bound by.
  • Target Locus' As used herein, the term “target locus” may refer to a specific site or location on a chromosome of interest. For example, a target locus may be a site to be “engineered” or “altered” by the hand of man.
  • an engineered polynucleotide comprises homology to a target locus in order to allow further alterations at a specific site (e.g., CCR5 as a target locus, whose homologous sequence may be part of a guide RNA to result in incorporation of an edit via CRISPR/Cas -mediated gene editing).
  • target locus may interchangeably refer to a target gene of interest for manipulation by man.
  • such target locus manipulation is or comprises a genetic manipulation, so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols).
  • new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols.
  • Target site refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur.
  • Therapeutic agent refers to an agent that, when administered to a subject, has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect.
  • a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.
  • transfected As used herein, the term “transfected” or “transformed” or “transduced” refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.
  • treat refers to partial or complete alleviation, amelioration, delay of onset of, inhibition, prevention, relief, and/or reduction in incidence and/or severity of one or more symptoms or features of a disease, disorder, and/or condition.
  • treatment may be administered to a subject who docs not exhibit signs or features of a disease, disorder, and/or condition (c.g., may be prophylactic).
  • treatment may be administered to a subject who exhibits only early or mild signs or features of the disease, disorder, and/or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.
  • treatment may be administered to a subject who exhibits established, severe, and/or late-stage signs of the disease, disorder, or condition.
  • Variant As used herein in the context of molecules, e.g., nucleic acids, proteins, or small molecules, the term “variant” refers to a molecule that shows significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or in the level of one or more biological or chemical moieties as compared to the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. In some embodiments, a variant differs structurally but performs the same or similar’ function (e.g., enzymatic function) as its reference molecule.
  • a variant differs structurally but performs the same or similar’ function (e.g., enzymatic function) as its reference molecule.
  • any biological or chemical reference molecule has certain characteristic structural elements.
  • a variant by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule.
  • a polypeptide may have a characteristic sequence element comprised of a plurality of amino acids having designated positions relative to one another in linear or three-dimensional space and/or contributing to a particular structural motif and/or biological function;
  • a nucleic acid may have a characteristic sequence element comprised of a plurality of nucleotide residues having designated positions relative to on another in linear or three-dimensional space.
  • a valiant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone).
  • moieties e.g., carbohydrates, lipids, phosphate groups
  • a variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%.
  • a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid.
  • a reference polypeptide or nucleic acid has one or more biological activities.
  • a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid.
  • a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid shows a reduced level of one or more biological activities as compared to the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid is a truncated form of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide that is a truncated form of the reference polypeptide may demonstrate comparable, identical, or greater levels of one or more biological activities as compared to the reference polypeptide or nucleic acid.
  • a polypeptide or nucleic acid of interest is considered to be a “variant” of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions.
  • Vector refers to a composition of matter that comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • the term “vector” includes an autonomously replicating plasmid or a virus.
  • the term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like.
  • viral vectors include, but are not limited to, adenoviral vectors, adeno- associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
  • the present disclosure encompasses culturing and engineering of B lineage cell populations in order to express an alkaline phosphatase (ALP) or fusion protein thereof, e.g., a fusion protein comprising ALP and a human immunoglobulin G fragment crystallizable region (IgG Fc; an ALP-Fc fusion protein).
  • ALP alkaline phosphatase
  • B lineage cell populations express a fusion protein comprising ALP and a human IgGl Fc.
  • an IgGl Fc of a fusion protein described herein comprises one or more substitutions.
  • an IgGl Fc comprises K392D, D399K, and/or K409D substitutions.
  • B lineage cell populations express a fusion protein comprising ALP and a human IgG4 Fc.
  • an IgG4 Fc of a fusion protein described herein comprises one or more substitutions.
  • an IgG4 Fc comprises K392D, D399K, and/or R409D substitutions.
  • an ALP is or comprises tissue non-specific alkaline phosphatase (TNSALP).
  • TNSALP tissue non-specific alkaline phosphatase
  • an ALP is or comprises a catalytic domain of TNSALP.
  • an ALP comprises of a glycosyl-phosphatidylinositol (GPI) linkage.
  • GPI glycosyl-phosphatidylinositol
  • an ALP does not comprise of a glycosyl-phosphatidylinositol (GPI) linkage.
  • an ALP of a fusion protein described herein comprises one or more substitutions.
  • an Fc of ALP-Fc fusion protein comprises S288P, F234A, and/or L235A substitutions.
  • an ALP-Fc fusion protein further comprises a deca-aspartate (DIO) tag.
  • an ALP-Fc fusion protein further comprises an octa-aspartate tag (D8).
  • an ALP-Fc fusion protein further comprises a linker between ALP and Fc components.
  • a linker comprises three continuous tetra-glycine and mono-serine (GGGGS) regions.
  • an ALP-Fc fusion protein comprises an IgGl Fc. In some embodiments, an ALP-Fc fusion protein comprises an IgGl Fc and an D8 tag. In some embodiments, an ALP-Fc fusion protein comprises: (1) an IgGl Fc comprising K392D, D399K, and K409D substitutions, (2) linker comprising three continuous GGGGS regions, and (3) a DIO tag.
  • an ALP-Fc fusion protein comprises: (1) an IgG4 Fc comprising K392D, D399K, and R409D substitutions, (2) an Fc of ALP-Fc fusion protein comprising S288P, F234A, and L235A substitutions, (3) linker comprising three continuous GGGGS regions, and (4) a DIO tag.
  • an ALP-Fc fusion protein comprises or has an amino acid sequence of any one of SEQ ID NOs: 62-75. In some embodiments, an ALP-Fc fusion protein is encoded by a nucleic acid sequence comprising any one of SEQ ID NOs: 55-61 and 87-90.
  • Hypophosphatasia is a rare, heritable disease that is distinguished by one or more autosomal recessive or dominant loss-of-function mutations in the gene ALPL, which encodes tissue-nonspecific alkaline phosphatase (TNALP; a.k.a. liver/bone/kidney type ALP).
  • TAALP tissue-nonspecific alkaline phosphatase
  • ALP Alkaline phosphatase
  • PPi depho sphorylating inorganic pyrophosphate
  • PPi inorganic pyrophosphate
  • HPP osteoblasts and chondrocytes impairs skeletal mineralization, leading to symptoms of varying severity, from rickets or osteomalacia to almost complete absence of bone mineralization in utero.
  • the current prevalence for HPP is unknown, but it is estimated that 1 in 100,000 live births and 1 in 200 Americans may be a carrier for HPP. HPP is also quite prevalent in other countries including Canada and Japan.
  • Subjects with persistently low ALP can experience debilitating symptoms that may progress with wide-ranging systemic complications.
  • Dysfunctional bone mineralization leads to skeletal and dental deformities with manifestation that depends on both subtype and age.
  • HPP There are two notable subtypes of HPP including severe HPP and moderate HPP.
  • Severe HPP is associated with deformed limbs, abnormally shaped chest/head, soft skulls, stunted growth, and life-threatening complications (still birth, brain pressure, seizures). Severe HPP is often diagnosed at perinatal and infantile stages.
  • Moderate HPP is associated with slow/stunted growth, delay in gross motor skills, bowed legs, enlarged joints, muscle weakness/pain, frequent bone injuries and fractures, and early tooth loss.
  • Moderate HPP is often diagnosed in childhood or adulthood.
  • Current treatment methods are limited toward enzyme replace therapy, which is only currently approved for pediatric-onset HPP and requires burdensome dosing frequency of six times per week. Further, the current dosing routine is associated with intensely painful injection with serious side effects, including but not limited to, lipodystrophy. As result, new treatments and therapeutics are strongly needed. The present disclosure sets out to address these serious health concerns with compositions and methods provided herein.
  • engineered B lineage cell populations that may be useful as a viable cell therapy option for treatment of HPP.
  • engineered B lineage cell populations prepared via methods disclosed herein express one or more therapeutic proteins, including alkaline phosphatase (ALP) or a fusion protein thereof, e.g., fusion proteins comprising ALP and a human immunoglobulin G fragment crystallizable region (IgG Fc), which are also referred to herein as “ALP-Fc fusion proteins.”
  • ALP-Fc fusion proteins comprise a Fc region derived from human immunoglobulin G1 (IgGl).
  • ALP-Fc fusion proteins comprise a Fc region derived from human immunoglobulin G4 (IgG4).
  • ALP-Fc fusion proteins comprise one or more substitutions.
  • an IgGl Fc of a fusion protein described herein comprises one or more substitutions.
  • an IgGl Fc comprises K392D, D399K, and/or K409D substitutions.
  • B lineage cell populations express a fusion protein comprising ALP and a human IgG4 Fc.
  • an IgG4 Fc of a fusion protein described herein comprises one or more substitutions.
  • an IgG4 Fc comprises K392D, D399K, and/or R409D substitutions.
  • an ALP is or comprises tissue non-specific alkaline phosphatase (TNSALP). In some embodiments, an ALP is or comprises the catalytic domain of TNSALP. In some embodiments, an ALP of a fusion protein described herein comprises one or more substitutions. In some embodiments, an Fc of ALP-Fc fusion protein comprises S288P, F234A, and/or L235A substitutions. In some embodiments, an ALP-Fc fusion protein further comprises a deca-aspartate tag (DIO). In some embodiments, an ALP-Fc fusion protein further comprises an octa-aspartate tag (D8). In some embodiments, an ALP-Fc fusion protein further comprises a linker between the ALP and Fc components. In some embodiments, a linker comprises three continuous tetra-glycine and mono- scrinc (GGGGS) regions.
  • Cell-based therapeutics are an emerging class of medicine that makes use of innate cellular machinery to combat disease. Unlike many traditional treatment methods, cell therapies make use of cellular localization, migration, and proliferation within the body, which can translate to improved biodistribution and targeted delivery of therapeutics. Cell therapies also benefit from cellular ability to sense and respond to various extrinsic signals within a subject, including, e.g., small molecules, other cells, physical forces, and/or marker proteins. Cellular persistence in vivo also enables cell therapies to survive, differentiate, function, etc. within a subject over extended time periods. These innate qualities may also lead to improved safety and efficacy for cell therapies as compared to other biologies or pharmaceutical compounds, providing long-lived, specifically targeted, adjustable, and/or responsive treatment for disease.
  • Cell-based therapies may have potential applications for a broad range of diseases, including those that have proved intractable or difficult to manage with traditional treatment options.
  • Diseases that have been targeted for cell-based treatment include, e.g., various cancers, autoimmune diseases, central nervous system (CNS) diseases, neurodegenerative disorders, inherited metabolic disorder, and cardiovascular diseases, among others.
  • CNS central nervous system
  • Cellular therapeutics offer an alternative over other treatment options for diseases where highly specific targeting (e.g., to a particular tissue type, area of the body, etc.) and/or longer-term treatment efficacy (e.g., enabling lower dosage frequency, single treatment options, etc.) are highly favored or necessary.
  • Cell therapies may employ a number of different cell types, which are typically modified to provide a therapeutic effect (e.g., transgene expression, reprogrammed cellular targeting, etc.). Although many cell types have potential to provide some form of therapeutic effect, recent therapies have heavily favored adaptive immune cells such as T lymphocytes and B lymphocytes (referred to interchangeably herein as T cells and B cells, respectively). For example, chimeric antigen receptor T (CAR-T) cells have been engineered to treat various cancers through recognition of one or more tumor cell markers, leading to cytotoxic destruction of tumor cells. CAR-T cells arc also being adapted to treat infectious diseases (e.g., HIV) through recognition of other targeted antigens. Recent engineering efforts are focused on enhancing CAR-T receptor functionality, reducing innate immune response to CAR-T, and developing allogeneic therapies that make use of donor cells.
  • CAR-T chimeric antigen receptor T
  • Monoclonal antibodies can be produced to target antigens within the body, which is valuable for treatment of diseases where such antibodies cannot be induced through natural processes (e.g., self-antigens for cancer and/or autoimmune diseases, antigens that fail to elicit natural immune response through infection and/or vaccination, etc.).
  • Current antibody therapies require frequent administration and are costly to produce.
  • researchers have attempted to address these issues through use of gene therapy, which makes use of various techniques (e.g., viral vectors, CRISPR/Cas9 editing) to deliver antibody payloads to endogenous cells in the body, leading to persistent antibody production within a subject.
  • these approaches can result in low levels of antibody expression and a counter- active response by the subject’s own immune system.
  • B lineage cells are a highly desirable cell-based target to secrete other payloads including, but not limited to, enzymes, complement proteins, cytokines, cytokine receptors, chimeric antigen receptors (CARs), anti-fibrotic molecules, anti- thrombotic molecules, antigens, both wild type and variant proteins, coagulation factors, glucose response elements, and fragments of antibodies, antigens, and proteins.
  • CARs chimeric antigen receptors
  • B lineage cells are an attractive option for development of cell-based therapies and may potentially offer improved therapeutic effects (e.g., payload delivery, targeting, long-term payload expression, reduced auto-immune response, etc.) as compared to traditional therapies (e.g., antibody -based therapeutics, other cell therapies, etc.).
  • a B lineage cell is a cell that expresses one or more B cell receptors (BCR) on a cell membrane.
  • BCR B cell receptors
  • a B lineage cell is a modified version or variant of a cell that expresses one or more B cell receptors (BCRs) on a cell membrane.
  • a B lineage cell is a naive or memory B cell.
  • a B lineage cell is a cell derived from a naive B cell (e.g., activated B lineage cell, plasmablast, plasma cell) or a valiant thereof.
  • a B lineage cell is an activated B lineage cell.
  • a B lineage cell is a plasmablast.
  • a B lineage cell is a plasma cell.
  • a B lineage cell population comprise naive B cells.
  • naive B cell populations are used as reference cell populations.
  • naive B cell populations express CD 19 (CD19 + ).
  • expression of CD 19 in naive B cell populations is used as a reference to assist in characterization of other B lineage cell populations.
  • naive B cell populations express CD20 (CD20 + ).
  • expression of CD20 in naive B cell populations is used as a reference to assist in characterization of other B lineage cell populations.
  • naive B cell populations express low amounts of CD27 (CD27 10 ).
  • expression of CD27 in naive B cell populations are as a reference to assist in characterization of other B lineage cell populations.
  • naive B cell populations express low amounts of CD38 (CD38 10 ).
  • expression of CD38 in naive B cell populations are used as a reference to assist in characterization of other B lineage cell populations.
  • naive B cell populations express low amounts of CD 138 (CD138 10 ).
  • expression of CD138 in naive B cell populations arc used as a reference to assist in characterization of other B lineage cell population.
  • a B lineage cell population comprises activated B lineage cells.
  • activated B lineage cell populations are used as reference cell populations.
  • activated B lineage cell populations are compared to a reference cell population (e.g., naive B cell population).
  • activated B lineage cell populations express lower amounts of CD 19 (CD19 10 ) as compared to a reference cell population (e.g., naive B cell populations).
  • activated B lineage cell populations express different e.g., higher or lower) amounts of CD 19 as compared to a reference cell population (e.g., differentiated B cell populations, etc.).
  • activated B lineage cell populations express lower amounts of CD20 (CD20 10 ) as compared to a reference cell population (e.g., naive B cell populations). In some embodiments, activated B lineage cell populations express different (e.g., higher or lower) amounts of CD20 as compared to a reference cell population (e.g., differentiated B cell populations, etc.). In some embodiments, activated B lineage cell populations express higher amounts of CD27 (CD27 hl ) as compared to a reference cell population (e.g., naive B cell populations).
  • activated B lineage cell populations express different (e.g., higher or lower) amounts of CD27 as compared to a reference cell population (e.g., differentiated B cell populations, etc.). In some embodiments, activated B lineage cell populations express lower amounts of CD38 (CD38 10 ) as compared to a reference cell population (e.g., naive B cell populations). In some embodiments, activated B lineage cell populations express different (e.g., higher or lower) amounts of CD38 as compared to a reference cell population (e.g., differentiated B cell populations, etc.).
  • activated B lineage cell populations express lower amounts of CD 138 (CD138 10 ) as compared to a reference cell population (e.g., naive B cell populations). In some embodiments, activated B lineage cell populations express different (e.g., higher or lower) amounts of CD138 as compared to a reference cell population (e.g., differentiated B cell populations, etc.).
  • a B lineage cell population may comprise plasmablast cells.
  • plasmablast cell populations are used as reference cell populations.
  • plasmablast cell populations are compared to a reference cell population (e.g., naive B cell population).
  • plasmablast cell populations express lower amounts of CD 19 (CD19 10 ) as compared to a reference cell population (e.g., naive B cell populations).
  • plasmablast cell populations express different (e.g., higher or lower) amounts of CD 19 as compared to a reference cell population (e.g., activated cell populations, plasma cell populations, etc.).
  • plasmablast cell populations express lower amounts of CD20 (CD20 10 ) as compared to a reference cell population (e.g., naive B cell populations). In some embodiments, plasmablast cell populations express different (e.g., higher or lower) amounts of CD20 as compared to a reference cell population (e.g., activated cell populations, plasma cell populations, etc.). In some embodiments, plasmablast cell populations express higher amounts of CD27 (CD27 hl ) as compared to a reference cell population (e.g., naive B cell populations).
  • plasmablast cell populations express different (e.g., higher or lower) amounts of CD27 as compared to a reference cell population (e.g., activated cell populations, plasma cell populations, etc.).
  • plasmablast cell populations express higher amounts of CD38 (CD38 hl ) as compared to a reference cell population (e.g., naive B cell populations).
  • plasmablast cell populations express different (e.g., higher or lower) amounts of CD38 as compared to a reference cell population (e.g., activated cell populations, plasma cell populations, etc.).
  • plasmablast cell populations express lower amounts of CD138 (CD138 10 ) as compared to a reference cell population (e.g., naive B cell populations). In some embodiments, plasmablast cell populations express different (e.g., higher or lower) amounts of CD138 as compared to a reference cell population (e.g., activated cell populations, plasma cell populations, etc.).
  • a B lineage cell population comprises plasma cells.
  • plasma cell populations are used as reference cell populations.
  • plasma cell populations are compared to a reference cell population (e.g., naive B cell population).
  • plasma cell populations express lower amounts of CD 19 (CD19 10 ) as compared to a reference cell population (e.g., naive B cell populations).
  • plasma cell populations express different (e.g., higher or lower) amounts of CD 19 as compared to a reference cell population (e.g., activated cell populations, plasmablast populations, etc.).
  • plasma cell populations express lower amounts of CD20 (CD20 10 ) as compared to a reference cell population (e.g., naive B cell populations). In some embodiments, plasma cell populations express different (e.g., higher or lower) amounts of CD20 as compared to a reference cell population (e.g., activated cell populations, plasmablast populations, etc.). In some embodiments, plasma cell populations express higher amounts of CD27 (CD27 hl ) as compared to a reference cell population (e.g., naive B cell populations). In some embodiments, plasma cell populations express different (e.g., higher or lower) amounts of CD27 as compared to a reference cell population (e.g., activated cell populations, plasmablast populations, etc.).
  • a reference cell population e.g., activated cell populations, plasmablast populations, etc.
  • engineered cells for various uses including, e.g., cell therapies is an active area of development. Genomic and epigenomic modifications, synthetic biology, and application of biomaterials may be employed to generate engineered cells with desired properties for therapeutic applications. Selection of an appropriate method for generating engineered cells is typically dependent upon the desired output and effects of cell therapy and requires optimization for different cell types, transgenes of interest, etc. It is understood in the art that engineering methods that are effective for a particular cell type may be less effective (or not viable) for another cell type. Furthermore, engineering methods may vary depending upon whether a therapy requires cellular localization, expression of an endogenous or exogenous protein, removal of an endogenous protein, etc.
  • Various B lineage cell engineering techniques are described in the art, including, e.g., CRISPR/Cas9, AAV, lentiviral, and recombinase-based methods. These methods are generally employed to introduce a payload (e.g., expression cassette, transgene, etc.) into naive B cells in order to express a protein of interest.
  • a payload may be designed for cpisomal expression, integration into a specific target locus (e.g., endogenous target gene locus), or integration into a non-specific locus (e.g., endogenous random or non-target gene locus).
  • Payloads may be designed such that an endogenous target gene locus continues to produce functional protein and/or fulfill its natural function (non-disruptive integration). Pay loads may also be designed to intentionally disrupt an endogenous target gene locus to produce lowered or non-detectable levels of functional protein and/or some amount of non-functional protein.
  • Methods for integration of a pay load (e.g., expression cassette comprising one or more transgenes) into an endogenous target locus may comprise site-specific cleavage with a targeted nuclease (e.g., Cas protein, including Cas9), followed by integration of a transgene (e.g., ALP or a fusion protein thereof, such as ALP-Fc fusion protein) through an endogenous repair pathway (e.g., homologous recombination, homology-directed repair, etc.).
  • a pay load e.g., expression cassette comprising one or more transgenes
  • a transgene e.g., ALP or a fusion protein thereof, such as ALP-Fc fusion protein
  • endogenous repair pathway e.g., homologous recombination, homology-directed repair, etc.
  • methods for integration of an expression cassette comprising an ALP or a fusion protein thereof (e.g., an ALP-Fc fusion protein) encoding transgene comprise site-specific cleavage at a target locus (e.g., CCR5) with a guide RNA / Cas9 complex, followed by integration of an ALP or a fusion protein thereof at the target locus through homologous recombination.
  • a method of B lineage cell engineering is or comprises administration of a ribonuclcoprotcin (RNP) to a cell population.
  • RNP ribonuclcoprotcin
  • a method of B lineage cell engineering is or comprises administration of a composition comprising a Cas protein complexed with guide RNA (gRNA) to a cell population.
  • a method of B lineage cell engineering is or comprises administration of a composition comprising a Cas9/guide RNA complex to a cell population.
  • a method of B lineage cell engineering is or comprises administration of a composition comprising a Cas9/guide RNA complex to a cell population.
  • a method of B lineage cell engineering is or comprises administration of a composition comprising a payload (e.g., expression cassette comprising one or more transgenes) of interest to a cell population.
  • a payload e.g., expression cassette comprising one or more transgenes
  • a method of B lineage cell engineering is or comprises administration of a composition comprising a payload (e.g., expression cassette comprising one or more transgenes) to a cell population through use of a viral vector.
  • a method of B lineage cell engineering is or comprises administration of a composition comprising a payload (e.g., expression cassette comprising one or more transgenes) encapsulated within an AAV capsid (e.g., AAV2, AAV3, AAV5, AAV6, AAV8, etc.) to a cell population.
  • AAV capsid e.g., AAV2, AAV3, AAV5, AAV6, AAV8, etc.
  • a method of B lineage cell engineering is or comprises administration of a composition comprising a transgene encapsulated within an AAV capsid (e.g., AAV2, AAV3, AAV5, AAV6, AAV8, etc.) in combination with or in addition to administration of a composition comprising a Cas9/gRNA complex.
  • AAV capsid e.g., AAV2, AAV3, AAV5, AAV6, AAV8, etc.
  • a method of B lineage cell engineering comprises a step of electroporation to facilitate cellular uptake of one or more engineering components. In some embodiments, a method of B lineage cell engineering comprises a step of electroporation to facilitate cellular uptake of a Cas9/gRNA complex. In some embodiments, a method of B lineage cell engineering comprises a step of electroporation to facilitate cellular uptake of a Cas9/gRNA complex and a payload.
  • a method of B lineage cell engineering may comprise a step of electroporation to facilitate cellular uptake of a Cas9/gRNA complex and a pay load (e.g., expression cassette comprising one or more transgenes) encapsulated in an AAV capsid (AAV2, AAV3, AAV5, AAV6, AAV8, etc.).
  • a method of B lineage cell engineering comprises a step of electroporation to facilitate cellular uptake of a Cas9/gRNA complex and a payload (e.g., expression cassette comprising one or more transgcncs) encapsulated in an AAV6 capsid.
  • a method of B lineage cell engineering comprises a step of viral transduction to facilitate cellular uptake of a payload (e.g., expression cassette comprising one or more transgenes).
  • a method of B lineage cell engineering comprises a step of viral transduction to facilitate cellular' uptake of a payload (e.g., transgene) encapsulated in an AAV capsid (AAV2, AAV3, AAV5, AAV6, AAV8, etc.).
  • a method of B lineage cell engineering comprises a step of viral transduction to facilitate cellular uptake of a pay load (e.g., expression cassette comprising one or more transgenes) encapsulated in an AAV6 capsid.
  • a method of B lineage cell engineering comprises one or more steps of: (i) electroporation to facilitate cellular uptake of a Cas9/gRNA complex; and (ii) viral transduction to facilitate cellular' uptake of a payload encapsulated in an AAV capsid.
  • a method of cell engineering is particularly effective for one type of cell (e.g., T cell) and less effective for another type of cell (e.g., B cell).
  • a method of B lineage cell engineering provides improved genome editing efficiency in B lineage cells as compared to other cell types (e.g., T cell).
  • a method of B cell engineering provides at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% editing efficiency.
  • a method of B lineage cell engineering provides at least about 1E6, 1.5E6, 2E6, 2.5E6, 3E6, 3.5E6, 4E6, 4.5E6, 5E6, 5.5E6, 6E6, 6.5E6, 7E6, 7.5E6, 8E6, 8.5E6, 9E6, 9.5E6, 1E7, 1.5E7, 2E7, 2.5E7, 3E7, 3.5E7, 4E7, 4.5E7, or 5E7 edited B lineage cells.
  • a method of B lineage cell engineering comprises a step of editing activated B cells.
  • a method of B lineage cell engineering comprises a step of editing B lineage cells after an activation step of about 1, 2, 3, 4, or 5 day(s).
  • a method of B lineage cell engineering comprises a step of editing B lineage cells after an activation step of 2 days.
  • a method of B lineage cell engineering comprises a step of editing B lineage cells after an activation step of about 1, 2, 3, 4, or 5 day(s) and expanding edited B lineage cells in the activation media for an additional period of about 1, 2, 3, 4, 5, 6, 7, or 8 days.
  • a method of B lineage cell engineering comprises a step of editing B lineage cells after an activation step of 2 days and expanding edited B lineage cells in the activation media for an additional 6 days.
  • a gRNA is or comprises a polynucleotide sequence in Table 8.
  • a gRNA is or comprises SEQ ID NO: 79.
  • a gRNA is or comprises SEQ ID NO: 80.
  • a gRNA is or comprises SEQ ID NO: 81.
  • a gRNA is or comprises SEQ ID NO: 82.
  • a gRNA is or comprises SEQ ID NO: 83.
  • a gRNA is or comprises SEQ ID NO: 84.
  • a gRNA is or comprises SEQ ID NO: 85.
  • a gRNA is or comprises SEQ ID NO: 86. In some embodiments, a gRNA is or comprises SEQ ID NO: 129. In some embodiments, a gRNA is or comprises SEQ ID NO: 130. In some embodiments, a gRNA is or comprises SEQ ID NO: 131. In some embodiments, a gRNA is or comprises SEQ ID NO: 132.
  • engineered cells comprising one or more payloads.
  • engineered cells e.g., B lineage cell populations, etc.
  • any of a variety of payloads may be used (e.g., those with a therapeutic or monitoring purpose), alone or in combination.
  • a payload is or comprises a polynucleotide sequence encoding a peptide or polypeptide.
  • a payload is or comprises one or more transgenes.
  • a pay load is or comprises one or more homology arm sequences.
  • a pay load is or comprises a transgene flanked by one or more homology sequences.
  • a payload may be or comprise a polynucleotide sequence, which comprises an expression cassette.
  • an expression cassette comprises one or more polynucleotide sequence elements (e.g., promoters, enhancers, transgenes, termination elements, homology arms, biomarkers, signal peptide sequences, internal ribosome entry site elements, self-cleaving peptide sequences, ubiquitous chromatin opening element, etc.).
  • an expression cassette comprises one or more polynucleotide sequence elements (e.g., promoters, enhancers, transgenes, termination elements, homology arms, biomarkers, signal peptide sequences, internal ribosome entry site elements, self-cleaving peptide sequences, ubiquitous chromatin opening element, etc.) in a particular configuration and/or combination.
  • polynucleotide sequence elements e.g., promoters, enhancers, transgenes, termination elements, homology arms, biomarkers, signal peptide sequences, internal ribosome entry site elements, self-cleaving peptide sequences, ubiquitous chromatin opening element, etc.
  • an expression cassette comprises one or more polynucleotide sequence elements (e.g., promoters, enhancers, transgenes, termination elements, homology arms, biomarkers, signal peptide sequences, internal ribosome entry site elements, self-cleaving peptide sequences, ubiquitous chromatin opening element, etc.) in a particular configuration and/or combination in order to promote expression of a transgene in a cell population.
  • polynucleotide sequence elements e.g., promoters, enhancers, transgenes, termination elements, homology arms, biomarkers, signal peptide sequences, internal ribosome entry site elements, self-cleaving peptide sequences, ubiquitous chromatin opening element, etc.
  • an expression cassette comprises one or more polynucleotide sequence elements (e.g., promoters, enhancers, transgenes, termination elements, homology arms, biomarkers, signal peptide sequences, internal ribosome entry site elements, self-cleaving peptide sequences, ubiquitous chromatin opening element, etc.) in a particular configuration and/or combination in order to promote expression of a transgene in an engineered B lineage cell population.
  • polynucleotide sequence elements e.g., promoters, enhancers, transgenes, termination elements, homology arms, biomarkers, signal peptide sequences, internal ribosome entry site elements, self-cleaving peptide sequences, ubiquitous chromatin opening element, etc.
  • an expression cassette comprises one or more polynucleotide sequences encoding one or more signal peptides.
  • a polynucleotide sequence encoding a signal peptide is or comprises a sequence of ATGCCGTCTTCTGTCTCGTGGGGCATCCTCCTGCTGGCAGGCCTGTGCTGCCTGGTCC CTGTCTCCCTGGCT (SEQ ID NO: 143).
  • a polynucleotide sequence encoding a signal peptide is or comprises a sequence with at least 70% sequence identity to SEQ ID NO: 143.
  • a polynucleotide sequence encoding a signal peptide is or comprises a sequence with at least 75% sequence identity to SEQ ID NO: 143. In some embodiments, a polynucleotide sequence encoding a signal peptide is or comprises a sequence with at least 80% sequence identity to SEQ ID NO: 143. In some embodiments, a polynucleotide sequence encoding a signal peptide is or comprises a sequence with at least 85% sequence identity to SEQ ID NO: 143. In some embodiments, a polynucleotide sequence encoding a signal peptide is or comprises a sequence with at least 90% sequence identity to SEQ ID NO: 143.
  • a polynucleotide sequence encoding a signal peptide is or comprises a sequence with at least 95% sequence identity to SEQ ID NO: 143. In some embodiments, a polynucleotide sequence encoding a signal peptide is or comprises a sequence with at least 99% sequence identity to SEQ ID NO: 143. In some embodiments, a polynucleotide sequence encoding a signal peptide consists of a polynucleotide sequence of SEQ ID NO: 143. In some embodiments, a signal peptide is or comprises a polypeptide sequence of MPSSVSWGILLLAGLCCLVPVSLA (SEQ ID NO: 144).
  • a signal peptide is or comprises a polypeptide sequence with at least 70% sequence identity to SEQ ID NO: 144. In some embodiments, a signal peptide is or comprises a polypeptide sequence with at least 75% sequence identity to SEQ ID NO: 144. In some embodiments, a signal peptide is or comprises a polypeptide sequence with at least 80% sequence identity to SEQ ID NO: 144. In some embodiments, a signal peptide is or comprises a polypeptide sequence with at least 85% sequence identity to SEQ ID NO: 144. In some embodiments, a signal peptide is or comprises a polypeptide sequence with at least 90% sequence identity to SEQ ID NO: 144.
  • a signal peptide is or comprises a polypeptide sequence with at least 95% sequence identity to SEQ ID NO: 144. In some embodiments, a signal peptide is or comprises a polypeptide sequence with at least 99% sequence identity to SEQ ID NO: 144. In some embodiments, a signal peptide consists of a polypeptide sequence of SEQ ID NO: 144.
  • an expression cassette comprises one or more polynucleotide sequences encoding one or more promoters (e.g., MND, CMV, FEEK I, SFFV, PGK, EF-la, B2M, etc.).
  • an expression cassette comprises one or more polynucleotide sequences encoding one or more exogenous promoters.
  • an expression cassette comprises one or more polynucleotide sequences encoding one or more endogenous promoters.
  • an expression cassette does not comprise one or more promoters.
  • an expression cassette does not comprise one or more exogenous promoters.
  • an expression cassette does not comprise one or more endogenous promoters.
  • a promoter is or comprises one or more polynucleotide sequences in Table 4.
  • a promoter is or comprises SEQ ID NO: 76.
  • a promoter is or comprises SEQ ID NO: 125.
  • a promoter is or comprises SEQ ID NO: 126.
  • a promoter is or comprises SEQ ID NO: 127.
  • a promoter is or comprises SEQ ID NO: 128.
  • a promoter is or comprises a polynucleotide sequence of SEQ ID NO: 76. In some embodiments, a promoter is or comprises a polynucleotide sequence having at least 70% sequence identity to SEQ ID NO: 76. In some embodiments, a promoter is or comprises a polynucleotide sequence having at least 75% sequence identity to SEQ ID NO: 76. In some embodiments, a promoter is or comprises a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 76. In some embodiments, a promoter is or comprises a polynucleotide sequence having at least 85% sequence identity to SEQ ID NO: 76.
  • a promoter is or comprises a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 76. In some embodiments, a promoter is or comprises a polynucleotide sequence having at least 95% sequence identity to SEQ ID NO: 76. In some embodiments, a promoter is or comprises a polynucleotide sequence having at least 99% sequence identity to SEQ ID NO: 76. In some embodiments, a promoter consists of a polynucleotide sequence of SEQ ID NO: 76.
  • an expression cassette comprises one or more polynucleotide sequences encoding a translation initiation site (e.g., Kozak consensus sequence, ribosomal binding site, etc.). In some embodiments, an expression cassette comprises one or more exogenous polynucleotide sequences encoding a translation initiation site. In some embodiments, an expression cassette comprises one or more endogenous polynucleotide sequences encoding a translation initiation site. In some embodiments, an expression cassette does not comprise one or more polynucleotide sequences encoding a translation initiation site.
  • an expression cassette does not comprise one or more exogenous polynucleotide sequences encoding a translation initiation site. In some embodiments, an expression cassette does not comprise one or more endogenous polynucleotide sequences encoding a translation initiation site.
  • an expression cassette comprises one or more polynucleotide sequences encoding one or more enhancers (e.g., WPRE, beta-globin, Ep etc.) or a variant thereof.
  • an expression cassette comprises one or more polynucleotide sequences encoding one or more exogenous enhancers.
  • an expression cassette comprises one or more polynucleotide sequences encoding one or more endogenous enhancers.
  • an enhancer may be viral (e.g., WPRE, etc.) or non-viral.
  • an expression cassette does not comprise one or more enhancers.
  • an expression cassette does not comprise one or more exogenous enhancers.
  • an expression cassette does not comprise one or more endogenous enhancers.
  • one or more enhancers is an Ep enhancer or a variant thereof. In some embodiments, one or more enhancers is a murine Ep enhancer or a variant thereof. In some embodiments, one or more enhancers is a human Ep enhancer or a variant thereof. In some embodiments, one or more enhancers is a primate Ep enhancer or a variant thereof. In some embodiments, a Ep enhancer is a truncated, extended, and/or mutated version of a wild-type Ep enhancer. In some embodiments, an Ep enhancer may be or comprise a fusion with one or more additional sequence elements (e.g., promoter, enhancer, signal peptide, polyA, etc.).
  • additional sequence elements e.g., promoter, enhancer, signal peptide, polyA, etc.
  • an expression cassette comprises an Ep enhancer in combination with another enhancer e.g., WPRE, beta-globin, Ep etc.).
  • an Ep enhancer may be oriented in a forward (e.g., 5’ to 3’) or reverse (e.g., 3’ to 5’) orientation within a payload.
  • an Ep enhancer may be in different positions within a payload.
  • an Ep enhancer may be upstream of (e.g., 5’ to) a transgene in a payload.
  • an Ep enhancer may be downstream of (e.g., 3’ to) a transgene in a payload.
  • an enhancer is or comprises a polynucleotide sequence in Table 9.
  • an enhancer is or comprises a polynucleotide sequence of SEQ ID NO: 95. In some embodiments, an enhancer is or comprises a polynucleotide sequence with at least 70% sequence identity to SEQ ID NO: 95. In some embodiments, an enhancer is or comprises a polynucleotide sequence with at least 75% sequence identity to SEQ ID NO: 95. In some embodiments, an enhancer is or comprises a polynucleotide sequence with at least 80% sequence identity to SEQ ID NO: 95. In some embodiments, an enhancer is or comprises a polynucleotide sequence with at least 85% sequence identity to SEQ ID NO: 95.
  • an enhancer is or comprises a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 95. In some embodiments, an enhancer is or comprises a polynucleotide sequence with at least 95% sequence identity to SEQ ID NO: 95. In some embodiments, an enhancer is or comprises a polynucleotide sequence with at least 99% sequence identity to SEQ ID NO: 95. In some embodiments, an enhancer consists of a polynucleotide sequence of SEQ ID NO: 95. [0169] In some embodiments, an enhancer is or comprises a polynucleotide sequence of SEQ ID NO: 96.
  • an enhancer is or comprises a polynucleotide sequence with at least 70% sequence identity to SEQ ID NO: 96. In some embodiments, an enhancer is or comprises a polynucleotide sequence with at least 75% sequence identity to SEQ ID NO: 96. In some embodiments, an enhancer is or comprises a polynucleotide sequence with at least 80% sequence identity to SEQ ID NO: 96. In some embodiments, an enhancer is or comprises a polynucleotide sequence with at least 85% sequence identity to SEQ ID NO: 96. In some embodiments, an enhancer is or comprises a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 96.
  • an enhancer is or comprises a polynucleotide sequence with at least 95% sequence identity to SEQ ID NO: 96. In some embodiments, an enhancer is or comprises a polynucleotide sequence with at least 99% sequence identity to SEQ ID NO: 96. In some embodiments, an enhancer consists of a polynucleotide sequence of SEQ ID NO: 96.
  • an enhancer is or comprises a polynucleotide sequence of SEQ ID NO: 97. In some embodiments, an enhancer is or comprises a polynucleotide sequence with at least 70% sequence identity to SEQ ID NO: 97. In some embodiments, an enhancer is or comprises a polynucleotide sequence with at least 75% sequence identity to SEQ ID NO: 97. In some embodiments, an enhancer is or comprises a polynucleotide sequence with at least 80% sequence identity to SEQ ID NO: 97. In some embodiments, an enhancer is or comprises a polynucleotide sequence with at least 85% sequence identity to SEQ ID NO: 97.
  • an enhancer is or comprises a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 97. In some embodiments, an enhancer is or comprises a polynucleotide sequence with at least 95% sequence identity to SEQ ID NO: 97. In some embodiments, an enhancer is or comprises a polynucleotide sequence with at least 99% sequence identity to SEQ ID NO: 97. In some embodiments, an enhancer consists of a polynucleotide sequence of SEQ ID NO: 97.
  • an enhancer is or comprises a polynucleotide sequence of SEQ ID NO: 98. In some embodiments, an enhancer is or comprises a polynucleotide sequence with at least 70% sequence identity to SEQ ID NO: 98. In some embodiments, an enhancer is or comprises a polynucleotide sequence with at least 75% sequence identity to SEQ ID NO: 98. In some embodiments, an enhancer is or comprises a polynucleotide sequence with at least 80% sequence identity to SEQ ID NO: 98. In some embodiments, an enhancer is or comprises a polynucleotide sequence with at least 85% sequence identity to SEQ ID NO: 98.
  • an enhancer is or comprises a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 98. In some embodiments, an enhancer is or comprises a polynucleotide sequence with at least 95% sequence identity to SEQ ID NO: 98. In some embodiments, an enhancer is or comprises a polynucleotide sequence with at least 99% sequence identity to SEQ ID NO: 98. In some embodiments, an enhancer consists of a polynucleotide sequence of SEQ ID NO: 98.
  • an enhancer is or comprises a polynucleotide sequence of SEQ ID NO: 99. In some embodiments, an enhancer is or comprises a polynucleotide sequence with at least 70% sequence identity to SEQ ID NO: 99. In some embodiments, an enhancer is or comprises a polynucleotide sequence with at least 75% sequence identity to SEQ ID NO: 99. In some embodiments, an enhancer is or comprises a polynucleotide sequence with at least 80% sequence identity to SEQ ID NO: 99. In some embodiments, an enhancer is or comprises a polynucleotide sequence with at least 85% sequence identity to SEQ ID NO: 99.
  • an enhancer is or comprises a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 99. In some embodiments, an enhancer is or comprises a polynucleotide sequence with at least 95% sequence identity to SEQ ID NO: 99. In some embodiments, an enhancer is or comprises a polynucleotide sequence with at least 99% sequence identity to SEQ ID NO: 99. In some embodiments, an enhancer consists of a polynucleotide sequence of SEQ ID NO: 99.
  • an expression cassette comprises one or more polynucleotide sequences encoding one or more terminators (e.g., polyA, including, e.g., BGH polyA, SV40 polyA, etc.). In some embodiments, an expression cassette comprises one or more polynucleotide sequences encoding one or more exogenous terminators. In some embodiments, an expression cassette comprises one or more polynucleotide sequences encoding one or more endogenous terminators. In some embodiments, an expression cassette does not comprise one or more terminators. In some embodiments, an expression cassette does not comprise one or more exogenous terminators. In some embodiments, an expression cassette does not comprise one or more endogenous terminators.
  • terminators e.g., polyA, including, e.g., BGH polyA, SV40 polyA, etc.
  • an expression cassette comprises one or more polynucleotide sequences encoding one or more exogenous terminators.
  • an expression cassette does not comprise one or more termin
  • a terminator is or comprises a polynucleotide sequence of SEQ ID NO: 78. In some embodiments, a terminator is or comprises a polynucleotide sequence having at least 70% sequence identity to SEQ ID NO: 78. In some embodiments, a terminator is or comprises a polynucleotide sequence having at least 75% sequence identity to SEQ ID NO: 78. In some embodiments, a terminator is or comprises a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 78. In some embodiments, a terminator is or comprises a polynucleotide sequence having at least 85% sequence identity to SEQ ID NO: 78.
  • a terminator is or comprises a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 78. In some embodiments, a terminator is or comprises a polynucleotide sequence having at least 95% sequence identity to SEQ ID NO: 78. In some embodiments, a terminator is or comprises a polynucleotide sequence having at least 99% sequence identity to SEQ ID NO: 78. In some embodiments, a terminator consists of a polynucleotide sequence of SEQ ID NO: 78.
  • an expression cassette comprises a polynucleotide sequence encoding a transgene (e.g., ALP or a fusion protein thereof, such as an ALP-Fc fusion protein) or variant thereof.
  • a transgene e.g., ALP or a fusion protein thereof, such as an ALP-Fc fusion protein
  • an expression cassette comprises a polynucleotide sequence encoding ALP, or a variant thereof.
  • an expression cassette comprises a polynucleotide sequence encoding a functional ALP valiant.
  • an expression cassette comprises a polynucleotide sequence encoding ALP or a fusion protein thereof e.g., an ALP-Fc fusion protein or a variant thereof).
  • an expression cassette comprises a polynucleotide sequence encoding a fusion protein comprising ALP and a human IgGl Fc.
  • an IgGl Fc of a fusion protein described herein comprises one or more substitutions.
  • an IgGl Fc comprises K392D, D399K, and/or K409D substitutions.
  • an expression cassette comprises a polynucleotide sequence encoding a fusion protein comprising ALP and a human IgG4 Fc.
  • an IgG4 Fc of a fusion protein described herein comprises one or more substitutions.
  • an IgG4 Fc comprises K392D, D399K, and/or R409D substitutions.
  • an ALP is or comprises tissue non-specific alkaline phosphatase (TNSALP).
  • an ALP is or comprises the catalytic domain of TNSALP.
  • an ALP of a fusion protein described herein comprises one or more substitutions.
  • an Fc of ALP-Fc fusion protein comprises S288P, F234A, and/or L235A substitutions.
  • an ALP-Fc fusion protein further comprises a deca-aspartate tag (DIO).
  • an ALP-Fc fusion protein further comprises an octa-aspartate tag (D8).
  • an ALP-Fc fusion protein further comprises a linker between the ALP and Fc components.
  • a linker is or comprises three continuous tetra-glycine and mono-serine (GGGGS) regions.
  • a linker is encoded by a polynucleotide sequence that is or comprises CTGAAGGAGAGCAAGTACGGCCCCCCC (SEQ ID NO: 140).
  • a linker is encoded by a polynucleotide sequence having at least about 70% sequence identity to SEQ ID NO: 140.
  • a linker is encoded by a polynucleotide sequence having at least about 75% sequence identity to SEQ ID NO: 140.
  • a linker is encoded by a polynucleotide sequence having at least about 80% sequence identity to SEQ ID NO: 140. In some embodiments, a linker is encoded by a polynucleotide sequence having at least about 85% sequence identity to SEQ ID NO: 140. In some embodiments, a linker is encoded by a polynucleotide sequence having at least about 90% sequence identity to SEQ ID NO: 140. In some embodiments, a linker is encoded by a polynucleotide sequence having at least about 95% sequence identity to SEQ ID NO: 140.
  • a linker is encoded by a polynucleotide sequence having at least about 99% sequence identity to SEQ ID NO: 140. In some embodiments, a linker is encoded by a polynucleotide sequence consisting of SEQ ID NO: 140. In some embodiments, a linker comprises a polypeptide sequence that is or comprises LKESKYGPP (SEQ ID NO: 141). In some embodiments, a linker is or comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 141. In some embodiments, a linker is or comprises a polypeptide sequence having at least 75% sequence identity to SEQ ID NO: 141.
  • a linker is or comprises a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 141. In some embodiments, a linker is or comprises a polypeptide sequence having at least 85% sequence identity to SEQ ID NO: 141 . In some embodiments, a linker is or comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 141. In some embodiments, a linker is or comprises a polypeptide sequence having at least 95% sequence identity to SEQ ID NO: 141 . In some embodiments, a linker is or comprises a polypeptide sequence having at least 99% sequence identity to SEQ ID NO: 141. In some embodiments, a linker consists of a polypeptide sequence of SEQ ID NO: 141.
  • an expression cassette is or comprises a polynucleotide sequence in Table 2.
  • an expression cassette is or comprises SEQ ID NO: 55.
  • an expression cassette is or comprises SEQ ID NO: 56.
  • an expression cassette is or comprises SEQ ID NO: 57.
  • an expression cassette is or comprises SEQ ID NO: 58.
  • an expression cassette is or comprises SEQ ID NO: 59.
  • an expression cassette is or comprises SEQ ID NO: 60.
  • an expression cassette is or comprises SEQ ID NO: 61.
  • an expression cassette is or comprises SEQ ID NO: 87.
  • an expression cassette is or comprises SEQ ID NO: 88. In some embodiments, an expression cassette is or comprises SEQ ID NO: 89. In some embodiments, an expression cassette is or comprises SEQ ID NO: 90. In some embodiments, an expression cassette is or comprises SEQ ID NO: 100. In some embodiments, an expression cassette is or comprises SEQ ID NO: 102. In some embodiments, an expression cassette is or comprises SEQ ID NO: 103. In some embodiments, an expression cassette is or comprises SEQ ID NO: 104. In some embodiments, an expression cassette is or comprises SEQ ID NO: 105. In some embodiments, an expression cassette is or comprises SEQ ID NO: 106. In some embodiments, an expression cassette is or comprises SEQ ID NO: 107. In some embodiments, an expression cassette is or comprises SEQ ID NO: 108.
  • an expression cassette is or comprises a polynucleotide sequence of SEQ ID NO: 100. In some embodiments, an expression cassette is or comprises a polynucleotide sequence having at least 70% sequence identity to SEQ ID NO: 100. In some embodiments, an expression cassette is or comprises a polynucleotide sequence having at least 75% sequence identity to SEQ ID NO: 100. In some embodiments, an expression cassette is or comprises a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 100. In some embodiments, an expression cassette is or comprises a polynucleotide sequence having at least 85% sequence identity to SEQ ID NO: 100.
  • an expression cassette is or comprises a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 100. In some embodiments, an expression cassette is or comprises a polynucleotide sequence having at least 95% sequence identity to SEQ ID NO: 100. In some embodiments, an expression cassette is or comprises a polynucleotide sequence having at least 99% sequence identity to SEQ ID NO: 100. In some embodiments, an expression cassette consists of a polynucleotide sequence of SEQ ID NO: 100.
  • an expression cassette is or comprises a polynucleotide sequence of SEQ ID NO: 149. In some embodiments, an expression cassette is or comprises a polynucleotide sequence having at least 70% sequence identity to SEQ ID NO: 149. In some embodiments, an expression cassette is or comprises a polynucleotide sequence having at least 75% sequence identity to SEQ ID NO: 149. In some embodiments, an expression cassette is or comprises a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 149. In some embodiments, an expression cassette is or comprises a polynucleotide sequence having at least 85% sequence identity to SEQ ID NO: 149.
  • an expression cassette is or comprises a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 149. In some embodiments, an expression cassette is or comprises a polynucleotide sequence having at least 95% sequence identity to SEQ ID NO: 149. In some embodiments, an expression cassette is or comprises a polynucleotide sequence having at least 99% sequence identity to SEQ ID NO: 149. In some embodiments, an expression cassette consists of a polynucleotide sequence of SEQ ID NO: 149.
  • an expression cassette encodes a polypeptide sequence that is or comprises SEQ ID NO: 101. In some embodiments, an expression cassette encodes a polypeptide sequence that is or comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 101. In some embodiments, an expression cassette encodes a polypeptide sequence that is or comprises a polypeptide sequence having at least 75% sequence identity to SEQ ID NO: 101. In some embodiments, an expression cassette encodes a polypeptide sequence that is or comprises a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 101.
  • an expression cassette encodes a polypeptide sequence that is or comprises a polypeptide sequence having at least 85% sequence identity to SEQ ID NO: 101. In some embodiments, an expression cassette encodes a polypeptide sequence that is or comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 101 . In some embodiments, an expression cassette encodes a polypeptide sequence that is or comprises a polypeptide sequence having at least 95% sequence identity to SEQ ID NO: 101. In some embodiments, an expression cassette encodes a polypeptide sequence that is or comprises a polypeptide sequence having at least 99% sequence identity to SEQ ID NO: 101. In some embodiments, an expression cassette encodes a polypeptide sequence that consists of SEQ ID NO: 101.
  • an expression cassette comprises a polynucleotide sequence that comprises one or more of:
  • transgene that is or comprises SEQ ID NO: 139;
  • an expression cassette comprises a polynucleotide sequence that comprises one or more of:
  • transgene that is or comprises SEQ ID NO: 145;
  • an expression cassette comprises a polynucleotide sequence that comprises one or more of: (i) a 5’ homology arm that is or comprises SEQ TD NO: 39;
  • transgene that is or comprises SEQ ID NO: 146;
  • an expression cassette comprises a polynucleotide sequence that comprises one or more of:
  • transgene that is or comprises SEQ ID NO: 145;
  • a terminator that is or comprises SEQ ID NO: 78;
  • an expression cassette comprises a polynucleotide sequence that comprises one or more of:
  • an expression cassette comprises a polynucleotide sequence that comprises one or more of:
  • transgene that is or comprises SEQ ID NO: 145;
  • transgene that is or comprises SEQ ID NO: 146;
  • an expression cassette encodes a polypeptide that comprises one or more of:
  • transgene that is or comprises SEQ ID NO: 147;
  • a linker encoded hat is or comprises SEQ ID NO: 141.
  • an expression cassette encodes a polypeptide that comprises one or more of:
  • transgene that is or comprises SEQ ID NO: 148.
  • an expression cassette encodes a polypeptide that comprises one or more of: (i) a signal peptide encoded that is or comprises SEQ TD NO: 144; and/or
  • transgene that is or comprises SEQ ID NO: 142.
  • an expression cassette encodes a polypeptide that comprises one or more of:
  • transgene that is or comprises SEQ ID NO: 147;
  • transgene that is or comprises SEQ ID NO: 148.
  • a transgene is a corrective gene chosen to improve one or more signs and/or symptoms of a disease, disorder, or condition.
  • transgenes are functional versions of disease associated genes (i.e., gene isoform(s) which are associated with the manifestation or worsening of a disease, disorder or condition) found in a subject.
  • one or more transgenes are optimized versions of disease- associated genes found in a subject (e.g., codon optimized or expression-optimized variants).
  • transgenes are variants of disease-associated genes found in a subject (e.g., a functional gene fragment or variant thereof).
  • a transgcnc is a gene that causes expression of a peptide that is normally expressed in one or more healthy tissues.
  • a transgene is a gene that causes expression of an altered protein with a gain- or loss-of-function mutation. In some embodiments, a transgene is a gene that causes expression of a fusion protein. In some embodiments, a transgene is a gene that causes expression of an antibody agent. In some embodiments, a transgene is a gene that causes expression of a multispecific antibody. In some embodiments, a transgene is a fragment of an antibody, antigen, or protein. In some embodiments, a transgene is a gene that causes expression an enzyme (e.g., for enzyme replacement therapy). In some embodiments, a transgene is a gene that causes expression of a cytokine.
  • a transgcnc is a gene that causes expression of a cytokine receptor.
  • a transgene is a gene that causes expression of a chimeric antigen receptor (CAR).
  • a transgene is a gene that causes expression of an anti-thrombotic molecule.
  • a transgene is a gene that causes expression of a coagulation factor.
  • a transgcnc is a gene that causes expression of a glucose response element.
  • a transgene is a gene that causes expression of a nanobody.
  • a transgene is a gene that causes expression of fusion protein or a variant thereof.
  • a transgene is a gene that causes expression of ALP or a fusion protein thereof. In some embodiments, a transgene is a gene that causes expression of ALP that comprises of a glycosylphosphatidylinositol (GPI) linkage. In some other embodiments, a transgene is a gene that causes expression of ALP that does not comprise of a glycosyl-phosphatidylinositol (GPI) linkage. In some embodiments, a transgene is a gene that causes expression of fusion protein comprising ALP and human immunoglobulin fragment crystallizable region (“ALP-Fc”) or a variant thereof.
  • ALP-Fc human immunoglobulin fragment crystallizable region
  • a transgene is or comprises a gene encoding a functional nucleic acid.
  • a therapeutic agent is or comprises an agent that has a therapeutic effect upon a host cell or subject (including, e.g., a ribozyme, guide RNA (gRNA), antisense oligonucleotide (ASO), miRNA, siRNA, and/or shRNA).
  • a therapeutic agent promotes a biological process to treat a medical condition, e.g., at least one symptom of a disease, disorder, or condition, such as Hypophosphatasia (HPP).
  • HPP Hypophosphatasia
  • transgene expression in a subject results substantially from integration at a target locus.
  • 75% or more e.g., 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, 99.5% or more
  • 25% or less e.g., 20% or less, 15% or less, 10% or less, 5% or less, 1% or less, 0.5% or less, 0.1% or less
  • of total transgene expression in a subject is from a source other than transgene integration at a target locus (e.g., episomal expression, integration at a non-target locus).
  • a transgene is or comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity to a corresponding wild-type reference nucleotide sequence (e.g., a wild-type gene sequence).
  • a transgene is or comprises a sequence having a least 80%, 85%, 90%, 95%, 99%, or 100% identity to a portion of a corresponding wild-type reference nucleotide sequence (e.g., a wild-type gene sequence).
  • a polynucleotide sequence encoding a transgene is or comprises one or more sequences in Table 5.
  • a polynucleotide sequence encoding a transgene is or comprises SEQ ID NO: 77. In some embodiments, an amino acid sequence encoding a transgene is or comprises SEQ ID NO: 133. In some embodiments, an amino acid sequence encoding a transgene is or comprises SEQ ID NO: 134. In some embodiments, an amino acid sequence encoding a transgene is or comprises SEQ ID NO: 135. In some embodiments, an amino acid sequence encoding a transgene is or comprises SEQ ID NO: 139. In some embodiments, a polynucleotide sequence encoding a transgene is or comprises one or more sequences in Table 6.
  • an amino acid sequence encoding a transgene is or comprises SEQ ID NO: 120. In some embodiments, an amino acid sequence encoding a transgene is or comprises SEQ ID NO: 136. In some embodiments, an amino acid sequence encoding a transgene is or comprises SEQ ID NO: 137. In some embodiments, an amino acid sequence encoding a transgene is or comprises SEQ ID NO: 138.
  • a transgene is or comprises a polynucleotide sequence that is or comprises SEQ ID NO: 133. In some embodiments, a transgene is or comprises a polynucleotide sequence having at least 70% sequence identity to SEQ ID NO: 133. In some embodiments, a transgene is or comprises a polynucleotide sequence having at least 75% sequence identity to SEQ ID NO: 133. In some embodiments, a transgene is or comprises a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 133.
  • a transgene is or comprises a polynucleotide sequence having at least 85% sequence identity to SEQ ID NO: 133. In some embodiments, a transgene is or comprises a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 133. In some embodiments, a transgene is or comprises a polynucleotide sequence having at least 95% sequence identity to SEQ ID NO: 133. In some embodiments, a transgene is or comprises a polynucleotide sequence having at least 99% sequence identity to SEQ ID NO: 133. In some embodiments, a transgene consists of a polynucleotide sequence of SEQ ID NO: 133.
  • a transgene is or comprises a polynucleotide sequence that is or comprises SEQ ID NO: 139. In some embodiments, a transgene is or comprises a polynucleotide sequence having at least 70% sequence identity to SEQ ID NO: 139. In some embodiments, a transgene is or comprises a polynucleotide sequence having at least 75% sequence identity to SEQ ID NO: 139. In some embodiments, a transgene is or comprises a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 139.
  • a transgene is or comprises a polynucleotide sequence having at least 85% sequence identity to SEQ ID NO: 139. In some embodiments, a transgene is or comprises a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 139. In some embodiments, a transgene is or comprises a polynucleotide sequence having at least 95% sequence identity to SEQ ID NO: 139. In some embodiments, a transgene is or comprises a polynucleotide sequence having at least 99% sequence identity to SEQ ID NO: 139. In some embodiments, a transgene consists of a polynucleotide sequence of SEQ ID NO: 139.
  • a transgene is or comprises a polynucleotide sequence that is or comprises SEQ ID NO: 145. In some embodiments, a transgene is or comprises a polynucleotide sequence having at least 70% sequence identity to SEQ ID NO: 145. In some embodiments, a transgene is or comprises a polynucleotide sequence having at least 75% sequence identity to SEQ ID NO: 145. In some embodiments, a transgene is or comprises a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 145.
  • a transgene is or comprises a polynucleotide sequence having at least 85% sequence identity to SEQ ID NO: 145. In some embodiments, a transgene is or comprises a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 145. In some embodiments, a transgene is or comprises a polynucleotide sequence having at least 95% sequence identity to SEQ ID NO: 145. In some embodiments, a transgene is or comprises a polynucleotide sequence having at least 99% sequence identity to SEQ ID NO: 145. In some embodiments, a transgene consists of a polynucleotide sequence of SEQ ID NO: 145.
  • a transgene is or comprises a polynucleotide sequence that is or comprises SEQ ID NO: 146. In some embodiments, a transgene is or comprises a polynucleotide sequence having at least 70% sequence identity to SEQ ID NO: 146. In some embodiments, a transgene is or comprises a polynucleotide sequence having at least 75% sequence identity to SEQ ID NO: 146. In some embodiments, a transgene is or comprises a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 146.
  • a transgene is or comprises a polynucleotide sequence having at least 85% sequence identity to SEQ ID NO: 146. In some embodiments, a transgene is or comprises a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 146. In some embodiments, a transgcnc is or comprises a polynucleotide sequence having at least 95% sequence identity to SEQ ID NO: 146. In some embodiments, a transgene is or comprises a polynucleotide sequence having at least 99% sequence identity to SEQ ID NO: 146. In some embodiments, a transgene consists of a polynucleotide sequence of SEQ ID NO: 146.
  • a transgene encodes a polypeptide that is or comprises SEQ ID NO: 142. In some embodiments, a transgene encodes a polypeptide that is or comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 142. In some embodiments, a transgene encodes a polypeptide that is or comprises a polypeptide sequence having at least 75% sequence identity to SEQ ID NO: 142. In some embodiments, a transgene encodes a polypeptide that is or comprises a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 142.
  • a transgene encodes a polypeptide that is or comprises a polypeptide sequence having at least 85% sequence identity to SEQ ID NO: 142. In some embodiments, a transgene encodes a polypeptide that is or comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 142. In some embodiments, a transgene encodes a polypeptide that is or comprises a polypeptide sequence having at least 95% sequence identity to SEQ ID NO: 142. In some embodiments, a transgene encodes a polypeptide that is or comprises a polypeptide sequence having at least 99% sequence identity to SEQ ID NO: 142. In some embodiments, a transgene encodes a polypeptide that consists of SEQ ID NO: 142.
  • a transgene encodes a polypeptide that is or comprises SEQ ID NO: 147. In some embodiments, a transgene encodes a polypeptide that is or comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 147. In some embodiments, a transgene encodes a polypeptide that is or comprises a polypeptide sequence having at least 75% sequence identity to SEQ ID NO: 147. In some embodiments, a transgene encodes a polypeptide that is or comprises a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 147.
  • a transgene encodes a polypeptide that is or comprises a polypeptide sequence having at least 85% sequence identity to SEQ ID NO: 147. In some embodiments, a transgene encodes a polypeptide that is or comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 147. In some embodiments, a transgene encodes a polypeptide that is or comprises a polypeptide sequence having at least 95% sequence identity to SEQ ID NO: 147. In some embodiments, a transgcnc encodes a polypeptide that is or comprises a polypeptide sequence having at least 99% sequence identity to SEQ ID NO: 147. In some embodiments, a transgene encodes a polypeptide that consists of SEQ ID NO:
  • a transgene encodes a polypeptide that is or comprises SEQ ID NO: 148. In some embodiments, a transgene encodes a polypeptide that is or comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 148. In some embodiments, a transgene encodes a polypeptide that is or comprises a polypeptide sequence having at least 75% sequence identity to SEQ ID NO: 148. In some embodiments, a transgene encodes a polypeptide that is or comprises a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 148.
  • a transgene encodes a polypeptide that is or comprises a polypeptide sequence having at least 85% sequence identity to SEQ ID NO: 148. In some embodiments, a transgene encodes a polypeptide that is or comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 148. In some embodiments, a transgene encodes a polypeptide that is or comprises a polypeptide sequence having at least 95% sequence identity to SEQ ID NO: 148. In some embodiments, a transgene encodes a polypeptide that is or comprises a polypeptide sequence having at least 99% sequence identity to SEQ ID NO: 148. In some embodiments, a transgene encodes a polypeptide that consists of SEQ ID NO:
  • a pay load may comprise one or more flanking polynucleotide sequences with significant sequence homology to a target locus (e.g., homology arms).
  • homology arms flank a polynucleotide sequence encoding a payload (e.g., one homology arm is 5’ to a payload (also referred to herein as a 5’ homology arm) and one homology arm is 3’ to a pay load (also referred to herein as a 3’ homology arm)).
  • homology arms direct site-specific integration of a payload.
  • homology arms are between 400 and 1000 nt in length. In some embodiments, homology arms are between 800 and 1500 nt in length. In some embodiments, homology arms are at least 500 nt in length. Tn some embodiments, homology arms arc less than 1500 nt in length. In some embodiments, homology arms arc 1000 nt in length. In some embodiments, homology arms are at least 50 nt in length. In some embodiments, homology arms are at least 600 nt in length. In some embodiments, homology arms are of the same length. In some embodiments, homology are not of the same length. In some embodiments, homology arms have at least 70% sequence homology to a target locus.
  • homology arms have at least 80% sequence homology to a target locus. In some embodiments, homology arms have at least 90% sequence homology to a target locus. In some embodiments, homology arms have at least 95% sequence homology to a target locus. In some embodiments, homology arms have at least 99% sequence homology to a target locus. In some embodiments, homology arms have 100% sequence homology to a target locus. In some embodiments, homology arms have at least 70% sequence identity to a target locus. In some embodiments, homology arms have at least 80% sequence identity to a target locus. In some embodiments, homology arms have at least 90% sequence identity to a target locus. In some embodiments, homology arms have at least 95% sequence identity to a target locus. In some embodiments, homology arms have at least 99% sequence identity to a target locus. In some embodiments, homology arms have 100% sequence identity to a target locus.
  • constructs comprising homology arms provide rates of target site integration of at least 5%. In some embodiments, constructs comprising homology arms provide rates of target site integration of 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more. In some embodiments, constructs comprising homology arms provide rates of target site integration of 30% or more. In some embodiments, constructs comprising homology arms provide rates of target site integration of 35% or more. In some embodiments, a homology arm is or comprises a sequence in Table 1. In some embodiments, a homology arm is or comprises SEQ ID NO: 39.
  • a homology arm is or comprises SEQ ID NO: 40. In some embodiments, a homology arm is or comprises SEQ ID NO: 41. In some embodiments, a homology arm is or comprises SEQ ID NO: 42. In some embodiments, a homology arm is or comprises SEQ ID NO: 43. In some embodiments, a homology arm is or comprises SEQ ID NO: 44. Tn some embodiments, a homology arm is or comprises SEQ ID NO: 45. In some embodiments, a homology ami is or comprises SEQ ID NO: 46. In some embodiments, a homology arm is or comprises SEQ ID NO: 47. In some embodiments, a homology arm is or comprises SEQ ID NO: 48.
  • a homology arm is or comprises SEQ ID NO: 49. In some embodiments, a homology arm is or comprises SEQ ID NO: 50. In some embodiments, a homology arm is or comprises SEQ ID NO: 51. In some embodiments, a homology arm is or comprises SEQ ID NO: 52. In some embodiments, a homology arm is or comprises SEQ ID NO: 53. In some embodiments, a homology arm is or comprises SEQ ID NO: 54. In some embodiments, a homology arm is or comprises SEQ ID NO: 118. In some embodiments, a homology arm is or comprises SEQ ID NO: 119. In some embodiments, a homology aim is or comprises SEQ ID NO: 121. In some embodiments, a homology arm is or comprises SEQ ID NO: 122. In some embodiments, a homology arm is or comprises SEQ ID NO: 123. In some embodiments, a homology arm is or comprises SEQ ID NO: 124.
  • a 5’ homology arm is or comprises a polynucleotide sequence of SEQ ID NO: 39. In some embodiments, a 5’ homology arm is or comprises a polynucleotide sequence having at least 70% sequence identity to SEQ ID NO: 39. In some embodiments, a 5’ homology arm is or comprises a polynucleotide sequence having at least 75% sequence identity to SEQ ID NO: 39. In some embodiments, a 5’ homology arm is or comprises a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 39.
  • a 5’ homology arm is or comprises a polynucleotide sequence having at least 85% sequence identity to SEQ ID NO: 39. In some embodiments, a 5’ homology arm is or comprises a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 39. In some embodiments, a 5’ homology arm is or comprises a polynucleotide sequence having at least 95% sequence identity to SEQ ID NO: 39. In some embodiments, a 5’ homology arm is or comprises a polynucleotide sequence having at least 99% sequence identity to SEQ ID NO: 39. In some embodiments, a 5’ homology arm consists of a polynucleotide sequence of SEQ ID NO: 39.
  • a 3’ homology arm is or comprises a polynucleotide sequence of SEQ ID NO: 44. In some embodiments, a 3’ homology arm is or comprises a polynucleotide sequence having at least 70% sequence identity to SEQ ID NO: 44. In some embodiments, a 3’ homology arm is or comprises a polynucleotide sequence having at least 75% sequence identity to SEQ ID NO: 44. In some embodiments, a 3’ homology arm is or comprises a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 44.
  • a 3’ homology arm is or comprises a polynucleotide sequence having at least 85% sequence identity to SEQ ID NO: 44. In some embodiments, a 3’ homology arm is or comprises a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 44. In some embodiments, a 3’ homology arm is or comprises a polynucleotide sequence having at least 95% sequence identity to SEQ ID NO: 44. In some embodiments, a 3’ homology arm is or comprises a polynucleotide sequence having at least 99% sequence identity to SEQ ID NO: 44. In some embodiments, a 3’ homology arm consists of a polynucleotide sequence of SEQ ID NO: 44.
  • compositions disclosed herein direct integration of a payload (e.g., a transgene) at a target locus (e.g., an endogenous gene).
  • a payload e.g., a transgene
  • compositions and constructs provided herein direct integration of a payload at a target locus in a specific cell type (e.g., naive B cells, B lineage cell populations, etc.).
  • compositions and constructs provided herein direct integration of a pay load (e.g., expression cassette comprising a transgene encoding an ALP-Fc fusion protein) at a target locus in a specific cell type (e.g, naive B cells, B lineage cell populations, etc.).
  • a payload is or comprises a transgene or variant thereof encoding ALP or a fusion protein or a variant thereof, e.g., an ALP-Fc fusion protein or a variant thereof.
  • compositions and constructs provided herein direct integration of a payload at a target locus that is considered a safe-harbor site (e.g., CCR5, AAVS1).
  • a target locus is selected from any genomic site appropriate for use with methods and compositions provided herein.
  • a target locus encodes a polypeptide.
  • a target locus encodes a polypeptide that is highly expressed in a subject e.g., a subject not suffering from a disease, disorder, or condition, or a subject suffering from a disease, disorder, or condition).
  • a target locus is selected from one or more of CD19, CD20, IGH, B2M, CCR5, JCHAIN, PAX5, IRF4, IRF8, BACH2, EZH2, XBP1, CARD11, PRDM1, and BAFF.
  • B lineage cell culture methods [0211] Various methods for culturing and long-term maintenance of B lineage cells in vitro arc described in the art (Sec, Rawlings ct al. 1995, Rawlings ct al 1997, and Fluckingcr ct al. 1998, each of which is incorporated by reference herein in its entirety). B lineage cell culturing conditions can significantly affect normal human B lineage development and for production of mature Ig- secreting B cells.
  • B lineage cell activation and proliferation in vitro employed CD40L-expressing feeder cell layer systems. Such feeder cell systems were described as difficult to standardize and often unreliable for providing consistent levels of activation and proliferation of B lineage cells. Recent advances have shifted to protocols for in vitro activation and proliferation of B lineage cells in specific culture systems comprising cytokines or other components in the absence of feeder cells (See Jourdan et al., 2009 and Hartweger et al., 2019, each of which is incorporated herein by reference in its entirety).
  • methods for B lineage cell activation comprise contacting cells with media comprising one or more components of the present disclosure.
  • methods for B lineage cell activation comprise contacting cells with media comprising one or more cytokines and/or oligonucleotides (e.g., multimeric human CD40L, IL-2, IL- 10, IL- 15, IL-21 and/or CpG).
  • cytokines and/or oligonucleotides e.g., multimeric human CD40L, IL-2, IL- 10, IL- 15, IL-21 and/or CpG.
  • methods for B cell activation comprise contacting cells with media comprising at least about 5 ng/mL, 10 ng /mL, 15 ng/mL, 20 ng/mL, 25 ng/mL, 30 ng/mL, 35 ng/mL, 40 ng/mL, 45 ng/mL, 50 ng/mL, 55ng/mL, 60 ng/mL, 65 ng/mL, 70 ng/mL, 75 ng/mL, 80 ng/mL, 85 ng/mL, 90 ng/mL, 95 ng/mL, 100 ng/mL, 150 ng/mL, 200 ng/mL, 500 ng/mL, 600 ng/mL, 700 ng/mL, 800 ng/mL, 900 ng/mL, or 1 ug/mL of one or more cytokines and/or oligonucleotides (e.g., multimeric human CD40L, IL-2
  • methods for B lineage cell activation comprise contacting cells with media comprising at least about 0.1 ug/mL, 0.2 ug/mL, 0.3 ug/mL, 0.4 ug/mL, 0.5 ug/mL, 0.6 ug/mL, 0.7 ug/mL, 0.8 ug/mL, 0.9 ug/mL, 1 ug/mL, 1.5 ug/mL, 2 ug/mL, 2.5 ug/mL, 3 ug/mL, 3.5 ug/mL, 4 ug/mL, 4.5 ug/mL, or 5 ug/mL of one or more cytokines and/or oligonucleotides (e.g., multimeric human CD40L, IL-2, IL- 10, IL-15, IL-21 and/or CpG).
  • cytokines and/or oligonucleotides e.g., multimeric human
  • methods for B lineage cell activation comprise a step of contacting cells with media comprising one or more components of the present disclosure for at least about 1, 2, 3, 4, 5, 6, or 7 days.
  • methods for B lineage cell activation comprise a step of contacting cells with media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL- 10, IL- 15, IL-21 and/or CpG) for at least about 1 , 2, 3, 4, 5, 6, or 7 days.
  • cytokines and/or oligonucleotides e.g., CD40L, IL-2, IL- 10, IL- 15, IL-21 and/or CpG
  • methods for B lineage cell activation comprise a step of contacting cells with media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG) for at least 2 days.
  • methods for B lineage cell activation comprise a step of contacting cells with media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL- 10, IL- 15, IL-21 and/or CpG) for at least 2 days, followed by a step of gene editing.
  • methods for B lineage cell activation comprise a step of contacting cells with media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG) for at least 2 days, followed by a step of B lineage cell expansion.
  • media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG) for at least 2 days, followed by a step of B lineage cell expansion.
  • cytokines and/or oligonucleotides e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG
  • methods for B lineage cell activation comprise a step of contacting cells with media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG) for at least 2 days, followed by a step of B lineage cell expansion for at least about 1, 2, 3, 4, 5, 6, 7, or 8 days.
  • media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG) for at least 2 days, followed by a step of B lineage cell expansion for at least about 1, 2, 3, 4, 5, 6, 7, or 8 days.
  • cytokines and/or oligonucleotides e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG
  • methods described herein result in an activated B lineage cell population.
  • Plasmablasts are short-lived, rapidly produced effector cells that are primarily present in an early antibody response and are one potential product of terminal B cell differentiation. Plasmablasts are capable of secreting antibodies, including IgM subtype antibodies, in order to mount an immediate response to certain antigens in the body. Differentiation of B cells into plasmablasts in vitro may be promoted through use of certain signaling molecules, including one or more cytokines (e. ., IL-2, IL-6, IL- 10, and/or IL- 15). A variety of methods for differentiation of B cells into plasmablasts are known, but not limited to, those outlined in WO/2018/170150, incorporated herein by reference in its entirety. Plasmablasts produced by such methods may be characterized as cells that are CD27 + / CD38 + / CD138'.
  • methods for B cell differentiation into plasmablasts comprise contacting cells with media comprising one or more components.
  • methods for B cell differentiation into plasmablasts comprise contacting activated B lineage cells with media comprising one or more cytokines (e.g., IL-2, IL-6, IL- 10, and/or IL- 15).
  • cytokines e.g., IL-2, IL-6, IL- 10, and/or IL- 15.
  • methods for B cell differentiation into plasmablasts comprise contacting activated B lineage cells with media comprising at least about 0.5 ng/mL, 1 ng/mL, 1.5 ng/mL, 2 ng/mL, 2.5 ng/mL, 3 ng/mL, 3.5 ng/mL, 4 ng/mL, 4.5 ng/mL, 5 ng/mL, 10 ng /mL, 15 ng/mL, 20 ng/mL, 25 ng/mL, 30 ng/mL, 35 ng/mL, 40 ng/mL, 45 ng/mL, 50 ng/mL, 55 ng/mL, 60 ng/mL, 65 ng/mL, 70 ng/mL, 75 ng/mL, 80 ng/mL, 85 ng/mL, 90 ng/mL, 95 ng/mL, or ng/mL of one or more cytokines (e.g., IL-2,
  • methods for B cell differentiation into plasmablasts comprise a step of contacting cells with media comprising one or more components of the present disclosure for at least about 1 , 2, 3, or 4 days.
  • methods for B cell differentiation into plasmablasts comprise a step of contacting activated B lineage cells with media comprising one or more cytokines (e.g., IL-2, IL-6, IL- 10, and/or IL- 15) for at least about 1, 2, 3, or 4 days.
  • cytokines e.g., IL-2, IL-6, IL- 10, and/or IL- 15
  • methods for B cell differentiation into plasmablasts comprise a step of contacting activated B lineage cells with media comprising one or more cytokines (e.g., IL-2, IL-6, IL- 10, and/or IL- 15) for at least 3 days.
  • methods for B cell differentiation into plasmablasts comprise a step of contacting activated B lineage cells with media comprising one or more cytokines (e.g., IL-2, IL-6, IL-10, and/or IL-15) for at least 3 days, followed by a step of plasmablast expansion.
  • Plasmablasts secrete more antibodies than naive B cells, they are shorter-lived and secrete fewer antibodies than plasma cells (PCs).
  • Long-lived plasma cells (LLPCs, used interchangeably throughout with plasma cells) localize to bone marrow in the body and are capable of secreting high levels of antibodies and surviving for decades in the absence of proliferation (See, Hammerland et al., 2017 and Khodadadi et al., 2019, both of which incorporated herewith in their entirety).
  • Differentiation of plasmablasts to long-lived plasma cells can be triggered by certain events in the body, including, e.g., activity of transcription factors Blimp- 1/PRDM1 and IRF4.
  • Plasmablasts to plasma cells in vitro may be promoted through use of certain signaling molecules, including one or more cytokines (e.g., IL- 6, IL-15, and/or IFNa-2P).
  • cytokines e.g., IL- 6, IL-15, and/or IFNa-2P.
  • a variety of methods for differentiation of plasmablasts into plasma cells are known, but not limited to, those outlined in Jourdan et al. 2019 and WO/2018/170150 (each of which incorporated herein by reference in its entirety).
  • Plasma cells produced by such methods may be characterized as cells that are CD27 + / CD38 + / CD138 + .
  • methods for plasmablast differentiation into plasma cells comprise contacting cells with media comprising one or more cytokines (e.g., IL-6, IL- 15, and/or IFNa-2P). In some embodiments, methods for plasmablast differentiation into plasma cells comprise contacting plasmablasts with media comprising one or more cytokines (e.g., IL-6, IL- 15, and/or IFNa-2P).
  • cytokines e.g., IL-6, IL- 15, and/or IFNa-2P.
  • methods for plasmablast differentiation into plasma cells comprise contacting plasmablasts with media comprising at least about 0.5 ng/mL, 1 ng/mL, 1.5 ng/mL, 2 ng/mL, 2.5 ng/mL, 3 ng/mL, 3.5 ng/mL, 4 ng/mL, 4.5 ng/mL, 5 ng/mL, 10 ng /mL, 15 ng/mL, 20 ng/mL, 25 ng/mL, 30 ng/mL, 35 ng/mL, 40 ng/mL, 45 ng/mL, 50 ng/mL, 55 ng/mL, 60 ng/mL, 65 ng/mL, 70 ng/mL, 75 ng/mL, 80 ng/mL, 85 ng/mL, 90 ng/mL, 95 ng/mL, or ng/mL of one or more cytokines (e.g., IL-6, IL-15, and
  • methods for plasmablast differentiation into plasma cells comprise a step of contacting cells with media comprising one or more components of the present disclosure for at least about 1, 2, 3, or 4 days.
  • methods for plasmablast differentiation into plasma cells comprise a step of contacting plasmablasts with media comprising one or more cytokines (e.g., IL-6, IL-15, and/or IFNa-2P) for at least about 1, 2, 3, or 4 days.
  • methods for plasmablast differentiation into plasma cells comprise a step of contacting plasmablasts with media comprising one or more cytokines (e.g., IL-6, IL- 15, and/or IFNa-2P) for at least 3 days.
  • methods for plasmablast differentiation into plasma cells comprise a step of contacting plasmablasts with media comprising one or more cytokines (e.g., IL-6, IL- 15, and/or IFNa-2 ) for at least 3 days, followed by a step of cell isolation.
  • methods for plasmablast differentiation into plasma cells comprise a step of contacting plasmablasts with media comprising one or more cytokines (e.g., IL-6, IL- 15, and/or IFNa-2 ) for at least 3 days, followed by a step of administration to a subject.
  • engineered cell preparations comprising populations of cells modified to perform one or more desired functions.
  • engineered cell preparations are compositions comprising genetically modified immune cell populations (e.g., B cell, T cell).
  • engineered cell preparations arc compositions comprising genetically modified B lineage cell populations (e.g., B cell, plasmablast, plasma cell).
  • engineered cell preparations are compositions comprising genetically modified plasmablast cell populations.
  • engineered cell preparations are compositions comprising genetically modified plasma cell populations.
  • engineered cell preparations are genetically modified to express a payload (e.g., transgene) of interest.
  • engineered cell preparations are genetically modified to express a transgene from an expression cassette (e.g., comprising additional polynucleotide sequence elements).
  • engineered cell preparations comprise genetically modified cells that express a transgene of interest (e.g., therapeutic protein, antibody, etc.). In some embodiments, engineered cell preparations comprise genetically modified cells that express a transgene of interest (e.g., therapeutic protein, antibody, etc.) from an endogenous gene locus. In some embodiments, engineered cell preparations comprise genetically modified cells that express a transgene of interest (e.g., therapeutic protein, antibody, etc.) from an endogenous gene locus under control of an endogenous promoter.
  • a transgene of interest e.g., therapeutic protein, antibody, etc.
  • engineered cell preparations comprise genetically modified cells that express a transgene of interest (e.g., therapeutic protein, antibody, etc.) from an endogenous gene locus under control of an exogenous promoter.
  • engineered cell preparations comprise genetically modified cells that express a transgcnc of interest (e.g., therapeutic protein, antibody, etc.) from an endogenous gene locus without disrupting endogenous gene expression and/or function.
  • engineered cell preparations comprise genetically modified cells that express a transgene of interest (e.g., therapeutic protein, antibody, etc.) from an endogenous gene locus and partially or fully disrupt endogenous gene expression and/or function.
  • engineered cell preparations are genetically modified to express a transgene from an expression cassette, wherein the expression cassette further comprises one or more polynucleotide sequences encoding one or more promoters. In some embodiments, engineered cell preparations are genetically modified to express a transgene from an expression cassette, wherein the expression cassette further comprises one or more polynucleotide sequences encoding one or more enhancers. In some embodiments, engineered cell preparations are genetically modified to express a transgene from an expression cassette, wherein the expression cassette further comprises one or more polynucleotide sequences encoding one or more terminators.
  • engineered cell preparations are genetically modified to express a transgene from an expression cassette, wherein the expression cassette further comprises one or more polynucleotide sequences encoding one or more homology arms. In some embodiments, engineered cell preparations are genetically modified to express a transgene from an expression cassette, wherein the expression cassette further comprises one or more polynucleotide sequences encoding one or more promoters, one or more enhancers, one or more terminators, and/or one or more homology arms.
  • engineered cell preparations are genetically modified to express an ALP-Fc fusion protein transgene, or variant thereof, from an expression cassette, wherein the expression cassette further comprises one or more polynucleotide sequences encoding a MND promoter, a WPRE enhancer, a BGH poly A, a 5’ homology arm, and a 3’ homology arm.
  • engineered cell preparations are genetically modified to express an ALP-Fc fusion protein transgene, or variant thereof, from an expression cassette, wherein the expression cassette further comprises one or more polynucleotide sequences encoding a MND promoter, a BGH polyA, a 5’ homology arm, and a 3’ homology arm.
  • engineered cell preparations are genetically modified to express an ALP-Fc fusion protein transgene, or variant thereof, from an expression cassette, wherein the expression cassette further comprises one or more polynucleotide sequences encoding a MND promoter, a WPRE enhancer, a SV40 polyA, a 5’ homology arm, and a 3’ homology arm.
  • engineered cell preparations arc genetically modified to express an ALP-Fc fusion protein transgene, or variant thereof, from an expression cassette, wherein the expression cassette further comprises one or more polynucleotide sequences encoding a MND promoter, a SV40 polyA, a 5’ homology arm, and a 3’ homology arm.
  • engineered cell preparations are genetically modified to express an ALP-Fc fusion protein transgene, or variant thereof, from an expression cassette, wherein the expression cassette further comprises one or more polynucleotide sequences encoding an EF-la promoter, a WPRE enhancer, a BGH polyA, a 5’ homology arm, and a 3’ homology arm.
  • engineered cell preparations are genetically modified to express an ALP-Fc fusion protein transgene, or variant thereof, from an expression cassette, wherein the expression cassette further comprises one or more polynucleotide sequences encoding an EF-la promoter, a BGH polyA, a 5’ homology arm, and a 3’ homology arm.
  • engineered cell preparations are genetically modified to express an ALP-Fc fusion protein transgene, or variant thereof, from an expression cassette, wherein the expression cassette further comprises one or more polynucleotide sequences encoding an EF-la promoter, a SV40 polyA, a 5’ homology arm, and a 3’ homology arm.
  • a B lineage cell preparation comprises both engineered and non-engineered cells.
  • an engineered B lineage cell population comprises plasmablasts.
  • an engineered B lineage cell population comprises plasma cells.
  • an engineered B lineage cell population comprises long-lived plasma cells.
  • an engineered B lineage cell population comprises plasmablasts, plasma cells, and/or long-lived plasma cells and/or any mixtures or combinations thereof disclosed herein.
  • an engineered B lineage cell preparation comprises plasmablasts.
  • plasmablasts arc rapidly produced and short-lived effector cells of the early antibody response.
  • Plasmablasts can be generated from activated B lineage cells using methods described herein.
  • engineered plasmablasts may be generated from engineered activated B lineage cells.
  • plasmablast cell populations may be contacted with media comprising one or more components of the present disclosure (e.g., IL-6, IL- 15, and/or lFNa-2P) in order to initiate differentiation into plasma cell populations.
  • methods for plasmablast cell population differentiation into a plasma cell population comprise contacting plasmablast cell population with media comprising one or more cytokines (e.g., IL-6, IL- 15, and/or IFNa-2P).
  • methods for a plasmablast cell population differentiation into a plasma cell population comprise contacting plasmablast cell population with media comprising at least about 0.5 ng/mL, 1 ng/mL, 1.5 ng/mL, 2 ng/mL, 2.5 ng/mL, 3 ng/mL, 3.5 ng/mL, 4 ng/mL, 4.5 ng/mL, 5 ng/mL, 10 ng /mL, 15 ng/mL, 20 ng/mL, 25 ng/mL, 30 ng/mL, 35 ng/mL, 40 ng/mL, 45 ng/mL, 50 ng/mL, 55 ng/mL, 60 ng/mL, 65 ng/mL, 70 ng/mL, 75 ng/mL, 80 ng/mL, 85 ng/mL, 90 ng/mL, 95 ng/mL, or ng/mL of one or more cytokines e.g.,
  • methods for plasmablast cell population differentiation into a plasma cell population comprise a step of contacting cells with media comprising one or more components of the present disclosure for at least about 1, 2, 3, or 4 days.
  • methods for plasmablast cell population differentiation into a plasma cell population comprise a step of contacting plasmablast cell population with media comprising one or more cytokines (e.g., IL-6, IL-15, and/or IFNa-2P) for at least about 1, 2, 3, or 4 days.
  • cytokines e.g., IL-6, IL-15, and/or IFNa-2P
  • methods for plasmablast cell population differentiation into a plasma cell population comprise a step of contacting plasmablast cell population with media comprising one or more cytokines (e.g., IL-6, IL-15, and/or IFNa-2P) for at least 3 days.
  • methods for plasmablast cell population differentiation into a plasma cell population comprise a step of contacting plasmablast cell population with media comprising one or more cytokines (e.g., IL-6, IL- 15, and/or IFNa-2P) for at least 3 days, followed by a step of cell isolation.
  • methods for plasmablast cell population differentiation into a plasma cell population comprise a step of contacting plasmablasts with media comprising one or more cytokines e.g., IL-6, IL- 15, and/or IFNa-2P) for at least 3 days, followed by a step of administration to a subject.
  • cytokines e.g., IL-6, IL- 15, and/or IFNa-2P
  • an engineered B lineage cell preparation comprises a plasma cell population.
  • a plasma cell population comprises predetermined engineered plasma cell precursors (e.g., plasma blasts) that, upon administration to a subject, further differentiates into a mature plasma cell population.
  • a mature plasma cell population comprises quiescent, non-dividing cells of the humoral immune response that are capable of secreting large amounts of antibodies.
  • a mature plasma cell population can comprise short-lived plasma cells and/or long-lived plasma cells (LLPCs) and/or any combination thereof.
  • Cell therapies provide a number of potential advantages over traditional treatment options, at least in part due to the unique properties of viable cells within the body.
  • it has been recognized by those in the art that successful clinical outcomes require that cell therapies be quickly and effectively delivered to a subject.
  • preservation and storage techniques to maintain a standardized efficacy until they can be transported and administered to a subject.
  • it is equally crucial to recognize and identify conditions that will increase viability and function of the cell population, as each cell population has different structural considerations for preservation (See, Meneghel, 2020, incorporated herein by reference in its entirety). Numerous people have worked hard to try to address these concerns and develop useful preservation technologies, but these again are very cell-specific and can lead to various inadequacies.
  • cryopreservation processes are well-established for certain cell types, such as hematopoietic stem cells (HSCs) for transplantation, with over 47,000 procedures carried out in Europe in 2018 (See, Passweg et al, 2020, incorporated herein by reference in its entirety).
  • HSCs hematopoietic stem cells
  • cryopreservation protocols must be clear and precise, with specific requirements for each step of freezing, storage, and thawing prior to patient administration.
  • Cryoprcscrvation may also be used at multiple points in a process chain for cell therapy, e.g. multiple times between isolation of cells and patient administration.
  • cryopreservation conditions may vary between cell therapies depending on specific requirements and features of each therapy. For example, in some embodiments, cryopreservation conditions that preserve activity of one type of cell therapy do not maintain activity of another type of cell therapy. Similarly, in some embodiments, cryopreservation conditions that preserve viability of a particular cell type do not maintain viability and/or activity of a cell therapy employing an engineered cell of the same cell type.
  • the present disclosure provides certain technologies that achieve effective cryopreservation of certain cell populations (e.g., of certain B lineage cell populations).
  • Cry oprote dive agents Cry oprote dive agents
  • Cryopreservation may cause a number of cellular injuries, including, e.g., potentially harmful changes in cell morphology, characteristics (e.g., adhesion, cell surface markers, protein expression profile), metabolic activity (e.g., proliferative ability, potency), and/or function (e.g., immunomodulation, signal responsiveness), and may also lead to cell death.
  • Temperature changes associated with cryopreservation methods may cause an adverse response in cells, including, e.g., activation of certain stress response pathways upon initial freezing and/or activation of apoptotic and necrotic pathways upon thawing.
  • Ice crystal formation in media during cryopreservative freezing can also cause significant cellular damage through, e.g., physical interruption of cellular functions and processes and through solute exclusion and increased localized solute concentration, which may result in increased osmolarity and osmotic stress (See, Meneghel et al, 2020). Temperature changes may also lead to intracellular ice formation, which can cause cell damage and death.
  • cryoprotective agents In order to mitigate cellular' injuries as a result of temperature changes, additive compounds known as cryoprotective agents (also referred to interchangeably herein as cryoprotectants) may be added to cell media.
  • Cryoprotective agents Cryoprotective agents (CPAs) have relatively low toxicity to cells, particularly at lower temperatures. CPAs may also exclude ice crystals and maintain a larger, unfrozen extracellular portion of media at certain temperatures, reducing osmotic effects and stress on cells (See, Meneghel et al, 2020). CPAs (e.g., DMSO) may also promote cell dehydration, limiting probability of intracellular ice formation and interacting with I stabilizing cellular membranes (See, Gao et al, 2000).
  • CPAs that diffuse through the outer cell membrane may also be capable of altering intracellular osmotic concentration, reducing and/or regulating intracellular ice formation, dehydration, etc (See, Whaley et al 2021). Mixtures of permeable and non-permeable CPAs may be employed to provide desired effects and can be optimized for particular cell types (e.g., cell therapies) (See, Balci and Can 2013). Certain CPAs may also be optimized for exposure time, exposure temperature, loading time, loading temperature, etc., to balance cryoprotective effects with potential toxicity to cells.
  • permeable CPAs e.g., DMSO
  • cryopreserved cell preparations provided and/or utilized in accordance with the present disclosure comprise one or more CPAs disclosed herein.
  • cryopreserved cell preparations comprise one or more permeable CPAs.
  • cryopreserved cell preparations comprise DMSO.
  • cryopreserved cell preparations comprise glycerol.
  • cryopreserved cell preparations comprise ethylene glycol.
  • cryopreserved cell preparations comprise propylene glycol.
  • cryopreserved cell preparations comprise one or more non- pcrmcablc CPAs.
  • cryoprcscrvcd cell preparations comprise glucose.
  • cryopreserved cell preparations comprise sucrose.
  • cryopreserved cell preparations comprise trehalose.
  • cryopreserved cell preparations comprise raffinose.
  • cryopreserved cell preparations comprise hydroxyethyl starch (HES).
  • cryopreserved cell preparations comprise polyvinylpyrrolidone (PVP).
  • cryopreserved cell preparations comprise polyvinyl alcohol (PVA). In some embodiments, cryopreserved cell preparations comprise polyethylene glycol (PEG). In some embodiments, cryopreserved cell preparations comprise dextran. In some embodiments, cryopreserved cell preparations comprise one or more albumin proteins. In some embodiments, cryopreserved cell preparations comprise human serum albumin (HSA). In some embodiments, cryopreserved cell preparations comprise blood serum. In some embodiments, cryopreserved cell preparations comprise poloxamers (e.g., P188). In some embodiments, cryopreserved cell preparations comprise mannitol.
  • PVA polyvinyl alcohol
  • cryopreserved cell preparations comprise polyethylene glycol (PEG). In some embodiments, cryopreserved cell preparations comprise dextran. In some embodiments, cryopreserved cell preparations comprise one or more albumin proteins. In some embodiments, cryopreserved
  • cryopreserved cell preparations comprise glutathione. In some embodiments, cryopreserved cell preparations comprise thioredoxin. In some embodiments, cryopreserved cell preparations comprise glutathione. In some embodiments, cryopreserved cell preparations comprise ascorbic acid. In some embodiments, cryopreserved cell preparations comprise mitoquinone. In some embodiments, cryopreserved cell preparations comprise salidroside. In some embodiments, cryopreserved cell preparations comprise reservatrol. In some embodiments, cryopreserved cell preparations comprise N-acetyl-L-cysteine.
  • cryopreserved cell preparations comprise catalase. In some embodiments, cryopreserved cell preparations comprise a-tocopheryl acetate. In some embodiments, cryopreserved cell preparations comprise lactated ringers. In some embodiments, cryopreserved cell preparations comprise dextrose.
  • cryopreserved cell preparations comprise one or more permeable CPAs and one or more non-permeable CPAs.
  • cryopreserved cell preparations comprise DMSO and HSA.
  • cryopreserved cell preparations comprise lactated ringers and dextrose.
  • cryoprotectants are added to cell preparations in a particular order to facilitate cryopreservation (e.g., HSA followed by the addition of DMSO).
  • one or more non-permeable CPAs is added first, followed by a permeable CPA.
  • one or more non-pcrmcablc CPAs is added first, followed by another non-permeable CPA.
  • one or more permeable CPAs is added first, followed by a non-permeable CPA.
  • one or more permeable CPAs is added first, followed by another permeable CPA.
  • cryoprotectants are added to cell preparations at a particular temperature in order to facilitate cryopreservation.
  • cryoprotectants are added at a temperature of about -5 °C, -4 °C, -3 °C, -2 °C, -1 °C, 0 °C, 1 °C, 2 °C, 3 °C, 4 °C, 5°C, 6 °C, 7 °C, 8 °C, 9 °C, or 10 °C.
  • cryoprotectants are added at a temperature of about 4 °C.
  • cryoprotectants are added at a temperature of about 10 °C, 11 °C, 12 °C, 13 °C, 14 °C, 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, or 30 °C. In some embodiments, cryoprotectants are added at a temperature of about 20 °C.
  • cryoprotectants are added at a temperature of about 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, or 45 °C. In some embodiments, cryoprotectants are added at a temperature of about 37 °C.
  • cryoprotectants are added to cell preparations and allowed to equilibrate during an incubation period to facilitate cryoprcscrvation.
  • cell preparations are incubated with cryoprotectants at a temperature of about 5 °C, -4 °C, -3 °C, - 2 °C, -1 °C, 0 °C, 1 °C, 2 °C, 3 °C, 4 °C, 5°C, 6 °C, 7 °C, 8 °C, 9 °C, or 10 °C.
  • cell preparations are incubated with cryoprotectants at a temperature of about 4 °C.
  • cell preparations are incubated with cryoprotectants at a temperature of about 10 °C, 11 °C, 12 °C, 13 °C, 14 °C, 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, or 30 °C. In some embodiments, cell preparations are incubated with cryoprotectants at a temperature of about 20 °C.
  • cell preparations are incubated with cryoprotectants at a temperature of about 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, or 45 °C. In some embodiments, cell preparations are incubated with cryoprotectants at a temperature of about 37 °C.
  • cell preparations are incubated with cryoprotectants for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes. In some embodiments, cell preparations arc incubated with cryoprotectants for at least about 10 minutes. In some embodiments, cell preparations are incubated with cryoprotectants for at least about 30, 31, 32, 33, 34, 35, or 36 minutes. In some embodiments, cell preparations are incubated with cryoprotectants for at least about at least about 36 minutes. In some embodiments, cell preparations are incubated with cryoprotectants for at least about 120, 121, 122, 123, 124, 125, 126, or 127 minutes. In some embodiments, cell preparations are incubated with cryoprotectants for at least about 127 minutes.
  • cell preparations are exposed to cryoprotectants for a certain fixed time period prior to a freezing process. In some embodiments, cell preparations are exposed to cryoprotectants for no more than about 20, 21, 22, 23, 24, or 25 minutes. In some embodiments, cell preparations are exposed to cryoprotectants for no more than about 25 minutes. In some embodiments, cell preparations are exposed to cryoprotectants for no more than about 45, 46, 47, 48, 49, 50, or 51 minutes. In some embodiments, cell preparations are exposed to cryoprotectants for no more than about 51 minutes.
  • cell preparations are exposed to cryoprotectants for no more than about 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, or 142 minutes. In some embodiments, cell preparations are exposed to cryoprotectants for no more than about 142 minutes.
  • Cooling rate is another factor for consideration when optimizing cryopreservation protocols.
  • Appropriate freezing rates may vary between cell types and can also be affected by media components and cryoprotectants.
  • Cellular injury during freezing may arise from both extracellular and intracellular ice formation and controlled cooling rate can affect when and where ice forms (See, Meneghel et al 2020).
  • Relatively slow cooling rates can result in ice forming first within an extracellular media, excluding solutes from the ice crystals and leading to increased osmolarity and potential osmotic shock to the cells. These changes in osmolarity can also lead to partial dehydration of cells, which can provide a beneficial effect by reducing probability of intracellular ice formation and/or stabilizing cell membranes.
  • Relatively high cooling rates may prevent cellular dehydration from occurring prior to freezing, leading to increased intracellular ice levels and cellular damage.
  • the present disclosure recognizes that differences exist between various cell types in viability and function under different cooling rates.
  • the present disclosure employs a process comprising of both heating and cooling phases in a process known as controlled cooling.
  • controlled cooling comprises a step of cooling a chamber until a cell preparation reaches about ⁇ l-10°C. In some embodiments, controlled cooling comprises a step of cooling the chamber at a rate of ⁇ 1-25°C / minute until the cry opreservation cell preparation reaches about ⁇ l-20°C. In some embodiments, controlled cooling comprises a step of cooling the chamber at a rate of ⁇ 1-25°C I minute until the chamber reaches about ⁇ l-40°C. In some embodiments, controlled cooling comprises a step of heating the chamber at a rate of about ⁇ l-10°C I minute until the chamber reaches about ⁇ 1-12°C.
  • controlled cooling comprises a step of cooling the chamber at a rate of ⁇ l-10°C I minute until the chamber reaches ⁇ l-40°C. In some embodiments, controlled cooling comprises a step of cooling the chamber at a rate of ⁇ 1- 10°C I minute until the chamber reaches ⁇ I-90°C.
  • controlled cooling comprises a step of cooling a chamber until a cell preparation reaches 4°C. In some embodiments, controlled cooling comprises a step of cooling the chamber at a rate of 1°C / minute until the cryopreservation cell preparation reaches -4°C. In some embodiments, controlled cooling comprises a step of cooling the chamber at a rate of 25°C I minute until the chamber reaches -40°C. In some embodiments, controlled cooling comprises a step of heating the chamber at a rate of 10°C I minute until the chamber reaches -12°C.
  • controlled cooling comprises a step of cooling the chamber at a rate of 1°C / minute until the chamber reaches -40°C. In some embodiments, controlled cooling comprises a step of cooling the chamber at a rate of 10°C I minute until the chamber reaches -90°C.
  • cryopreservation and storage methods described herein provides numerous advantages over conventional short-term storage, including but not limited to, availability of preparations of consistent viability and functionality, flexibility and predictability for delivery, less cellular waste, reproducibility, and minimization of contamination.
  • B lineage cell preparations may be stored in sterile cryovials (e.g., 1-50 ml). In some embodiments, such cryovials may be hermetically sealed. In some embodiments, B lineage cell preparations may be stored in a cryobag. In some embodiments, such cryobags may be hermetically sealed. In some embodiments, cryobags may additionally be stored within an overwrap bag. In some embodiments, cryobags may be stored in a metallic casing. In some embodiments, cryobags stored within an overwrap bag may be additionally stored in a metallic casing. In some embodiments, B lineage cell preparations may be stored in syringes.
  • B lineage cell preparations are stored at -170 °C. In some embodiments, B lineage cell preparations are stored in ultra-cold freezers (e.g., vapor-based liquid nitrogen-based freezer). In some embodiments, B lineage cell preparations are stored in liquid nitrogen-based containers. In some embodiments, B lineage cell preparations are stored in vapor phase above liquid nitrogen. In some embodiments, B lineage cell preparations are stored in cryogenic storage.
  • B lineage cell preparations are transported through one or more methods to preserve a temperature of about -170°C. In some embodiments, B lineage cell preparations are transported in vapor phase above liquid nitrogen. In some embodiments, B lineage cell preparations are transported on dry ice.
  • the present disclosure describes cryopreserved engineered B lineage cell populations that can be used in both pre-clinical and clinical settings.
  • Appropriate thawing rate must be considered in order to both successfully cryopreserved a cell population as well as for administration of said cell population.
  • Thawing rate goes hand-in-hand with controlled cooling rate, as these are co-dependent processes. Rapid cooling will result in the amount of ice within the cryopreserved product to be lower than anticipated.
  • a relatively rapid rate of warmthing during thawing is required in order to obviate any potential cellular injuries to occur via further ice crystallization.
  • the cryoprcscrvcd B lineage cell preparation is transferred from a cryogenic storage into a thawing mechanism.
  • a thawing mechanism is a dry heater.
  • a thawing mechanism is a water bath.
  • the present disclosure provides various methods for characterization of engineered cell populations (e.g., B lineage cell populations).
  • methods disclosed herein are used to segregate and/or characterize naive B cell subpopulations within a B lineage cell population.
  • naive B cell subpopulations are characterized through one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic- activated cell sorting (MACS), and/or affinity chromatography.
  • FACS fluorescence-activated cell sorting
  • MCS magnetic- activated cell sorting
  • affinity chromatography affinity chromatography
  • one or more methods of characterizing naive B cell subpopulations are employed during each step of a controlled cooling method.
  • methods of characterization are employed to segregate and/or characterize engineered naive B cell subpopulations.
  • engineered naive B cell subpopulations are characterized through one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and/or affinity chromatography.
  • FACS fluorescence-activated cell sorting
  • MCS magnetic-activated cell sorting
  • affinity chromatography affinity chromatography
  • one or more methods of characterizing engineered naive B cell subpopulations are employed during each step of a controlled cooling method.
  • methods disclosed herein are used to segregate and/or characterize activated B lineage cell subpopulations within a B lineage cell population.
  • activated B lineage cell subpopulations are characterized through one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and/or affinity chromatography.
  • FACS fluorescence-activated cell sorting
  • MCS magnetic-activated cell sorting
  • affinity chromatography affinity chromatography
  • one or more methods of characterizing activated B lineage cell subpopulations are employed during each step of a controlled cooling method.
  • methods disclosed herein are used to segregate and/or characterize engineered activated B lineage cell subpopulations.
  • engineered activated B lineage cell subpopulations are characterized through one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and/or affinity chromatography. In some embodiments, one or more methods of characterizing engineered activated B lineage cell subpopulations are employed during each step of a controlled cooling method.
  • methods disclosed herein are used to segregate and/or characterize plasmablast cell subpopulations within a B lineage cell population.
  • plasmablast cell subpopulations are characterized through one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and/or affinity chromatography.
  • FACS fluorescence-activated cell sorting
  • MCS magnetic-activated cell sorting
  • affinity chromatography e.g., affinity chromatography.
  • one or more methods of characterizing plasmablast cell subpopulations are employed during each step of a controlled cooling method.
  • methods disclosed herein are used to segregate and/or characterize engineered plasmablast cell subpopulations.
  • engineered plasmablast cell subpopulations are characterized through one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and/or affinity chromatography. In some embodiments, one or more methods of characterizing engineered plasmablast cell subpopulations are employed during each step of a controlled cooling method.
  • methods disclosed herein are used to segregate and/or characterize plasma cell precursor subpopulations within a B lineage cell population.
  • plasma cell precursor subpopulations are characterized through one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and/or affinity chromatography.
  • FACS fluorescence-activated cell sorting
  • MCS magnetic-activated cell sorting
  • affinity chromatography e.g., affinity chromatography.
  • one or more methods of characterizing plasma cell precursor subpopulations are employed during each step of a controlled cooling method.
  • methods disclosed herein are used to segregate and/or characterize engineered plasma cell precursor subpopulations.
  • engineered plasma cell precursor subpopulations are characterized through one or more of flow cytometry, fluorescence-activated cell sorting (FACs), magnetic-activated cell sorting (MACs), and/or affinity chromatography. In some embodiments, one or more methods of characterizing engineered plasma cell precursor subpopulations are employed during each step of a controlled cooling method.
  • methods disclosed herein are used to segregate and/or characterize plasma cell subpopulations within a B lineage cell population.
  • plasma cell subpopulations may be characterized through one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and/or affinity chromatography.
  • FACS fluorescence-activated cell sorting
  • MCS magnetic-activated cell sorting
  • affinity chromatography chromatography
  • one or more methods of characterizing plasma cell subpopulations are employed during each step of a controlled cooling method.
  • methods disclosed herein are used to segregate and/or characterize engineered plasma cell subpopulations.
  • engineered plasma cell subpopulations are characterized through one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and/or affinity chromatography. In some embodiments, one or more methods of characterizing engineered plasma cell subpopulations are employed during each step of a controlled cooling method.
  • methods disclosed herein are used to segregate and/or characterize short-lived plasma cell populations within a B lineage cell population.
  • short-lived plasma cell subpopulations are characterized through one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and/or affinity chromatography.
  • FACS fluorescence-activated cell sorting
  • MCS magnetic-activated cell sorting
  • affinity chromatography affinity chromatography
  • one or more methods of characterizing short-lived plasma cell subpopulations are employed during each step of a controlled cooling method.
  • methods disclosed herein are used to segregate and/or characterize engineered short-lived plasma cell subpopulations.
  • engineered short-lived plasma cell subpopulations are characterized through one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and/or affinity chromatography. In some embodiments, one or more methods of characterizing engineered short-lived plasma cell subpopulations are employed during each step of a controlled cooling method.
  • methods disclosed herein may be used to segregate and/or characterize long-lived plasma cell subpopulations with a B lineage cell population.
  • long-lived plasma cell subpopulations are characterized through one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and/or affinity chromatography.
  • FACS fluorescence-activated cell sorting
  • MCS magnetic-activated cell sorting
  • affinity chromatography affinity chromatography
  • one or more methods of characterizing long-lived plasma cell subpopulations are employed during each step of a controlled cooling method.
  • methods disclosed herein are used to segregate and/or characterize engineered long-lived plasma cell subpopulations.
  • engineered long-lived plasma cell subpopulations are characterized through one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and/or affinity chromatography. In some embodiments, one or more methods of characterizing engineered long-lived plasma cell subpopulations are employed during each step of a controlled cooling method.
  • the present disclosure provides methods of treating a disease, disorder, or condition (e.g., a disease, disorder, or condition described herein) in a subject comprising administering a pharmaceutical composition described herein.
  • a therapeutically effective amount of a pharmaceutical composition described herein is administered to a subject having a disease or disorder.
  • Pharmaceutical compositions described herein can be for use in the manufacture of a medicament for treating a disease, disorder, or condition (e.g., a disease, disorder, or condition described herein) in a subject.
  • compositions described herein can comprise one or more B lineage cells selected from a population of genetically modified B lineage cells described herein.
  • B lineage cell populations are engineered to express or comprise a payload.
  • a payload is or comprises an expression cassette.
  • an expression cassette comprises a transgene encoding ALP or a fusion protein thereof, e.g., an ALP-Fc fusion protein.
  • a subject to be treated with methods described herein can be a mammal, e.g., a primate, e.g., a human (e.g., a patient having, or at risk of having, a disease, disorder, or condition described herein).
  • a subject has Hypophosphatasia (HPP).
  • HPP Hypophosphatasia
  • a subject can be an adult subject.
  • engineered B lineage cells are administered to a pediatric subject.
  • Administration of pharmaceutical compositions described herein may be carried out in any convenient manner (c.g., injection, ingestion, transfusion, inhalation, implantation, or transplantation).
  • a pharmaceutical composition described herein is administered by injection or infusion.
  • compositions described herein may be administered to a subject intravenously transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, or intraperitoneally.
  • a pharmaceutical composition described herein is administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or intramuscularly).
  • a pharmaceutical composition described herein is administered by intravenous infusion or injection.
  • a pharmaceutical composition described herein is administered by intramuscular or subcutaneous injection.
  • a pharmaceutical composition described herein is administered at a pharmaceutically suitable dosage to a subject.
  • a pharmaceutical composition described herein is administered monthly.
  • a pharmaceutical composition described herein is administered once every other month.
  • a pharmaceutical composition described herein is administered once every three months.
  • a pharmaceutical composition described herein is administered once every six months.
  • a pharmaceutical composition described herein is administered once a year.
  • a pharmaceutical composition described herein is administered to a subject having Hypophosphatasia (HPP).
  • HPP Hypophosphatasia
  • a pharmaceutical composition described herein is administered to a subject with mild-to-severe HPP.
  • the methods disclosed herein comprise measuring and/or monitoring treatment of Hypophosphatasia (HPP).
  • HPP Hypophosphatasia
  • a sample is collected from a subject treated with a pharmaceutical composition described herein.
  • sample collection is or comprises venipuncture.
  • sample collection is or comprises tissue collection.
  • levels of an ALP-Fc fusion protein are measured from sample collection.
  • lipids are assessed from a collected sample.
  • engraftment of one or more B lineage cells of a population of genetically modified B lineage cells described herein is measured in a non-clinical species (e. ., hIL6-/NOG mice).
  • engraftment of one or more B lineage cells of a population of genetically modified B lineage cells described herein is measured in a clinical species (e.g., a human subject). In some embodiments, engraftment is measured by use of bioluminescence. In some embodiments, engraftment is measured by use of ELISpot. In some embodiments, levels of plasma IgG are determined to measure engraftment. In some embodiments, levels of plasma IgM are determined to measure engraftment. In some embodiments, levels of a transgene (e.g., ALP or a fusion protein thereof, such as ALP-Fc fusion protein) are determined to measure engraftment.
  • ALP a transgene
  • engraftment of one or more B lineage cells of an engineered B lineage cell population occurs in a non-clinical species (e.g., hIL6-/NOG mice) with or without preconditioning of a non-clinical subject (e.g., chemotherapy, immunosuppressive treatment, etc. of a subject prior to administering B lineage cells as described herein).
  • engraftment of one or more B lineage cells of an engineered B lineage cell population described herein occurs in a clinical species (e.g., human subject) with or without preconditioning of a clinical subject (e.g., chemotherapy, immunosuppressive treatment, etc. of a subject prior to administering B lineage cells as described herein).
  • a B lineage cell population (e.g., an engineered B lineage cell population) described herein is cryopreserved prior to administration to a subject.
  • a cryopreservation method is described herein and/or described in WO 2024/173502, which is incorporated herein by reference in its entirety.
  • Example 1 Materials and Methods [0270] The present Example demonstrates in vitro assay methods that may be implemented to certain cell populations, including, but not limited to, engineered B lineage cell populations.
  • Cell populations can comprise or be engineered B lineage cell populations that express an ALP or a fusion protein thereof, such as an ALP-Fc fusion protein.
  • Engineered B lineage cell populations can comprise or be naive B cells.
  • Engineered B lineage cell populations can comprise or be engineered activated B lineage cells.
  • Engineered B lineage cell populations may comprise or be engineered plasmablast.
  • Engineered B lineage cell populations may comprise or be engineered plasma cells.
  • the present application provides methods that result in engineered B lineage cell populations. These methods include, but are not limited to, serum variation methods described herein.
  • B lineage cells are isolated and cultured in media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL-10, IL-15, 1L- 21 and/or CpG) for at least about 1, 2, 3, 4, 5, 6, or 7 days.
  • methods for B lineage cell activation comprise a step of contacting cells with media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG) for at least 2 days.
  • methods for B lineage cell activation comprise a step of contacting cells with media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL- 10, IL- 15, IL-21 and/or CpG) for at least 2 days, followed by a step of gene editing.
  • media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL- 10, IL- 15, IL-21 and/or CpG) for at least 2 days, followed by a step of gene editing.
  • cytokines and/or oligonucleotides e.g., CD40L, IL-2, IL- 10, IL- 15, IL-21 and/or CpG
  • B lineage cells are contacted with media lacking serum (e.g., human serum, bovine serum, horse serum, newborn calf serum, goat serum, rabbit serum, porcine serum, chicken serum, or a combination thereof) during gene editing.
  • B lineage cells subsequently undergo a wash step with base media (e.g., Excellerate) with or without cytokines.
  • base media e.g., Excellerate
  • B lineage cells are contacted with media lacking serum (e.g., human serum, bovine serum, horse serum, newborn calf serum, goat serum, rabbit serum, porcine serum, chicken scrum, or a combination thereof) and then incubated at 37°C for up to 24 hours.
  • B lineage cells are contacted with media substantially free of serum (e.g., human serum, bovine scrum, horse scrum, newborn calf scrum, goat scrum, rabbit scrum, porcine scrum, chicken serum, or a combination thereof) and then incubated at 37 °C for up to 24 hours.
  • serum e.g., human serum, bovine scrum, horse scrum, newborn calf scrum, goat scrum, rabbit scrum, porcine scrum, chicken serum, or a combination thereof.
  • B lineage cells are replated in media comprising one or more cytokines and/or oligonucleotides e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG) and expanded for at least 5 days.
  • cytokines and/or oligonucleotides e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG
  • Absolute ALP quantification was evaluated using targeted mass spectrometry.
  • This technique employs tandem mass spectrometry (MS/MS) using high-resolution mass spectrometers, and enabled quantification of ALP-specific peptide sequences.
  • samples were analyzed using single-point calibration against an ALP-specific standard isotopically labeled (SIL) peptide.
  • SIL isotopically labeled
  • sample preparation began with reduction with Tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP) and heat followed by alkylation with iodoacetamide. Protein was then precipitated using methanol/chloroform, and total protein content was assessed with a BCA Protein assay.
  • TCEP Tris-(2-carboxyethyl)-phosphine hydrochloride
  • the protein sample was digested with trypsin and SIL peptides were spiked into the sample.
  • tryptic peptides were fractionated using basic reverse phase liquid chromatography.
  • the mobile phase consisted of 10 mm triethylamine bicarbonate at pH 7.5 (TEAB) in water as solvent A, and 10 mm TEAB in 90% acetonitrile as solvent B .
  • the peptide fragments were then analyzed via selected reaction monitoring (SRM) on a mass spectrometer. Retention time was determined from prior analyses of synthetic peptide standards and found to be about 10.5 minutes.
  • Sample peptide signal intensities were monitored against that of the SIL peptide.
  • Area under the curve (AUC) for sample and SIL peptides were calculated in Skyline.
  • Sample concentration was mathematically determined based on a ratio of SIL AUC/SIL concentration.
  • a mouse preosteoblast cell line (MC3T3-E1 Subclone 4) was used to assess the ability of B lineage cells to influence in vitro mineralization.
  • MC3T3 base media which is composed of Alpha Minimum Essential Medium with nucleosides (a-MEM), 10% Fetal Bovine serum (FBS), and 1% Penicillin-Streptomycin.
  • a-MEM Alpha Minimum Essential Medium with nucleosides
  • FBS Fetal Bovine serum
  • Penicillin-Streptomycin 1% Penicillin-Streptomycin.
  • osteogenesis was initiated by the addition of ascorbic acid and P-glycerophosphate to a final concentration of 200 M and 100 pM, respectively.
  • PPi inorganic pyrophosphate
  • ARS staining After 21 days (about 3 weeks) of osteogenesis, mineralization was assessed by Alizarin Red S (ARS) staining or by total calcium levels.
  • ARS staining cells were washed with PBS and subsequently fixed with 4% paraformaldehyde (PFA) for 15 minutes. After fixation, cells were washed 3X with diH2O, allowing the water to sit for 5 to 10 minutes at each wash. Next, cells were submerged in ARS and incubated for 20 minutes at room temperature. Following staining, plates were washed three times with distilled water while gently rocking. Finally, fresh distilled water was added to prevent drying, and staining was imaged on a Cytation5 Plate Reader and Imager.
  • PFA paraformaldehyde
  • Calcium deposition was measured using a colorimetric calcium assay. At first, calcium deposits were extracted by incubating the samples with 0.5 N hydrochloric acid (HC1) for 4 hours. HC1 buffer was collected, and samples and standards then were plated in duplicate in a 96-well flat bottom plate. Chromogenic reagent and assay buffer were added to samples and standards. Plates developed for 5 to 10 minutes protected from light and read at 575 nm on a Cytation5 Plate Reader. Sample calcium levels were calculated using linear’ regression to a standard curve.
  • HC1 buffer 0.5 N hydrochloric acid
  • MSD immunoassay plates were precoated with capture IgG and IgM antibodies on independent and well-defined spots. Next, the MSD plates were first blocked using IX PBS with 1% BSA blocking buffer, prior to the addition of calibration standards, quality control samples, and test samples. Mouse plasma or serum samples were diluted in diluent 100 to a minimum of 64-fold before adding to the MSD plate.
  • MSD streptavidin immunoassay plates were used to detect human ALP and ALP- Fc fusion proteins in mouse serum or plasma samples. Plates were first blocked using IX PBS with 1% BSA blocking buffer and incubated for one hour room temperature shaking at 700 rpm. Then, plates were washed, and biotinylated anti-human ALP antibody was added to the plate and incubated for one hour room temperature shaking at 700 rpm. Plates were washed before adding standards, quality control samples, and test samples. Recombinant human ALP was used as a reference standard for ALP containing samples. For ALP-Fc containing samples, internal standards were generated.
  • Mouse plasma or serum samples were diluted in diluent 100 to a minimum of 5 -fold before adding to the MSD plate. All samples were incubated for one hour at room temperature, shaking at 700 rpm, then the plate was washed and sulfo-tag labelled antihuman ALP detection antibody was added to all wells. Next, samples were incubated for one hour at room temperature while shaking at 700 rpm. Analytes in sample bind to capture antibodies immobilized on the working electrode surface and recruitment of the detection antibodies by the bound analytes completed the sandwich. After another wash, read buffer is added to all wells, which provides an appropriate chemical environment for electrochemiluminescence. The plate was loaded onto the instrument, where a voltage applied to the plate electrodes caused captured labels to emit light. The instrument measured the intensity of emitted light to provide quantitative measure of analytes in the sample. Human ALP and ALP- Fc fusion proteins concentrations were calculated by five parameter logistic regression model to appropriate reference protein standards.
  • Methods for quantifying cytokines on flow cytometers was used per vendor recommendation to assess ALP or ALP-Fc fusion protein concentration in engineered B lineage cell culture supernatant or mouse plasma samples.
  • Samples from relevant experiments were harvested and frozen at -80°C prior to testing. All samples and reagents were brought to room temperature prior to the initiation of the assay.
  • ALP antibody coated capture beads, LEGENDPlex assay buffer, and standard/sample were added to a 96 well V-bottom plate. Plates were incubated (protected from light) for 2 hours at room temperature while shaking vigorously (at -800 rpm). Following incubation, plates were washed twice by addition of LEGENDPlex wash buffer and centrifuged for 5 minutes at 250 g at 23°C. Supernatant was removed by flicking.
  • ALP and ALP-Fc containing samples were analyzed using different reference standards. Recombinant human ALP was used as a reference standard for ALP containing samples. For ALP-Fc containing samples, an internal standard was generated and described herein. Bulk BCM cell culture supernatant containing ALP-Fc was harvested, aliquoted, banked, and stored at -80°C. A thawed frozen aliquot was analyzed for absolute ALP quantification using targeted mass spectrometry (TMS; described above). Banked engineered B lineage cell supernatant containing a known amount of ALP-Fc (based on the TMS-derived concentration) was serially diluted and used as a reference standard for ALP-Fc BCM samples.
  • TMS targeted mass spectrometry
  • Immunoglobulin G (IgG) and Immunoglobulin M (IgM) ELISpot of engineered B lineage cell population supernatant
  • 96 well multiscreen filter plates were briefly washed with 35% ethanol for 40-60 seconds until the membrane turned gray from white. Plates were then washed 3 times with Dulbccco’s PBS (dPBS), and subsequently coated with the capture antibody (anti-human IgG or anti-human IgM) at 10 pg/mL in PBS overnight at 4°C. Next day, the plates were washed 3 times with an automated plate washer. The antibody coated plates were then blocked against non-specific binding with Iscove's Modified Dulbecco's Medium (IMDM) with 1% FBS for 2 hours at 37°C. Next, cells were counted and added to the wells of the plate.
  • IMDM Iscove's Modified Dulbecco's Medium
  • the plates were incubated in an incubator at 37°C for 16-24 hours. Next day, the plates were washed for an additional 3 times. Plates were then blocked with 1% BSA at room temperature for 1 hour and washed 3 more times. Next, plate-bound hlgG or hlgM were detected by a secondary biotinylated antibody (anti-human IgG or anti-human IgM) at 1:1000 dilution in 1% BSA and were incubated for 2 hours at room temperature. At the end of incubation, plates were washed as previously described and then with exposed to streptavidin alkaline phosphatase at 1:5000 dilution in 1% BSA.
  • a secondary biotinylated antibody anti-human IgG or anti-human IgM
  • ALP enzymatic activity analysis for engineered B lineage cell population supernatant [0283] In order to evaluate the ALP activity of engineered B lineage cell populations as disclosed herein, enzymatic activity was assessed by measuring the hydrolysis of a synthetic substrate 4-methylumbelliferone phosphate (4-MUP), into its fluorescent product, 4- methylumbelliferone (4-MU). All reagents were brought to room temperature prior to the initiation of the assay. B lineage cell timed supernatant samples or standards were added to a black 384 well plate. The reaction was initiated by the addition of 4-MUP, then immediately placed into a Cytation5 plate reader set to 37°CFluorescent readings at 360/440 nm were taken in a kinetic fashion every minute for one hour.
  • B lineage Cell-derived ALP enzymatic activity at a point of linearity was calculated by linear regression to a reference 4-MU product standard.
  • activity was normalized to percent HDR as determined by ddPCR.
  • Sample ALP enzymatic activity was calculated by linear regression at a sample dilution that reaches linearity at 15 minutes to the activity of a reference 4-MU product standard. Activity was converted to Units, where 1 unit is equal to 1 pmole of 4-MUP hydrolyzed/minute, then converted to Units/Liter of serum.
  • B lineage Cell-derived ALP enzymatic activity was calculated by linear regression to the activity of a reference 4-MU product standard. Activity was converted to Units, where 1 unit is equal to 1 pmole of 4-MUP hydrolyzed/minute, then converted to mUnits/Liter of sample.
  • gCCR5_232 ICE Reverse primer AGGATTCCCGAGTAGCAGATGAC (SEQ ID NO: 1).
  • gJchain_31 ICE Forward (and sequencing) primer TTCAGACTTCAACCACAGTTG (SEQ ID NO: 4).
  • gJchain3_l ICE Reverse primer AACCACAGACTCAGTGATAGG (SEQ ID NO: 5).
  • gJchain_54 ICE Forward primer: TTGGCATTCATAGAAAGGCC (SEQ ID NO: 6).
  • gJchain_54 ICE Reverse (and sequencing) primer AAAAGCCCTGGTTTCAAATTCATTG (SEQ ID NO: 7).
  • gJtoIGHM_23 ICE Forward (and sequencing) primer: GAATGGCCACTCTAGGGCCTTTG (SEQ ID NO: 8).
  • gJtoIGHM_23 ICE Reverse primer: TGATTTATGATGGTCAAAACGCAG (SEQ ID NO: 9).
  • gCD 19_5 ICE Forward primer GAAACAGAGTCTCACTCTGG (SEQ ID NO: 12).
  • CCRL2_ddPCR Reverse primer for pairing with gJCHAIN_54: GCTGTATGAATCCAGGTCC (SEQ ID NO: 39); and [0324] CCRL2_ddPCR Probe (HEX) (for pairing with gJCHAIN_54): TGTTTCCTCCAGGATAAGGCAGCTGT (SEQ ID NO: 40).
  • FcyRIIa binding assay was performed using the Lumit FcyRIIa (H131) Binding Immunoassay kit according to the manufacturer’s instructions (Promega). Briefly, an 8-point, 4 fold titration series was generated for the human IgG negative control, human IgA positive control and test samples stalling at 0.25 mg/mL. Next, 25 pL of prepared Tracer-LgBiT solution was added per well into the microplate wells, and 25 pL of control antibodies standards or protein samples were added to their respective wells. Next, 50 pL of the prepared FcyRIIa (H131)-SmBiT solution was added per well.
  • the plate was then covered with a foil plate seal and incubated with gentle mixing on a plate shaker (300 rpm) for 45 minutes at room temperature. 25 pL of prepared Lumit Detection Reagent per well was then added to the microplatc. The plate was subsequently incubated at room temperature for 5 minutes and then read on a plate luminometer.
  • FcyRI binding assay was performed using the Lumit FcyRI Binding Immunoassay kit according to the manufacturer’s instructions (Promega). Briefly, an 8-point, 4 fold titration series was generated for the human IgG negative control, human IgA positive control and test samples starting at 0.25 mg/mL. Next, 25 pL of prepared Tracer-LgBiT solution was added per well into microplatc wells and 25 pL of the control antibodies standards or protein samples were added to their respective wells. 50 pL of the prepared FcyRI-SmBiT solution was added per each reaction well.
  • the plate was covered with a foil plate seal and then incubated with gentle mixing on a plate shaker (300 rpm) for 60 minutes at room temperature.
  • 25 pL of prepared Lumit Detection per well was added to the microplate. The plate was then incubated at room temperature for 5 minutes and read on a plate luminometer.
  • FcRn binding assay was performed using the FcRn AlphaLISA Kit according to the manufacturer’s instructions (Revvity).
  • a lx AlphaLISA MES buffer was prepared fresh using Milli-Q water and buffer concentrate.
  • serial dilutions of human IgG positive control, human IgA negative control antibody and protein samples were generated in lx AlphaLISA MES buffer.
  • 10 pL of control antibodies or test proteins were added to their appropriate wells and spun at 400xg for 1 minute.
  • 10 pL of 800 ng/mL human FcRn was added to each well of the plate and span plate at 400xg for 1 minute.
  • N-linked glycans were released from the protein sample using PNGaseF (New England BioLabs) following the manufacturer’s instruction, and were labeled with 2- aminobenzamide (2-AB) (Sigma- Aldrich)
  • the released N-linked glycans were subsequently loaded onto a Waters XBridge Amide column (4.6 x 150 mm, 3.5 pm) with mobile phases A and B made of 0.05% trifluoroacetic acid in acetonitrile and 0.05% trifluoroacetic acid in water, respectively.
  • the water gradient (mobile phase B) of 28 to 42% was delivered from timepoints 5 to 42 minutes onto the column, then changed to water gradient of 42 to 90% from timepoint 42 to 43 minutes at a flow rate of 1.0 mL/minute.
  • the 2-AB labeled glycans were then detected by fluorescence (330 nm excitation, 420 nm emission).
  • the present Example demonstrates that B lineage cell populations engineered with one or more expression cassettes (e.g., ALP or a fusion protein thereof, such as ALP-Fc fusion protein containing expression cassette) may integrate a polynucleotide sequence encoding for a transgene and express said transgene (e.g., ALP or a fusion protein thereof, such as ALP-Fc fusion protein). Further, the present Example demonstrates that substitutions within the immunoglobulin fragment crystallizable region (Fc) may reduce undesirable heterodimer formation with endogenous Fc while maintaining enzymatic function.
  • ALP or a fusion protein thereof such as ALP-Fc fusion protein containing expression cassette
  • B lineage cells were engineered using methods described herein with a histidine tagged alkaline phosphatase (ALP) and immunoglobulin fragment crystallizable region (ALP- IgGlFc-His) or guide alone. Protein from engineered B lineage cell populations was then histidine purified and ran on a Western blot using the methods described in Example 1. Use of anti-alkaline phosphatase antibody revealed two distinct bands in Western blot. Anti-IgG-CHl antibody blotting revealed IgG heterodimerization, possibly corresponding to the secondary band in the anti- ALP blot ( Figure 1A).
  • ALP histidine tagged alkaline phosphatase
  • ALP- IgGlFc-His immunoglobulin fragment crystallizable region
  • ALP-Fc fusion protein comprised three substitutions (K392D/D399K7K409D) in the Fc domain (ALP-IgGlFc-3xsubst). Both anti- ALP and anti-IgG CHI Western blots showed reduced heterodimer formation in engineered B lineage cells expressing the ALP-Fc fusion protein ( Figure IB).
  • One mutant ALP-Fc fusion protein comprised three substitutions (K392D/D399K/K409D) in the Fc domain (ALP-IgG4Fc-3xsubst).
  • Anti- ALP Western blot showed reduced heterodimer formation in engineered B lineage cells expressing ALP-IgG4Fc-3xsubst fusion protein ( Figure 26).
  • B lineage cells may be engineered to express a transgene which encodes an alkaline phosphatase (ALP) and immunoglobulin fragment crystallizable region fusion protein (ALP-Fc).
  • ALP alkaline phosphatase
  • ALP-Fc immunoglobulin fragment crystallizable region fusion protein
  • the Fc region is derived from human immunoglobulin 1 (IgGlFc).
  • the Fc region is derived from human immunoglobulin 4 (IgG4Fc).
  • one or more substitutions may be introduced within the Fc region of the ALP-Fc fusion protein.
  • one or more substitutions within the Fc region comprise K392D, D399K, and/or K409D.
  • one or more substitutions within the IgGlFc region comprise K392D, D399K, and/or K409D. In some embodiments, one or more substitutions within the IgG4Fc comprise K392D, D399K, and/or R409D. In some embodiments, incorporation of one or more substitutions within the Fc region of an ALP-Fc fusion protein may reduce formation of heterodimers with endogenous Fc. In some embodiments, incorporation of one or more substitutions within the Fc region of an ALP-Fc fusion protein may reduce formation of heterodimers with endogenous Fc while maintaining comparable enzymatic activity.
  • ALP-Fc fusions may be further optimized to reduce formation of heterodimers with endogenous Fc, while maintaining comparable enzymatic activity. In some embodiments, ALP-Fc fusions may be further optimized to reduce formation of heterodimers with endogenous Fc, while maintaining improving enzymatic activity.
  • substitutions within the Fc region including but not limited to, K392D, D399K, and/or K409D of IgGlFc or K392D, D399K, and/or R409D of IgG4Fc may reduce heterodimerization of the fusion protein with endogenous Fc.
  • Example 3 Engineered B lineage cell populations can integrate, express and/or secrete proteins of interest
  • B lineage cell populations engineered with one or more expression cassettes e.g., ALP or a fusion protein thereof, such as ALP-Fc fusion protein containing expression cassette) may integrate a polynucleotide sequence encoding for a transgene and express said transgene (e.g., ALP-Fc fusion protein).
  • Subjects treated with engineered cell populations described herein may demonstrate persistent levels of transgene expression (e.g., tissue non-specific alkaline phosphatase) over an extended period of time (e.g., one or more weeks, one or more months, etc.).
  • Osteogenic stimulation assay was performed with osteoblast precursor (MC3T3) cells, which were cultured in ascorbic acid and beta-glycerolphosphate over a time course of 21 days as described in Example 1 ( Figure 2A). Osteoblast precursor cells were treated at days 6-21 with either media alone, media with pyrophosphate (PPi), or media containing cultured supernatant from un-engineered B lineage cells ( Figure 2B).
  • osteogenic stimulation assay was performed (Figure 3A) involving osteoblast precursor cells (MC3T3) with 250 or 500 ng/mL of either ALP-Fc fusion proteins with a deca-aspartate tag (ALP-Fc-DlO) or alkaline phosphatase with a deca-aspartate tag (ALP-D10) ( Figure 3B).
  • Osteogenic stimulation assay was performed involving osteoblast precursor (MC3T3) cells contacted with media alone, media with pyrophosphate (PPi), or 50 ng/mL of either media containing cultured supernatant from B lineage cells engineered to express ALP or electroporated control (EPC) B lineage cells. (Figure 27).
  • a 4-MUP activity assay was used to evaluate the enzymatic activity of recombinant ALP (rALP) and engineered B lineage cell derived ALP fusion proteins; ALP-Fc, ALP-D10, or ALP- Fc-DlO using methods described in Example 1 ( Figure 6). Subsequently, ALP enzymatic activity was assessed for engineered B lineage cell derived alkaline phosphatase and immunoglobulin fragment crystallizable region fusion protein (ALP-IgGlFc) or ALP- IgGlFc with the K392D, D399K, and K409D substitutions (ALP-IgGlFc-3xsubst) ( Figure 7 and Figure 12).
  • Engineered B lineage cells expressing ALP-D10 were administered to NOG-IL6 mice. Levels of secreted ALP were measured in mice upon administration (day 0) of ALP-BCMs through day 84 post-administration ( Figure 34D). Results were compared to NOG-IL6 mice to which control cells electroporated without vector were administered.
  • NOG-IL6 mice were either intravenously or intraperitoneally treated with engineered B lineage cells expressing ALP-Fc fusion protein and measured at days 7-14 post- engraftment for levels of either ALP or ALP-Fc in their serum (Figure 8A).
  • Human immunoglobulin G was measured in mouse serum using methods described in Example 1. Results were compared to a media alone control (PlasmaLyte I.P.) ( Figure 8B).
  • Engineered B lineage cells expressing ALP-Fc were administered to N0G-IL6 mice. Levels of secreted ALP-Fc were measured in mice upon administration (day 0) of ALP- BCMs through day 84 post-administration ( Figure 25A).
  • ALP-Fc was isolated at day 84 postadministration from mice to which ALP-BCMs were administered and assessed for activity in serum (Figure 25B). Levels of human immunoglobulin G were assessed in mice to which ALP- BCMs were administered using methods described in Example 1 ( Figure 25C). Mouse weight was also assessed ( Figure 25D). Results were compared to NOG-IL6 mice to which control cells electroporated without vector (EPC) were administered.
  • B lineage cells engineered with HPPpO215 (ALP-LvL-lgG4Fc-D10) were cryopreserved and expanded as described in Example 1 and were administered to NOG-IL6 mice.
  • Levels of secreted ALP-Fc were measured in mice upon administration (day 0) of ALP- BCMs through day 14 post-administration ( Figure 30). Results were compared to NOG-IL6 mice to which control cells electroporated without vector (EPC) were administered.
  • Cryoprcscrvcd B lineage cells engineered with HPPpO215 were administered to NOG-IL6 mice.
  • Levels of secreted ALP-Fc were measured in mice upon administration (day 0) of ALP-BCMs through day 58 post-administration ( Figure 31 A).
  • hALP activity was assessed at day 58 post-administration ( Figure 3 IB).
  • Results were compared to NOG-IL6 mice to which control cells electroporated without vector (EPC) were administered.
  • the present disclosure demonstrates that engineered B cells expressing ALP or variants thereof (e.g., ALP-Fc fusion proteins) were effective in restoring bone mineralization (i.e., calcium levels).
  • ALP-Fc fusion proteins e.g., ALP-Fc fusion proteins
  • the present disclosure demonstrates that engineered B lineage cells were able to express functional ALP-Fc fusion proteins.
  • cryopreserved engineered B lineage cells were able to express functional ALP-Fc fusion proteins.
  • cryoprcscrvcd engineered B lineage cells expanded with various expansion processes were able to express functional ALP-Fc fusion proteins.
  • the present disclosure shows that engineered B lineage cells expressing ALP-Fc fusion proteins were capable of engrafting (e.g., in bone marrow) in a subject (e.g., mouse) for long-term protein expression.
  • the present disclosure demonstrates that engineered B lineage cells expressing ALP-Fc fusion proteins were capable of engrafting (e.g., in bone marrow) in a subject (e.g., mouse) and to produce and secrete functional ALP (e.g., in serum) for an extended time period.
  • the present disclosure demonstrates that engineered B lineage cells expressing ALP-IgG4-Fc-D10 fusion proteins were capable of engrafting (e.g., in bone marrow) in a subject (e.g., mouse) and to produce and secrete functional ALP (e.g., in serum) for an extended time period.
  • the present disclosure shows that treatment of a subject (e.g., mouse) with engineered B lineage cells expressing ALP-Fc fusion proteins does not result in adverse effects in the subject.
  • the present disclosure shows that treatment of a subject (e.g., mouse) with engineered B lineage cells expressing ALP-Fc fusion proteins could restore bone mineralization.
  • such engineered B lineage cell populations are capable of producing clinically relevant doses of ALP or a fusion protein thereof, such as ALP-Fc fusion protein. In some embodiments, such engineered B lineage cell populations are capable of engraftment in a subject without pre-conditioning. In some embodiments, such engineered B lineage cell populations are capable of long-term expression of a protein (e.g., a protein described herein) in a subject.
  • a protein e.g., a protein described herein
  • Example 4 Engineered B lineage cell populations expressing ALP-Fc fusion proteins with variant linkers may have improved enzymatic activity
  • the present Example demonstrates that B lineage cell populations engineered with one or more expression cassettes (e.g., ALP or a fusion protein thereof, such as ALP-Fc fusion protein containing expression cassette) may integrate a polynucleotide sequence encoding for a transgene and express said transgene (e.g., ALP-Fc fusion protein). Further, the present Example demonstrates that integration of a ‘long linker’ (also interchangeably referred to herein as “LL”) comprising three continuous regions of tetra-glycine and mono-serine (GGGGSx3) may improve enzymatic activity (Figure 16A).
  • LL long linker
  • the present Example demonstrates that integration of a linker comprising two continuous regions of tetra-glycine and mono-serine followed by a mono-glycine (GGGGSx2-G) may both improve enzymatic activity and allow for flexibility of ALP.
  • ALP-Fc fusion proteins comprising different linkers between the ALP and Fc domains were engineered. These ALP-Fc fusion proteins comprise: (1) a linker comprising a leucine and a lysine (LK), or (2) a linker comprising three continuous regions of tetra-glycine and mono-serine (GGGGSx3).
  • LK leucine and a lysine
  • GGGGSx3 mono-serine
  • ALP-Fc fusion proteins comprising a Fc derived from human immunoglobulin 4 (IgG4) and S288P, F234A, and L235A (PAA) substitutions was assessed with and without addition of the long linker (LL) and the K392D, D399K, and R409D substitutions (3xsubst) indicated a potential increase in enzymatic activity (Figure 9D).
  • LL long linker
  • substitutions 3xsubst
  • design, testing, and/or selection of ALP-Fc fusion protein sequences may comprise various methods including, but not limited to, use of software (e.g., AI- assisted software, e.g., AlphaFold2).
  • software e.g., AlphaFold2
  • such software may provide information on projected protein structure and/or function.
  • such software may provide information to assist with selection of one or more linkers (e.g., linkers between domains, such as, e.g., ALP and Fc domains) ( Figure 16B and Figure 16C).
  • ALP-Fc fusion proteins comprising different linkers between ALP and Fc domains were tested using software modeling. Linker length was systematically decreased, and GGGGS(x2)-G and GGGGS(x2)-GS linkers were identified as allowing for flexibility of ALP in ALP-Fc fusion proteins while maintaining conformation.
  • such engineered B lineage cell populations are capable of producing ALP or a fusion protein thereof, such as ALP-Fc fusion protein. In some embodiments, such engineered B lineage cell populations are capable of enzymatic activity.
  • ALP-Fc fusion proteins may further comprise of a linker.
  • a linker may comprise of three continuous regions of tetra-glycine and monoserine (GGGGS). In some embodiments, incorporation of the linker comprising GGGGSx3 may improve enzymatic activity. In some embodiments, incorporation of the linker comprising GGGGSx3 may improve activity of the ALP in the ALP-Fc fusion protein.
  • a linker may comprise of two continuous regions of tetra-glycine and mono-serine (GGGGS) followed by an additional C-terminal mono-glycine (G). In some embodiments, a linker may comprise of two continuous regions of tetra-glycine and mono-serine (GGGGS) followed by an additional C-terminal mono-glycine and mono-serine (GS). In some embodiments, incorporation of the linker comprising GGGGSx2-G may allow for flexibility of ALP and improve activity of the ALP in the ALP-Fc fusion protein while maintaining conformation.
  • B lineage cell populations engineered with one or more expression cassettes may integrate a polynucleotide sequence encoding for a transgene and express said transgene (e.g., ALP-Fc fusion protein).
  • expression cassettes may comprise one or more sequence elements (e.g., enhancers, promoters, etc.) providing enhanced integration, secretion, and/or activity of a transgene (e.g., ALP-Fc fusion protein) as compared to a reference (e.g., expression cassettes that do not comprise one or more sequence elements).
  • expression cassettes comprise one or more enhancers (e.g., Ep) or variants thereof providing enhanced integration, secretion, and/or activity of a transgene (e.g., ALP-Fc fusion protein).
  • expression cassettes may comprise a codon-optimized variant of a transgene (c.g., ALP-Fc) that provides enhanced integration, secretion, and/or activity of transgcnc (c.g., ALP-Fc fusion protein) as compared to a reference (e.g., expression cassettes that do not comprise a codon-optimized transgene variant).
  • ALP-Fc a transgene
  • expression cassettes may comprise a codon-optimized variant of a transgene (c.g., ALP-Fc) that provides enhanced integration, secretion, and/or activity of transgcnc (c.g., ALP-Fc fusion protein) as compared to a reference (e.g., expression cassettes that do not comprise a codon-optimized transgene variant).
  • the present Example demonstrates use of different gRNAs that may result in integration, secretion, and activity of transgenes (e.g., ALP-Fc
  • ALP-Fc fusion protein comprising different codon optimized regions were engineered using GeneArt (“CO-1”), or an alternative method (“CO-2”).
  • B lineage cells were engineered with either CO-1 , CO-2, or non-codon optimized (Unoptimized) expression cassettes as described in Example 1.
  • ALP-Fc secretion from the supernatant of engineered B lineage cells was measured using LegendPlex assay as described within Example 1 ( Figure 13).
  • B lineage cells were engineered with expression cassettes comprising either ALP signal peptide (“SP-1”) or A1AT signal peptide (“SP-2”). Measurements of ALP-Fc secretion from collected supernatant was analyzed using methods described herein ( Figure 14).
  • B lineage cells were engineered with expression cassettes described herein using gRNAs targeting either CCR5 or JCHAIN1 (“Locus B”) ( Figure 11).
  • ALP-Fc fusion protein secretion was measured from collected supernatant and assessed ( Figure 15). Further assessment of ALP-Fc fusion protein production from B lineage cells engineered using guides targeting CCR5, JCHAIN173 (“Locus A”), or JCHAIN1 was conducted and compared to an electroporated control (“EPC”) ( Figure 17). Both Bulk ( Figure 18) and Specific (Figure 19) ALP-Fc activity was measured in these engineered B lineage cell populations using a 4-MUP activity assay as described in Example 1.
  • B lineage cells engineered with ALP-Fc expression cassettes comprising an MND promoter with a shortened Ep enhancer element or variant thereof in vivo B lineage cells engineered with ALP-Fc expression cassettes comprising a MND promoter with a small Ep enhancer (p00983) or without a Ep enhancer (HPPpO215) were administered intravenously to NOG-IL6 mice.
  • Levels of secreted ALP-Fc were measured in mice upon administration (day 0) of ALP-BCMs through day 35 postadministration (Figure 32A).
  • hALP activity was assessed at day 35 post-administration ( Figure 32B). Results were compared to NOG-IL6 mice to which control cells electroporated without vector were administered (“unengineered”).
  • B lineage cells engineered with ALP-Fc expression cassettes comprising an MND promoter with a shortened Ep enhancer element or variant thereof in vivo B lineage cells engineered with ALP-Fc expression cassettes comprising a MND promoter with a small Ep enhancer were administered intravenously to NOG-IL6 mice.
  • Levels of secreted ALP-Fc were measured in mice upon administration (day 0) of ALP-BCMs through day 42 post-administration ( Figure 36). Results were compared to NOG- IL6 mice to which control cells electroporated without vector were administered (“unengineered”).
  • expression cassettes may comprise one or more elements to increase expression and/or secretion of ALP-Fc fusion proteins, or variants thereof.
  • expression cassettes may comprise one or more signal peptides (e.g., ALP signal peptide, A1AT signal peptide, etc.) that increase expression and/or secretion of ALP-Fc fusion proteins, or variants thereof.
  • expression cassettes may comprise a polyaspartate tag.
  • expression cassettes may comprise one or more promoters (e.g., EF- la promoter, MND promoter, etc.) that increase expression and/or secretion of ALP-Fc fusion proteins, or variants thereof.
  • expression cassettes may comprise one or more enhancers (e.g., Ep) or variants thereof that increase transgene integration, expression, and/or secretion of ALP-Fc fusion proteins, or variants thereof.
  • expression cassettes may comprise one or more Ep enhancers or variants thereof that provide increased protein secretion.
  • expression cassettes may comprise one or more enhancers (e.g., Ep) or shortened variants thereof that alter transgene integration, expression, and/or secretion of ALP-Fc fusion proteins, or variants thereof.
  • expression cassettes may comprise one or more Ep enhancers or shortened variants thereof that provide altered protein secretion.
  • ALP-Fc fusion proteins were purified from cells expressing ALP-Fc expression cassettes as described in Example 1.
  • ALP-IgG4Fc-D10 was purified from the supernatant of BCM engineered to express ALP-Fc and assessed via size exclusion chromatography as described in Example 1 ( Figure 39A).
  • ALP-IgG4Fc-D10 was purified from the supernatant of BCM engineered to express ALP-Fc after three frcczc-thaw cycles and assessed via size exclusion chromatography (Figure 39B). Size exclusion chromatography peaks for ALP-IgG4Fc- D10 from freeze-thaw and non-freeze-thaw BCMs were compared ( Figure 39C).
  • ALP-Fc fusion proteins were purified from cells expressing ALP-Fc expression cassettes as described in Example 1.
  • ALP-IgG4Fc-D10 was purified from the supernatant of BCM engineered to express ALP-Fc and assessed using mass spectrometry as described in Example 1 ( Figure 40).
  • FgyRI binding assay was performed on purified proteins produced from various ALP-Fc fusion protein constructs as described in Example 1. FgyRI binding was measured on proteins produced from ALP-IgG4-D10, ALP-IgG4, ALP-IgGl-DlO, IgG (positive control), or IgA (negative control) expression cassettes ( Figure 42). FgyRI binding was measured on proteins produced from ALP-IgG4-D10, ALP-IgGl-DlO, or IgG expression cassettes ( Figure 46).
  • FcRn binding assay was performed on purified proteins produced from various ALP-Fc fusion protein constructs as described in Example 1. FcRn binding was measured on proteins produced from ALP-IgG4-D10, ALP-IgG4, ALP-IgGl-DlO, IgG (positive control), or IgA (negative control) expression cassettes ( Figure 43). FcRn binding was measured on proteins produced from ALP-IgG4-D10, ALP-IgGl-DlO, IgA, or IgG expression cassettes ( Figure 47)
  • N-glycan analysis was performed on purified proteins produced from various ALP-Fc fusion protein constructs as described in Example 1. Sialic acid residues were compared between ALP-IgG4-D10 and ALP-IgGl-DlO ( Figure 44). Sialic acid residues were compared between ALP-IgG4-D10 and ALP-IgGl -DlO ( Figure 48).
  • Alp-Fc levels were measured over time in serum from Tg32 mice dosed with purified proteins produced from ALP-Fc fusion protein constructs as described in Example 1.
  • ALP-Fc levels were measured in samples from mice dosed with ALP-lgG4Fc-D10, ALP-IgG4Fc and ALP-IgGlFc-DlO purified from HEK293 cells ( Figure 49A).
  • ALP-Fc levels were measured in samples from mice dosed with ALP-IgG4Fc-D10 purified from HEK293 cells, ALP-IgG4Fc- D10 from BCM and ALP-IgGlFc-DlO from HEK293 ( Figure 50A).
  • the present disclosure demonstrates that purified ALP- IgG4Fc with substitutions in the Fc region (ALP-IgG4-Fc-PAA) exhibits similar biochemical spectra to ALP-IgGl-Fc. In some embodiments, the present disclosure demonstrates that purified ALP-IgG4Fc exhibits similar’ biochemical spectra when produced by fresh BCM or BCM after freeze-thaw cycles.
  • the present disclosure demonstrates that ALP-IgG4Fc with substitutions in the Fc region (ALP-IgG4-Fc-PAA) exhibits higher binding to FcRn than ALP- IgGl-Fc. In some embodiments, the present disclosure demonstrates that ALP-IgG4-Fc-PAA shows lower binding to FcyRIIa than ALP-IgGl-Fc. In some embodiments, the present disclosure demonstrates that ALP-IgG4-Fc-PAA shows lower binding to FcyRI than ALP-IgGl- Fc. In some embodiments, the present disclosure demonstrates that ALP-IgG4-Fc-PAA contains increased sialic acid residues when compared to ALP-IgGl-Fc.
  • the present disclosure demonstrates that ALP-IgG4Fc produced by HEK cells has comparable pharmacokinetics in serum to ALP-IgGlFc. In some embodiment, the present disclosure demonstrates that ALP-IgG4Fc produced by BCM exhibits higher ALP levels and ALP activity over time in serum when compared to ALP-IgGlFc.
  • ALP-IgG4-Fc-PAA fusion proteins may be associated with decreased immune activation as compared to ALP-IgGl-Fc fusion proteins. In some embodiments, ALP-IgG4-Fc-PAA fusion proteins may be associated with increased cellular recycling as compared to ALP-IgGl-Fc fusion proteins. In some embodiments, ALP-IgG4-Fc- PAA fusion proteins may be associated with decreased cndocytosis as compared to ALP-IgGl- Fc fusion proteins. In some embodiments, AEP-IgG4-Fc-PAA fusion proteins may evade receptor-mediated degradation.
  • AEP-IgG4-Fc-PAA fusion proteins may experience lower receptor-mediated degradation as compared to AEP-IgGl-Fc fusion proteins.
  • ALP-IgG4-Fc-PAA fusion proteins may have extended half-life in vivo as compared to AEP-IgGl-Fc fusion proteins.

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Abstract

La présente invention concerne des procédés et des compositions pour la culture, la modification et l'administration de populations de cellules de lignée B modifiées qui produisent une ALP ou une protéine de fusion de celle-ci, telle qu'une protéine de fusion ALP-Fc comprenant une phosphatase alcaline et une région cristallisable de fragment d'immunoglobuline humaine.
PCT/US2024/054285 2023-11-02 2024-11-01 Préparations cellulaires modifiées pour le traitement de l'hypophosphatasie Pending WO2025097067A2 (fr)

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