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WO2025165787A1 - Sélection sans contact de thérapies cellulaires modifiées par un gène par l'intermédiaire d'insertions d'introns trac - Google Patents

Sélection sans contact de thérapies cellulaires modifiées par un gène par l'intermédiaire d'insertions d'introns trac

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Publication number
WO2025165787A1
WO2025165787A1 PCT/US2025/013455 US2025013455W WO2025165787A1 WO 2025165787 A1 WO2025165787 A1 WO 2025165787A1 US 2025013455 W US2025013455 W US 2025013455W WO 2025165787 A1 WO2025165787 A1 WO 2025165787A1
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WIPO (PCT)
Prior art keywords
cell
cells
car
rna
intron
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Inventor
Ansuman Satpathy
Theo ROTH
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Leland Stanford Junior University
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Leland Stanford Junior University
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    • 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
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] 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
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • 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)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C12N9/222—Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes
    • C12N9/226—Class 2 CAS enzyme complex, e.g. single CAS protein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]

Definitions

  • Sequence Listing is provided herewith as a Sequence Listing XML file, “STAN- 2097WO”, created on January 23, 2025, and having a size of 581 ,334 bytes.
  • the contents of the Sequence Listing XML file are incorporated by reference herein in their entirety.
  • Genetically modified cell therapies such as CAR-T therapies have revolutionized cancer care and hundreds are in active clinical development.
  • the process of making the necessary genetic modifications to these cells especially targeted genetic editing using CRISPR/Cas9 systems, which have shown greater efficacy than randomly integrating viral vectors, can be inefficient, leaving a mixed cell population of some edited cells along with many unedited or incorrectly edited cells.
  • CRISPR/Cas9 systems which have shown greater efficacy than randomly integrating viral vectors
  • compositions and methods are provided for genetically modifying a T cell to express a chimeric antigen receptor (CAR) and/or additional new genes with knockout of an endogenous T cell receptor (TCR).
  • the subject methods utilize a donor polynucleotide encoding a CAR that is integrated into an intron of a TCR gene.
  • the successfully edited T cells produce a mature mRNA with a nucleotide sequence encoding the CAR spliced in between the exon(s) encoding the TCR protein, resulting in expression of the CAR and knockout of the TCR.
  • the unsuccessfully edited T cells retain expression of their TCR.
  • the T cell population can be enriched for successfully edited TCR negative T cells expressing the CAR by using negative selection to remove unsuccessfully edited TCR positive T cells with binding agents that bind to the TCR marker.
  • a method of genetically modifying a T cell to express a chimeric antigen receptor comprising: introducing a donor polynucleotide into the T cell, wherein the donor polynucleotide comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence flanking a synthetic exon comprising a nucleotide sequence encoding the CAR, wherein the CAR can specifically bind to a target antigen on a target cell, wherein at least a portion of the 5' genomic target sequence and at least a portion of the 3' genomic target sequence are located within an intron of a gene encoding an endogenous ?
  • CAR chimeric antigen receptor
  • TCR cell receptor
  • RNA-guided nuclease into the T cell
  • introducing a guide RNA into the T cell wherein the guide RNA forms a complex with the RNA-guided nuclease such that the guide RNA directs the RNA-guided nuclease to a genomic target sequence in the intron of the gene encoding the endogenous TCR protein chain, wherein the RNA-guided nuclease creates a doublestranded break in the genomic target sequence in the intron, wherein the donor polynucleotide is integrated into the intron by homology directed repair (HDR); and culturing the T cell under suitable conditions for transcription, wherein a pre-messenger RNA (mRNA) transcript encoding the CAR is produced, wherein splicing of the pre- mRNA transcript generates a mature mRNA comprising the synthetic exon encoding the CAR in between exons of the gene encoding the endogenous TCR protein chain,
  • mRNA
  • the gene encoding the endogenous TCR protein chain is a TCR alpha chain (TRAC) gene.
  • TCR TCR alpha chain
  • the intron is between exon 1 and exon 2 of the TRAC gene.
  • At least a portion of the 5' genomic target sequence and at least a portion of the 3' genomic target sequence are located within the intron.
  • the CAR-T cell is in a sample comprising T cells expressing the endogenous TCR, wherein the method further comprises performing negative selection to remove the T cells expressing the endogenous TCR from the sample.
  • performing negative selection comprises using an agent that selectively binds to the endogenous TCR.
  • the agent comprises an antibody, an antibody mimetic, an aptamer, or a ligand that selectively binds to the endogenous TCR.
  • the antibody is attached to a solid support.
  • the solid support is a magnetic bead, wherein the T cells comprising the endogenous TCR are removed from the sample by magnetic separation.
  • the donor polynucleotide, the RNA-guided nuclease, and the guide RNA are provided by one or more vectors.
  • the one or more vectors are viral vectors or plasmids.
  • the viral vectors are lentivirus vectors, retrovirus vectors, or adeno-associated virus vectors.
  • the donor polynucleotide and the RNA-guided nuclease are provided by separate vectors.
  • the donor polynucleotide and the RNA-guided nuclease are provided by the same vector.
  • the guide RNA and the RNA-guided nuclease are provided by the same vector. In some embodiments, the guide RNA and the RNA-guided nuclease are provided by different vectors. In some embodiments, the one or more vectors are introduced into the T cell by transient transfection or stable transfection. In some embodiments, the one or more vectors are introduced into the T cell by electroporation, nucleofection, or lipofection.
  • the RNA-guided nuclease and the guide RNA are provided by a recombinant polynucleotide that is integrated into the genome of the T cell.
  • RNA-guided nuclease and/or the guide RNA is inducible.
  • the RNA-guided nuclease is provided by a mRNA encoding the RNA-guided nuclease, wherein translation of the mRNA results in production of the RNA-guided nuclease in the T cell.
  • the RNA-guided nuclease is a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease.
  • CRISPR regularly interspaced short palindromic repeats
  • Cas Cas-associated nuclease.
  • the Cas nuclease is Cas9 or Cas12a.
  • the T cell is a helper CD4 + T cell, a cytotoxic CD8 + T cell, a natural killer T cell, or a gamma delta T cell that has been genetically modified to express the chimeric antigen receptor.
  • the CAR-T cell is a helper CD4 + T cell, a cytotoxic CD8 + T cell, a natural killer T cell, or a gamma delta T cell that has been genetically modified to express the chimeric antigen receptor, and wherein expression of the endogenous TCR is eliminated.
  • the chimeric antigen receptor comprises a transmembrane domain linked to an extracellular antigen binding domain and an intracellular signaling domain, wherein the extracellular antigen-binding domain specifically binds to an antigen on the target cell.
  • the extracellular antigen binding domain comprises a single chain variable fragment (scFv), an antigenbinding fragment (Fab), a nanobody, a heavy chain variable (VH) domain, a light chain variable (VL) domain, a single domain antibody (sdAb), a shark variable domain of a new antigen receptor (VNAR), a single variable domain on a heavy chain (VHH), a bispecific antibody, a diabody, or a functional fragment thereof that binds specifically to the antigen.
  • the intracellular signaling domain is a CD3-zeta intracellular signaling domain or a ZAP-70 intracellular signaling domain.
  • the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM).
  • ITAM immunoreceptor tyrosine-based activation motif
  • the transmembrane domain is a CD8, Megfl O, FcRy, Bail , MerTK, TIM4, Stabilin-1 , Stabilin-2, RAGE, CD300f, integrin subunit av, integrin subunit 5, CD36, LRP1 , SCARF1 , C1 Qa, Axl, CD45, or CD86 transmembrane domain.
  • the chimeric antigen receptor further comprises a costimulatory domain.
  • the costimulatory domain is a 4-1 BB, CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, or HVEM costimulatory domain.
  • the target cell is a cancer cell, a tumor cell, an activated fibroblast, an autoreactive immune cell, a pathogen, or a diseased cell.
  • the antigen on the target cell is a tumor antigen or tumor- associated antigen.
  • the pathogen is a virus, a bacterium, a fungus, or a parasite.
  • the antigen on the target cell is a viral antigen, a bacterial antigen, a fungal antigen or a parasite antigen.
  • the autoreactive immune cell is an autoreactive T cell or B cell.
  • the antigen on the target cell is an antigen on the autoreactive T cell or B cell.
  • the chimeric antigen receptor of the CAR-T cell specifically binds to a tumor antigen or tumor-associated antigen.
  • a method of treating cancer in a subject comprising administering a therapeutically effective amount of the CAR-T cell comprising the chimeric antigen receptor that specifically binds to the tumor antigen or tumor- associated antigen.
  • the chimeric antigen receptor of the CAR-T cell specifically binds to a viral antigen, a bacterial antigen, a fungal antigen or a parasite antigen.
  • a method of treating an infection in a subject comprising administering a therapeutically effective amount of the CAR-T cell comprising the chimeric antigen receptor that specifically binds to the viral antigen, bacterial antigen, fungal antigen or parasite antigen.
  • the chimeric antigen receptor of the CAR-T cell specifically binds to an antigen on an autoreactive T cell or B cell.
  • a method of treating an autoimmune disease in a subject comprising administering a therapeutically effective amount of the CAR-T cell comprising the chimeric antigen receptor that specifically binds to the antigen on an autoreactive T cell or B cell.
  • the guide RNA comprises a sequence selected from the group consisting of SEQ ID NOS:2-23.
  • the donor polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOS:36-79.
  • the synthetic exon encoding the CAR is flanked by a synthetic splice acceptor site and a synthetic splice donor site.
  • the donor polynucleotide further comprises a 2A multicistronic element at the 5’ end of the synthetic exon.
  • the donor polynucleotide further comprises an exonic splicing silencer (ESS) or an exonic splicing enhancer (ESE) at the 5’ end of the synthetic exon.
  • ESS exonic splicing silencer
  • ESE exonic splicing enhancer
  • the synthetic exon is expressed from an endogenous promoter.
  • the donor polynucleotide further comprises an exogenous promoter, wherein the synthetic exon is expressed from the exogenous promoter.
  • the donor polynucleotide further comprises one or more additional synthetic exons.
  • one or more additional synthetic exons encode one or more fluorescent proteins, one or more detectable cell surface reporters, or a combination thereof.
  • a composition comprising: a donor polynucleotide, wherein the donor polynucleotide comprises a 5' homology arm that can hybridize to a 5' genomic target sequence and a 3' homology arm that can hybridize to a 3' genomic target sequence flanking a synthetic exon comprising a nucleotide sequence encoding a CAR that specifically binds to a target antigen, wherein at least a portion of the 5' genomic target sequence and at least a portion of the 3' genomic target sequence are located within an intron of a gene encoding an endogenous T cell receptor (TCR) protein chain; a recombinant polynucleotide encoding an RNA-guided nuclease; and a recombinant polynucleotide encoding a guide RNA, wherein the guide RNA can form a complex with the RNA-guided nuclease such that the guide RNA directs
  • TCR T cell receptor
  • kits comprising a composition, described herein, and instructions for producing a genetically modified T cell expressing a chimeric antigen receptor is provided.
  • the kit further comprises a transfection agent.
  • a genetically modified T cell expressing a chimeric antigen receptor produced according to a method, described herein, is provided.
  • composition comprising a CAR-T cell produced according to a method, described herein and a pharmaceutically acceptable excipient is provided.
  • a method of performing cellular therapy comprising administering a therapeutically effective amount of a composition comprising a CAR-T cell produced according to a method, described herein, to a subject.
  • the CAR-T cell is autologous or allogeneic.
  • a method of genetically modifying a cell comprising: introducing a donor polynucleotide into the cell, wherein the donor polynucleotide comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence flanking a synthetic exon, wherein at least a portion of the 5' genomic target sequence and at least a portion of the 3' genomic target sequence are located within an intron of an endogenous gene; introducing an RNA-guided nuclease into the cell; introducing a guide RNA into the cell, wherein the guide RNA forms a complex with the RNA-guided nuclease such that the guide RNA directs the RNA-guided nuclease to a genomic target sequence in the intron of the endogenous gene, wherein
  • the endogenous gene encodes a T cell receptor (TCR) protein chain, CD3 epsilon subunit of T-cell receptor complex (CD3E), beta-2 microglobulin (B2M), or CD47.
  • TCR T cell receptor
  • CD3E CD3 epsilon subunit of T-cell receptor complex
  • B2M beta-2 microglobulin
  • the intron is intron 4 or intron 5 of CD3E, intron 1 of B2M, or intron 2 or intron 4 of CD47.
  • the guide RNA comprises a sequence selected from the group consisting of SEQ ID NOS:24-35.
  • the donor polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOS:80-85.
  • the synthetic exon is flanked by a synthetic splice acceptor site and a synthetic splice donor site.
  • the donor polynucleotide further comprises a 2A multicistronic element at the 5’ end of the synthetic exon.
  • the donor polynucleotide further comprises an exonic splicing silencer (ESS) or an exonic splicing enhancer (ESE) at the 5’ end of the synthetic exon.
  • ESS exonic splicing silencer
  • ESE exonic splicing enhancer
  • the synthetic exon is expressed from an endogenous promoter.
  • the donor polynucleotide further comprises an exogenous promoter, wherein the synthetic exon is expressed from the exogenous promoter.
  • the donor polynucleotide further comprises one or more additional synthetic exons.
  • the one or more additional synthetic exons encode one or more fluorescent proteins, one or more detectable cell surface reporters, or a combination thereof.
  • the cell is a T cell.
  • FIGS. 1A-1 D Non-viral TRAC intron knockins and negative selection of successfully edited CAR T cells.
  • FIG. 1A Traditional endogenous gene targeting approaches integrate a new transgene into endogenous exons, causing knockout of the endogenous gene, as shown by knockin of a GFP-CAR multicistronic cassette into the first exon of TRAC, causing TCR loss in Homology Directed Repair (HDR) edited cells. The majority of cells lacking a gene knockin show knockout of the TCR, due to Non- Homologous End Joining (NHEJ) and large deletions. Unedited Wild Type (WT) cells remain TCR positive.
  • NHEJ Non- Homologous End Joining
  • WT Unedited Wild Type
  • FIG. 1 B Efficient knockins across TRAC Intron 1.
  • TRAC intron knockin CD19-28z CAR T cells kill Nalm6 target cells at same efficiency as TRAC exon knockin CAR T cells 48 hours post mixing at a 1 :2 E:T ratio.
  • FIGS. 2A-2E Introduction of >5 kb large synthetic exons with TRAC intron knockins.
  • FIG. 2A Synthetic exons of increasingly large sizes from 2.5 kb to 5.3 kb targeted to TRAC intron 1 .
  • FIG. 2B Flow cytometric plots demonstrating knockin efficiency of large synthetic exons at TRAC intron 1 .
  • FIG. 2C Increasing synthetic exon size shows decreasing transgene expression levels. Mean fluorescence intensity of GFP expression measured by flow cytometry in knockin positive cells after integration of increasingly large synthetic exons to TRAC intron 1.
  • FIG. 2E In Vitro target cell killing with large synthetic exons.
  • FIGS. 3A-3E Engineered control of synthetic exon splicing using intron knockins.
  • FIG. 3A After intron knockin, successful splicing of a synthetic exon into the endogenous mRNA transcript disrupts the endogenous gene (“synthetic splicing”). If the synthetic exon is not spliced into the mature endogenous mRNA transcript, the new gene contained within the synthetic exon will not be expressed but endogenous gene expression is maintained (“endogenous splicing”). See Extended Data Fig 3a.
  • FIG. 3A After intron knockin, successful splicing of a synthetic exon into the endogenous mRNA transcript disrupts the endogenous gene (“synthetic splicing”). If the synthetic exon is not spliced into the mature endogenous mRNA transcript, the new gene contained within the synthetic exon will not be expressed but endogenous gene expression is maintained (“endogenous splicing”). See Extended Data Fig 3a. FIG.
  • FIG. 3B Replacing the synthetic exon’s 3’ splice donor sequence with a polyA creates unbalanced splicing, resulting in the majority of cells expressing both the synthetic gene, e.g., GFP (“synthetic splicing”, Blue Box) while also expressing the targeted endogenous gene, e.g., TCR/CD3 (“endogenous splicing”, Orange Box).
  • FIG. 3C Varying the 3’ splicing architecture of synthetic exons allows different balances of synthetic splicing only (GFP+ TCR-) compared to synthetic and endogenous splicing (GFP+ TCR+). See Extended Data Fig 3b.
  • FIG. 3D Altering degenerate bases of the synthetic exon’s 5' end (within the 2A multicistronic element necessary to separate the synthetic gene from the preceding endogenous exon’s translation) to include either Exonic Splice Silencer (ESS) or Exonic Splice Enhancer (ESE) elements similarly enables control over the degree of synthetic vs alternative splicing.
  • ESS Exonic Splice Silencer
  • ESE Exonic Splice Enhancer
  • FIG. 3E Frequency of cells expressing both the synthetic knockin gene and the endogenous gene can be controlled by inclusion of ESS or ESE elements with the 5’ splicing architecture of synthetic exons. Note, the SV40 polyA construct in FIGS.
  • FIGS. 4A-4E Biallelic TRAC intron knockins demonstrate alternative splicing of synthetic exons.
  • FIG. 4A Biallelic knockin of synthetic exons containing either GFP or RFP with balanced splice sites. Gating on dual positive cells showed almost none of the dual GFP positive I RFP positive cells still expressed the endogenous gene (TCR, measured by CD3 staining).
  • FIG. 4B Biallelic knockin of a synthetic exon containing Exonic Splicing Silencing elements at its 5’ end and a polyA at its 3’ end.
  • FIG. 4C Biallelic knockin of a synthetic exon containing Exonic Splicing Enhancer elements at its 5’ end. Gating on dual positive cells showed almost none of the dual GFP positive / RFP positive cells still expressed the endogenous gene (TCR, measured by CD3 staining).
  • the degree of off-target integrations is important for interpreting biallelic integration data, as dual positive cells also expressing the endogenous gene (GFP I RFP I TCR positive cells) could be due to one knockin allele, one unedited endogenous allele, and then an off-target integration of the second fluorescent protein.
  • the degree of off-target vs on-target integrations was assessed by electroporation of the GFP DNA Homology Directed Repair Template used in FIG. 4B without its accompanying RNP.
  • FIGS. 5A-5D Efficient non-viral intronic knockins across three genomic loci and distinct T cell types.
  • FIG. 6 Intron knockins enable flexible endogenous gene targeting with simplified selection. Intron knockins expand the toolbox for gene targeting methods. Intron knockins with 5’ and 3’ architectures that can alternatively splice between synthetic and endogenous exons allow more flexible targeting of synthetic genes under endogenous regulatory control, and intron knockins with splicing architectures resulting in only synthetic splicing enable negative selection of successfully gene edited cells.
  • FIGS. 7A-7F Efficient knockin of a synthetic exon across TRAC intronic sites.
  • FIG. 7A Observed efficiency of TCR knockout (measured by flow cytometric surface staining for CD3) after gene editing with Cas9 or Cas12a RNPs containing gRNAs targeting TRAC exon 1 5 or 18 distinct targets within TRAC intron 1 (avoiding a highly repetitive region within the intron). TCR knockout was highly efficient for the exon targeting gRNA, but significantly lower for intronic guides.
  • FIG. 7B Percentage of total cells expressing the knockin gene cassette (GFP+) following CD3 depletion (removing TCR positive cells) after intron knockin at 18 unique sites throughout TRAC intron 1 .
  • FIG. 7C TCR knockout (measured by flow cytometric surface staining for CD3) after gene editing with Cas12a RNPs containing gRNAs targeting TRAC intron 2.
  • FIG. 7D Knockin efficiency at four unique sites within TRAC intron 2.
  • FIG. 7E Percentage of total cells expressing the knockin gene cassette (GFP+) following CD3 depletion (removing TCR positive cells) after intron targeting at 4 unique sites throughout TRAC intron 2.
  • FIG. 7E Percentage of total cells expressing the knockin gene cassette (GFP+) following CD3 depletion (removing TCR positive cells) after intron targeting at 4 unique sites throughout TRAC intron 2.
  • FIG. 7F Across four TRAC intron 1 target sites targeted with Cas9 RNPs, no major difference in knockin efficiency and successful protein expression from the integrated synthetic exon was observed when using the endogenous splice acceptor and donor sites from the adjacent endogenous TRAC exons compared to synthetic consensus splice acceptor and donor sites.
  • FIG. 7A-7F, n 2-4 unique primary human T cell donors.
  • FIGS. 8A-8E Viability, editing, and activation metrics with non-viral intron editing and negative selection in human T cells.
  • FIG. 8A Timeline of primary human T cell editing using non-viral intron knockins, followed by negative selection. T cells are isolated and activated on Day 0, followed by electroporation based non-viral intron editing on Day 2. CD3 Negative selection was performed on Day 8, six days followed editing.
  • FIG. 8B Total T cell counts on Day 1 and Day 4 post electroporation relative to no electroporation controls following non-viral intron knockins by electroporation of a TRAC intron targeting CRISPR-Cas9 RNP along with a DNA Homology Directed Repair Template.
  • FIG. 8D At Day 8 post activation, TRAC intron knockin T cells were negatively selected by binding magnetic beads to the CD3 complex.
  • FIG. 8E After either negative selection or no selection, the activation status of TRAC intron knockin CD19- 28z CAR T cells was assessed through in vitro activation by Nalm6 target cells at a 1 :4 EffectonTarget cell ratio.
  • FIGS. 9A-9H Direct comparison of common edited primary human T cell selection methods.
  • FIG. 9A Intron knockin of a tNGFR-CAR-PuroR synthetic exon enabled a single population of edited cells to be compatible with four common selection methods.
  • FIG. 9B Representative flow cytometric plots of TRAC intron CAR T cells after purification with four distinct selection methods.
  • Knockin of a synthetic exon containing a tNGFR-CAR-PuroR multicistronic cassette to TRAC intron 1 enabled successfully edited cells to be negatively selected by CD3 Depletion (removal of TCR positive cells), positively selected by streptavidin magnetic bead enrichment after binding of anti-tNGFR biotinylated antibodies, fluorescence-activated cell sorting after binding of anti-tNGFR fluorescent antibodies, or drug selection after culture in puromycin.
  • Negative selection by CD3 depletion yields predominantly a successfully edited CAR+ T cell population without the endogenous TCR, although rarer TCR negative I knockin negative cells are present (likely due to the RNP induced double stranded break within the intron causing a large deletion that included a portion of one or both adjacent exons, instead of the more common NHEJ repair outcome of smaller indels).
  • Positive selection, sorting, and drug selection in contrast remove all knockin negative cells, but retain a population of TCR positive / knockin positive cells (likely due to successful HDR mediated knockin to one TRAC allele with either no editing or a small indel removed during mRNA splicing on the second TRAC allele; while in some T cells one TCRa loci is silenced, numerous T cells express functional TCRa chains from both alleles 50 ). Only negative selections do not require additional genetic material (e.g. no selection marker or resistance gene necessary) and leave cells untouched post-selection.
  • FIG. 9C Percentage of residual TCR positive cells following four different selection methods. TRAC intron knockin followed by CD3 Negative Selection (Blue) leaves almost no detectable TCR+ cells remaining.
  • FIGS. 10A-10D Variable 5’ and 3’ splicing architectures enable control of alternative splicing of synthetic exons.
  • FIG. 10A Correlation between observed cellular protein phenotypes by flow cytometry with inferred splicing behavior. Cells expressing both the protein encoded by the synthetic exon (e.g. GFP) and the endogenous protein (e.g. TCR) must be undergoing both synthetic splicing and endogenous splicing.
  • the synthetic exon e.g. GFP
  • TCR endogenous protein
  • FIG. 10B Variable 3’ synthetic exon splicing architectures lead to controllable degrees of alternative splicing.
  • Balanced splicing (2A-SD, far left) results in the majority of knockin (GFP) cells being negative for the endogenous gene (TCR-), with the residual GFP+ TCR+ cells likely due to one knockin allele and one WT or NHEJ edited allele.
  • Exonic Splicing Silencer (ESS) DNA sequences adjacent to the synthetic exon’s 5’ splice acceptor resulted in increased numbers of dual positive cells showing evidence of alternative splicing.
  • the short 6-8 bp ESS sequences were introduced into the 2A multicistronic element at the 5' end of the synthetic exon (necessary to separate the new synthetic gene’s protein translation from the translation of the preceding endogenous exon) using degenerate bases to maximize the number and strength of the ESS sequences present 36 .
  • an Exonic Splicing Silencer 5’ architecture paired with a short SV40 polyA 3’ architecture proved optimal.
  • an Exonic Splicing Enhancer (ESE) 5’ architecture paired with any of the tested polyA 3’ architectures showed approximately equivalent degrees of synthetic splicing.
  • FIGS. 11A-11 D Generalized endogenous gene targeting with exon and intron knockins.
  • FIG. 11 A A new synthetic gene can be introduced under endogenous regulatory control of an existing gene without also loosing expression of the existing gene only be integration at the N terminus (immediately before the Start codon) or C terminus (immediately before the Stop codon) of the targeted gene. This limitation in target sites means that efficient gRNAs for gene knockin may not be present for many genes, as observed in previous studies 6 .
  • FIG. 11 B Intron knockins offer greater flexibility for placing a synthetic gene under endogenous regulatory control without disrupting the endogenous gene.
  • a synthetic exon can be introduced throughout the intronic regions of the endogenous gene, with alternative splicing resulting in two separate mature mRNA transcripts, one encoding the new synthetic protein and a second encoding the endogenous gene. Orders of magnitude more gRNA target sites are available within intronic regions than only at the very N or C terminus of a gene.
  • FIG. 11C Exonic targeting of a new synthetic gene within the coding sequence of an endogenous gene disrupts the endogenous gene’s sequence, resulting in a multicistronic mRNA transcript encoding the new synthetic protein and two partial fragments of the endogenous protein, causing knockout of the endogenous gene.
  • FIG. 11 D Intron targeting with a synthetic exon containing optimized 5’ and 3’ splicing architectures to induce synthetic splicing only also results in endogenous gene knockout.
  • endogenous gene knockout unlike in exonic targeting, where alleles with unsuccessful knockins largely have NHEJ induced indels resulting in frameshift mutations and protein knockout, with intron targeting NHEJ induced indels reside within an intronic sequence that is largely tolerant of short DNA changes which will be spliced out of the final mRNA transcript (more rare but detectable larger deletions that include parts of the surrounding exons induced by double stranded breaks can still result in endogenous protein knockout).
  • the targeted endogenous gene is a surface receptor, then intron knockins offer the unique ability to perform negative selections to purify successfully edited cells.
  • compositions and methods are provided for genetically modifying a T cell to express a chimeric antigen receptor (CAR) with knockout of an endogenous T cell receptor (TCR).
  • the subject methods utilize a donor polynucleotide encoding a CAR that is integrated into an intron of a TCR gene.
  • the successfully edited T cells produce a mature mRNA with a nucleotide sequence encoding the CAR spliced in between the endogenous exons of the TCR protein, resulting in expression of the CAR and knockout of the TCR.
  • the unsuccessfully edited T cells retain expression of their TCR.
  • the T cell population can be enriched for successfully edited TCR negative T cells expressing the CAR by using negative selection to remove unsuccessfully edited TCR positive T cells with binding agents that bind to the TCR marker.
  • immune cells generally includes white blood cells (leukocytes) which are derived from hematopoietic stem cells (HSC) produced in the bone marrow.
  • HSC hematopoietic stem cells
  • Biocompatible or “cytocompatible” as used herein, refers to a property of a material that allows for prolonged contact with a cell or tissue without causing toxicity or significant damage.
  • Exemplary human interventions comprise transfection with a heterologous polynucleotide, molecular cloning resulting in a deletion, insertion, modification and/or rearrangement with respect to a naturally occurring sequence such as a naturally occurring sequence in a T cell, gene, nucleic acid and/or protein herein described.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the agents calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
  • the specifications for the unit dosage forms for use in the present invention depend on the particular compound employed and the effect to be achieved, the pharmacodynamics associated with each compound in the host, and the like.
  • biological sample encompasses a clinical sample, including, but not limited to, a bodily fluid, tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, fine needle aspirate, lymph node aspirate, cystic aspirate, a paracentesis sample, a thoracentesis sample, and the like.
  • the terms “obtained” or “obtaining” as used herein can also include the physical extraction or isolation of a biological sample (e.g., comprising immune cells) from a subject. Accordingly, a biological sample comprising immune cells can be isolated from a subject (and thus “obtained”) by the same person or same entity that subsequently isolates immune cells from the sample.
  • a biological sample is “extracted” or “isolated” from a first party or entity and then transferred (e.g., delivered, mailed, etc.) to a second party, the sample was “obtained” by the first party (and also “isolated” by the first party), and then subsequently “obtained” (but not “isolated”) by the second party. Accordingly, in some embodiments, the step of obtaining does not comprise the step of isolating a biological sample.
  • the step of obtaining comprises the step of isolating a biological sample.
  • Methods and protocols for isolating various biological samples e.g., a blood sample, a biopsy sample, an aspirate, etc. will be known to one of ordinary skill in the art and any convenient method may be used to isolate a biological sample.
  • isolated refers to an entity of interest that is in an environment different from that in which it may naturally occur. “Isolated” is meant to include entities that are within samples that are substantially enriched for the entity of interest and/or in which the entity of interest is partially or substantially purified.
  • substantially purified generally refers to isolation of a component of a sample (e.g., cell or substance), such that the component comprises the majority percent of the sample in which it resides.
  • a substantially purified component comprises at least 70%, preferably at least 80%-85%, more preferably at least 90-99% of the sample.
  • Subjects include, but are not limited to, mammals, including human and non-human mammals such as non-human primates, including chimpanzees and other apes and monkey species; laboratory animals such as mice, rats, rabbits, hamsters, guinea pigs, and chinchillas; domestic animals such as dogs and cats; farm animals such as sheep, goats, pigs, horses and cows.
  • the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; primates, and transgenic animals.
  • rodents including mice, rats, and hamsters; primates, and transgenic animals.
  • the term "subject” generally refers to an individual who will be administered or who has been administered one or more compositions described herein (e.g., cellular therapy with cells screened according to the methods described herein).
  • treatment used herein to generally refer to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect can be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease.
  • treatment encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease and/or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease and/or symptom(s), i.e., arresting their development; or (c) relieving the disease symptom(s), i.e., causing regression of the disease and/or symptom(s).
  • Those in need of treatment include those already inflicted as well as those in which prevention is desired, including those with a genetic predisposition or increased susceptibility to developing a disease.
  • a therapeutic treatment is one in which the subject is inflicted prior to administration and a prophylactic treatment is one in which the subject is not inflicted prior to administration.
  • the subject has an increased likelihood of becoming inflicted or is suspected of being inflicted prior to treatment.
  • the subject is suspected of having an increased likelihood of becoming inflicted.
  • a “therapeutically effective amount” or “therapeutic dose” is an amount sufficient to effect desired clinical results (i.e., achieve therapeutic efficacy).
  • a therapeutically effective dose or amount can be administered in one or more administrations.
  • “Pharmaceutically acceptable excipient or carrier” refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.
  • “Pharmaceutically acceptable salt” includes, but is not limited to, amino acid salts, salts prepared with inorganic acids, such as chloride, sulfate, phosphate, diphosphate, bromide, and nitrate salts, or salts prepared from the corresponding inorganic acid form of any of the preceding, e.g., hydrochloride, etc., or salts prepared with an organic acid, such as malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, para-toluenesulfonate, palmoate, salicylate and stearate, as well as estolate, gluceptate and lactobionate salts.
  • salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).
  • polynucleotide and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
  • this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • Homology refers to the percent identity between two polynucleotide or two polypeptide molecules.
  • Two nucleic acid, or two polypeptide sequences are “substantially homologous” to each other when the sequences exhibit at least about 50% sequence identity, preferably at least about 75% sequence identity, more preferably at least about 80% 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% 98% sequence identity over a defined length of the molecules.
  • substantially homologous also refers to sequences showing complete identity to the specified sequence.
  • identity refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M.O. Dayhoff ed., 5 Suppl.
  • nucleotide sequence identity is available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wl) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent identity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.
  • Another method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages, the Smith Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the “Match” value reflects "sequence identity.”
  • Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters.
  • homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single stranded specific nuclease(s), and size determination of the digested fragments.
  • DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra', DNA Cloning, supra', Nucleic Acid Hybridization, supra.
  • Recombinant as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature.
  • the term "recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide.
  • the gene of interest is cloned and then expressed in transformed organisms, as described further below. The host organism expresses the foreign gene to produce the protein under expression conditions.
  • transformation refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion. For example, direct uptake, transduction or f-mating are included.
  • the exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.
  • Recombinant host cells refer to cells which can be, or have been, used as recipients for recombinant vector or other transferred DNA, and include the original progeny of the original cell which has been transfected.
  • a "coding sequence” or a sequence which "encodes" a selected polypeptide is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or “control elements”).
  • the boundaries of the coding sequence can be determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
  • a coding sequence can include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and even synthetic DNA sequences.
  • a transcription termination sequence may be located 3' to the coding sequence.
  • Typical "control elements,” include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3' to the translation stop codon), sequences for optimization of initiation of translation (located 5’ to the coding sequence), and translation termination sequences.
  • operably linked refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
  • a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper enzymes are present.
  • the promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked" to the coding sequence.
  • Expression cassette or "expression construct” refers to an assembly which is capable of directing the expression of the sequence(s) or gene(s) of interest.
  • An expression cassette generally includes control elements, as described above, such as a promoter which is operably linked to (so as to direct transcription of) the sequence(s) or gene(s) of interest, and often includes a polyadenylation sequence as well.
  • the expression cassette described herein may be contained within a plasmid construct.
  • the plasmid construct may also include, one or more selectable markers, a signal which allows the plasmid construct to exist as single stranded DNA (e.g., a M13 origin of replication), at least one multiple cloning site, and a "mammalian" origin of replication (e.g., a SV40 or adenovirus origin of replication).
  • a signal which allows the plasmid construct to exist as single stranded DNA e.g., a M13 origin of replication
  • at least one multiple cloning site e.g., a "mammalian" origin of replication (e.g., a SV40 or adenovirus origin of replication).
  • Polynucleotide refers to a polynucleotide of interest or fragment thereof which is essentially free, e.g., contains less than about 50%, preferably less than about 70%, and more preferably less than about at least 90%, of the protein with which the polynucleotide is naturally associated.
  • Techniques for purifying polynucleotides of interest include, for example, disruption of the cell containing the polynucleotide with a chaotropic agent and separation of the polynucleotide(s) and proteins by ion-exchange chromatography, affinity chromatography and sedimentation according to density.
  • transfection is used to refer to the uptake of foreign DNA by a cell.
  • a cell has been "transfected” when exogenous DNA has been introduced inside the cell membrane.
  • transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (2001 ) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill, and Chu et al. (1981 ) Gene 13:197.
  • Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells.
  • the term refers to both stable and transient uptake of the genetic material, and includes uptake of peptide- or antibody-linked DNAs.
  • a “vector” is capable of transferring nucleic acid sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes).
  • target cells e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes.
  • vector construct e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes.
  • expression vector e transfer vector
  • the term includes cloning and expression vehicles, as well as viral vectors.
  • hybridization refers to the specific binding of a nucleic acid to a complementary nucleic acid via Watson-Crick base pairing.
  • a polynucleotide "derived from" a designated sequence refers to a polynucleotide sequence which comprises a contiguous sequence of approximately at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about I Q- 12 nucleotides, and even more preferably at least about 15-20 nucleotides corresponding, i.e., identical or complementary to, a region of the designated nucleotide sequence.
  • the derived polynucleotide will not necessarily be derived physically from the nucleotide sequence of interest, but may be generated in any manner, including, but not limited to, chemical synthesis, replication, reverse transcription or transcription, which is based on the information provided by the sequence of bases in the region(s) from which the polynucleotide is derived. As such, it may represent either a sense or an antisense orientation of the original polynucleotide.
  • a “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes.
  • one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence.
  • Cas9 encompasses type II clustered regularly interspaced short palindromic repeats (CRISPR) system Cas9 endonucleases from any species, and also includes biologically active fragments, variants, analogs, and derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks).
  • CRISPR type II clustered regularly interspaced short palindromic repeats
  • a Cas9 endonuclease binds to and cleaves DNA at a site comprising a sequence complementary to its bound guide RNA (gRNA).
  • a gRNA may comprise a sequence "complementary" to a target sequence (e.g., in an intron of a TCR gene), capable of sufficient base-pairing to form a duplex (i.e., the gRNA hybridizes with the target sequence).
  • the gRNA may comprise a sequence complementary to a PAM sequence, wherein the gRNA also hybridizes with the PAM sequence in a target DNA.
  • a Cas9 polynucleotide, nucleic acid, oligonucleotide, protein, polypeptide, or peptide refers to a molecule derived from any source. The molecule need not be physically derived from an organism, but may be synthetically or recombinantly produced. Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries for Cas9 from: Streptococcus pyogenes (WP 002989955, WP 038434062, WP_01 1528583); Campylobacter jejuni (WP_022552435,
  • Corynebacterium diphtheria (NC_016782, NC_016786); Enterococcus faecalis (WP 033919308); Spiroplasma syrphidicola (NC_021284); Prevotella intermedia (NC_017861 ); Spiroplasma taiwanense (NC_021846); Streptococcus iniae (NC.018721 ); P 061046374, WP 024786433); Listeria innocua (NP 472073); Listeria monocytogenes (WP_061665472); Legionella pneumophila (WP_062726656); Staphylococcus aureus (WP_O01573634); Francisella tularensis (WP_032729892, WP_014548420),
  • Enterococcus faecalis WP 033919308; Lactobacillus rhamnosus (WP 048482595, WP 032965177); and Neisseria meningitidis (WP_061704949, YP_002342100); all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference.
  • sequences or a variant thereof comprising a sequence having at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing, as described herein, wherein the variant retains biological activity, such as Cas9 site-directed endonuclease activity. See also Fonfara et al. (2014) Nucleic Acids Res.
  • a gRNA will bind to a substantially complementary sequence and not to unrelated sequences.
  • a gRNA that selectively binds to a particular target DNA sequence will selectively direct binding of Cas9 to a substantially complementary sequence at the target site and not to unrelated sequences.
  • donor polynucleotide refers to a polynucleotide that provides a sequence of an intended edit to be integrated into the genome at a target locus by homology directed repair (HDR).
  • HDR homology directed repair
  • a "target site” or “target sequence” is the nucleic acid sequence recognized (i.e., sufficiently complementary for hybridization) by a guide RNA (gRNA) or a homology arm of a donor polynucleotide.
  • gRNA guide RNA
  • the target site may be in an exon or an intron or a specific allele.
  • homology arm is meant a portion of a donor polynucleotide that is responsible for targeting the donor polynucleotide to the genomic sequence to be edited in a cell.
  • the donor polynucleotide typically comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence flanking a nucleotide sequence comprising the intended edit to the genomic DNA.
  • the homology arms are referred to herein as 5' and 3' (i.e., upstream and downstream) homology arms, which relates to the relative position of the homology arms to the nucleotide sequence comprising the intended edit within the donor polynucleotide.
  • the 5' and 3' homology arms hybridize to regions within the target locus in the genomic DNA to be modified, which are referred to herein as the "5' target sequence” and "3' target sequence,” respectively.
  • the nucleotide sequence comprising the intended edit is integrated into the genomic DNA by HDR or recombineering at the genomic target locus recognized (i.e., sufficiently complementary for hybridization) by the 5' and 3' homology arms.
  • complementary refers to polynucleotides that are able to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in an anti-parallel orientation between polynucleotide strands. Complementary polynucleotide strands can base pair in a Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil (U) rather than thymine (T) is the base that is considered to be complementary to adenosine.
  • uracil when a uracil is denoted in the context of the present invention, the ability to substitute a thymine is implied, unless otherwise stated.
  • “Complementarity” may exist between two RNA strands, two DNA strands, or between a RNA strand and a DNA strand. It is generally understood that two or more polynucleotides may be “complementary” and able to form a duplex despite having less than perfect or less than 100% complementarity.
  • Two sequences are "perfectly complementary” or “100% complementary” if at least a contiguous portion of each polynucleotide sequence, comprising a region of complementarity, perfectly base pairs with the other polynucleotide without any mismatches or interruptions within such region.
  • Two or more sequences are considered “perfectly complementary” or “100% complementary” even if either or both polynucleotides contain additional non- complementary sequences as long as the contiguous region of complementarity within each polynucleotide is able to perfectly hybridize with the other.
  • "Less than perfect" complementarity refers to situations where less than all of the contiguous nucleotides within such region of complementarity are able to base pair with each other.
  • a gRNA may comprise a sequence "complementary" to a target sequence (e.g., in an intron), capable of sufficient base-pairing to form a duplex (i.e., the gRNA hybridizes with the target sequence). Additionally, the gRNA may comprise a sequence complementary to a PAM sequence, wherein the gRNA also hybridizes with the PAM sequence in a target DNA.
  • administering a nucleic acid, such as a viral vector or a CRISPR system (expressing, e.g., a donor polynucleotide, guide RNA, Cas protein (e.g., Cas9, Cas12a (Cpf1 ), Cas12d, or Cas13)) to a cell comprises transducing, transfecting, electroporating, translocating, fusing, phagocytosing, shooting or ballistic methods, etc., i.e., any means by which a nucleic acid can be transported across a cell membrane.
  • a nucleic acid such as a viral vector or a CRISPR system (expressing, e.g., a donor polynucleotide, guide RNA, Cas protein (e.g., Cas9, Cas12a (Cpf1 ), Cas12d, or Cas13)
  • a nucleic acid such as a viral vector or a CRISPR system (expressing, e.g
  • a "barcode” refers to one or more nucleotide sequences that are used to identify a nucleic acid or cell with which the barcode is associated. Barcodes can be 3-1000 or more nucleotides in length, preferably 10-250 nucleotides in length, and more preferably 10-30 nucleotides in length, including any length within these ranges, such as 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides in length.
  • Barcodes may be used, for example, to identify a single cell, subpopulation of cells, colony, or sample from which a nucleic acid originated. Barcodes may also be used to identify the position (i.e., positional barcode) of a cell, colony, or sample from which a nucleic acid originated, such as the position of a colony in a cellular array, the position of a well in a multi-well plate, or the position of a tube, flask, or other container in a rack. In particular, a barcode may be used to identify a genetically modified cell from which a nucleic acid originated. In some embodiments, a barcode is used to identify a donor T cell from which a CAR-T cell originated.
  • a unique barcode may be used to identify each guide-RNA and donor polynucleotide used in multiplexed or multi-step genome editing.
  • multiple barcodes can be used in combination to identify different features of a nucleic acid or cell.
  • positional barcoding e.g., to identify the position of a cell, colony, culture, or sample in an array, multi-well plate, or rack
  • polypeptide “peptide,” and “protein” are used interchangeably herein to refer to any compound comprising naturally occurring or synthetic amino acid polymers or amino acid-like molecules including but not limited to compounds comprising amino and/or imino molecules. No particular size is implied by use of the terms “polypeptide,” “peptide,” and “protein” and these terms are used interchangeably.
  • the “terms include post-expression modifications of the polypeptide, peptide, or protein such as glycosylation, acetylation, phosphorylation, and the like.
  • polypeptides, peptides, or proteins, as described herein may include additional molecules such as labels (e.g., fluorescent, bioluminescent, or radioactive), tags (e.g., histidine tag, epitope tag), or other chemical moieties.
  • labels e.g., fluorescent, bioluminescent, or radioactive
  • tags e.g., histidine tag, epitope tag
  • antibodies and immunoglobulin include antibodies or immunoglobulins of any isotype, fragments of antibodies which retain specific binding to an antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, monoclonal antibodies, hybrid antibodies, chimeric antibodies, humanized antibodies, single-chain antibodies, single-domain antibodies, nanobodies, bispecific antibodies, tri-specific antibodies, and other multi-specific antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein.
  • Antibody fragments comprise a portion of an intact antibody, for example, the antigen binding or variable region of the intact antibody.
  • antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily.
  • Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
  • Single-chain Fv or “sFv” antibody fragments comprise the VH and V domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the Vn and VL domains, which enables the sFv to form the desired structure for antigen binding.
  • the terms “specific binding,” “specifically binds,” and the like, refer to non-covalent or covalent preferential binding to a molecule relative to other molecules or moieties in a solution or reaction.
  • the affinity of one molecule for another molecule to which it specifically binds is characterized by a KD (dissociation constant) of 10 -5 M or less (e.g., 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 11 M or less, 10 12 M or less).
  • KD dissociation constant
  • affinity is determined by surface plasmon resonance (SPR), e.g., as used by Biacore systems.
  • SPR surface plasmon resonance
  • the affinity of one molecule for another molecule is determined by measuring the binding kinetics of the interaction, e.g., at 25°C.
  • antigen-binding fragment refers to any antibody fragment that specifically binds to a target antigen including, but not limited to, a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), an scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody including one or more complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR).
  • CDRs complementarity-determining regions
  • FR framework
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a p-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the p-sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991 )).
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
  • VL and VH sequences can be reformatted as fragments, as single chain binding domains, linked to chimeric antigen receptors, and the like.
  • the term “antigen binding domain (ABD)” refers to a domain that specifically binds to a target antigen.
  • the antigen binding domain region of an antibody may comprise a heavy-chain variable domain (VH) and a light-chain variable domain (VL) in non-covalent association as a single polypeptide or as a dimer.
  • the three complementaritydetermining regions of the heavy chain variable domain (CDR H1 , H2, H3) and three complementarity-determining regions of the light chain variable domain (CDR L1 , L2, L3) interact to define an antigen-binding site on the surface of an antibody.
  • CDR H1 , H2, H3 three complementarity-determining regions of the light chain variable domain
  • CDR L1 , L2, L3 interact to define an antigen-binding site on the surface of an antibody.
  • An antigen binding domain region of a CAR may comprise all six CDRs of an antibody or a single variable domain or half of an Fv fragment comprising only three CDRs specific for an antigen, which still retains the ability to recognize and bind the target antigen.
  • the antigen-binding domain binds to one or more target antigens expressed on the surface of a target cell (e.g., cell surface markers).
  • T cell includes all types of immune cells expressing CD3 including T- helper cells (CD4 + cells), cytotoxic T-cells (CD8 + cells), natural killer T cells, T-regulatory cells (T reg) and gamma-delta T cells.
  • T cell also includes genetically modified T cells, including T cells engineered to express a chimeric antigen receptor (CAR) and T cells from which the gene encoding the endogenous T cell receptor has been inactivated or deleted (i.e., TCR gene knockout).
  • CAR chimeric antigen receptor
  • T cell receptor and “TCR” are used interchangeably and generally refer to a receptor found on the surface of T cells or T lymphocytes that is responsible for recognizing antigenic peptides bound to major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • the TCR is a membrane-anchored heterodimeric protein comprising two different protein chains. In the majority of human T cells, the TCR consists of an alpha (a) chain and a beta (P) chain (encoded by TRA and TRB genes, respectively). In about 5% of human T cells, the TCR consists of gamma and delta (y/8) chains (encoded by TRG and TRD genes, respectively).
  • T cells expressing a TCR comprising alpha and beta chains are referred to as T cells, and T cells expressing a TCR comprising gamma and delta chains are referred to as yS T cells
  • the ratio of T cells to y8 T cells differs between species and may be altered by disease (such as leukemia).
  • the variable domains of the TCR a-chain and p-chain each have three hypervariable or complementarity-determining regions (CDRs).
  • CDR 1 and CDR3 bind to the antigenic peptide.
  • CDR2 recognizes the MHC.
  • the constants domains of the TCR a-chain and p- chain each have a cysteine that forms a disulfide bond that links the two chains.
  • the TCR receptor a and p chains associate with six additional adaptor proteins, including a delta chain, a gamma chain, two epsilon chains, and two zeta chains to form an octameric complex.
  • the adaptor proteins comprise signaling motifs involved in TCR signaling.
  • Chimeric antigen receptor (CAR) may have any suitable architecture, as known in the art, comprising an antigen binding domain, usually provided in an scFv format, linked to T cell receptor effector functions.
  • the term refers to artificial multimodule molecules capable of triggering or inhibiting the activation of an immune cell.
  • a CAR will generally comprise an antigen binding domain, linker, transmembrane domain and cytoplasmic signaling domain. In some instances, a CAR will include one or more co-stimulatory domains and/or one or more co-inhibitory domains.
  • the antigen-binding domain of the CAR may include any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a target antigen of interest.
  • the binding region is an antigen-binding region, such as an antibody or functional binding domain or antigen-binding fragment thereof.
  • the antigen-binding region of the CAR can include any domain that binds to the antigen and may include, but is not limited to, a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, a single-chain antibody, and any antigen-binding fragment thereof.
  • the antigen binding domain portion includes a mammalian antibody or an antigen-binding fragment thereof.
  • An antigen-binding domain may comprise an antigenbinding fragment (Fab), a single-chain variable fragment (scFv), a nanobody, a VH domain, a VL domain, a single domain antibody (sdAb), a shark variable domain of a new antigen receptor (VNAR), a single variable domain on a heavy chain (VHH), a bispecific antibody, or a diabody; or a functional antigen-binding fragment thereof.
  • the antigen-binding domain is derived from the same cell type or the same species in which the CAR will ultimately be used.
  • the antigen-binding domain of the CAR may include a human antibody, a humanized antibody, or an antigen-binding fragment thereof.
  • the antigen binding domain is derived from a single chain antibody that selectively binds to a target antigen.
  • the antigen binding domain is provided by a single chain variable fragment (scFv).
  • scFv is a recombinant molecule in which the variable regions of the light and heavy immunoglobulin chains are connected in a single fusion polypeptide.
  • the VH and VL sequences are joined by a linker sequence. See, for example, Ahmad (2012) Clinical and Developmental Immunology Article ID 980250, herein specifically incorporated by reference. In principle, there are no particular limitations to the length and/or amino acid composition of the linker peptide joining the VH and VL sequences.
  • any arbitrary single-chain peptide including about 1 to 100 amino acid residues can be used as a peptide linker.
  • the linker peptide sequence includes about 5 to 50, about 10 to 60, about 20 to 70, about 30 to 80, about 40 to 90, about 50 to 100, about 60 to 80, about 70 to 100, about 30 to 60, about 20 to 80, about 30 to 90 amino acid residues.
  • the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25, about 20 to 40, about 30 to 50, about 40 to 60, about 50 to 70 amino acid residues. In some embodiments, the linker peptide sequence includes about 40 to 70, about 50 to 80, about 60 to 80, about 70 to 90, or about 80 to 100 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25 amino acid residues.
  • the transmembrane domain may be derived either from a natural or a synthetic source. Where the source is natural, the domain may be derived from any membranebound or transmembrane protein.
  • the transmembrane domain comprises at least the stalk and/or transmembrane region(s) of CD8, Megf10, FcRy, Bail , MerTK, TIM4, Stabilin-1 , Stabilin-2, RAGE, CD300f, integrin subunit av, Integrin subunit P5, CD36, LRP1 , SCARF1 , C1 Qa, Axl, CD45, and/or CD86.
  • the CAR transmembrane domain is derived from a type I membrane protein, such as, but not limited to, CD3£ CD4, CD8, or CD28.
  • the transmembrane domain is synthetic, in which case it will include predominantly hydrophobic residues such as leucine, isoleucine, valine, phenylalanine, tryptophan, and alanine.
  • a triplet of phenylalanine, tryptophan and valine will be inserted at each end of a synthetic transmembrane domain.
  • the CAR further comprises one or more linkers/spacers.
  • an extracellular spacer region may link the antigen binding domain to the transmembrane domain and/or an intracellular spacer region may link an intracellular signaling domain to the transmembrane domain.
  • a spacer (linker) region linking the antigen binding domain to the transmembrane domain should be flexible enough to allow the antigen binding domain to orient in different directions to facilitate antigen recognition.
  • the linker includes a peptide linker/spacer sequence.
  • the spacer comprises the hinge region from an immunoglobulin, e.g., the hinge from any one of lgG1 , lgG2a, lgG2b, lgG3, lgG4, particularly the human protein sequences.
  • Alternatives include the CH2CH3 region of immunoglobulin and portions of CD3. For many scFv based constructs, an IgG hinge is effective.
  • a linker peptide sequence comprises about 1 to 100 amino acid residues, including any number of residues within this range such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues.
  • the linker peptide sequence includes about 5 to 50, about 10 to 60, about 20 to 70, about 30 to 80, about 40 to 90, about 50 to 100, about 60 to 80, about 70 to 100, about 30 to 60, about 20 to 80, about 30 to 90 amino acid residues.
  • the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25, about 20 to 40, about 30 to 50, about 40 to 60, about 50 to 70 amino acid residues. In some embodiments, the linker peptide sequence includes about 40 to 70, about 50 to 80, about 60 to 80, about 70 to 90, or about 80 to 100 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25 amino acid residues. In some embodiments, the linker peptide sequence may include up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.
  • a short oligo- or polypeptide linker preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the intracellular engulfment signaling domain or extracellular antigen binding domain of the CAR.
  • the linker comprises the amino acid sequence (G4S)n where n is 1 , 2, 3, 4, 5, etc., and in some embodiments, n is 3.
  • a cytoplasmic signaling domain such as those derived from the T cell receptor - chain, is employed as part of the CAR in order to produce stimulatory signals for T lymphocyte proliferation and effector function following engagement of the chimeric receptor with the target antigen. Endodomains from co-stimulatory molecules may be included in the cytoplasmic signaling portion of the CAR.
  • co-stimulatory domain refers to a stimulatory domain, typically an endodomain, of a CAR that provides a secondary non-specific activation mechanism through which a primary specific stimulation is propagated.
  • co-stimulation include antigen nonspecific T cell co-stimulation following antigen specific signaling through the T cell receptor and antigen nonspecific B cell co-stimulation following signaling through the B cell receptor.
  • Co-stimulation, e.g., T cell co-stimulation, and the factors involved have been described in Chen & Flies. Nat Rev Immunol (2013) 13(4):227-42, the disclosure of which are incorporated herein by reference in their entirety.
  • suitable co-stimulatory polypeptides include, but are not limited to, 4-1 BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.
  • co-inhibitory domain refers to an inhibitory domain, typically an endodomain, derived from a receptor that provides secondary inhibition of primary antigen-specific activation mechanisms which prevents co-stimulation.
  • Co-inhibition e.g., T cell co-inhibition
  • co-inhibitory domains homodimerize.
  • a co-inhibitory domain can be an intracellular portion of a transmembrane protein.
  • suitable co-inhibitory polypeptides include, but are not limited to, CTLA-4 and PD-1 .
  • a first-generation CAR transmits the signal from antigen binding through only a single signaling domain, for example a signaling domain derived from the high-affinity receptor for IgE FccRIy, or the CD3£ chain.
  • the domain contains one or three immunoreceptor tyrosine-based activating motif(s) [ITAM(s)] for antigen-dependent T- cell activation.
  • ITAM-based activating signal endows T-cells with the ability to lyse the target tumor cells and secret cytokines in response to antigen binding.
  • Second-generation CARs include a co-stimulatory signal in addition to the CD3 signal. Coincidental delivery of the delivered co-stimulatory signal enhances cytokine secretion and antitumor activity induced by CAR-transduced T-cells.
  • the co-stimulatory domain will usually be membrane proximal relative to the CD3 ⁇ domain.
  • Third-generation CARs include a tripartite signaling domain, comprising for example a CD28, CD3 , 0X40 or 4-1 BB signaling region.
  • fourth generation, or “armored car” CAR-T cells CAR-T cells are further genetically modified to express or block molecules and/or receptors to enhance immune activity.
  • CAR variants include split CARs wherein the extracellular portion, the ABD and the cytoplasmic signaling domain of a CAR are present on two separate molecules.
  • CAR variants also include ON-switch CARs which are conditionally activatable CARs, e.g., comprising a split CAR wherein conditional hetero-dimerization of the two portions of the split CAR is pharmacologically controlled.
  • ON-switch CARs which are conditionally activatable CARs, e.g., comprising a split CAR wherein conditional hetero-dimerization of the two portions of the split CAR is pharmacologically controlled.
  • CAR molecules and derivatives thereof i.e., CAR variants are described, e.g., in PCT Application Nos. US2014/016527, US1996/017060, US2013/063083; Fedorov et al.
  • CAR variants also include bispecific or tandem CARs, which include a secondary CAR binding domain that can either amplify or inhibit the activity of a primary CAR.
  • CAR variants also include inhibitory chimeric antigen receptors (iCARs) which may, e.g., be used as a component of a bispecific CAR system, where binding of a secondary CAR binding domain results in inhibition of primary CAR activation.
  • Tandem CARs (TanCAR) mediate bispecific activation of T cells through the engagement of two chimeric receptors designed to deliver stimulatory or costimulatory signals in response to an independent engagement of two different tumor associated antigens.
  • iCARs use the dual antigen targeting to shout down the activation of an active CAR through the engagement of a second suppressive receptor equipped with inhibitory signaling domains
  • Inhibitory CARs are designed to regulate CAR-T cells activity through inhibitory receptor signaling module activation. This approach combines the activity of two CARs, one of which generates dominant negative signals limiting the responses of CAR-T cells activated by the activating receptor. iCARs can switch off the response of the counteracting activator CAR when bound to a specific antigen expressed only by normal tissues. In this way, iCARs-T cells can distinguish cancer cells from healthy ones, and reversibly block functionalities of transduced T cells in an antigen-selective fashion. CTLA-4 or PD-1 intracellular domains in iCARs trigger inhibitory signals on T lymphocytes, leading to less cytokine production, less efficient target cell lysis, and altered lymphocyte motility.
  • An ABD can be provided as a “chimeric bispecific binding member”, i.e., a chimeric polypeptide having dual specificity to two different binding partners (e.g., two different antigens).
  • chimeric bispecific binding members include bispecific antibodies, bispecific conjugated monoclonal antibodies (mab)2, bispecific antibody fragments (e.g., F(ab)2, bispecific scFv, bispecific diabodies, single chain bispecific diabodies, etc.), bispecific T cell engagers (BiTE), bispecific conjugated single domain antibodies, micabodies and mutants thereof, and the like.
  • Non-limiting examples of chimeric bispecific binding members also include those chimeric bispecific agents described in Kontermann.
  • a chimeric bispecific binding member may be a bispecific T cell engager (BiTE).
  • a BiTE is generally made by fusing a specific binding member (e.g., a scFv) that binds an antigen to a specific binding member (e.g., a scFv) with a second binding domain specific for a T cell molecule such as CD3.
  • a chimeric bispecific binding member may be a CAR-T cell adapter.
  • CAR-T cell adapter an expressed bispecific polypeptide that binds the antigen recognition domain of a CAR and redirects the CAR to a second antigen.
  • a CAR-T cell adapter will have two binding regions, one specific for an epitope on the CAR to which it is directed and a second epitope directed to a binding partner which, when bound, transduces the binding signal activating the CAR.
  • Useful CAR-T cell adapters include but are not limited to e.g., those described in Kim et al. J Am Chem Soc.
  • Effector CAR-T cells include autologous or allogeneic immune cells having cytolytic activity against a target cell.
  • a T cell is engineered to express a CAR.
  • the term “T cells” refers to mammalian immune effector cells that may be characterized by expression of CD3 and/or a T cell antigen receptor.
  • the CAR-T cells are engineered from a complex mixture of immune cells, e.g., tumor infiltrating lymphocytes (TILs) isolated from an individual in need of treatment. See, for example, Yang and Rosenberg (2016) Adv Immunol. 130:279-94, “Adoptive T Cell Therapy for Cancer; Feldman et al (2015) Semin Oncol.
  • TILs tumor infiltrating lymphocytes
  • the engineered T cell is allogeneic with respect to the individual that is treated, e.g. see clinical trials NCT03121625; NCT03016377; NCT02476734; NCT02746952; NCT02808442. See for review Graham et al. (2016) Cells. 7(10) E155.
  • an allogeneic engineered T cell is fully HLA matched. However not all patients have a fully matched donor, and a cellular product suitable for all patients independent of HLA type provides an alternative.
  • Allogeneic T cells may be administered in combination with intensification of lymphodepletion to allow CAR-T cells to expand and clear malignant cells prior to host immune recovery, e.g., by administration of Alemtuzumab (monoclonal anti-CD52), purine analogs, etc.
  • the allogeneic T cells may be modified for resistance to Alemtuzumab.
  • Gene editing can be used to prevent expression of HLA class I molecules on CAR-T cells, e.g. by deletion of p2-microglobulin.
  • induced pluripotent stem (iPS) cell-derived CAR- T cells can be used.
  • donor T cells can be transduced with reprogramming factors to restore pluripotency, and then re-differentiated into T effector cells.
  • T cells for engineering collected from a subject or a donor, may be separated from a mixture of cells by techniques that enrich for desired cells, or may be engineered and cultured without separation.
  • An appropriate solution may be used for dispersion or suspension.
  • Such solution will generally be a balanced salt solution, e.g. normal saline, PBS, Hank’s balanced salt solution, etc., conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from 5-25 mM.
  • Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc.
  • Techniques for affinity separation may include magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, e.g., complement and cytotoxins, and "panning" with antibody attached to a solid matrix, e.g., a plate, or other convenient technique.
  • Techniques providing accurate separation include fluorescence activated cell sorters, which can have varying degrees of sophistication, such as multiple color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc.
  • the cells may be selected against dead cells by employing dyes associated with dead cells (e.g., propidium iodide).
  • the affinity reagents may be specific receptors or ligands for the cell surface molecules indicated above.
  • peptide- MHC antigen and T cell receptor pairs may be used; peptide ligands and receptor; effector and receptor molecules, and the like.
  • the separated cells may be collected in any appropriate medium that maintains the viability of the cells, usually having a cushion of serum at the bottom of the collection tube.
  • Various media are commercially available and may be used according to the nature of the cells, including dMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., frequently supplemented with fetal calf serum (FCS).
  • FCS fetal calf serum
  • the collected and optionally enriched cell population may be used immediately for genetic modification, or may be frozen at liquid nitrogen temperatures and stored, being thawed and capable of being reused.
  • the cells will usually be stored in 10% DMSO, 50% FCS, 40% RPMI 1640 medium.
  • Engineered CAR-T cells may be infused into a subject in any physiologically acceptable medium by any convenient route of administration, normally intravascularly, though CAR-T cells may also be introduced by other routes, where the cells may find an appropriate site for growth. Usually, at least 1 x10 6 cells/kg will be administered, at least 1x10 7 cells/kg, at least 1 x10 8 cells/kg, at least 1 x10 9 cells/kg, at least 1 x10 1 ° cells/kg, or more, usually being limited by the number of T cells that are obtained during collection.
  • chimeric antigen receptor (CAR)-T cells that are T-cells that have been genetically engineered to express a CAR.
  • a coding sequence encoding a CAR may be introduced on an expression vector into a cell to be engineered.
  • a CAR coding sequence may be introduced into the genome at the site of an endogenous T cell receptor gene.
  • cells are further engineered to delete an endogenous T cell receptor (i.e., TCR knockout).
  • a CRISPR/Cas9 system is used to genetically modify a T cell.
  • a CRISPR/Cas9 system can be introduced into cells by transfection with mRNA or a plasmid that encodes Cas9 and a gRNA or by viral delivery of CRISPR components, e.g., using lentiviral, retroviral vectors, or non-integrating viruses, such as adenovirus and adeno-associated virus (AAV).
  • AAV adeno-associated virus
  • binding-triggered transcriptional switch or “BTSS”, it is intended to mean a synthetic modular polypeptide or system of interacting polypeptides having an extracellular domain that includes a second member of a specific binding pair that binds a first member of the specific binding pair (e.g., an antigen), a binding-transducer and an intracellular domain.
  • a first member of the specific binding pair e.g., an antigen
  • binding-transducer e.g., an antigen
  • intracellular domain e.g., an antigen
  • the binding signal is transduced to the intracellular domain such that the intracellular domain becomes activated and performs a function, e.g., transcription activation, within the cell that it does not perform in the absence of the binding signal.
  • Examples of BTSS include the synNotch system, the modular extracellular sensor architecture (MESA) system, the TANGO system, the A2 Notch system, and the synthetic intramembrane proteolysis receptor (SNIPR) system, etc.
  • the synNotch receptor may be for example as described in U.S. Patent No. 9,670,281 and described in more detail below.
  • the MESA system may be as described in WO 2018/081039 A1 and comprises a self-containing sensing and signal transduction system, such that binding of a ligand (first member of the specific binding pair) to the receptor (second member of the specific binding pair) induces signaling to regulate expression of a target gene.
  • the TANGO system may be as described in Barnea et al., 2008 Proc. Natl. Acad. Sci. U.S.A., 105(1 ): 64-9. Briefly, the TANGO system sequesters a transcription factor to the cell membrane by physically linking it to a membrane-bound receptor (e.g., GPCRs, receptor kinases, Notch, steroid hormone receptors, etc.).
  • a membrane-bound receptor e.g., GPCRs, receptor kinases, Notch, steroid hormone receptors, etc.
  • the A2 Notch system may be as described in WO 2019099689 A1. Briefly, the A2 Notch system incorporates a force sensor cleavage domain which, upon cleavage induced upon binding of a ligand to the receptor, releases the intracellular domain into the cell.
  • the SNIPR system may be described as in Zhu et al. (2022) Cell 185(8) :1431 -1443. e16; herein incorporated by reference.
  • the SNIPR system uses a synthetic RiP receptor comprising an ectodomain comprising an extracellular regulatory element that specifically binds a ligand, a transmembrane domain, a juxtamembrane domain, and a transcription factor that can be cleaved from the SNIPR by a protease in response to binding of a ligand to the extracellular regulatory element.
  • the second binding member may be present on the surface of a genetically engineered cell, such as, a cell expressing a BTTS and a CAR under the control of the BTTS. In certain embodiments, the second binding member may be present on the surface of a genetically engineered cell, such as, a cell expressing the BTTS and a CAR under control of the BTTS.
  • the first binding member may bind to a synNotch receptor as described in U.S. Patent No. 9,670,281 .
  • the synNotch receptor may include an extracellular domain that includes the second binding member, where the second binding member is a single-chain Fv (scFv) or a nanobody and the first binding member present on the particles is an antigen to which the single-chain Fv (scFv) or a nanobody binds.
  • the second binding member may be an anti-CD19, anti- mesothelin, anti-GFP antibody, scFv, or a nanobody and the first binding member may be CD19, mesothelin, GFP, respectively.
  • the BTTS is a chimeric Notch polypeptide comprising, from N-terminus to C-terminus and in covalent linkage: a) an extracellular domain comprising the second member of the specific-binding pair that is not naturally present in a Notch receptor polypeptide and that specifically binds to the first member of the specific-binding pair; b) a Notch regulatory region comprising a Lin 12-Notch repeat, an S2 proteolytic cleavage site, and a transmembrane domain comprising an S3 proteolytic cleavage site; c) an intracellular domain comprising a transcriptional activator or a transcriptional repressor that is heterologous to the Notch regulatory region and replaces a naturally-occurring intracellular Notch domain, wherein binding of the first member of the specific-binding pair to the second member of the specific-binding pair induces cleavage at the S2 and S3 proteolytic cleavage sites, thereby releasing the intracellular domain
  • the cell may be a T-cell, such as, those described in U.S. Patent No. 9,670,281 , which is herein incorporated by reference.
  • Methods are provided for genetically modifying a T cell to express a chimeric antigen receptor (CAR) with simultaneous knockout of the endogenous TCR.
  • the gene editing technique involves creating a double-strand break (DSB) in the genomic DNA of a T cell at a target site in an intron of a gene encoding a TCR protein chain using an RNA-guided nuclease.
  • a donor polynucleotide encoding a CAR is subsequently integrated into the intron at the target site by homologous recombination (HR).
  • the successfully edited cells By knocking in a synthetic exon expressing the CAR into an intron, the successfully edited cells produce a mature mRNA with a nucleotide sequence encoding the CAR spliced in and exon(s) encoding the TCR protein chain splice out, resulting in expression of the CAR and knockout of the endogenous TCR.
  • Unsuccessfully edited cells which predominantly have an indel at the intronic target site, still maintain expression of their endogenous TCR, as the mutated base pairs are spliced out during mRNA processing, unlike the frameshift mutations commonly seen with traditional exonic targeting of the CAR coding sequence.
  • the donor polynucleotide encoding the CAR is integrated into an intron of an endogenous TCR gene.
  • the TCR consists of an alpha (a) chain (TRAC), encoded by a TRA gene, and a TCR beta (0) chain (TRBC), encoded by a TRB gene, wherein the donor polynucleotide encoding the CAR can be integrated into an intron at the TRAC or TRBC locus.
  • the TCR consists of a gamma chain (TRGC), encoded by a TRG gene, and a delta chain (TRDC), encoded by a TRD gene, wherein the donor polynucleotide encoding the CAR can be integrated into an intron at the TRGC or TRDC locus.
  • TRGC gamma chain
  • TRDC delta chain
  • the nucleotide sequence encoding the CAR is flanked by a pair of homology arms responsible for targeting the donor polynucleotide to the intron where the CAR is integrated into the genome of a T cell.
  • the donor polynucleotide typically comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence.
  • the homology arms are referred to herein as 5' and 3' (i.e., upstream and downstream) homology arms, which relates to the relative position of the homology arms to the nucleotide sequence encoding the CAR within the donor polynucleotide.
  • the 5' and 3' homology arms hybridize to regions within the target locus in the genomic DNA to be modified, which are referred to herein as the "5' target sequence" and "3' target sequence,” respectively.
  • the homology arm must be sufficiently complementary for hybridization to the target sequence to mediate homologous recombination between the donor polynucleotide and genomic DNA at the target locus.
  • a homology arm may comprise a nucleotide sequence having at least about 80-100% sequence identity to the corresponding genomic target sequence, including any percent identity within this range, such as at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein the nucleotide sequence encoding the CAR is integrated into the genomic DNA by HDR at the genomic target locus recognized (i.e., sufficiently complementary for hybridization) by the 5' and 3' homology arms.
  • the corresponding homologous nucleotide sequences in the genomic target sequence flank a specific site for cleavage and/or a specific site for introducing the nucleotide sequence encoding the CAR.
  • the distance between the specific cleavage site and the homologous nucleotide sequences can be several hundred nucleotides. In some embodiments, the distance between a homology arm and the cleavage site is 200 nucleotides or less (e.g., 0, 10, 20, 30, 50, 75, 100, 125, 150, 175, and 200 nucleotides).
  • the donor polynucleotide is substantially identical to the target genomic sequence, across its entire length except for the sequence changes to be introduced to a portion of the genome that encompasses both the specific cleavage site and the portions of the genomic target sequence to be altered.
  • a homology arm can be of any length, e.g., 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 300 nucleotides or more, 350 nucleotides or more, 400 nucleotides or more, 450 nucleotides or more, 500 nucleotides or more, 1000 nucleotides (1 kb) or more, 5000 nucleotides (5 kb) or more, 10000 nucleotides (10 kb) or more, etc.
  • the 5' and 3' homology arms are substantially equal in length to one another, e.g.
  • the 5' and 3' homology arms are substantially different in length from one another, e.g. one may be 40% shorter or more, 50% shorter or more, sometimes 60% shorter or more, 70% shorter or more, 80% shorter or more, 90% shorter or more, or 95% shorter or more than the other homology arm.
  • RNA-guided nuclease can be targeted to a particular genomic sequence (i.e., genomic target sequence to be modified) by altering its guide RNA sequence.
  • a targetspecific guide RNA comprises a nucleotide sequence that is complementary to a genomic target sequence, and thereby mediates binding of the nuclease-gRNA complex by hybridization at the target site.
  • the gRNA can be designed with a sequence complementary to a sequence of the intron to target the nuclease-gRNA complex to a target site within the intron.
  • the RNA-guided nuclease used for genome modification is a clustered regularly interspersed short palindromic repeats (CRISPR) system Cas nuclease.
  • CRISPR clustered regularly interspersed short palindromic repeats
  • Any RNA-guided Cas nuclease capable of catalyzing site-directed cleavage of DNA to allow integration of donor polynucleotides by the HDR mechanism can be used in genome editing, including CRISPR system type I, type II, or type III Cas nucleases.
  • Cas proteins include Cas1 , Cas1 B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1 , Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Csy1 , Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1 , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1 , Cmr3, Cmr4, Cmr5, Cmr6, Csb1 , Csb2, Csb3, Csx17, Csx14, Csx10,
  • a type II CRISPR system Cas9 endonuclease is used.
  • Cas9 nucleases from any species, or biologically active fragments, variants, analogs, or derivatives thereof that retain Cas9 endonuclease activity i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks
  • the Cas9 need not be physically derived from an organism, but may be synthetically or recombinantly produced.
  • Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database.
  • sequences or a variant thereof comprising a sequence having at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing, as described herein. See also Fonfara et al. (2014) Nucleic Acids Res. 42(4):2577-90; Kapitonov et al. (2015) J. Bacteriol.
  • the CRISPR-Cas system naturally occurs in bacteria and archaea where it plays a role in RNA-mediated adaptive immunity against foreign DNA.
  • the bacterial type II CRISPR system uses the endonuclease, Cas9, which forms a complex with a guide RNA (gRNA) that specifically hybridizes to a complementary genomic target sequence, where the Cas9 endonuclease catalyzes cleavage to produce a double-stranded break.
  • gRNA guide RNA
  • Targeting of Cas9 typically further relies on the presence of a 5' protospacer-adjacent motif (PAM) in the DNA at or near the gRNA-binding site.
  • PAM 5' protospacer-adjacent motif
  • the genomic target site will typically comprise a nucleotide sequence that is complementary to the gRNA, and may further comprise a protospacer adjacent motif (PAM).
  • the target site comprises 20-30 base pairs in addition to a 3 base pair PAM.
  • the first nucleotide of a PAM can be any nucleotide, while the two other nucleotides will depend on the specific Cas9 protein that is chosen.
  • Exemplary PAM sequences are known to those of skill in the art and include, without limitation, NNG, NGN, NAG, and NGG, wherein N represents any nucleotide.
  • the intron sequence of the TCR gene targeted by a gRNA comprises a mutation that creates a PAM within the intron, wherein the PAM promotes binding of the Cas9-gRNA complex to the intron.
  • the gRNA is 5-50 nucleotides, 10-30 nucleotides, 15-25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides in length, or any length between the stated ranges, including, for example, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, or 35 nucleotides in length.
  • the guide RNA may be a single guide RNA comprising crRNA and tracrRNA sequences in a single RNA molecule, or the guide RNA may comprise two RNA molecules with crRNA and tracrRNA sequences residing in separate RNA molecules.
  • Cas12a the CRISPR nuclease from Prevotella and Francisella 1 (Cpf1 ) also referred to as CRISPR associated protein 12a (Cas12a) may be used.
  • Cas12a is another class II CRISPR/Cas system RNA-guided nuclease with similarities to Cas9 and may be used analogously. Unlike Cas9, Cas12a does not require a tracrRNA and only depends on a crRNA in its guide RNA, which provides the advantage that shorter guide RNAs can be used with Cas12a for targeting than Cas9.
  • Cas12a is capable of cleaving either DNA or RNA.
  • the PAM sites recognized by Cas12a have the sequences 5'-YTN-3' (where "Y” is a pyrimidine and “N” is any nucleobase) or 5'-TTN-3', in contrast to the G-rich PAM site recognized by Cas9.
  • Cas12a cleavage of DNA produces double-stranded breaks with sticky-ends having a 4 or 5 nucleotide overhang.
  • Ledford et al. (2015) Nature. 526 (7571 ):17-17, Zetsche et al. (2015) Cell. 163 (3):759-771 Murovec et al. (2017) Plant Biotechnol. J. 15(8):917-926, Zhang et al. (2017) Front. Plant Sci. 8:177, Fernandes et al. (2016) Postepy Biochem. 62(3):315-326; herein incorporated by reference.
  • C2c1 is another class II CRISPR/Cas system RNA-guided nuclease that may be used.
  • C2c1 similarly to Cas9, depends on both a crRNA and tracrRNA for guidance to target sites.
  • C2c1 see, e.g., Shmakov et al. (2015) Mol Cell. 60(3):385-397, Zhang et al. (2017) Front Plant Sci. 8:177; herein incorporated by reference.
  • RNA-guided Fokl nucleases comprise fusions of inactive Cas9 (dCas9) and the Fokl endonuclease (Fokl-dCas9), wherein the dCas9 portion confers guide RNA- dependent targeting on Fokl.
  • dCas9 inactive Cas9
  • Fokl-dCas9 Fokl endonuclease
  • engineered RNA-guided Fokl nucleases see, e.g., Havlicek et al. (2017) Mol. Ther. 25(2):342-355, Pan et al. (2016) Sci Rep. 6:35794, Tsai et al. (2014) Nat Biotechnol. 32(6):569-576; herein incorporated by reference.
  • the RNA-guided nuclease can be provided in the form of a protein, such as the nuclease complexed with a gRNA, or provided by a nucleic acid encoding the RNA- guided nuclease, such as an RNA (e.g., messenger RNA) or DNA (expression vector such as a plasmid or viral vector). Codon usage may be optimized to improve production of an RNA-guided nuclease in a particular cell or organism.
  • RNA e.g., messenger RNA
  • DNA expression vector such as a plasmid or viral vector
  • a nucleic acid encoding an RNA-guided nuclease can be modified to substitute codons having a higher frequency of usage in a human T cell or a non-human mammalian T cell, such as a nonhuman primate T cell, a rodent cell, a mouse cell, a rat cell, or any other host T cell of interest, as compared to the naturally occurring polynucleotide sequence.
  • a nucleic acid encoding the gRNA and/or RNA-guided nuclease is introduced into T cells, the gRNA and/or RNA-guided nuclease can be transiently, conditionally, or constitutively expressed in the cell.
  • Recombinant nucleic acids encoding the gRNA, RNA-guided nuclease, and/or donor polynucleotide can be introduced into a T cell using any suitable transfection technique such as, but not limited to electroporation, nucleofection, or lipofection.
  • a ribonucleoprotein complex of the gRNA and the RNA-guided nuclease may be introduced into a T cell by microinjection into the cytoplasm or nucleus.
  • the CRISPR/Cas9 system is introduced into T cells with a viral vector that encodes Cas9 and a guide RNA (gRNA).
  • gRNA guide RNA
  • Viral delivery of CRISPR components has been demonstrated using lentiviral, retroviral, adenovirus, and adeno- associated virus (AAV) vectors.
  • AAV adeno-associated virus
  • a gRNA and a messenger RNA encoding the Cas9 can be introduced into T cells, wherein the Cas9 is produced by translation of the mRNA in the cytoplasm.
  • the gRNA and Cas9 then form a complex in the cytoplasm and enter the nucleus.
  • RNA transfection of T cells can be performed using electroporation, cationic- lipid-mediated transfection, or using liposomes or lipid nanoparticles (LNPs) encapsulating the gRNA and mRNA. See, e.g., Billingsley et al. (2022) Nano Lett 22(1 ):533-542, Tchou et al. (2017) Cancer Immunol Res.
  • Donor polynucleotides and gRNAs are readily synthesized by standard techniques, e.g., solid phase synthesis via phosphoramidite chemistry, as disclosed in U.S. Patent Nos. 4,458,066 and 4,415,732, incorporated herein by reference; Beaucage et al., Tetrahedron (1992) 48:2223-2311 ; and Applied Biosystems User Bulletin No. 13 (1 April 1987).
  • gRNA-donor polynucleotide cassettes can be produced by standard oligonucleotide synthesis techniques and subsequently ligated into vectors.
  • a CAR-T cell is further engineered to comprise a binding- triggered transcriptional switch (BTSS) that regulates expression of the chimeric antigen receptor or activity of the CAR-T cell.
  • BTSS binding- triggered transcriptional switch
  • BTSS is intended to mean a synthetic modular polypeptide or system of interacting polypeptides having an extracellular domain that includes a second member of a specific binding pair that binds a first member of the specific binding pair (e.g., an antigen), a binding-transducer and an intracellular domain.
  • the binding signal Upon binding of the first member of the specific binding pair to the BTTS the binding signal is transduced to the intracellular domain such that the intracellular domain becomes activated and performs a function, e.g., transcription activation, within the cell that it does not perform in the absence of the binding signal.
  • the second binding member may be present on the surface of a genetically engineered cell, such as, a cell expressing a BTTS and a CAR under the control of the BTTS.
  • binding-triggered transcriptional switches include the synNotch system, the modular extracellular sensor architecture (MESA) system, the TANGO system, the A2 Notch system, and the synthetic intramembrane proteolysis receptor (SNIPR) system, etc.
  • the synNotch receptor may be for example as described in U.S. Patent No. 9,670,281 and described in more detail below.
  • the MESA system may be as described in WO 2018/081039 A1 and comprises a self-containing sensing and signal transduction system, such that binding of a ligand (first member of the specific binding pair) to the receptor (second member of the specific binding pair) induces signaling to regulate expression of a target gene.
  • the TANGO system may be as described in Barnea et al., 2008 Proc. Natl. Acad. Sci. U.S.A., 105(1 ): 64-9. Briefly, the TANGO system sequesters a transcription factor to the cell membrane by physically linking it to a membrane-bound receptor (e.g., GPCRs, receptor kinases, Notch, steroid hormone receptors, etc.).
  • a membrane-bound receptor e.g., GPCRs, receptor kinases, Notch, steroid hormone receptors, etc.
  • the A2 Notch system may be as described in WO 2019099689 A1. Briefly, the A2 Notch system incorporates a force sensor cleavage domain which, upon cleavage induced upon binding of a ligand to the receptor, releases the intracellular domain into the cell.
  • the SNIPR system may be described as in Zhu et al. (2022) Cell 185(8):1431 -1443.e16; herein incorporated by reference.
  • the SNIPR system uses a synthetic RIP receptor comprising an ectodomain comprising an extracellular regulatory element that specifically binds a ligand, a transmembrane domain, a juxtamembrane domain, and a transcription factor that can be cleaved from the SNIPR by a protease in response to binding of a ligand to the extracellular regulatory element.
  • the first binding member may bind to a synNotch receptor as described in U.S. Patent No. 9,670,281 .
  • the synNotch receptor may include an extracellular domain that includes the second binding member, where the second binding member is a single-chain Fv (scFv) or a nanobody and the first binding member present on the particles is an antigen to which the single-chain Fv (scFv) or a nanobody binds.
  • the second binding member may be an anti-CD19, anti- mesothelin, anti-GFP antibody, scFv, or a nanobody and the first binding member may be CD19, mesothelin, GFP, respectively.
  • the BTTS is a chimeric Notch polypeptide comprising, from N-terminus to C-terminus and in covalent linkage: a) an extracellular domain comprising the second member of the specific-binding pair that is not naturally present in a Notch receptor polypeptide and that specifically binds to the first member of the specific-binding pair; b) a Notch regulatory region comprising a Lin 12-Notch repeat, an S2 proteolytic cleavage site, and a transmembrane domain comprising an S3 proteolytic cleavage site; c) an intracellular domain comprising a transcriptional activator or a transcriptional repressor that is heterologous to the Notch regulatory region and replaces a naturally-occurring intracellular Notch domain, wherein binding of the first member of the specific-binding pair to the second member of the specific-binding pair induces cleavage at the S2 and S3 proteolytic cleavage sites, thereby releasing the intracellular domain
  • a CAR-T cell is further engineered to express one or more additional genes that improve CAR-T cell function.
  • the CAR-T cell may be further engineered to express a dominant negative receptor such as a PD-1 dominant negative receptor, a dominant negative TGF- type II receptor, or a Fas dominant negative receptor; a chimeric switch receptor such as an IL-4 chimeric switch receptor, a costimulatory signaling domain, a cytokine such as IL-12, IL-18, IL-7, IL-15, and IL-21 ; and/or a chemokine receptor to promote migration to a targeted cell (e.g., that binds chemokines secreted in a targeted tumor) such as CXCR2, CXCR1 , CXCR3, CCR4, CCR2b, and/ CXCL9.
  • a targeted cell e.g., that binds chemokines secreted in a targeted tumor
  • the subject methods using intronic targeting of the CAR coding sequence, yield a population of T cells in which the successfully edited T cells do not have an endogenous TCR, whereas the unsuccessfully edited T cells all have the endogenous TCR.
  • This allows the T cell population to be enriched for the successfully edited T cells expressing the CAR using negative selection with a binding agent (e.g., antibody, antibody mimetic, aptamer, or ligand) that specifically binds to the endogenous TCR to remove the unsuccessfully edited TCR positive T cells from the T cell population, leaving only the successfully edited TCR negative T cells behind for further research or clinical use without having to bind any reagents to the successfully edited T cells expressing the CAR.
  • a binding agent e.g., antibody, antibody mimetic, aptamer, or ligand
  • Binding agents may comprise, but are not limited to, proteins, peptides, antibodies, antibody fragments, antibody mimetics, aptamers, or ligands that specifically bind to a an endogenous TCR on a T cell.
  • the phrase “specifically (or selectively) binds” refers to a binding reaction that is determinative of the presence of the endogenous TCR on a T cell in a heterogeneous population of successfully and unsuccessfully edited T cells and other biologies.
  • the specified binding agents bind to the endogenous TCR on unsuccessfully edited T cells at least two times the background and do not substantially bind in a significant amount to CARs present on successfully edited T cells in the sample.
  • the binding agent binds to the endogenous TCR with high affinity.
  • the binding agent may be immobilized on a solid support to facilitate removal of T cells comprising the endogenous TCR from a liquid sample.
  • the binding agent may be associated with the solid support either directly or indirectly.
  • Binding agents may be immobilized on the surface of a solid support, such as, but not limited to, a non-magnetic bead, magnetic bead, rod, particle, plate, slide, wafer, strand, disc, membrane, film, or the inner surface of a tube, channel, column, flow cell device, or microfluidic device.
  • a solid support may comprise various materials, including, but not limited to glass, quartz, silicon, metal, ceramic, plastic, nylon, polyacrylamide, agarose, resin, porous polymer monoliths, hydrogels, and composites thereof. Additionally, a substrate may be added to the surface of a solid support to facilitate attachment of a binding agent.
  • the capture agent comprises an antibody that specifically binds to the endogenous TCR on a T cell.
  • Any type of antibody may be used, including polyclonal and monoclonal antibodies, hybrid antibodies, altered antibodies, chimeric antibodies and, humanized antibodies, as well as: hybrid (chimeric) antibody molecules (see, for example, Winter et al. (1991 ) Nature 349:293-299; and U.S. Pat. No. 4,816,567); F(ab')2 and F(ab) fragments; F v molecules (noncovalent heterodimers, see, for example, Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662; and Ehrlich et al.
  • the binding agent comprises an aptamer that specifically binds to the endogenous TCR on a T cell.
  • aptamer Any type of aptamer may be used, including a DNA, RNA, xeno-nucleic acid (XNA), or peptide aptamer that specifically binds to the target antibody isotype.
  • XNA xeno-nucleic acid
  • Such aptamers can be identified, for example, by screening a combinatorial library.
  • Nucleic acid aptamers e.g., DNA or RNA aptamers
  • that bind selectively to a target antibody isotype can be produced by carrying out repeated rounds of in vitro selection or systematic evolution of ligands by exponential enrichment (SELEX).
  • Peptide aptamers that bind to a target antibody isotype may be isolated from a combinatorial library and improved by directed mutation or repeated rounds of mutagenesis and selection.
  • Aptamers Tools for Nanotherapy and Molecular Imaging (R.N. Veedu ed., Pan Stanford, 2016)
  • Nucleic Acid and Peptide Aptamers Methods and Protocols (Methods in Molecular Biology, G. Mayer ed., Humana Press, 2009)
  • Nucleic Acid Aptamers Selection, Characterization, and Application (Methods in Molecular Biology, G.
  • the binding agent comprises an antibody mimetic.
  • Any type of antibody mimetic may be used, including, but not limited to, affibody molecules (Nygren (2008) FEBS J. 275 (1 1 ):2668-2676), affilins (Ebersbach et al. (2007) J. Mol. Biol. 372 (1 ):172-185), affimers (Johnson et al. (2012) Anal. Chem. 84 (15):6553-6560), affitins (Krehenbrink et al. (2008) J. Mol. Biol. 383 (5):1058-1068), alphabodies (Desmet et al.
  • a magnetic separation method is used, wherein the T cell population is contacted with a binding agent (e.g., antibody, antibody mimetic, aptamer, or ligand) that selectively binds to the endogenous TCR and not the CAR.
  • a binding agent e.g., antibody, antibody mimetic, aptamer, or ligand
  • the binding agent is linked to a magnetic particle, which allows the unsuccessfully edited T cells expressing the endogenous TCR to be separated from the successfully edited T cells expressing the CAR using a magnet.
  • CAR-T cells engineered as described herein, express a chimeric antigen receptor (CAR) that specifically binds to a target antigen.
  • the CAR localizes T cells to sites where target cells are present that express the target antigen. Binding of a CAR-T cell to a target antigen on the surface of a cell activates the T cell resulting in secretion of cytokines, which regulate other immune cells, and killing of target cells.
  • CAR-T cells may be engineered to target an antigen that is expressed on the surface of tumors but not on healthy cells to selectively kill tumor cells.
  • CAR-T cells may also be engineered to target an antigen that is expressed on the surface of activated fibroblasts or fibrotic tissue, which may be used to selectively eliminate fibrotic tissue.
  • CAR-T cells may also be engineered to target an antigen that is expressed on the surface of a pathogen (e.g., bacterium, virus, fungus, or parasite) to eradicate a pathogen.
  • CAR-T cells may be engineered to target an antigen that is expressed on the surface of an autoreactive immune cell (e.g., autoreactive T cell or B cell) to eliminate autoreactive immune cells.
  • CAR-T cells may be used for the treatment of various diseases, including cancer, fibrosis, infections such as bacterial infections (e.g., multidrug resistant bacteria), viral infections, fungal infections, and parasitic infections, and autoimmune diseases.
  • the T cell from which the CAR-T cell is derived, may be autologous or allogeneic.
  • the CAR-T cell is an effector T cell (e.g., a helper CD4 + T cell, a cytotoxic CD8 + T cell, a natural killer T cell, or a gamma delta T cell) or a regulatory T cell (Treg) that has been genetically modified to express a CAR.
  • an effector T cell e.g., a helper CD4 + T cell, a cytotoxic CD8 + T cell, a natural killer T cell, or a gamma delta T cell
  • Reg regulatory T cell
  • a CAR may have any suitable architecture, known in the art, wherein the CAR comprises an antigen binding domain linked to T cell receptor effector functions.
  • CAR refers to an artificial multi-module molecule capable of triggering or inhibiting the activation of an immune cell.
  • a CAR will generally comprise an antigen binding domain, linker, transmembrane domain and cytoplasmic signaling domain.
  • a CAR includes one or more co-stimulatory domains and/or one or more co- inhibitory domains.
  • the antigen-binding domain of a CAR may include any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a target antigen of interest.
  • the binding region is an antigen-binding region, such as an antibody or functional binding domain or antigen-binding fragment thereof.
  • the antigen-binding region of the CAR can include any domain that binds to the antigen and may include, but is not limited to, a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, a single-chain antibody, and any antigen-binding fragment thereof.
  • the antigen binding domain portion includes a mammalian antibody or an antigen-binding fragment thereof.
  • An antigen-binding domain may comprise an antigenbinding fragment (Fab), a single-chain variable fragment (scFv), a nanobody, a VH domain, a VL domain, a single domain antibody (sdAb), a shark variable domain of a new antigen receptor (VNAR), a single variable domain on a heavy chain (VHH), a bispecific antibody, or a diabody; or a functional antigen-binding fragment thereof.
  • the antigen-binding domain is derived from the same cell type or the same species in which the CAR will ultimately be used.
  • the antigen-binding domain of the CAR may include a human antibody, a humanized antibody, or an antigen-binding fragment thereof.
  • the antigen binding domain is derived from a single chain antibody that selectively binds to a target antigen.
  • the antigen binding domain is provided by a single chain variable fragment (scFv).
  • scFv is a recombinant molecule in which the variable regions of the light and heavy immunoglobulin chains are connected in a single fusion polypeptide.
  • the VH and VL sequences are joined by a linker sequence. See, for example, Ahmad (2012) Clinical and Developmental Immunology Article ID 980250, herein specifically incorporated by reference. In principle, there are no particular limitations to the length and/or amino acid composition of the linker peptide joining the VH and VL sequences.
  • any arbitrary single-chain peptide including about 1 to 100 amino acid residues can be used as a peptide linker.
  • the linker peptide sequence includes about 5 to 50, about 10 to 60, about 20 to 70, about 30 to 80, about 40 to 90, about 50 to 100, about 60 to 80, about 70 to 100, about 30 to 60, about 20 to 80, about 30 to 90 amino acid residues.
  • the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25, about 20 to 40, about 30 to 50, about 40 to 60, about 50 to 70 amino acid residues. In some embodiments, the linker peptide sequence includes about 40 to 70, about 50 to 80, about 60 to 80, about 70 to 90, or about 80 to 100 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25 amino acid residues. [00200]
  • the transmembrane domain may be derived either from a natural or a synthetic source. Where the source is natural, the domain may be derived from any membranebound or transmembrane protein.
  • the transmembrane domain comprises at least the stalk and/or transmembrane region(s) of CD8, Megfl O, FcRy, Bail , MerTK, TIM4, Stabilin-1 , Stabilin-2, RAGE, CD300f, integrin subunit av, Integrin subunit (35, CD36, LRP1 , SCARF1 , C1 Qa, Axl, CD45, and/or CD86.
  • the CAR transmembrane domain is derived from a type I membrane protein, such as, but not limited to, CD3 , CD4, CD8, or CD28.
  • the transmembrane domain is synthetic, in which case it will include predominantly hydrophobic residues such as leucine, isoleucine, valine, phenylalanine, tryptophan, and alanine.
  • a triplet of phenylalanine, tryptophan and valine will be inserted at each end of a synthetic transmembrane domain.
  • the CAR further comprises one or more linkers/spacers.
  • an extracellular spacer region may link the antigen binding domain to the transmembrane domain and/or an intracellular spacer region may link an intracellular signaling domain to the transmembrane domain.
  • a spacer (linker) region linking the antigen binding domain to the transmembrane domain should be flexible enough to allow the antigen binding domain to orient in different directions to facilitate antigen recognition.
  • the linker includes a peptide linker/spacer sequence.
  • the spacer comprises the hinge region from an immunoglobulin, e.g., the hinge from any one of lgG1 , lgG2a, lgG2b, lgG3, lgG4, particularly the human protein sequences.
  • Alternatives include the CH2CH3 region of immunoglobulin and portions of CD3. For many scFv based constructs, an IgG hinge is effective.
  • a linker peptide sequence comprises about 1 to 100 amino acid residues, including any number of residues within this range such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues.
  • the linker peptide sequence includes about 5 to 50, about 10 to 60, about 20 to 70, about 30 to 80, about 40 to 90, about 50 to 100, about 60 to 80, about 70 to 100, about 30 to 60, about 20 to 80, about 30 to 90 amino acid residues.
  • the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25, about 20 to 40, about 30 to 50, about 40 to 60, about 50 to 70 amino acid residues. In some embodiments, the linker peptide sequence includes about 40 to 70, about 50 to 80, about 60 to 80, about 70 to 90, or about 80 to 100 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25 amino acid residues. In some embodiments, the linker peptide sequence may include up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.
  • a short oligo- or polypeptide linker preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the intracellular engulfment signaling domain or extracellular antigen binding domain of the CAR.
  • the linker comprises the amino acid sequence (G4S) n where n is 1 , 2, 3, 4, 5, etc., and in some embodiments, n is 3.
  • a cytoplasmic signaling domain such as those derived from the T cell receptor chain, is employed as part of the CAR in order to produce stimulatory signals for T lymphocyte proliferation and effector function following engagement of the chimeric receptor with the target antigen. Endodomains from co-stimulatory molecules may be included in the cytoplasmic signaling portion of the CAR.
  • co-stimulatory domain refers to a stimulatory domain, typically an endodomain, of a CAR that provides a secondary non-specific activation mechanism through which a primary specific stimulation is propagated.
  • co-stimulation include antigen nonspecific T cell co-stimulation following antigen specific signaling through the T cell receptor and antigen nonspecific B cell co-stimulation following signaling through the B cell receptor.
  • Co-stimulation, e.g., T cell co-stimulation, and the factors involved have been described in Chen & Flies, Nat Rev Immunol (2013) 13(4):227-42, the disclosure of which is incorporated herein by reference in its entirety.
  • suitable co-stimulatory polypeptides include, but are not limited to, 4-1 BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.
  • co-inhibitory domain refers to an inhibitory domain, typically an endodomain, derived from a receptor that provides secondary inhibition of primary antigen-specific activation mechanisms which prevents co-stimulation.
  • Co-inhibition e.g., T cell co-inhibition
  • co-inhibitory domains homodimerize.
  • a co-inhibitory domain can be an intracellular portion of a transmembrane protein.
  • a first-generation CAR transmits the signal from antigen binding through only a single signaling domain, for example a signaling domain derived from the high-affinity receptor for IgE FceRly, or the CD3£ chain.
  • the domain contains one or three immunoreceptor tyrosine-based activating motif(s) [ITAM(s)] for antigen-dependent T- cell activation.
  • ITAM-based activating signal endows T-cells with the ability to lyse the target tumor cells and secret cytokines in response to antigen binding.
  • Second-generation CARs include a co-stimulatory signal in addition to the CD3 signal. Coincidental delivery of the delivered co-stimulatory signal enhances cytokine secretion and antitumor activity induced by CAR-transduced T-cells.
  • the co-stimulatory domain will usually be membrane proximal relative to the CD3 ⁇ domain.
  • Third-generation CARs include a tripartite signaling domain, comprising for example a CD28, CD3 , 0X40 or 4-1 BB signaling region.
  • fourth generation, or “armored car” CAR-T cells CAR-T cells are further genetically modified to express or block molecules and/or receptors to enhance immune activity.
  • CAR variants include split CARs wherein the extracellular portion, the ABD and the cytoplasmic signaling domain of a CAR are present on two separate molecules.
  • CAR variants also include ON-switch CARs which are conditionally activatable CARs, e.g., comprising a split CAR wherein conditional hetero-dimerization of the two portions of the split CAR is pharmacologically controlled.
  • CAR molecules and derivatives thereof i.e., CAR variants
  • CAR variants also include bispecific or tandem CARs, which include a secondary CAR binding domain that can either amplify or inhibit the activity of a primary CAR.
  • CAR variants also include inhibitory chimeric antigen receptors (iCARs) which may, e.g., be used as a component of a bispecific CAR system, where binding of a secondary CAR binding domain results in inhibition of primary CAR activation.
  • Tandem CARs (TanCAR) mediate bispecific activation of T cells through the engagement of two chimeric receptors designed to deliver stimulatory or costimulatory signals in response to an independent engagement of two different tumor associated antigens.
  • iCARs use the dual antigen targeting to shout down the activation of an active CAR through the engagement of a second suppressive receptor equipped with inhibitory signaling domains.
  • Inhibitory CARs are designed to regulate CAR-T cell activity through inhibitory receptor signaling module activation. This approach combines the activity of two CARs, one of which generates dominant negative signals limiting the responses of CAR-T cells activated by the activating receptor. iCARs can switch off the response of the counteracting activator CAR when bound to a specific antigen expressed only by normal tissues. In this way, iCARs-T cells can distinguish cancer cells from healthy ones, and reversibly block functionalities of transduced T cells in an antigen-selective fashion. CTLA-4 or PD-1 intracellular domains in iCARs trigger inhibitory signals on T lymphocytes, leading to less cytokine production, less efficient target cell lysis, and altered lymphocyte motility.
  • An ABD can be provided as a “chimeric bispecific binding member”, i.e., a chimeric polypeptide having dual specificity to two different binding partners (e.g., two different antigens).
  • chimeric bispecific binding members include bispecific antibodies, bispecific conjugated monoclonal antibodies (mab)2, bispecific antibody fragments (e.g., F(ab)2, bispecific scFv, bispecific diabodies, single chain bispecific diabodies, etc.), bispecific T cell engagers (BITE), bispecific conjugated single domain antibodies, micabodies and mutants thereof, and the like.
  • Non-limiting examples of chimeric bispecific binding members also include those chimeric bispecific agents described in Kontermann.
  • a chimeric bispecific binding member may be a bispecific T cell engager (BiTE).
  • a BiTE is generally made by fusing a specific binding member (e.g., a scFv) that binds an antigen to a specific binding member (e.g., a scFv) with a second binding domain specific for a T cell molecule such as CD3.
  • a chimeric bispecific binding member may be a CAR-T cell adapter.
  • CAR-T cell adapter an expressed bispecific polypeptide that binds the antigen recognition domain of a CAR and redirects the CAR to a second antigen.
  • a CAR-T cell adapter will have two binding regions, one specific for an epitope on the CAR to which it is directed and a second epitope directed to a binding partner which, when bound, transduces the binding signal activating the CAR.
  • Useful CAR-T cell adapters include but are not limited to e.g., those described in Kim et al. J Am Chem Soc.
  • Effector CAR-T cells include autologous or allogeneic immune cells having cytolytic activity against a target cell.
  • a patient's own T cells or T cells from a donor are engineered to express a CAR.
  • the CAR-T cells are engineered from a complex mixture of immune cells, e.g., tumor infiltrating lymphocytes (TILs) isolated from an individual in need of treatment.
  • TILs tumor infiltrating lymphocytes
  • stem cells differentiated into T cells, are engineered to express a CAR.
  • iPSC induced pluripotent stem cell
  • a biological sample comprising T cells, from which CAR-T cells are generated may be collected from a subject or a donor.
  • the biological sample may include, without limitation, blood, lymphoid tissue (e.g., bone marrow, spleen, tonsils, lymph nodes), mucosal tissue (e.g., lungs, small intestine, and large intestine), skin, or a tissue where T cells have infiltrated.
  • lymphoid tissue e.g., bone marrow, spleen, tonsils, lymph nodes
  • mucosal tissue e.g., lungs, small intestine, and large intestine
  • the T cells may be separated from a mixture of cells prior to engineering the T cells to generate CAR-T cells.
  • T cells may be engineered and cultured without separation from other cells.
  • T cells may be separated from other cells using any suitable cell separation technique such as, but not limited to, centrifugation-based cell separation, positive or negative selection against surface markers on cells (e.g., with antibody-coated beads), affinity chromatography, panning and immunopanning techniques, fluorescence activated cell sorting (FACS), or magnetic-activated cell sorting (MACS).
  • FACS fluorescence activated cell sorting
  • MCS magnetic-activated cell sorting
  • Affinity reagents may be employed comprising specific receptors or ligands specific for cell surface molecules.
  • the T cells may be separated from dead cells by employing viability dyes (e.g., propidium iodide). Any technique may be employed which is not unduly detrimental to the viability of the T cells.
  • the cells may be collected in any appropriate medium that maintains the viability of the cells.
  • Various media are commercially available and may be used according to the nature of the cells, including dMEM, HBSS, dPBS, RPMI, Iscove’s medium, etc., which may be supplemented with fetal calf serum (FCS).
  • FCS fetal calf serum
  • the collected cells may be used immediately or frozen (e.g., at liquid nitrogen temperatures) prior to use.
  • CAR-T cells are expanded in culture prior to screening, as described further below, or use in therapy.
  • the CAR-T cells require activation for expansion in vitro or ex vivo, which can be accomplished by co-incubating T cells with natural antigen-presenting cells (e.g., dendritic cells) or artificial antigen-presenting cells or particles that present antigen and/or activating signals to the CAR-T cells.
  • natural antigen-presenting cells e.g., dendritic cells
  • artificial antigen-presenting cells or particles that present antigen and/or activating signals to the CAR-T cells.
  • CAR-T cells from multiple donors wherein the CAR-T cells have knockouts of their endogenous TCRs, as described herein, can be pooled and tested simultaneously in multiplexed assays. See, e.g., co-owned Provisional Patent Application, entitled “Massively Parallel Mixed Lymphocyte Reactions,” filed even date herewith, the disclosure of which is hereby incorporated by reference herein in its entirety.
  • Activation of CAR-T cells can be determined by measuring cell proliferation, expression of activation markers (e.g., detection of CD69, HLA-DR, IL2RA, and/or CD25), and production of effector cytokines (e.g., IFN-y, TNF-a, TNF-p, IL-1 , IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, IL- 12, IL-13, and IL-25).
  • Multiplexed screening of CAR-T cell cytotoxic activity can be performed in vitro to validate activity against target cells before further testing individual CAR-T cell in vivo in animal models and human clinical trials.
  • Cytotoxicity of CD8+ CAR-T cells involves exocytosis of granules containing the pore-forming toxin, perforin, proapoptotic serine proteases, and granzymes that lyse target cells. Cytotoxicity of CD4+ CAR-T cells involves secretion of cytokines and apoptotic factors such as TNF-a, INF-y, and TRAIL that induce apoptosis of target cells or activate macrophages to engulf tumor cells.
  • cytokines and apoptotic factors such as TNF-a, INF-y, and TRAIL that induce apoptosis of target cells or activate macrophages to engulf tumor cells.
  • Perforin, proapoptotic serine proteases, granzymes, cytokines, and apoptotic factors can be measured, for example, using a multiplexed enzyme-linked immunosorbent assay (ELISA). Cytolysis can be assayed in vitro based on the release of compounds containing radioactive isotopes such as 51 Cr from radiolabeled target cells. Alternatively a membrane-permeable live-cell labeling dye such as calcein acetoxymethyl ester of calcein (Calcein/AM) can be used to distinguish live cells from dead cells.
  • a membrane-permeable live-cell labeling dye such as calcein acetoxymethyl ester of calcein (Calcein/AM) can be used to distinguish live cells from dead cells.
  • LDH lactate dehydrogenase
  • Flow cytometry can also be used to assess cell proliferation, activation, and cytotoxicity.
  • the percentage of target cells that are live, apoptotic, or dead can be determined by staining target cells with viability dyes such that the live and dead cell populations can be distinguished based on differences in fluorescence.
  • viability dyes such as Annexin V-FITC
  • Propidium iodide can be used to label target cells that are at a late stage of apoptosis or dead.
  • Lipophilic dyes, such as PKH67 and PKH26 can be used to label the cell membranes of target cells for measuring proliferation of CAR-T cells by flow cytometry.
  • T cell activation can also be detected by immunofluorescent labeling of activation markers such as CD69, HLA-DR, IL2RA, and CD25.
  • activation markers such as CD69, HLA-DR, IL2RA, and CD25.
  • Cell proliferation can also be detected and quantified, for example, using a cell counter or staining of CAR-T cells with a fluorescent tracking dye, such as carboxyfluorescein succinimidyl ester (CFSE).
  • CFSE carboxyfluorescein succinimidyl ester
  • the CAR-T cells may be further tested for efficacy in treating a disease in vivo, e.g., in an animal.
  • CAR-T cells can be tested for cytotoxicity against cancerous cells in an animal with solid tumors.
  • human xenograft tumors are implanted in animals, followed by administration of CAR-T cells, and evaluation of antitumor responses.
  • An exemplary animal model of cancer is a NOD Scid Gamma (NSG) mouse transplanted with human tumors. NSG mice are completely deficient in adaptive immunity and severely deficient in innate immunity, which avoids transplant rejection of CAR-T cells and patient-derived xenografts.
  • NSG mice are completely deficient in adaptive immunity and severely deficient in innate immunity, which avoids transplant rejection of CAR-T cells and patient-derived xenografts.
  • Antitumor responses can be evaluated by various methods known in the art.
  • the volume of a subcutaneous tumor can be measured by using a digital caliper.
  • Internal tumors can be measured by x-ray imaging, computed tomography (CT), ultrasound (US), magnetic resonance imaging (MRI), positron emission tomography (PET), or singlephoton emission computed tomography (SPECT).
  • CT computed tomography
  • US ultrasound
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • SPECT singlephoton emission computed tomography
  • the CAR-T cells are further modified to express a bioluminescent protein such as luciferase to allow monitoring of tumors by bioluminescence imaging or a fluorescent protein such as green fluorescent protein to allow monitoring of tumors by fluorescence imaging.
  • tumors can be removed from the animals and measured after the treatment with CAR-T cells is completed. Immunohistochemistry of tumor specimens can be used to detect T cell infiltration into tumors and quantitate target antigen expression. Cytokine profiling of tumors treated with CAR-T cells can also be performed.
  • CAR-T cells can be tested for cytotoxicity against activated fibroblasts or fibrotic tissue in an animal with fibrosis.
  • the extent of fibrosis can be monitored in an animal in vivo, for example, by x-ray imaging, computed tomography (CT), ultrasound (US), magnetic resonance imaging (MRI), positron emission tomography (PET), or single-photon emission computed tomography (SPECT).
  • CT computed tomography
  • US ultrasound
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • fibrotic tissue can be removed from the animals and measured after the treatment with CAR-T cells is completed. Immunohistochemistry of fibrotic tissue specimens can be used to detect T cell infiltration into fibrotic tissue and quantitate target antigen expression. Cytokine profiling of fibrotic tissue treated with CAR-T cells can also be performed.
  • An animal model can be used not only to determine efficacy but also the toxicity or side effects of treatment with a CAR-T cell. Furthermore, this disclosure pertains to uses of CAR-T cells, identified by the above-described screening assays for treatment of a disease such as, but not limited to, cancer, fibrosis, an infection, or an autoimmune disease.
  • a CAR-T cell identified by the above-described screening assays for treatment of a disease, may be expanded in culture in the presence of a natural antigen-presenting cell (e.g., dendritic cell) or an artificial antigen-presenting cell or particle under selective conditions prior to formulation into a pharmaceutical composition and administration.
  • a natural antigen-presenting cell e.g., dendritic cell
  • an artificial antigen-presenting cell or particle under selective conditions prior to formulation into a pharmaceutical composition and administration.
  • compositions comprising CAR-T cells generated as described herein, can be prepared by formulating the CAR-T cells into dosage forms by known pharmaceutical methods.
  • a pharmaceutical composition comprising CAR- T cells can be formulated for parenteral administration, as capsules, liquids, film-coated preparations, suspensions, emulsions, and injections (such as venous injections, drip injections, and the like).
  • the CAR-T cells can be combined as appropriate, with pharmaceutically acceptable carriers or media, in particular, sterile water and physiological saline, vegetable oils, resolvents, bases, emulsifiers, suspending agents, surfactants, stabilizers, vehicles, antiseptics, binders, diluents, tonicity agents, soothing agents, bulking agents, disintegrants, buffering agents, coating agents, lubricants, coloring agents, solution adjuvants, or other additives.
  • pharmaceutically acceptable carriers or media in particular, sterile water and physiological saline, vegetable oils, resolvents, bases, emulsifiers, suspending agents, surfactants, stabilizers, vehicles, antiseptics, binders, diluents, tonicity agents, soothing agents, bulking agents, disintegrants, buffering agents, coating agents, lubricants, coloring agents, solution adjuvants, or other additives.
  • the CAR-T cells may also be used in combination with other therapeutic agents for treating a disease.
  • CAR-T cells may be used in combination with anti-cancer agents such as, but not limited to: chemotherapeutic agents such as cyclophosphamide, doxorubicin, vincristine, methotrexate, cytarabine, ifosfamide, etoposide, adriamycin, bleomycin, vinblastine, dacarbazine, chlormethine, oncovin, and procarbazine; immunotherapeutic agents such as antibodies (e.g., rituximab), cytokines (e.g., interferons, including type I (IFNa and IFN
  • chemotherapeutic agents such
  • the pharmaceutical composition comprising the CAR-T cells is a sustained-release formulation, or a formulation that is administered using a sustained-release device.
  • sustained-release devices include, for example, transdermal patches, and miniature implantable pumps that can provide for delivery of the CAR-T cells over time in a continuous, steady-state fashion at a variety of doses to achieve a sustained-release effect with a non-sustained-release pharmaceutical composition.
  • the subject who receives the CAR-T cells is also the subject from whom the original T cells (i.e., before genetic modification to express a CAR specific for a target cell) are harvested or obtained, which provides the advantage that the cells are autologous.
  • T cells can be obtained from another subject (i.e., donor), a culture of cells from a donor, or from established cell culture lines and genetically modified, as described herein.
  • T cells may be obtained from the same or a different species than the subject to be treated, but preferably are of the same species, and more preferably of the same immunological profile as the subject.
  • Such cells can be obtained, for example, from a biological sample comprising T cells from a close relative or matched donor, genetically modified to express a CAR, and administered to a subject in need of treatment.
  • the patients or subjects who donate or receive the T cells are typically mammalian, and usually human. However, this need not always be the case, as veterinary applications are also contemplated.
  • the CAR-T cells administered to a subject are autologous or allogeneic.
  • CAR-T cells are administered to a subject in a therapeutically effective amount.
  • therapeutically effective amount refers to the administration of the CAR-T cells to a subject, either alone or as a part of a pharmaceutical composition and either in a single dose or as part of a series of doses, in an amount that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease, disorder or condition when administered to a patient.
  • the therapeutically effective amount can be ascertained by measuring relevant physiological effects. For example, in the case of cancer, a therapeutically effective amount of the CAR-T cells provides an anti-tumor effect, as defined herein.
  • a positive therapeutic response would refer to one or more of the following improvements in the disease: (1 ) reduction in tumor size; (2) reduction in the number of cancer cells; (3) inhibition (i.e., slowing to some extent, preferably halting) of tumor growth; (4) inhibition (i.e., slowing to some extent, preferably halting) of cancer cell infiltration into peripheral organs; (5) inhibition (i.e., slowing to some extent, preferably halting) of tumor metastasis; and (6) some extent of relief from one or more symptoms associated with the cancer.
  • Such therapeutic responses may be further characterized as to degree of improvement.
  • an improvement may be characterized as a complete response.
  • antigen-presenting cells e.g., dendritic cells
  • artificial antigen-presenting cells or particles are used to stimulate proliferation and expansion of CAR-T cells in vitro or ex vivo prior to administration.
  • the ex vivo method comprises contacting a population of T cells comprising a CAR-T cell with the antigen-presenting cells or artificial antigen-presenting cells or particles, wherein the population of T cells have been obtained from the subject to be treated, then genetically modified to express a CAR with an endogenous TCR knockout, as described herein. After one or more rounds of antigen-stimulation with the antigen-presenting cells or artificial antigen-presenting cells or particles and expansion of the CAR-T cells in culture, the autologous CAR-T cells are subsequently administered to the subject.
  • stimulation of proliferation and expansion of CAR-T cells with antigen-presenting cells are carried out in vitro.
  • the in vitro method comprises contacting a population of T cells comprising a CAR-T cell with the antigen-presenting cells (e.g., dendritic cells) or artificial antigen-presenting cells or particles, wherein the T cells have been obtained from a donor, a culture of cells from a donor, or from established cell culture lines, then genetically modified to express a CAR with an endogenous TCR knockout, as described herein.
  • the T cells may be obtained from the same or a different species than the subject to be treated, but preferably are of the same species, and more preferably of the same immunological profile as the subject. Such cells can be obtained, for example, from a blood sample comprising T cells from a close relative or matched donor. After one or more rounds of antigen-stimulation with the antigen-presenting cells (e.g., dendritic cells) or artificial antigen-presenting cells or particles and expansion of the CAR-T cells in culture, the CAR-T cells may be subsequently administered to a subject.
  • the antigen-presenting cells e.g., dendritic cells
  • the CAR-T cells may be subsequently administered to a subject.
  • proliferation and expansion of CAR-T cells occurs in vivo either by stimulation with an endogenous antigen-presenting cell or by coadministration of antigen-presenting cells or artificial antigen-presenting cells or particles with the CAR- T cells to the subject.
  • the subject may have cancer, wherein the CAR-T cells comprise a CAR that specifically binds to an antigen expressed on a cancerous cell.
  • the antigen is a tumor-specific antigen or a tumor-associated antigen expressed on a cancerous cell, wherein the antigen is used to activate a CAR-T cell designed for therapeutic use against a cancerous cell.
  • tumor-specific antigens and tumor-associated antigens include, without limitation, oncogene protein products, mutated or dysregulated tumor suppressor proteins, oncovirus proteins, oncofetal antigens, mutated or dysregulated differentiation antigens, overexpressed or aberrantly expressed cellular proteins (e.g., mutated or aberrantly expressed growth factors, mitogens, receptor tyrosine kinases, cytoplasmic tyrosine kinases, serine/threonine kinases and their regulatory subunits, G proteins, and transcription factors), and altered cell surface glycolipids and glycoproteins on cancerous cells.
  • oncogene protein products mutated or dysregulated tumor suppressor proteins, oncovirus proteins, oncofetal antigens, mutated or dysregulated differentiation antigens, overexpressed or aberrantly expressed cellular proteins (e.g., mutated or aberrantly expressed growth factors, mitogens, receptor tyrosine kinases, cytoplasmic tyrosine
  • tumor-specific antigens and tumor-associated antigens may include without limitation, dysregulated or mutated RAS, WNT, MYC, ERK, TRK, CTAG1 B, MAGEA1 , Bcr-Abl, p53, c-Sis, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), HER2/neu, Src-family, Syk-ZAP-70 family proteins, and BTK family of tyrosine kinases, Abl, Raf kinase, cyclin-dependent kinases, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, MUC-1 , epithelial tumor antigen (ETA), tyrosinase, melanoma- associated antigen (MAGE), and other abnormal or dysregulated proteins expressed on cancerous cells.
  • RAS dysregulated or mutated RAS
  • WNT
  • the subject has leukemia, lymphoma, myeloma, prostate cancer, breast cancer, lung cancer, kidney cancer, lung cancer, ovarian cancer, intestine cancer, or glioblastoma.
  • the subject has fibrosis, wherein the CAR-T cells comprise a CAR that specifically binds to a fibrosis antigen expressed on activated fibroblasts or fibrotic tissue such as fibroblast activation protein (FAP).
  • FAP fibroblast activation protein
  • the subject is undergoing or has previously undergone CAR-T cell immunotherapy.
  • the present disclosure contemplates the administration of the CAR-T cells, and compositions thereof, in any appropriate manner.
  • Suitable routes of administration include parenteral (e.g., intramuscular, intravenous, subcutaneous (e.g., injection or implant), intraperitoneal, intracisternal, intraarticular, intraperitoneal, intracerebral (intraparenchymal) and intracerebroventricular), oral, nasal, vaginal, sublingual, intraocular, rectal, topical (e.g., transdermal), sublingual, inhalation, local, e.g., injection directly into a target organ or tissue such as a tumor or fibrotic tissue.
  • parenteral e.g., intramuscular, intravenous, subcutaneous (e.g., injection or implant), intraperitoneal, intracisternal, intraarticular, intraperitoneal, intracerebral (intraparenchymal) and intracerebroventricular
  • oral nasal, vaginal, sublingual, intraocular,
  • the CAR-T cells may comprise a binding-triggered transcriptional switch.
  • the method may further include activating a T cell such as a T cell expressing a chimeric Notch polypeptide, as described herein.
  • the method of the present disclosure may be used for inducing T-cell proliferation without significantly increasing cytokine production by the T cell.
  • the method may include administering a T cell expressing a chimeric Notch polypeptide and CAPP having a protein displayed on the surface, where the protein binds to the Notch polypeptide resulting in expression of a cancer associated CAR on the cell surface.
  • the CAPP further includes an antigen that binds the cancer associated CAR, where binding of the antigen on the particle to the cancer associated CAR results in activation of the T cell in absence of significant expression of cytokines.
  • the level of cytokines produced by the T cells in the absence of cancer cells expressing the CAR antigen is substantially lower than the level of the cytokines produced by the T cells in the presence of cancer cells expressing the CAR antigen.
  • particles functionalized with both a protein that binds to the chimeric Notch polypeptide and an antigen that binds to the CAR expressed in response to the binding of the protein to the chimeric Notch polypeptide provides for proliferation of the T-cells while having a substantially lower production of cytokines by the activated T cell.
  • contacting a CAR-T cell expressing a BTTS, e.g., a chimeric Notch receptor polypeptide, as described herein with the CAPP of the present disclosure may modulate an activity of the CAR-T cell.
  • release of the intracellular domain modulates proliferation of the cell or of cells surrounding the cell.
  • release of the intracellular domain modulates apoptosis in the cell or in cells surrounding the cell.
  • release of the intracellular domain induces cell death by a mechanism other than apoptosis.
  • release of the intracellular domain modulates gene expression in the cell through transcriptional regulation, chromatin regulation, translation, trafficking or post-translational processing.
  • release of the intracellular domain modulates differentiation of the cell. In some cases, release of the intracellular domain modulates migration of the cell or of cells surrounding the cell. In some cases, release of the intracellular domain modulates the expression and secretion of a molecule from the cell. In some cases, release of the intracellular domain modulates adhesion of the cell to a second cell or to an extracellular matrix. In some cases, release of the intracellular domain induces de novo expression a gene product in the cell.
  • the gene product is a transcriptional activator, a transcriptional repressor, a chimeric antigen receptor, a second chimeric Notch receptor polypeptide, a translation regulator, a cytokine, a hormone, a chemokine, or an antibody.
  • Donor polynucleotides encoding one or more synthetic exons and guide RNAs can be designed to target an intron of an endogenous gene, wherein the donor polynucleotide is integrated into the intron by HDR. Subsequent transcription of the integrated donor polynucleotide generates a pre-mRNA transcript comprising the one or more synthetic exons, wherein splicing of the pre-mRNA transcript generates a mature mRNA comprising the one or more synthetic exons in between exons of the endogenous gene. Translation of the mature mRNA results in expression of the one or more synthetic exons by the cell without expression of the endogenous gene.
  • the targeted endogenous gene is a surface receptor. See Example 1 , for a description of exemplary gene knockins into intron 4 and intron 5 of the CD3 epsilon subunit of T-cell receptor complex (CD3E) gene, intron 1 of the beta- 2 microglobulin (B2M) gene, and intron 2 and intron 4 of the CD47 gene.
  • CD3E CD3 epsilon subunit of T-cell receptor complex
  • B2M beta- 2 microglobulin
  • Exemplary guide RNAs for performing such gene knockins comprise a sequence selected from the group consisting of SEQ ID NOS:24-35, and exemplary donor polynucleotides for performing such gene knockins comprise a sequence selected from the group consisting of SEQ ID NOS:80-85.
  • this method provides the ability of using negative selection to enrich for cells with successful intron knockins.
  • a binding agent that specifically binds to endogenous CD3E, B2M, or CD47 on cells can be used to perform negative selection to remove unsuccessfully edited cells from a sample.
  • intron knockins can be programmed by engineering the 5’ and 3’ end sequences of the integrated synthetic exons.
  • intron knockins can be alternatively spliced into a coding transcript, resulting in gene knockout, or skipped, enabling the endogenous mRNA to be expressed along with the synthetic knockin mRNA by adding exonic splicing silencers (ESS) or exonic splicing enhancers (ESE) into the constructs (see Example 1 ).
  • ESSs and ESEs have short ⁇ 6-8 bp sequences that are bound by SR proteins to control the efficiency of mRNA splicing at adjacent splice acceptor sites.
  • Kits are provided containing any of the compositions described herein for generating intron knockins, including donor polynucleotides and guide RNAs for inserting synthetic exons into the intron of a TCR gene for generating CAR-T cells, or donor polynucleotides and guide RNAs for inserting synthetic exons into the introns of the CD3E, B2M, or CD47 genes or other genes of interest to genetically modify a cell as desired.
  • the kit comprises a CRISPR system for genetically modifying T cells by insertion of a donor polynucleotide encoding a chimeric antigen receptor into an intron of a TCR gene, as described herein.
  • a kit may also include a binding agent that specifically binds an endogenous TCR on T cells for performing negative selection to remove unsuccessfully edited T cells from a sample.
  • the binding agent may be a magnetic bead comprising an antibody specific for the endogenous TCR to allow removal of unsuccessfully edited T cells using magnetic separation.
  • a kit may further comprise media suitable for culturing CAR-T cells.
  • the kit may include transfection agents, buffers, tissue culture plates, flasks, test tubes, vials, and the like, and optionally one or more other factors, such as cytokines (e.g., IL-2, IL-3, IL-6, IL-7, IL-15, TNFa, IFN-y, and GM-CSF), growth factors, antibiotics, or other media supplements, and the like.
  • cytokines e.g., IL-2, IL-3, IL-6, IL-7, IL-15, TNFa, IFN-y, and GM-CSF
  • growth factors e.g., IL-2, IL-3, IL-6, IL-7, IL-15, TNFa, IFN-y, and GM-CSF
  • antibiotics e.g., antibiotics, or other media supplements, and the like.
  • the kit comprises a guide RNA comprising a sequence selected from the group consisting of SEQ ID NOS:2-35.
  • the kit comprises a donor polynucleotide comprising a sequence selected from the group consisting of SEQ ID NOS:36-86.
  • kits generally will comprise, in suitable means, distinct containers for each individual reagent or solution.
  • suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes.
  • Containers can be formed from a variety of materials, including glass or plastic.
  • a container may have a sterile access port (for example, the container may be a vial having a stopper pierceable by a hypodermic injection needle).
  • kits may also provide a delivery device for administration of CAR-T cells to a patient.
  • kits may comprise a container having a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the kit can further comprise a container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It can also contain other materials useful to the end-user, including other pharmaceutically acceptable formulating solutions such as buffers, diluents, filters, needles, and syringes or other delivery device.
  • the subject kits may further include (in certain embodiments) instructions for practicing the subject methods.
  • These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit.
  • One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like.
  • Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), DVD, flash drive, and the like, on which the information has been recorded.
  • Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a removed site.
  • the binding agent comprises an antibody, an antibody mimetic, an aptamer, or a ligand that selectively binds to the endogenous TCR.
  • the viral vectors are lentivirus vectors, retrovirus vectors, or adeno-associated virus vectors
  • RNA-guided nuclease and the guide RNA are provided by a recombinant polynucleotide that is integrated into the genome of the T cell.
  • RNA-guided nuclease is provided by a mRNA encoding the RNA-guided nuclease, wherein translation of the mRNA results in production of the RNA-guided nuclease in the T cell.
  • RNA-guided nuclease is a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • T cell is a helper CD4 + T cell, a cytotoxic CD8 + T cell, a natural killer T cell, or a gamma delta T cell that has been genetically modified to express the chimeric antigen receptor.
  • the chimeric antigen receptor comprises a transmembrane domain linked to an extracellular antigen binding domain and an intracellular signaling domain, wherein the extracellular antigen-binding domain specifically binds to an antigen on the target cell.
  • the extracellular antigen binding domain comprises a single chain variable fragment (scFv), an antigen-binding fragment (Fab), a nanobody, a heavy chain variable (VH) domain, a light chain variable (VL) domain, a single domain antibody (sdAb), a shark variable domain of a new antigen receptor (VNAR), a single variable domain on a heavy chain (VHH), a bispecific antibody, a diabody, or a functional fragment thereof that binds specifically to the antigen.
  • scFv single chain variable fragment
  • Fab antigen-binding fragment
  • VH heavy chain variable
  • VL light chain variable
  • sdAb single domain antibody
  • VNAR shark variable domain of a new antigen receptor
  • VHH single variable domain on a heavy chain
  • bispecific antibody a diabody, or a functional fragment thereof that binds specifically to the antigen.
  • the intracellular signaling domain is a CD3-zeta intracellular signaling domain or a ZAP-70 intracellular signaling domain.
  • intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM).
  • ITAM immunoreceptor tyrosine-based activation motif
  • costimulatory domain is a 4-1 BB, CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, or HVEM costimulatory domain.
  • transmembrane domain is a CD8, Megfl O, FcRy, Bail , MerTK, TIM4, Stabilin-1 , Stabilin-2, RAGE, CD300f, integrin subunit av, integrin subunit P5, CD36, LRP1 , SCARF1 , C1 Qa, Axl, CD45, or CD86 transmembrane domain.
  • the target cell is a cancer cell, a tumor cell, an activated fibroblast, an autoreactive immune cell, a pathogen, or a diseased cell.
  • the antigen on the target cell is a tumor antigen or a tumor-associated antigen.
  • the pathogen is a virus, a bacterium, a fungus, or a parasite.
  • the antigen on the target cell is a viral antigen, a bacterial antigen, a fungal antigen or a parasite antigen.
  • the one or more additional exogenous genes are selected from the group consisting of a PD-1 dominant negative receptor, a dominant negative TGF-p type II receptor, a Fas dominant negative receptor, an IL-4 chimeric switch receptor, a costimulatory signaling domain, a cytokine, and a chemokine receptor.
  • T cell is further genetically modified to add a binding-triggered transcriptional switch that regulates expression of the chimeric antigen receptor or activation of the T cell.
  • binding-triggered transcriptional switch comprises a synthetic notch receptor, a modular extracellular sensor architecture (MESA), or a synthetic intramembrane proteolysis receptor (SNIPR).
  • MSA modular extracellular sensor architecture
  • SNIPR synthetic intramembrane proteolysis receptor
  • the synthetic notch receptor comprises i) an extracellular ligand-binding domain that specifically binds to a second target antigen on the target cell, and ii) an intracellular domain, wherein binding of the extracellular ligand-binding domain to the second target antigen results in cleavage of the intracellular domain to release a transcription factor from the intracellular domain, wherein the transcription factor that is released from the intracellular domain induces expression of the chimeric antigen receptor on the T cell.
  • the donor polynucleotide further comprises an exonic splicing silencer (ESS) or an exonic splicing enhancer (ESE) at the 5’ end of the synthetic exon.
  • ESS exonic splicing silencer
  • ESE exonic splicing enhancer
  • a composition comprising: a donor polynucleotide, wherein the donor polynucleotide comprises a 5' homology arm that can hybridize to a 5' genomic target sequence and a 3' homology arm that can hybridize to a 3' genomic target sequence flanking a synthetic exon comprising a nucleotide sequence encoding a CAR that specifically binds to a target antigen, wherein at least a portion of the 5' genomic target sequence and at least a portion of the 3' genomic target sequence are located within an intron of a gene encoding an endogenous T cell receptor (TCR) protein chain; a recombinant polynucleotide encoding an RNA-guided nuclease; and a recombinant polynucleotide encoding a guide RNA, where
  • composition of aspect 52, wherein the donor polynucleotide, the recombinant polynucleotide encoding the RNA-guided nuclease, and the recombinant polynucleotide encoding the guide RNA are provided by one or more vectors.
  • composition of aspect 53, wherein the one or more vectors are viral vectors.
  • composition of aspect 53 or 54 wherein the donor polynucleotide and the recombinant polynucleotide encoding the RNA-guided nuclease are provided by separate vectors.
  • 56. The composition of aspect 53 or 54, wherein the donor polynucleotide and the recombinant polynucleotide encoding the RNA-guided nuclease are provided by the same vector.
  • CRISPR regularly interspaced short palindromic repeats
  • composition of aspect 60, wherein the Cas nuclease is Cas9 or Cas12a.
  • kits comprising the composition of any one of aspects 52-61 and instructions for producing a genetically modified T cell expressing a chimeric antigen receptor.
  • kit of aspect 62 further comprising a binding agent that selectively binds to an endogenous TCR.
  • the binding agent comprises an antibody, an antibody mimetic, an aptamer, or a ligand that selectively binds to the endogenous TCR.
  • the binding agent is attached to a solid support.
  • kits of aspect 65 wherein the solid support is a magnetic bead, wherein the T cells comprising the endogenous TCR can be removed from the sample by magnetic separation.
  • a composition comprising the genetically modified T cell of aspect 68 and a pharmaceutically acceptable excipient.
  • a method of performing cellular therapy comprising administering a therapeutically effective amount of the composition of aspect 69 to a subject.
  • a method of genetically modifying a cell comprising: introducing a donor polynucleotide into the cell, wherein the donor polynucleotide comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence flanking a synthetic exon, wherein at least a portion of the 5' genomic target sequence and at least a portion of the 3' genomic target sequence are located within an intron of an endogenous gene; introducing an RNA-guided nuclease into the cell; introducing a guide RNA into the cell, wherein the guide RNA forms a complex with the RNA-guided nuclease such that the guide RNA directs the RNA-guided nuclease to a genomic target sequence in the intron of the endogenous gene, wherein the RNA- guided nuclease creates a double-stranded break in the genomic target sequence in the intron, wherein the donor
  • TCR T cell receptor
  • CD3E CD3 epsilon subunit of T-cell receptor complex
  • B2M beta-2 microglobulin
  • the donor polynucleotide further comprises an exonic splicing silencer (ESS) or an exonic splicing enhancer (ESE) at the 5’ end of the synthetic exon.
  • ESS exonic splicing silencer
  • ESE exonic splicing enhancer
  • An isolated polynucleotide comprising a sequence selected from the group consisting of SEQ ID NOS:1 -137, or a sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOS:1 -137.
  • RNA guided nucleases such as Cas9 and Cas12a have dramatically expanded applications of endogenous gene editing 1 2 .
  • new genetic sequences can be inserted into defined sites in the genomes of primary human cells through homology directed repair (HDR) 3 .
  • HDR homology directed repair
  • These targeted gene integrations have opened diverse research and therapeutic applications, from correcting disease causing mutations, to integrating new synthetic genes such as knockin of a chimeric antigen receptor (CAR) or T cell receptor (TCR) into the endogenous TCR alpha locus ( TRAC) in primary human T cells 4 5 .
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • TRAC TCR alpha locus
  • the majority of current gene targeting methods integrate new genetic material within exons, knocking out the endogenous gene by targeting the middle of the coding sequence. Endogenous gene expression can be maintained by targeting the N or C terminus, although this limits guide RNA (gRNA) target sites, resulting in effective editing in only a sub
  • successfully HDR edited cells may lose expression of the targeted gene, but the majority of non-HDR edited cells will acquire NHEJ mutations 16-18 or large deletions 19-21 which also results in target protein loss, as demonstrated by knockin of a CAR to the first exon of TRAC in primary human T cells (FIG. 1 A) 45 .
  • non-viral intron knockins where synthetic exons are knocked into intronic regions of endogenous genes in primary human T cells.
  • Successful knockin to an endogenous intron results in splicing of the inserted sequence into the mature endogenous mRNA transcript, disrupting the endogenous gene and resulting in loss of endogenous protein expression (e.g. a surface protein for selection purposes), as used historically in gene-trap random mutagenesis screening methods 26 27 .
  • endogenous protein expression e.g. a surface protein for selection purposes
  • NHEJ edits within introns are predominantly spliced out of mature mRNA transcripts 24 , resulting in continued protein expression (except for rarer large multi kb deletions 28 ).
  • non-viral intron knockins enabled negative selection of successfully gene edited primary human T cells.
  • Non-viral intron knockins were possible with even large synthetic exons, and detailed engineering of 5’ and 3’ splicing architectures enabled user control of the splicing behavior of the integrated synthetic exons, allowing for the targeted endogenous gene to be either knocked out or expressed depending on desired applications.
  • Intron knockins were successful across genomic target loci in primary human T cells and offer a generalized gene targeting method enabling both greater flexibility in target site choice while also allowing for simpler negative selections of successfully edited cells.
  • TRAC intron knockin CAR T cells by depletion of TCR (CD3) positive cells resulted in a dramatically enriched population of successfully-edited CAR T cells, with CAR positive cells increasing from 29.8% to 87.2% post selection.
  • the resulting purified TRAC intron knockin CAR T cell population was TCR negative, which is beneficial for clinical applications 4 ’ 5 14 .
  • Non-viral intron knockin of a CAR at the TRAC locus proved to be successful across 18 tested gRNAs targeted different regions of TRAC Intron 1 , and efficient gene integrations were possible using both Cas9 and Cas12a RNPs (FIG. 10 and FIG. 7).
  • TRAC intron knockins enabled negative selection of CAR T cells to greater than 90% purity (FIG. 1 D).
  • In vitro target cell killing assays showed that TRAC intron knockin CARs killed approximately 90% of target antigen positive Nalm6 leukemia cells within 24 hours post co-incubation, the same (p > 0.05) as TRAC exon CAR knockins (FIG.
  • Negative selection did not require antibody binding to the surface of the non-viral TRAC intron edited T cells, compared to previous positive selection methods that require antibody binding, which can induce TCR or CAR crosslinking depending on their target 32 .
  • Negatively selected CAR T cells showed a slight increase in in vitro proliferation after stimulation with target cells, and successfully killed target cells across tested E:T ratios (FIGS. 9C-9D).
  • TRAC intron knockins with negative selection enabled negative selection of successfully edited CAR T cells without integration of any additional expressed exogenous DNA.
  • Intron knockins can introduce large functional synthetic exons
  • intron knockins could be: (1 ) alternatively spliced into a coding transcript, resulting in gene knockout, or (2) skipped, enabling the endogenous mRNA to be expressed along with the synthetic knockin mRNA.
  • alternative splicing of intron knockins could be programmed through engineering of the 5’ and 3’ end sequences of integrated synthetic exons.
  • the splice sites present in the pre-mRNA become unbalanced, with the preceding endogenous exon’s splice donor able to splice with the integrated synthetic exon (“synthetic splicing”), or with the downstream endogenous exon (“endogenous splicing”; FIG. 3A and FIG. 10A).
  • intron knockins with balanced splice sites resulted predominantly in loss of the targeted endogenous gene (GFP+ / TCR-)
  • intron knockins with unbalanced splice sites caused most knockin positive cells to express both the gene encoded in the synthetic exon, as well as the targeted endogenous gene (GFP+ I TCR+; FIG. 3B).
  • splicing behavior of synthetic exons integrated through non- viral intron knockins could be controlled through engineering of splice sites, we designed a series of constructs testing different sequence architectures at synthetic exon’s 3’ ends to determine the degree to which endogenous vs synthetic splicing could be controlled.
  • Exonic Splicing Silencers (ESS) and Enhancers (ESE) are short ⁇ 6-8 bp degenerate sequences that are bound by SR proteins to control the efficiency of mRNA splicing at adjacent splice acceptor sites 34 35 .
  • ESS Exonic Splicing Silencers
  • ESE Enhancers
  • intron knockin of synthetic exons with balanced splice sites showed that less than 5% of the dual positive cells still expressed the targeted endogenous TCR gene (FIG. 4A).
  • intron knockin of synthetic exons containing optimized Exonic Splicing Silencing elements at the 5’ ends and a polyA at the 3’ end showed that almost 70% of the GFP+ 1 RFP+ positive cells were TCR+ (FIG. 4B).
  • both TRAC alleles possess a knocked in synthetic exon in the dual positive cells one or both alleles must be capable of alternative splicing to maintain expression of the endogenous TCR.
  • intron knockin of synthetic exons containing Exonic Splicing Enhancer elements at the 5’ end did not show any evidence of alternative splicing, with almost all dual GFP+ 1 RFP+ positive cells negative for the endogenous gene (FIG. 4C).
  • the ability of a handful of silent degenerate basepair changes at the 5’ end of the synthetic exon (ESS ESE element) to drastically change the degree of dual positive cells expressing the endogenous gene (TCR) further supports that a single allele with an intron knockin is capable of either expressing only the gene encoded in the synthetic exon (using splicing architectures in FIG. 4A and FIG. 4C), or is capable of alternative splicing and expression of both the synthetic gene and the endogenous gene (FIG. 4D).
  • Off-target integration analysis further confirmed that the biallelic intron knockin cells expressing the endogenous TCR (as in FIG. 4B) were due to alternative splicing rather than off-target effects (FIG. 4E).
  • intron knockin templates for three highly expressed surface receptors, the TCR complex member, CD3E, Beta-2 Microglobulin (B2M), and CD47 (FIG. 5). Efficient knockin was observed across all three targets, and the majority of cells that acquired a knockout of the targeted surface receptor expressed the knockin reporter tNGFR (FIGS. 5A-5C).
  • average knockin efficiency ranged from approximately 10% to 40% depending on the gRNA used, and the percentage of CD3 negative cells that were knockin positive relative to knockin negative ranged from approximately 75% to 97% (FIG. 5A).
  • TRAC intron knockin of a GFP cassette with expression driven by an exogenous promoter showed a twenty-fold increase in expression compared to expression from the endogenous TRAC promoter (p ⁇ 0.001 ), while maintaining the ability of negative selections to enrich for cells with successful intron knockins (FIG. 5D).
  • intron knockins offer greater flexibility in the control of endogenous gene expression through engineered control of alternative splicing of integrated synthetic exons. When expression of the targeted endogenous gene needs to be maintained, exon knockins can only target the N or C terminus, drastically limiting available gRNA options (FIG. 11 A). In contrast, Intron knockins with engineered alternative splicing allow for target site selection across a gene’s entire intronic region (FIG. 11 B).
  • Intron knockins uniquely enable successfully edited cells to be purified by negative selection.
  • intron knockins By using intron knockins to introduce a synthetic exon with engineered splicing architectures that prevent alternative splicing, the targeted endogenous gene is knocked out (FIGS. 11 C-11 D). While exonic knockins also generally cause knockout of the targeted endogenous gene, unsuccessfully edited cells without gene knockins largely have NHEJ mediated knockout of the targeted endogenous gene (FIG. 11C).
  • the target sites of intron knockins are spliced out of mature mRNA transcripts, allowing them to tolerant of small NHEJ mediated mutations while still maintaining endogenous gene expression.
  • intron knockins can result in successfully edited cells losing expression of the targeted endogenous gene while unedited cells or NHEJ edited cells maintain expression (FIG. 11 D).
  • successfully edited intron knockin cells can be negatively selected based on this selective loss of surface receptor expression.
  • Application of intron knockins in primary human T cells generated TRAC intron knockin CAR T cells that were negatively selected to >90% purity without the need for additional bulky transgenes or requiring disruption of essential endogenous genes, and with complete removal of TCR positive cells
  • non-viral TRAC intron knockins may offer a simpler clinical manufacturing method to select for pure clinical cell therapy products.
  • Intron knockins also offer potential advantages for future in vivo targeted editing applications. Unlike exonic knockins, intron knockins do not need to be inserted at an exact basepair target site, expanding the potential range of editors that can be used for integration to targeted but inexact transposases, integrases, and recombinases 45-49 .
  • Intron knockins offer a simple gene targeting strategy that overcomes key problems with prior selection methods for gene edited cellular therapies, while also dramatically expanding the flexibility of endogenous genetic manipulations.
  • PBMCs from healthy human blood donors were collected under an approved IRB protocol by the Stanford Blood Center and used to isolate human T cells.
  • leukoreduction chambers (LRS) from processing of platelet donations were used to isolate PBMCs using density centrifugation with Ficoll (Lymphoprep, StemCell) within SepMate tubes (StemCell) according to manufacturer’s instructions.
  • FLS leukoreduction chambers
  • stemCell SepMate tubes
  • primary human CD3 positive T cells were isolated by negative selection using Human CD3 T Cell Enrichment kit (StemCell) according to manufacturer's instructions.
  • Isolated primary human CD3 T cells were counted using an automated cell counter (Countess, Thermo), and activated using anti-human CD3/CD28 dynabeads (Cell Therapy Systems, Thermo) at a 1 :1 ratio in XVivo 15 media (Lonza) supplemented with 5% FBS (MilliporeSigma) and 50 U/mL of human IL-2 (Peprotech).
  • T cells were activated at 1 :1 ratio of cells to dynabeads, and initially cultured in standard tissue culture incubators at approximately 1 e6 cells / mL media. After gene editing/electroporations, T cells were counted and reseeded at approximately 1 e6 cells I mL XVivo 15 media with fresh IL-2 every 2-3 days.
  • T cells Two days after activation, human T cells were harvested, dynabeads were magnetically removed by incubating for two minutes at room temperature on a magnet (EasySep Magnet, StemCell), and cells were counted using an automated cytometer. For electroporations, one million T cells per editing condition were gently pelleted by centrifugation at 90G for 10 minutes, followed by careful aspiration of the supernatant. T cell pellets were resuspended in 20 uL per editing condition in P3 Buffer (Lonza) and then mixed with prepared RNP and DNA HDRT templates.
  • RNPs were prepared by first complexing the gRNA by mixing 0.375 uL of 200 uM tracrRNA (IDT) with 0.375 uL of 200 uM crRNA (IDT) and incubating for 15 minutes at room temperature. Next 0.25 uL of 100 mg/mL PGA (15-50 kDa poly(L-glutamic acid); MilliporeSigma) was then added to the complexed gRNA and mixed by pipetting up and down. Next 0.5 uL of 40 uM SpCas9 (UC Berkeley MacroLab) was then added, mixed by pipetting up and down, and incubated for 15 minutes at room temperature to form the final Cas9 RNP.
  • IDT uM tracrRNA
  • IDTT 200 uM crRNA
  • RNPs were prepared by first mixing 0.4 uL of 200 uM Cas12a gRNA (IDT) with 0.2 uL of 100 mg/mL PGA and pipetting up and down. Next 0.4 uL of 60 uM AsUltraCas12a (UC Berkeley MacroLab) was added and mixed by pipetting up and down, followed by incubation at room temperature for 10 minutes.
  • Cas12 a knockins 20 uL of T cells were mixed with 1 uL of Cas12a RNP (24 pmols total RNP) and 4 uL of plasmid DNA HDR Template at 1 ug/uL (4 ugs total HDRT).
  • T cells were electropoated on a Gen2 Lonza 4D electroporation/nucleofection system using 96 well plate attachment and 20 uL cuvettes, using pulse code EO-151.
  • Negative selections for successfully intron edited T cells were performed by CD3 depletion. Briefly, six days after non-viral gene editing by electroporation, subsequently expanded T cells were harvested, counted, and anti-CD3 biotinylated antibodies were added, following manufacturer’s instructions (EasySepTM Human CD3 Positive Selection Kit II, StemCell). After addition of magnetic beads and incubation at room temperature on a magnet, the supernatant containing untouched, CD3 negative cells was poured off into a new tube. Magnetic beads were added a second time according to manufacturer’s instructions, and again the supernatant was poured off after incubation on a magnet at room temperature, resulting in a final population of negatively selected, untouched, CD3 negative T cells.
  • T cells Bound T cells were then placed onto a magnet and incubated for 5 minutes before pouring off the supernatant. T cells were resuspended in FACS Buffer and placed on the magnet a second time. After pouring of the supernatant, T cells were resuspended in XVivo 15 culture media containing 1 uM free biotin (D-Biotin, Thermo) for 2 days to competitively compete for streptavidin binding sites and help to remove bound magnetic beads from the T cell surface before returning cells to standard culture conditions.
  • D-Biotin free biotin
  • Sorting based selection of successfully intron edited T cells was performed using standard fluorescence activated cell sorting six days after non-viral gene editing. Briefly, cells were bound with an anti-tNGFR fluorescent antibody (Table 3) for sorting as described for flow cytometric staining. A BD Aria Cell Sorter (Stanford Blood Center Flow Cytometry Core) was used for all cell sorting experiments. Cells were maintained at 4C throughout the duration of sorting, and sorted cells were collected into destination tubes of XVivo15 media mixed 1 :1 with FBS. After sorting, cells were centrifuged for 5 min at 300G prior to resuspension in culture media. Drug Selection with Puromycin
  • T cells were cultured in XVivo15 based media as described above with the addition of 5 ug/mL of puromycin (Puromycin Dihydrochloride, stock concentration of 10 mg/mL in 20 mM HEPES buffer, ThermoFisher). Concentrations used in dose titration experiments ranged from 1.0 ug/mL to 10 ug/mL as indicated in Extended Data Fig. 2b. After two days of puromycin selection, cells were centrifuged for 5 minutes at 300g and the supernatant was removed before resuspension in standard culture media without puromycin.
  • puromycin Puromycin Dihydrochloride
  • edited CAR T cells were mixed at indicated E:T ratios with target Nalm6 leukemia cells in 96 well plates, with 40,000 Nalm6 cells and varying numbers of T cells per well. Cell killing was assessed by flow cytometery at 48 hours, and the percentage of Nalm6 tumor cell killing was calculated by taking 1 - (# Nalm6 cells alive in experimental condition / # Nalm6 cells alive in no T cell conditions).
  • Nalm6 leukemia cells ATCC
  • Nalm6 leukemia cells were cultured in RPMI+10% FBS and passaged every 2- 3 days to maintain cell densities of approximately 1 e6 cells I mL. Nalm6 cells were cultured for up to ⁇ 20 passages before discarding cultures are returning to low passage number frozen aliquots of initial cell line stock acquired from ATCC.
  • T cells were expanded in XVivo15 media + 5% FBS with 50 U/mL human IL-2 over the next two weeks. Every 2-3 days, T cells were counted by automated cell counter, and reseeded at concentrations of approximately 1 e6 T cells I mL culture media. Total cumulative proliferation was calculated compared to the input number of CAR T cells for each of the four tested selection methods.
  • Transposase-CRISPR mediated targeted integration in the human genome. Sci. Rep. 12, 3390 (2022).
  • Table 1 gRNAs used in the study.

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Abstract

L'invention concerne des compositions et des procédés destinés à modifier génétiquement un lymphocyte T pour exprimer un récepteur antigénique chimérique (CAR) avec inactivation d'un récepteur de lymphocyte T (TCR) endogène. Les procédés selon l'invention utilisent un polynucléotide donneur codant pour un CAR qui est intégré dans un intron d'un gène TCR. En insérant un exon synthétique exprimant le CAR dans un intron, les lymphocytes T édités avec succès produisent un ARNm mature avec une séquence nucléotidique codant pour le CAR épissé entre le ou les exons codant pour la protéine TCR, conduisant à l'expression du CAR et à l'inactivation du TCR. Par contre, les lymphocytes T édités sans succès conservent l'expression de leur TCR. La population de lymphocytes T peut être enrichie pour des lymphocytes T négatifs TCR édités avec succès exprimant le CAR en utilisant une sélection négative pour éliminer des lymphocytes T positifs TCR édités sans succès avec des agents de liaison qui se lient au marqueur TCR.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200123516A1 (en) * 2017-06-30 2020-04-23 Precision Biosciences, Inc. Genetically-modified t cells comprising a modified intron in the t cell receptor alpha gene
US20230112075A1 (en) * 2020-03-13 2023-04-13 The Regents Of The University Of California Compositions and methods for modifying a target nucleic acid

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200123516A1 (en) * 2017-06-30 2020-04-23 Precision Biosciences, Inc. Genetically-modified t cells comprising a modified intron in the t cell receptor alpha gene
US20230112075A1 (en) * 2020-03-13 2023-04-13 The Regents Of The University Of California Compositions and methods for modifying a target nucleic acid

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