EP4132966A1 - Procédé de génération d'une bibliothèque de populations de cellules comprenant un transgène intégré dans le génome au niveau d'un locus cible - Google Patents

Procédé de génération d'une bibliothèque de populations de cellules comprenant un transgène intégré dans le génome au niveau d'un locus cible

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Publication number
EP4132966A1
EP4132966A1 EP21717956.3A EP21717956A EP4132966A1 EP 4132966 A1 EP4132966 A1 EP 4132966A1 EP 21717956 A EP21717956 A EP 21717956A EP 4132966 A1 EP4132966 A1 EP 4132966A1
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EP
European Patent Office
Prior art keywords
cells
car
transgene
locus
interest
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP21717956.3A
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German (de)
English (en)
Inventor
James SILLIBOURNE
Shaun CORDOBA
Sarah Brophy
Rosie WOODRUFF
Deimante NORMANTAITE
Martin PULÉ
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Autolus Ltd
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Autolus Ltd
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Publication of EP4132966A1 publication Critical patent/EP4132966A1/fr
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    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1079—Screening libraries by altering the phenotype or phenotypic trait of the host
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/06—Methods of screening libraries by measuring effects on living organisms, tissues or cells
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to methods for screening libraries of transgenes, which methods ensure that only a single transgene is inserted into the genome of each cell to avoid false positive or false negative identification of functional transgenes.
  • Cells for use in the present invention include, but are not limited to, T cells, NK cells, or NKT cells.
  • the transgene may encode, for example, a CAR or TOR.
  • Chimeric antigen receptors CARs
  • TCRs engineered T cell receptors
  • Chimeric antigen receptors consist of an antigen binding domain, typically a single chain antibody or ligand, fused to a spacer domain, transmembrane domain and intracellular signalling domains derived from coreceptors and O ⁇ 3z. Given their modular nature, it is possible to generate many iterations of a CAR from a single antigen-binding domain and the functional efficacy of each will vary. At present, there are no rules capable of predicting the efficacy of a CAR and empirical screening of individual CARs is required to identify the most efficacious, which is a costly and time-consuming process.
  • Methods currently employed to screen CARs for functional efficacy involve the transduction of T-cells with g-retroviral or lentiviral particles produced from a library of plasmids containing CAR encoding sequences.
  • MOI multiplicity of infection
  • An alternative to retroviral or lentiviral transduction is the insertion of a CAR coding sequence into the genome by homology-directed repair.
  • This approach typically involves generating a targeted DNA double stranded break in the genome using an endonuclease such as Cas9 (CRISPR-associated 9 - where CRIPSR stands for clustered regularly interspaced short palindromic repeats) (Eyquem, J. et al. Nature 543, 113-117 (2017); Osborn, M. J. et al. Mol. Ther. 24, 570-581 (2016); Roth, T. L. et al. Nature 559, 405 ⁇ 09 (2016)), Cas12a/Cpf1 (Dai, X. et al. Nat.
  • Cas9 CRISPR-associated 9 - where CRIPSR stands for clustered regularly interspaced short palindromic repeats
  • TALEN transcription activator-like effector nuclease
  • ZFN zinc finger nuclease
  • homing endonuclease Osborn, M. J. et al. Mol. Ther. 24, 570-581 (2016); MacLeod, D. T. et al. Mol. Ther.
  • the present invention relates to methods for screening libraries of transgenes, which methods ensure that only a single transgene is inserted into the genome of each cell to avoid false positive or false negative identification of functional transgenes.
  • Cells for use in the present invention include, but are not limited to, T cells, NK cells, or NKT cells.
  • the transgene may encode, for example, a CAR or TCR.
  • Targeting of the TRAC locus has distinct advantages over retroviral/lentiviral methods as the site of integration and the expression level of the inserted transgene are constant and the number of insertions is relatively controlled, although there will be a proportion of double positive cells expressing two transgenes due to a failure in allelic exclusion.
  • the present invention relates to insertion of transgenes into loci on the X chromosome that do not have a corresponding locus on the Y chromosome.
  • either male or female cells may be used.
  • Male cells only carry a single X chromosome, which ensures only a single copy of the transgene of interest is integrated into the genome.
  • Female cells carry two X chromosomes but many genes are subject to X chromosome inactivation, where one of the two alleles is silenced to maintain correct gene dosing and ensure that only a single allele is expressed. Genes subject to X chromosome inactivation are therefore suitable for use in this embodiment because only one copy of the transgene of interest will be expressed.
  • the present invention provides a method of generating a population of cells, wherein each cell comprises a transgene of interest integrated into the genome at a target locus, wherein the transgene of interest is selected from a library of transgenes of interest, the method comprising: i. introducing into the cells an exonuclease system which produces a DNA double-strand break at the target locus; and ii.
  • exogenous polynucleotide each comprising a 5' homology arm homologous to a portion of the target locus, a transgene of interest, and a 3' homology arm homologous to a portion of the target locus, wherein the exogenous polynucleotide is inserted into the genome at the target locus by homologous recombination, further wherein the target locus is either:
  • T cell receptor alpha constant region (a) the T cell receptor alpha constant region (TRAC)] or
  • the target locus is a locus on the X chromosome
  • only cells from male donors are used.
  • the cells may be any type of immune cell.
  • the cells may be T cells, NK cells, NKT cells, monocytes, or macrophages.
  • the homology arms are used to target the locus of interest and are typically 300-1000 bp in length. These sections of the exogenous polynucleotide contain sequences that are homologous to the target locus, which results in their incorporation via homology directed repair of double-strand breaks in genomic DNA.
  • the transgene of interest may be a chimeric antigen receptor (CAR) or T-cell receptor (TCR).
  • CAR chimeric antigen receptor
  • TCR T-cell receptor
  • the CAR may comprise one or more extracellular ligand-binding domains and one or more intracellular signalling domains.
  • the CAR may comprise an extracellular antigen binding domain, an optional spacer domain, a transmembrane domain, and an intracellular signalling domain.
  • the exonuclease system may comprise a recombinant meganuclease, a recombinant zinc- finger nuclease (ZFN), a recombinant transcription activator-like effector nuclease (TALEN), a CRISPR/Cas nuclease, or a megaTAL nuclease.
  • the exonuclease system comprises a CRISPR/Cas nuclease.
  • the exogenous polynucleotide may further comprise a marker gene or barcode sequence.
  • locus on the X chromosome may be selected from the group comprising HPRT1 , PGK1, CD40L, IL2RG, CXCR3, CETN2, OFD1 and SLC25A5.
  • Other loci on the X chromosome are also contemplated within the scope of the present invention.
  • Insertion may be targeted at exons or introns. Where exons are targeted, insertions may be made such that promoters at the site of insertion drive gene expression. Alternatively, the sequence to be inserted may include a separate promoter. Where introns are targeted the sequence to be inserted will typically include a promoter upstream of the transgene of interest. Sequences targeted to an intron may also include a polyadenylation signal sequence downstream of the gene of interest.
  • a method screening a library of CARs comprising: i. generating a population of cells according to the method of the first aspect, where the transgene of interest is a CAR; ii. assessing the resulting population of cells for activity of the transgene of interest.
  • the present invention provides a method of generating a population of cells, wherein each cell comprises a transgene of interest integrated into the genome at a target locus, wherein the transgene of interest is selected from a library of transgenes of interest, the method comprising: i. introducing into the cells an exonuclease system which produces a DNA double-strand break at the target locus; and ii. introducing into the cells a library of exogenous polynucleotides, each comprising a transgene of interest, wherein the exogenous polynucleotide is inserted into the genome at the target locus by non- homologous end joining (NHEJ), further wherein the target locus is either:
  • T cell receptor alpha constant region (a) the T cell receptor alpha constant region (TRAC)] or
  • Embodiments described with respect to the first aspect can also be applied to this further aspect mutatis mutandis.
  • FIG. 1 Insertion of CAR encoding sequence into the TRAC locus
  • a DNA double stranded break is created at the 5’ end of exon 1 of the TRAC locus. Repair of the DNA double stranded break can occur via non-homologous end joining or homolog- directed repair (HDR).
  • HDR can used to insert heterologous DNA sequences into the genome provided there are regions homologous to the target gene flanking it (left and right homology arms). These homology arms are typically 300-1000 bp in length.
  • the sequence encoding the CAR is inserted in-frame with the TCR a chain constant region and expression is facilitated by a self-cleaving peptide sequence placed upstream of the CAR. A stop codon and polyadenylation signal sequence placed downstream of the CAR terminate translation and transcription, respectively.
  • FIG. 2 Insertion of anti-CD19 and anti-CD22 CARs into the TRAC locus
  • Primary T-cells were nucleofected with sgRNA/Cas9 RNP complexes targeting the TRAC locus and HDR templates encoding an anti-CD19 CAR (FMC63) and an anti-CD22 CAR (LT22, 9A8 or inotuzamab). All CARs were inserted into the TRAC locus with varying efficiency. The reduced efficiency of the anti-CD22 CARs may be due in part to a detection issue related to the biophysical properties of the single chain antibodies.
  • a depiction of the HPRT1 gene and below are the proposed HDR templates to insert a CAR encoding sequence into the gene.
  • the HDR templates on the left are for CAR insertion at the 5’ end of the gene (exon 1) to produce either an in-frame insertion or an out of frame insertion that will disrupt expression of the HPRT1 gene product.
  • the out of frame insertion is achieved by placing a stop codon and polyadenylation sequence downstream of the CAR and deleting the initiating methionine from HPRT1.
  • the HDR template on the right makes an in-frame insertion in the HPRT1 gene (the CAR has a stop codon and polyadenylation sequence to facilitate expression).
  • Figure 4 The conversion of 6-thioguanine to thioguanine monophosphate.
  • FIG. 5 Flow cytometry of cells nucleofected with HDRT targeting the HPRT 1 gene exon 2 SupT 1 cells (which originate from a male donor) were nucleofected with one of four guide RNAs (sgRNAI, 2,3, or 4) and HDR templates comprising either CAT19 CAR or FMC63 CAR.
  • sgRNAI guide RNAs
  • FIG. 6 Flow cytometry of cells nucleofected with HDRT targeting the HPRT1 C terminus SupT 1 cells (which originate from a male donor) were nucleofected with one of two guide RNAs (92_fwd and 66_rev) and HDR templates comprising either CAT19 CAR or FMC63 CAR.
  • FIG. 1 Flow cytometry of cells nucleofected with HDRT targeting the CETN2 locus SupT 1 cells (which originate from a male donor) were nucleofected with one of four guide RNAs (59_rev, 60_fwd, 66_fwd, and 76_fwd) and HDR templates comprising either CAT19 CAR or FMC63 CAR.
  • FIG. 8 Flow cytometry of cells nucleofected with HDRT targeting the IL2RG locus SupT1 cells (which originate from a male donor) were nucleofected with one of three guide RNAs (48_rev, 69_rev, and 80_rev) and HDR templates comprising either CAT19 CAR or FMC63 CAR.
  • PBMCs from two healthy donors were treated with Cas9 RNP complex with sgRNA targeting PGK1 N-terminus (PGK1 81 forw) and a repair template for CAT19, FMC63 or both CAT19 and FMC63.
  • PGK1 81 forw sgRNA targeting PGK1 N-terminus
  • CAT19 expression and FMC63 expression were analysed by flow cytometry 7 days post nucleofection.
  • PBMCs from a healthy donor were treated with Cas9 RNP complex with sgRNA targeting PGK1 C-terminus (PGK1 28 forw) and repair templates for CAT19, FMC63 or both CAT19 and FMC63.
  • CAT19 expression and (a) FMC63 or (b) HA expression were analysed by flow cytometry 7 days post nucleofection.
  • FIG 11 Cytometric analysis of co-cultures of Raji cells and genome edited PBMCs Flow cytometry plots showing target cell populations from cytotoxicity assay using CAT19 and FMC63 integrated CAR-T cells after 24 hours.
  • PBMCs from two healthy donors D31 and D34
  • PGK1 81 forw sgRNA targeting PGK1 N-terminus
  • 8 days post nucleofection cells were incubated with Raji WT or Raji CD19 KO target cells at E:T ratios of 1:1 and 1:4 for 24 hours.
  • Cells were stained with CD2 and CD3s to distinguish effector and target populations. Samples with effectors alone or targets alone were used to determine gating.
  • FIG. 12 Cytometric analysis of co-cultures of Raji cells and genome edited PBMCs
  • PBMCs from two healthy donors were treated with Cas9 RNP complex with sgRNA targeting PGK1 N-terminus (PGK1 81 forw) and a repair template for CAT19, FMC63 or both CAT19 and FMC63.
  • 8 days post nucleofection cells were incubated with Raji WT or Raji CD19 KO target cells at E:T ratios of 1:1 and 1:4 for 24 hours. Cells were stained with CD2 and CD3s to distinguish effector and target populations. Samples with effectors alone or targets alone were used to determine gating.
  • FIG. 13 Percent survival of target cells normalised to the no DNA control PBMCs from two healthy donors (D31 and D34) were treated with Cas9 RNP complex with sgRNA targeting PGK1 N-terminus (PGK1 81 forw) and a repair template for CAT19, FMC63 or both CAT19 and FMC63. 8 days post nucleofection cells were incubated with Raji WT or Raji CD19 KO target cells at E:T ratios of 1 :1 and 1 :4 for (a) 24 and (b) 72 hours. Graphs show % survival of target cells normalized to the No DNA control.
  • PBMCs from three healthy donors were treated with Cas9 RNP complex with sgRNA targeting HPRT1 Exon2 (HPRT1 Synthego2) and a repair template for CAT19.
  • CAT19 expression was detected by flow cytometry 7 days post nucleofection using a biotinylated anti-idiotype antibody and PE-conjugated streptavidin. Insertion efficiency at exon 2 of the HPRT 1 gene ranged from 6 to 20%.
  • PBMCs from three healthy donors were treated with Cas9 RNP complex with sgRNA targeting HPRT1 C-terminus (HPRT1 92 forw) and a repair template for CAT19.
  • CAT19 expression was detected by flow cytometry 7 days post nucleofection using a biotinylated anti-idiotype antibody and PE-conjugated streptavidin. Integration efficiency at the final exon of the HPRT 1 gene ranged from 0.6 to 2.0%.
  • FIG 16 Flow cytometry of cells nucleofected with HDRT targeting the IL2RG locus PBMCs from three healthy donors (D063, D064 and D564) were treated with Cas9 RNP complex with sgRNA targeting IL2RG N-terminus (IL2RG 69 Rev) and a repair template for CAT19.
  • CAT19 expression was detected by flow cytometry 7 days post nucleofection using a biotinylated anti-idiotype antibody and PE-conjugated streptavidin. Integration efficiency was found to be dependent on the donor and ranged from 10 to 42%.
  • PBMCs were nucleofected with a HDR template encoding the anti-CD19 (CAT) CAR and RNP complexes formed with one of three sgRNAs targeting the 5’ end of the SLC25A5 gene. Insertion of the anti-CD19 (CAT) CAT was observed when PBMCs were nucleofected with an SLC25A5 targeting sgRNA but not the non-targeting sgRNA. Integration efficiency was up to 46%.
  • the present inventors use genome editing to insert transgene encoding sequences either into the TRAC locus or a gene located on the X chromosome.
  • Targeting genes located on the X chromosome ensures that only a single copy of the encoding transgene sequence is expressed and overcomes the problem of cells expressing multiple different copies of the transgene of interest.
  • the exonuclease system may comprise a recombinant meganuclease, a recombinant zinc-finger nuclease (ZFN), a recombinant transcription activator-like effector nuclease (TALEN), a CRISPR/Cas nuclease, or a megaTAL nuclease.
  • ZFN zinc-finger nuclease
  • TALEN transcription activator-like effector nuclease
  • CRISPR/Cas nuclease or a megaTAL nuclease.
  • a preferred exonuclease system is the CRISPR/Cas nuclease.
  • the CRISPR/Cas nuclease is used in conjunction with a guide RNA (gRNA) that targets the locus of interest.
  • gRNA guide RNA
  • These guide RNAs may also be termed “single guide RNA” or sgRNA.
  • the cell will attempt repair using native homology- directed repair mechanisms.
  • homology directed repair templates in the form of exogenous polynucleotides carrying suitable homology arms and a transgene of interest, insertions into the locus of interest can be achieved.
  • Example 1 provides data showing that it is possible to target the TRAC locus using Cas9 and insert one or two copies of a CAR into the genome ( Figures 1 and 2) by homology-directed repair (HDR).
  • HDR homology-directed repair
  • sequences are cloned into an HDR template and a library generated by amplifying the sequences individually from the plasmid library and combining the resulting PCR products into a single pool. These pooled PCR products provide the cells with templates for homology- directed repair, enabling the insertion of CAR-encoding sequences into their genome.
  • screening of the CARs can be carried out following the methods described below (screening of CAR T-cells). TARGETING GENES ON THE X CHROMOSOME
  • the problem of ensuring single copy gene insertion can be overcome by targeting the genes located on the X chromosome.
  • genes located on the X chromosome provided in Table 1, which facilitate the screening process or achieve desirable levels of CAR expression that are known to be efficacious in clinical trials.
  • the genes fall into three categories: house-keeping genes, immune-related, and cytoskeletal proteins (centriolar components).
  • the two preferred genes are the house-keeping genes hypoxanthine-guanine phosphoribosyltransferase 1 ( HPRT1 ) and phosphoglycerate kinase 1 ( PGK1 ).
  • Targeting insertions to the X chromosome may be carried out in either male or female cells.
  • the use of cells from male donors provides a convenient mechanism for single-copy insertion, since male cells carry only a single X chromosome.
  • Cells from female donors may also be used because of the phenomenon of X chromosome inactivation, in which one X chromosome is made transcriptionally silent. Female cells therefore only express one copy of X-linked genes.
  • HPRT is part of the non-essential purine salvage pathway and catalyses the conversion of hypoxanthine to inosine monophosphate and guanine to guanine monophosphate.
  • the cytotoxic compound 6-thioguanine is converted by HPRT to the nucleotide, which becomes incorporated into DNA and is methylated to form S 6 - methylthioguanine (Swann, P. F. et al. Science 273, 1109-1111 (1996); Karran, P. Br. Med. Bull. 79-80, 153-170 (2006)).
  • S 6 -methylthioguanine pairs with either cytosine or thymine. Mispairing of S 6 -methylthioguanine with thymine is recognised by the post-replicative mismatch repair pathway and causes apoptosis.
  • Targeting HPRT therefore not only enables the removal of non-edited cells from the pool prior to challenging CAR T-cells with antigen positive target cells (via 6-thioguanine selection), but also result in the production of CAR T-cells resistant to the chemotherapeutic agent 6-thioguanine.
  • the PGK 1 promoter is used in lentiviral and self-inactivating viral plasmids to drive the transcription of inserted transgenes.
  • the PGK1 promoter was used to drive transcription of the CAR (Ghorashian, S.et al. Br. J. Haematol. 169 (4), 463-478 (2015)).
  • pALL paediatric B-cell acute lymphoblastic leukaemia
  • adult B-cell ALL the PGK1 promoter was used to drive transcription of the CAR (Ghorashian, S.et al. Br. J. Haematol. 169 (4), 463-478 (2015)).
  • the level of CAR expressed from the PGK1 promoter is sufficient to confer functionality and to drive CAR T-cell persistence.
  • the level of CAR expression will be comparable to that from the promoter fragment cloned into self-inactivating g-retroviral and lentiviral constructs.
  • the lnterleukin-2 receptor gamma subunit (common g chain) is expressed in most lymphocytes and is therefore an attractive target for expression of a CAR, since lymphocytes are the target cell type for these molecules.
  • the centrin-2 protein is a component of the cytoskeleton and is therefore expressed in all cells.
  • Solute carrier family 25 member 5 is involved in the exchange of cytoplasmic ADP for ATP across the inner membrane of the mitochondrion.
  • a population of cells can be assessed for activity of the transgene of interest. This may include, for example, assessment of the expression of the transgene. In other cases, the activity of the transgene itself may be assessed. For example, where the transgene is an enzyme, the activity of the enzyme on its substrate can be measured. In the case of CARs, the assessment will typically be based on the ability of the resulting CAR-T cells to kill target cells.
  • bp 15 base pair
  • NGS next generation sequencing
  • identification of the most efficacious CARs may be carried out using two different approaches.
  • the fist approach involves the short-term challenge of CAR T-cells with antigen positive target cells and the isolation of responding T-cells, while the second approach requires the long-term culture of CAR T-cells with target cells to determine which CAR provides the most potent signal to the T-cell.
  • the short-term approach to determining CAR efficacy involves challenging CAR T-cells with antigen positive target cells and isolating responding T-cells that have upregulated CD69 and express this early activation marker on their cell surface. Identification of the CAR expressed by the responding cell can then carried out by extracting genomic DNA from the isolated cells, amplifying across the single chain antibody sequence of the CAR and analysing the PCR products by next generation sequencing (NGS).
  • NGS next generation sequencing
  • the second, long-term, approach involves repeatedly challenging a pool of CAR T-cells with target cells and sampling the population over time to identify the most frequently observed CARs to determine which ones confer to the T-cells proliferative capacity. The population will narrow over time as the most proliferative T-cells will outcompete the others in the culture.
  • identification of the CARs is carried out by extracting genomic DNA from the T-cells, amplifying across the single chain antibody and barcode sequence and analysing the resulting PCR products by NGS.
  • Classical CARs are chimeric type I trans-membrane proteins which connect an extracellular antigen-recognizing domain (binder) to an intracellular signalling domain (endodomain).
  • the binder is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it can be based on other formats which comprise an antibody-like antigen binding site or on a ligand for the target antigen.
  • mAb monoclonal antibody
  • a spacer domain may be necessary to isolate the binder from the membrane and to allow it a suitable orientation.
  • a common spacer domain used is the Fc of lgG1. More compact spacers can suffice e.g. the stalk from CD8a and even just the lgG1 hinge alone, depending on the antigen.
  • a trans-membrane domain anchors the protein in the cell membrane and connects the spacer to the endodomain.
  • TNF receptor family endodomains such as the closely related 0X40 and 4-1 BB which transmit survival signals.
  • CARs have now been described which have endodomains capable of transmitting activation, proliferation and survival signals.
  • CAR-encoding nucleic acids may be transferred to T cells using, for example, retroviral vectors.
  • retroviral vectors In this way, a large number of antigen-specific T cells can be generated for adoptive cell transfer.
  • the CAR binds the target-antigen, this results in the transmission of an activating signal to the T-cell it is expressed on.
  • the CAR directs the specificity and cytotoxicity of the T cell towards cells expressing the targeted antigen.
  • the transgene of interest may comprise a CAR. More particularly, a library of different CARs may be created and then screened using the methods described herein. The various components of the CAR are set out below in more detail below. Each of these components may be varied in the library, either independently or in combination with one another.
  • the antigen-binding domain is the portion of a classical CAR which recognizes antigen.
  • the antigen binding domain may comprise: a single-chain variable fragment (scFv) derived from a monoclonal antibody; a wild-type ligand of the target antigen; a peptide with sufficient affinity for the target; a single domain binder such as a camelid; an artificial binder single as a Darpin; or a single-chain derived from a T-cell receptor.
  • scFv single-chain variable fragment
  • tumour associated antigens are known, as shown in the following Table 2.
  • the antigen-binding domain used in the present invention may be a domain which is capable of binding a TAA as indicated therein.
  • the transmembrane domain is the sequence of a classical CAR that spans the membrane. It may comprise a hydrophobic alpha helix. The transmembrane domain may be derived from CD28, which gives good receptor stability.
  • the CAR or transgenic TCR for use in the present invention may comprise a signal peptide so that when it is expressed in a cell, such as a T-cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface, where it is expressed.
  • the core of the signal peptide may contain a long stretch of hydrophobic amino acids that has a tendency to form a single alpha-helix.
  • the signal peptide may begin with a short positively charged stretch of amino acids, which helps to enforce proper topology of the polypeptide during translocation.
  • At the end of the signal peptide there is typically a stretch of amino acids that is recognized and cleaved by signal peptidase.
  • Signal peptidase may cleave either during or after completion of translocation to generate a free signal peptide and a mature protein.
  • the free signal peptides are then digested by specific proteases.
  • the receptor may comprise a spacer sequence to connect the antigen-binding domain with the transmembrane domain.
  • a flexible spacer allows the antigen-binding domain to orient in different directions to facilitate binding.
  • the spacer sequence may, for example, comprise an lgG1 Fc region, an lgG1 hinge or a human CD8 stalk or the mouse CD8 stalk.
  • the spacer may alternatively comprise an alternative linker sequence which has similar length and/or domain spacing properties as an lgG1 Fc region, an lgG1 hinge or a CD8 stalk.
  • a human lgG1 spacer may be altered to remove Fc binding motifs.
  • the intracellular signalling domain is the signal-transmission portion of a classical CAR.
  • CD3-zeta endodomain which contains 3 ITAMs. This transmits an activation signal to the T cell after antigen is bound.
  • CD3-zeta may not provide a fully competent activation signal and additional co-stimulatory signalling may be needed.
  • chimeric CD28 and 0X40 can be used with CD3- Zeta to transmit a proliferative / survival signal, or all three can be used together.
  • the intracellular signalling domain may be or comprise a T cell signalling domain.
  • the intracellular signalling domain may comprise one or more immunoreceptor tyrosine-based activation motifs (ITAMs).
  • ITAM immunoreceptor tyrosine-based activation motifs
  • An ITAM is a conserved sequence of four amino acids that is repeated twice in the cytoplasmic tails of certain cell surface proteins of the immune system.
  • the motif contains a tyrosine separated from a leucine or isoleucine by any two other amino acids, giving the signature YxxL/l. Two of these signatures are typically separated by between 6 and 8 amino acids in the tail of the molecule (UCCI_/IC (6 -8 ) UCC ).
  • ITAMs are important for signal transduction in immune cells. Hence, they are found in the tails of important cell signalling molecules such as the CD3 and z-chains of the T cell receptor complex, the CD79 alpha and beta chains of the B cell receptor complex, and certain Fc receptors.
  • the tyrosine residues within these motifs become phosphorylated following interaction of the receptor molecules with their ligands and form docking sites for other proteins involved in the signalling pathways of the cell.
  • the intracellular signalling domain component may comprise, consist essentially of, or consist of the O ⁇ 3-z endodomain, which contains three ITAMs.
  • the O ⁇ 3-z endodomain transmits an activation signal to the T cell after antigen is bound.
  • the intracellular signalling domain may comprise additional co-stimulatory signalling.
  • 4-1 BB also known as CD137
  • CD28 and 0X40 can be used with O ⁇ 3-z to transmit a proliferative / survival signal.
  • the CAR may have the general format: antigen-binding domain-TCR element.
  • TCR element means a domain or portion thereof of a component of the TCR receptor complex.
  • the TCR element may comprise (e.g. have) an extracellular domain and/or a transmembrane domain and/or an intracellular domain e.g. intracellular signalling domain of a TCR element.
  • the TCR element may selected from TCR alpha chain, TCR beta chain, a CD3 epsilon chain, a CD3 gamma chain, a CD3 delta chain, CD3 epsilon chain.
  • T-cell receptor is a molecule found on the surface of T cells which is responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • the TCR is a heterodimer composed of two different protein chains.
  • the TCR in 95% of T cells the TCR consists of an alpha (a) chain and a beta (b) chain (encoded by TRA and TRB, respectively), whereas in 5% of T cells the TCR consists of gamma and delta (g/d) chains (encoded by TRG and TRD, respectively).
  • the T lymphocyte When the TCR engages with antigenic peptide and MHC (peptide/M HC), the T lymphocyte is activated through signal transduction.
  • antigens recognized by the TCR can include the entire array of potential intracellular proteins, which are processed and delivered to the cell surface as a peptide/MHC complex.
  • heterologous TCR molecules it is possible to engineer cells to express heterologous (i.e. non-native) TCR molecules by artificially introducing the TRA and TRB genes; or TRG and TRD genes into the cell using a vector.
  • the genes for engineered TCRs may be reintroduced into autologous T cells and transferred back into patients for T cell adoptive therapies.
  • Such ‘heterologous’ TCRs may also be referred to herein as ‘transgenic TCRs’.
  • the transgenic TCR for use in the present invention may recognise a tumour associated antigen (TAA) when fragments of the antigen are complexed with major histocompatibility complex (MHC) molecules on the surface of another cell.
  • TAA tumour associated antigen
  • MHC major histocompatibility complex
  • the transgenic TCR for use in the present invention may recognise a TAA listed in Table 2.
  • a library of transgenic TCRs may be created. This library may then be screened using the methods described herein.
  • polynucleotide As used herein, the terms “polynucleotide”, “nucleotide”, and “nucleic acid” are intended to be synonymous with each other.
  • nucleic acids may comprise DNA or RNA.
  • They may be single- stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the use as described herein, it is to be understood that the polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of interest.
  • variant in relation to a nucleotide sequence or amino acid sequence includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid(s) from or to the sequence.
  • CRISPR guide RNAs are supplied by Synthego. CRISPR guide RNAs are stored at -20°C unreconstituted and -80°C once reconstituted in re-suspension buffer.
  • HDRT Homology-directed repair templates
  • Transfection buffer was prepared by combining the following for each sample: SupT1-Cas9 cells (AU54111) were washed twice with an equal volume of PBS by centrifuging at 400g for 5 minutes to remove culture medium and RNases present in the serum that would rapidly degrade the gRNA duplexes. After 2x washes, cells were re-suspended in 21 pl_ of transfection buffer per sample to be nucleofected (e.g. 1x106 SupT1-Cas9 cells per nucleofection).
  • the total volume of cells was added to the sgRNA/HDR template and the mixture was transferred to the electroporation cuvette/plate. Cells were nucleofected using the CM- 150 pulse code. 80mI of prewarmed RPMI was added to each cuvette after electroporation and cells were left to recover in the incubator for 15 minutes.
  • a single anti-CD19 CAR (FMC63) and three different anti-CD22 CARs were chosen for integration into the TRAC locus.
  • the anti-CD22 CARs were: clone LT22, clone 9A8 and Inotuzumab.
  • the CARs were inserted into the TRAC locus either singly or in combination ( Figure 2).
  • the integration efficiency of the anti-CD19 CAR alone was approximately 30%, but this decreased by nearly half when simultaneously integrated into the genome with an anti- CD22 CAR. Integration of the anti-CD22 CARs appeared to be less efficient and ranged from 3-15% although these differences may be due in part to a detection issue related to the biophysical properties of the anti-CD22 single chain antibodies fused to the CARs.
  • Insertion of CARs into the HPRT1 gene should result in the constitutive expression of the CAR.
  • Several HDR templates were constructed to test the possibility of inserting CAR encoding sequences into the HPRT1 gene.
  • the first targets the 5’ end of the gene, specifically exon 2, with the CAR sequence placed in-frame with HPRT 1 or out of frame with the intention of disrupting the HPRT1 gene to prevent expression of a functional protein (Error! Reference source not found. Figure 3).
  • Figure 5 shows the results of nucleofecting SupT 1 cells (which originate from a male donor) with one of four guide RNAs (sgRNAI , 2,3, or 4) targeting exon 2 of the HPRT1 gene.
  • Cells were also nucleofected with HDR templates comprising either CAT19 CAR or FMC63 CAR.
  • Figure 6 shows the results of nucleofecting SupT 1 cells (which originate from a male donor) with one of two guide RNAs (92_fwd and 66_rev) targeting the C terminus of the HPRT 1 gene. Cells were also nucleofected with HDR templates comprising either CAT19 CAR or FMC63 CAR.
  • Figure 7 shows the results of nucleofecting SupT 1 cells (which originate from a male donor) with one of four guide RNAs (59_rev, 60_fwd, 66_fwd, and 76_fwd) targeting the CETN2 gene and HDR templates comprising either CAT19 CAR or FMC63 CAR.
  • SupT1 cells have the karyotype XXYY, some double insertions are seen when both HDR templates are used. However, expression from the desired locus using a mixture of templates has been achieved.
  • Figure 8 shows results of nucleofecting SupT 1 cells (which originate from a male donor) with one of three guide RNAs (48_rev, 69_rev, and 80_rev) targeting the IL2RG gene and HDR templates comprising either CAT19 CAR or FMC63 CAR.
  • SupT1 cells have the karyotype XXYY, some double insertions are seen when both HDR templates are used. However, expression from the desired locus using a mixture of templates has been achieved.
  • HDR templates were designed to insert the CAT19 CAR or HA-tagged FMC63 CAR in-frame at the 5’ and the 3’ end of the PGK1 gene.
  • the structure of these templates was similar to those described for HPRT1 ( Figure 3).
  • PBMCs Peripheral blood mononuclear cells
  • HDR templates encoding the CAT19 CAR and/or HA-tagged FMC63 CAR and staining carried out with anti- CAT19 CAR idiotype and anti-HA epitope tag antibodies.
  • the stained PBMCs were analysed by flow cytometry.
  • Figure 9 and Figure 10 show the flow cytometric analysis of PBMCs edited to introduce the CAT19 CAR or HA-tagged FMC63 CAR coding sequences by homology-directed repair to the 5’ or 3’ end of the PGK1 gene.
  • Figure 11 and Figure 12 show flow cytometric analysis of co-cultures of Raji cells and genome edited PBMCs, from two independent donors, stained with antibodies recognising the T cell-specific antigens CD2 and CD3s at 24 hours ( Figure 11) and 72 hours ( Figure 12). Staining with this combination of antibodies enabled the clear distinction of the different populations, which were enumerated.
  • Figure 13 shows the percent survival of target cells normalised to the no DNA control.
  • the data clearly show that the genome edited PBMCs expressing the CAT19 CAR or FMC63 CAR were able to lyse CD19 positive Raji cells, whereas only limited targeting of CD19 negative Raji cells was observed at the higher 1:1 ratio (effector: target).
  • the heterogeneous population of edited PBMCs, nucleofected with both the CAT19 and FMC63 CARs was able to lyse CD19 positive Raji cells with similar efficiency as the individual populations nucleofected with either the CAT 19 CAR or the FMC63 CAR, indicating that there was no loss in cytolytic activity when PBMCs were engineered using a heterogeneous pool of HDR templates.
  • Targeting HPRT1 gene in PBMCs insertion of CAT19 CAR
  • PBMCs from three donors were nucleofected with RNP complexes targeting exon 2 or the final exon of HPRT1 gene and HDR templates encoding the CAT19 CAR.
  • the edited PBMCs were cultured for 7 days before staining with the anti-CAT19 idiotype antibody and analysing them by flow cytometry.
  • Figure 14 and Figure 15 show the flow cytometric data of the edited PBMCs.
  • the insertion efficiency at exon 2 of the HPRT1 gene ranged from 6 to 20% ( Figure 14).
  • the integration efficiency at the final exon of the HPRT1 gene was considerably lower and ranged from 0.6 to 2.0% ( Figure 15).
  • nucleofections were carried using RNP complexes targeting the first exon of the IL- 2RG gene and the CAT19 CAR HDR template.
  • PBMCs edited to express the CAT19 CAR were stained with anti-CAT19 CAR idiotype antibody and analysed by flow cytometry.
  • Figure 16 shows the flow cytometric data and the integration efficiency was found to be dependent on the donor and ranged from 10 to 42%. The results of this experiment clearly indicated that it was possible to integrate CAR encoding sequences to the IL-2RG gene and this is viable target site for the use in screening libraries of CARs to determine which one has the optimal architecture eliciting potent cytolytic activity and cytokine secretion.
  • nucleofections were carried using RNP complexes targeting the 5’ end of the SLC25A5 gene and the CAT19 CAR HDR template.
  • PBMCs edited to express the CAT19 CAR were stained with anti-CAT19 CAR idiotype antibody and analysed by flow cytometry.
  • Figure 17 shows the flow cytometric data. Insertion of the anti-CD19 (CAT) CAT was observed when PBMCs were nucleofected with an SLC25A5 targeting sgRNA but not the non-targeting sgRNA. Integration efficiency was up to 46%. The results of this experiment clearly indicated that it was possible to integrate CAR encoding sequences to the SLC25A5 gene and this is viable target site for the use in screening libraries of CARs to determine which one has the optimal architecture eliciting potent cytolytic activity and cytokine secretion.
  • CAT anti-CD19

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Abstract

La présente invention concerne des procédés de criblage de bibliothèques de transgènes, lesquels procédés garantissent qu'un seul transgène est inséré dans le génome de chaque cellule pour éviter une identification par faux positifs ou faux négatifs de transgènes fonctionnels.
EP21717956.3A 2020-04-08 2021-04-07 Procédé de génération d'une bibliothèque de populations de cellules comprenant un transgène intégré dans le génome au niveau d'un locus cible Withdrawn EP4132966A1 (fr)

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WO2018073393A2 (fr) * 2016-10-19 2018-04-26 Cellectis Cellules allogéniques modifiées par une nucléase d'effecteur tal (talen) appropriées pour une thérapie
JP2020529834A (ja) * 2017-06-30 2020-10-15 プレシジョン バイオサイエンシズ，インク． Ｔ細胞受容体アルファ遺伝子の改変されたイントロンを含む遺伝子改変ｔ細胞
WO2019020733A1 (fr) * 2017-07-26 2019-01-31 Cellectis Procédés de sélection de cellule immunitaire du récepteur antigénique chimérique (car) dépendant de l'antigène
EP3775229A4 (fr) * 2018-03-27 2021-12-15 The Trustees Of The University Of Pennsylvania Cellules immunitaires modifiées ayant une fonction améliorée et procédés de criblage pour les identifier
AU2019247199A1 (en) * 2018-04-05 2020-10-15 Editas Medicine, Inc. T cells expressing a recombinant receptor, related polynucleotides and methods
KR20210008502A (ko) * 2018-05-11 2021-01-22 크리스퍼 테라퓨틱스 아게 암을 치료하기 위한 방법 및 조성물

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