Allogeneic t cells and methods of producing same

Abstract

The present invention provides methods for producing allogeneic T cells comprising the use of an engineered nuclease under the control of a controllable promoter. By preparing T cells with an inducible nuclease, a large number of cells can be prepared, each of the cells having the ability to produce a desired nuclease alone. These cells may then be modified as desired by introducing a gene of interest, or undesirable genes may be knocked out. Also provided herein are allogeneic T cells for use in various therapeutic applications.

Description

[0001] This application is a divisional application with application number 202080080542.8, application date November 4, 2020, and invention name “Allogeneic T cells and methods for producing them”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 930,617, filed on November 5, 2019, the disclosure of which is hereby incorporated by reference in its entirety. Technical Field

[0004] The present invention provides methods for generating allogeneic T cells, comprising the use of engineered nucleases under the control of a controllable promoter. By using inducible nucleases to generate T cells, large numbers of cells can be prepared, each of which has the ability to individually produce the desired nuclease. These cells can then be modified as needed by introducing genes of interest, or undesirable genes can be knocked out. Also provided herein are allogeneic T cells for various therapeutic applications. Background Art

[0005] As the clinical adoption of advanced cell therapies begins to gain traction, increased attention is turning to the underlying manufacturing strategies that will bring these therapies to patients worldwide. Successful results from immunotherapy trials using chimeric antigen receptor (CAR) T cells offer new hope for patients with previously incurable cancers. While cell therapies hold great promise in the clinic, high manufacturing costs relative to reimbursement are a significant barrier to commercialization.

[0006] One of the challenges facing CAR T-cell therapy is generating allogeneic cells that can be used in any patient. Scaling up allogeneic T-cell therapy can be very expensive and require a long development time due to the large amounts of viral vectors and / or recombinant endonucleases required.

[0007] What is needed to overcome these challenges are methods for generating T cell lines that do not require a substantial investment of time and money, yet still provide cell lines that can be used in multiple patient populations. The present invention satisfies these needs. Summary of the Invention

[0008] In some embodiments, provided herein is a method of generating a T cell line for allogeneic applications, the method comprising: introducing a nucleic acid molecule encoding an engineered nuclease under the control of a controllable promoter into a T cell line; integrating the nucleic acid molecule into the genome of the T cell line; and expanding the T cell line.

[0009] In another embodiment, a method for generating a genetically modified T cell line is provided, the method comprising: introducing a nucleic acid molecule encoding a CRISPR-associated nuclease under the control of a controllable promoter into a T cell line; integrating the nucleic acid molecule into the genome of the T cell line; expanding the T cell line; inducing expression of the CRISPR-associated nuclease by activating the controllable promoter; introducing a guide RNA and a gene of interest into the expanded T cell line; knocking out expression of a T cell receptor and introducing the gene of interest into the genome of the T cell line; and recovering the genetically modified T cell line.

[0010] Also provided herein is a method for generating a chimeric antigen receptor (CAR) T cell line, the method comprising: introducing a nucleic acid molecule encoding a Cas9 nuclease under the control of a controllable promoter into a T cell line; integrating the nucleic acid molecule into the genome of the T cell line; amplifying the T cell line; inducing expression of the Cas9 nuclease by activating the controllable promoter; introducing a guide RNA and a nucleic acid encoding a chimeric antigen receptor (CAR) into the amplified T cell line; knocking out expression of the T cell receptor and introducing the nucleic acid encoding the CAR into the genome of the T cell line; and recovering the CAR T cell line.

[0011] In additional embodiments, provided herein is an allogeneic T cell line comprising a CRISPR-associated (Cas) nuclease under the control of a controllable promoter integrated into the genome of the T cell line. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Schematic diagram showing autologous and allogeneic approaches to T cell therapy.

[0013] Figure 2 The steps for generating allogeneic T cells are shown.

[0014] Figure 3 Three exemplary stages of allogeneic T cell generation are shown.

[0015] Figures 4A to 4B An exemplary derepressible promoter system for use herein is shown.

[0016] Figures 4C to 4D Inducible vector systems used in the examples are shown.

[0017] Figure 4E The TRE3G Tet-On system used in the embodiments thereof is shown.

[0018] Figures 5A to 5C Three treatment schemes for transduction of T cells with Cas9 inducible vectors are shown.

[0019] Figures 6A to 6B Results are shown for T cells transduced with a Cas9 inducible vector. See the symbols in the legend to follow the line graph.

[0020] 7A to 7B The number of viable cells after selection and during cell expansion is shown.

[0021] Figure 8 Measurements of exhaustion markers, senescence markers, activation markers, and T cell markers are shown for the three treatment regimens.

[0022] Figure 9 Shown are the results of inducing Cas9 expression in T cells.

[0023] Figure 10A and Figure 10B T cell expansion after cryopreservation is shown.

[0024] Figure 11 Three TRAC gene knockout methods are shown.

[0025] Figures 12A-1 to 12C TRAC knockdown 4 days after nucleofection is shown.

[0026] Figures 13A-1 to 13C TRAC knockdown 7 days after nucleofection is shown.

[0027] Figures 14A-1 to 14C TRAC knockdown 14 days after nucleofection is shown.

[0028] FIG. 15A to FIG. 15B A compilation of knockout experiments is shown. DETAILED DESCRIPTION

[0029] In the claims and / or the specification, the use of the word "a" or "an" when used in conjunction with the term "comprising" may mean "one", but is also consistent with "one or more", "at least one", and "one or more than one".

[0030] Throughout this application, the term "about" is used to indicate that a value includes the inherent error variation of the method / device used to determine the value. Generally, the term is intended to encompass about or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% variability, as the case may be.

[0031] Use of the term "or" in the claims is intended to mean "and / or" unless explicitly stated to refer only to alternatives or the alternatives are mutually exclusive, although this disclosure supports definitions referring only to alternatives as well as to "and / or."

[0032] As used in this specification and claims, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of containing, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, system, host T cell, expression vector, and / or composition of the invention. Furthermore, the compositions, systems, cells, and / or nucleic acids of the invention can be used to implement any of the methods as described herein.

[0033] Chimeric antigen receptor T cells

[0034] Chimeric antigen receptor T cells or "CAR T cells" are T cells (also referred to herein as T cells) modified with chimeric antigen receptors (CARs) to more specifically target cancer cells. Typically, CAR comprises three parts: an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain is the region where the receptor is exposed to the extracellular fluid and comprises three parts: a signaling peptide, an antigen recognition region, and a spacer. The signaling peptide guides the nascent protein into the endoplasmic reticulum. In CAR, the signaling peptide is a single-chain variable fragment (scFv). scFv comprises a variable fragment of a light chain connected to a short linker peptide. In some embodiments, the linker comprises glycine and serine. In some embodiments, the linker comprises glutamic acid and lysine.

[0035] The transmembrane domain of CAR is a hydrophobic alpha helix that spans the membrane. In some embodiments, the transmembrane domain of CAR is a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain produces a highly expressed CAR. In some embodiments, the transmembrane domain of CAR is a CD3-ζ transmembrane domain. In some embodiments, the CD3-ζ transmembrane domain produces a CAR that is incorporated into a natural T cell receptor.

[0036] The intracellular domain of CAR is generally considered to be the "functional" end of the receptor. After the antigen recognition region of the extracellular domain recognizes the antigen, the CAR cluster and signal are delivered to the cell. In certain embodiments, the intracellular domain is a CD3-ζ intracellular domain, which includes 3 activation motifs (ITAMs) based on immunoreceptor tyrosine. In this case, after antigen binding, ITAM transmits activation signals to T cells, thereby triggering T cell immune responses. Other CAR designs known in the art can also be used in the practice of the methods described herein.

[0037] During the production of CAR T cells, T cells are taken out from human subjects, genetically modified, and reintroduced into patients to attack cancer cells. CAR T cells can be derived from the patient's own blood (autologous), or from another healthy donor (allogeneic). Typically, CAR T cells are developed to be specific for antigens that are overexpressed on tumors relative to healthy cells.

[0038] Methods for generating T cells for allogeneic applications

[0039] While autologous T cells represent a significant opportunity for treating a variety of diseases, and in particular cancer, the use of allogeneic cells, and in particular the generation of cell lines that can be modified according to the needs of each individual patient, represents a significant advance in T cell-based therapies. By generating T cell lines that can be used for any patient, not only is the supply of potential cells significantly increased, but the cost of generating such cells is also significantly increased.

[0040] After the cells are removed from the patient donor, the traditional method for generating allogeneic T cells includes a step of expanding the cells before introducing the desired CAR for necessary treatment. However, in this approach, because the T cell receptor is required for expansion but must be removed before introduction into the patient, all CAR introduction must occur after cell expansion. This requires not only large amounts of virus and the desired CAR, but also large amounts of any engineered nucleases that may be needed for gene editing. This significantly increases cost and complexity.

[0041] However, the methods described herein allow for the generation of T cells that contain engineered nucleases under the control of a controllable promoter within the cell, yet retain the natural T cell receptor that allows expansion. Once the cells are expanded, the desired CAR can be inserted, the T cell receptor removed, and the cells further processed and appropriately injected into the patient.

[0042] Figure 1The schematic diagram of the autologous method and allogeneic method of T cell therapy is shown.In autologous applications, T cells are separated from patients, CAR construct viral transduction is introduced into the T cells of patients, and then CAR T cells are reintroduced into the same patient.In allogeneic method, T cells are separated from healthy donors, and cells are subjected to viral transduction with desired CAR constructs.Meanwhile, T cell receptors are knocked out to prevent graft-versus-host disease (GvHD), which is a prerequisite for universal CAR T therapy (other genes can also be knocked out, including B2M and PD1 to help prevent GvHD).Now the allogeneic T cells comprising desired CAR can be introduced into any patient.As described herein, the T cell sources that can be expanded before introducing CAR constructs will allow scale to increase significantly, and also reduce required resources and costs.

[0043] Then in the examples, the present invention provides a method for producing a T cell line for allogeneic application. As used herein, a "T cell line" refers to a lymphocyte that develops in the thymus and contains a T cell receptor on its surface. T cells include immortalized T cells. As used herein, "allogeneic" or "allogeneic application" refers to the use of cells from one or more donor sources (usually healthy donors) for therapeutic applications in one or more patients, which may be unrelated to the donor source.

[0044] In embodiments, the methods described herein comprise introducing a nucleic acid molecule encoding an engineered nuclease under the control of a controllable promoter into a T cell line. Methods for introducing nucleic acid molecules into T cell lines include the use of various transduction or transfection systems, including various viral systems. For example, lentiviral vectors can be used to introduce nucleic acid molecules into T cell lines. Additional transduction or transfection systems include nucleofection, the use of exosome systems, the use of liposome systems, the use of polymer-based systems, and the like.

[0045] As used herein, "nucleic acid," "nucleic acid molecule," or "oligonucleotide" refers to a polymeric compound comprising covalently linked nucleotides. The term "nucleic acid" includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which can be single-stranded or double-stranded. DNA includes, but is not limited to, complementary DNA (cDNA), genomic DNA, plasmid or vector DNA, and synthetic DNA. RNA includes, but is not limited to, mRNA, tRNA, rRNA, snRNA, microRNA, miRNA, or MIRNA. Nucleic acid also includes RNA that is introduced into a cell and then reverse transcribed into DNA before being integrated into the cell genome.

[0046] As used herein, "gene" refers to an assembly of nucleotides encoding a polypeptide and includes cDNA and genomic DNA nucleic acid molecules. "Gene" also refers to a nucleic acid fragment that can serve as a regulatory sequence before (5' non-coding sequence) and after (3' non-coding sequence) the coding sequence. In some embodiments, a gene is integrated with multiple copies. In some embodiments, a gene is integrated with a predefined copy number.

[0047] As used herein, "transfection" means that an exogenous nucleic acid molecule comprising a vector is introduced into a cell. "Transfected" cells include exogenous nucleic acid molecules inside the cell, and "transformed" cells are cells in which the exogenous nucleic acid molecules in the cell induce phenotypic changes in the cell. The transfected nucleic acid molecules can be introduced into the cell as RNA, reverse transcribed into DNA by the cell, and then integrated into the genomic DNA of the host T cell and / or can be maintained by the cell temporarily or for a long time outside the chromosome. Host cells or organisms expressing exogenous nucleic acid molecules or fragments are referred to as "recombinant," "transformed," or "transgenic" organisms. Various transfection techniques are generally known in the art. See, for example, Graham et al., Virology, 52:456 (1973); Sambrook et al., Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratory, New York (1989); Davis et al., Basic Methods in Molecular Biology, Elsevier (1986); and Chu et al., Gene, 13:197 (1981). Suitably, T cells are transfected with one or more of the vectors described herein using a transfection agent, such as polyethyleneimine (PEI) or other suitable reagents, comprising various lipids and polymers, to integrate nucleic acids into the genomic DNA of the host T cells. In some embodiments, transfection comprises viral infection (also referred to as "transduction"), transposon, mRNA transfection, electroporation, or a combination thereof. In some embodiments, transfection comprises electroporation. In further embodiments, transfection comprises viral transduction. The vector can be a viral vector, such as a lentiviral vector, a gamma retroviral vector, an adeno-associated viral vector, or an adenoviral vector. In an embodiment, transfection comprises introducing the viral vector into activated T cells of a cell culture. In another embodiment, the vector is delivered as a viral particle.

[0048] like Figure 2As shown, T cells are appropriately contacted with nucleic acid molecules contained within viral particles to allow the nucleic acid to integrate into the genome of the T cell line. The nucleic acid is introduced as RNA, reverse transcribed into DNA by the cell, and then integrated into the genome of the cell. This provides a T cell line that contains a genetically integrated engineered nuclease under the control of a controllable promoter within each cell. These T cells can then be expanded as described herein to generate a T cell line for allogeneic applications.

[0049] As used herein, the term "engineered nuclease" refers to a nuclease that has been isolated, modified, mutated, and / or altered from its native nuclease state. A "nuclease" is an enzyme that is capable of cleaving DNA and / or RNA molecules. By engineering a nuclease, the specific location of cleavage can be designed and customized based on the desired cell type and / or gene of interest.

[0050] Exemplary engineered nucleases that can be inserted into T cells include, for example, meganucleases, methyltransferases, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), FokI nucleases, and CRISPR-associated nucleases. Typically, engineered nucleases use DNA binding proteins that have both the desired catalytic activity and the ability to bind to the desired target sequence in a manner similar to a restriction enzyme through protein-nucleic acid interactions. Examples include naturally occurring or engineered rare sequence cleavage enzymes, meganucleases, zinc finger nucleases (ZFNs), or transcription activator-like effector nucleases (TALENs), which contain FokI catalytic nuclease subunits connected to a modified DNA binding domain and can each cut a predetermined sequence. In ZFNs, the binding domain includes an amino acid chain folded into a custom zinc finger domain. Similarly, in TALENs, 34 amino acid repeats derived from transcription factors are folded into a huge DNA binding domain. In the case of gene targeting, these enzymes can cut genomic DNA to form double-strand breaks (DSBs) or generate nicks, which can be repaired by one of two repair pathways, non-homologous end joining (NHEJ) or homologous recombination (HR). The NHEJ pathway may cause specific mutations, deletions, insertions, or substitution events. The HR pathway causes the targeted sequence to be replaced by the supplied donor sequence. An exemplary FokI and methyltransferase-based system is described in U.S. Patent No. 10,220,052, the disclosure of which is incorporated herein by reference in its entirety.

[0051] In suitable embodiments, the CRISPR-associated nuclease is a Cas9 nuclease, or may be other Cas nucleases, such as Cas12 nuclease, Cas13 nuclease, Cas14 nuclease, etc. In an embodiment, the Cas9 nuclease is a Cas9 nuclease with reduced immunogenicity, as disclosed in U.S. Published Patent Application No. 2018-0319850, the disclosure of which is incorporated herein by reference in its entirety.

[0052] The clustered regularly interspaced short palindromic repeats (CRISPR) and associated proteins (CRISPR-associated nucleases or Cas proteins) comprising the CRISPR-Cas system were first identified in selected bacterial species and form part of the prokaryotic adaptive immune system. See Sorek et al., "CRISPR-a widespread system that provides acquired resistance against phages in bacteria and archaea," Nat. Rev. Microbiol. 6(3)181-6 (2008), which is incorporated herein by reference in its entirety. CRISPR-Cas systems are mainly classified into three types: type I, type II, and type III. The main defining feature of each type is the various Cas genes used and the corresponding proteins they encode. The cas1 and cas2 genes appear to be universal among the three main types, while cas3, cas9, and cas10 are considered to be specific to type I, type II, and type III systems. See, e.g., Barrangou, R. and Marraffini, LA, “CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity,” Mol. Cell. 54(2):234-44 (2014), which is incorporated herein by reference in its entirety.

[0053] Typically, CRISPR-Cas systems work by capturing short regions of invading viral or plasmid DNA and integrating the captured DNA into the host genome to form a so-called CRISPR array, which is spaced apart by repetitive sequences within the CRISPR locus. DNA is collected into the CRISPR array and then transcribed and processed into RNA.

[0054] Depending on the bacterial species, the CRISPR RNA processing performed is also different. For example, in the type II system originally described in the bacterium Streptococcus pyogenes, the transcribed RNA is paired with a transactivating RNA (tracrRNA) and then cut by RNaseIII to form a separate CRISPR-RNA (crRNA). The crRNA is further processed after binding to the Cas9 nuclease to produce mature crRNA. The crRNA / Cas9 complex then binds to DNA containing a sequence complementary to the captured region (called a protospacer). The Cas9 protein then cuts the two chains of DNA in a site-specific manner, forming a double-strand break (DSB). This provides DNA-based memory, resulting in rapid degradation of viral or plasmid DNA upon repeated exposure and / or infection. The natural CRISPR system has been comprehensively reviewed (see, for example, Barrangou, R. and Marraffini, LA, 2014).

[0055] Since its initial discovery, multiple groups have conducted extensive research on the potential applications of the CRISPR system in genetic engineering, including gene editing (Jinek et al., "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity", Science, 337(6096):816-21 (2012); Cong et al., "Multiplex genome engineering using CRISPR / Cas systems", Science, 339(6121):819-23 (2013); and Mali et al., "RNA-guided human genome engineering via Cas9", Science, 339(6121):823-26; each of which is herein incorporated by reference in its entirety). A major development is the use of chimeric RNA to target Cas9 protein, which is designed around a single unit from a CRISPR array fused to tracrRNA. This produces a single RNA species, referred to as a small guide RNA (gRNA), in which modifications to the sequence in the protospacer region can site-specifically target the Cas9 protein. Considerable work has been done to understand the nature of the base pairing interaction between chimeric RNA and the target site and its tolerance to mispairing, which is highly relevant to predicting and evaluating off-target effects (see, for example, Fu et al., "Improving CRISPR-Cas nucleases using Truncated guide RNAs," Nature Biotechnology 32(3):279-84(2014), and carrier materials, which are incorporated herein by reference in their entirety).

[0056] The CRISPR-Cas9 gene editing system has been successfully used in a variety of organisms and cell lines, and can use wild-type Cas9 protein to induce double-strand break formation, or can use a mutant protein called Cas9n / Cas9 D10A to cut a single DNA strand (see, for example, Mali et al., (2013) and Sander and Joung, "CRISPR-Cas systems for editing, regulating and targeting genomes", Nature Biotechnology 32(4):347-55 (2014), each of which is incorporated herein by reference in its entirety). Although the formation of double-strand breaks (DSBs) can lead to small insertions and deletions (indels) that may disrupt gene function, the Cas9n / Cas9D10A nickase avoids the generation of indels (the result of repair by non-homologous end joining) while stimulating the endogenous homologous recombination machinery. Therefore, the Cas9n / Cas9 D10A nickase can be used to insert DNA regions into the genome with high fidelity.

[0057] As described herein, suitably, the CRISPR-associated nuclease inserted into the T cell genome is a Cas9 nuclease. By placing the Cas9 nuclease under the control of a controllable promoter, the nuclease can remain dormant or silent before it is used as a gene editing tool. As used herein, a "controllable promoter" refers to a promoter that can be turned on or off, depending on the desired control of the gene under the control of the promoter.

[0058] In addition to Cas9 nuclease, Cas12, Cas13 and Cas14 nucleases can also be used in the methods described herein. Cas12 nuclease produces staggered cuts in dsDNA (5 nucleotide 5' overhang dsDNA breaks). Cas12 processes its own guide RNA, thereby improving multiplexing capabilities. Cas13t targets RNA, not DNA. Once it is activated by an ssRNA sequence complementary to its crRNA spacer, it releases nonspecific RNase activity and destroys all nearby RNA, regardless of its sequence. See, for example, Yan et al., "CRISPR-Cas12 and Cas13: the lesser known siblings of CRISPR Cas9", Cell Biology and Toxicology, pages 1 to 4 (August 29, 2019), the disclosure of which is incorporated herein by reference in its entirety.

[0059] In further embodiments, an inactivated Cas9 (dCas9) enzyme can be linked to an active endonuclease and used in the methods described herein, including, for example, a dCas9-Fok1 fusion.

[0060] As used herein, "under the control of ..." refers to a gene being regulated by a "promoter," "promoter sequence," or "promoter region," which refers to a DNA regulatory region / sequence that is capable of binding to RNA polymerase and initiating transcription of a downstream coding gene sequence or non-coding gene sequence. In other words, the promoter is operably combined or operably linked to the gene. As used herein, the terms "in an operable combination," "in an operable order," and "operably linked" refer to nucleic acid sequences being connected in a manner that produces a promoter that can direct the transcription of a given gene and / or the synthesis of a desired protein molecule. The term also refers to connecting amino acid sequences in a manner that produces a functional protein.

[0061] In some examples of the present disclosure, the promoter sequence comprises a transcription initiation site and extends upstream to include the minimum number of bases or elements necessary to initiate transcription at a detectable level above background. In some embodiments, the promoter sequence comprises a transcription initiation site and a protein binding domain responsible for binding RNA polymerase. Eukaryotic promoters will often, but not always, contain a "TATA" box and a "CAT" box.

[0062] Various promoters, including inducible promoters, can be used, for example, to drive gene expression in host T cells or vectors disclosed herein. In some embodiments, the promoter is not a leaky promoter, that is, the promoter does not constitutively express any gene product as described herein. In other embodiments as described herein, the promoter is a constitutive promoter that initiates mRNA synthesis without being affected by external regulation. In an exemplary embodiment, the promoter used to control engineered nucleases is an inducible promoter. "Inducible promoter" refers to a group of promoters that can enhance exogenous gene expression under the stimulation of specific physical, chemical or pathogen signals. In the embodiments herein, exemplary inducible promoters that can be used to control engineered nucleases include, but are not limited to, 4HT inducible promoters, rapamycin inducible promoters, hormone response elements, TET-on systems, or glutamate inducible promoters.

[0063] Suitably, the promoter for controlling engineered nucleases is a derepressible promoter. As used herein, a "derepressible promoter" refers to a structure comprising a functional promoter and another element or sequence that can bind a repressor element to cause functional promoter repression. "Repression" refers to reducing or inhibiting the transcription of downstream coding or non-coding gene sequences by the initiator. A "repressor element" refers to a protein or polypeptide that can bind a promoter (or near a promoter) to reduce or inhibit promoter activity. The repressor element can interact with the substrate or binding partner of the repressor element so that the repressor element undergoes conformational change. This conformational change of the repressor element takes away the ability of the repressor element to reduce or inhibit the promoter, thereby resulting in "derepression" of the promoter, thereby allowing the promoter to continue to initiate transcription. A "functional promoter" refers to a promoter that will be able to initiate transcription in the absence of a repressor element. Various functional promoters that can be used in the practice of the present invention are known in the art and include, for example, promoters of PCMV, PH1, P19, P5, P40, and adenovirus helper genes (e.g., E1A, E1B, E2A, E4Orf6, and VA).

[0064] Exemplary repressor elements and their corresponding binding partners that can be used as derepressible promoters are known in the art and include, for example, the cumate gene-switch system (CuO operator, CymR repressor, and cumate binding partner) (see, e.g., Mullick et al., "The cumate gene-switch: a system for regulated expression in mammalian cells," BMC Biotechnology 6:43(1-18) (2006), the disclosure of which is incorporated herein by reference in its entirety, including the disclosure of the derepressible promoter system described therein) and the TetO / TetR system described herein (see, e.g., Yao et al., "Tetracycline Repressor, tetR, rather than the tetR-Mammalian Cell Transcription Factor Fusion Derivatives, Regulates Inducible Gene Expression in Mammalian Cells." Cells", Human Gene Therapy 9:1939-1950 (1998), the disclosure of which is incorporated herein by reference in its entirety). In exemplary embodiments, the derepressible promoter comprises a functional promoter and either two tetracycline operator sequences (TetO or TetO2). In such embodiments, the nucleic acid introduced into the T cell further comprises a tetracycline repressor protein to control the TetO derepressible system.

[0065] In an exemplary embodiment, as Figure 3 As shown, in stage 1, T cells can be transfected with inducible Cas9 (iCas9) (or Cas9 under the control of a derepressible promoter) using a viral system, such as lentivirus. This will generate T cells containing genetically integrated Cas9 (or other engineered nucleases).

[0066] In stage 2, if Figure 3As shown, suitably, iCas9 T cells (or T cells containing another engineered nuclease) can be amplified using various cell amplification methods. The amplification method of T cells described herein can utilize any suitable reactor, including but not limited to stirred tank bioreactors, airlift, fibers, microfibers, hollow fibers, ceramic matrices, fluidized beds, fixed beds and / or spouted bed bioreactors. As used herein, "reactor" can include fermentor tanks or fermentation units or any other reaction vessels, and the terms "reactor" and "fermentor tanks" are used interchangeably. The terms fermentor tank or fermentation refer to both microbial cultures and mammalian cultures. For example, in some aspects, the example bioreactor unit can perform one or more or all of the following: feeding of nutrients and / or carbon sources, injection of suitable gases (e.g., oxygen), inlet and outlet flows of fermentation or cell culture media, separation of gas phase and liquid phase, maintenance of temperature, maintenance of oxygen and CO2 levels, maintenance of pH levels, agitation (e.g., stirring) and / or cleaning / sterilization. In one embodiment, the bioreactor can be used for the fermentation of a cell or a mixture of a plurality of reactors. The fermentation cell can contain a plurality of reactors in the cell, for example, the cell can have 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 or more bioreactors and / or facilities in each cell, and the plurality of units have single or multiple reactors in the facility. In various embodiments, the bioreactor can be suitable for batch, semi-fed batch, fed batch, perfusion and / or continuous fermentation processes. Any suitable reactor diameter can be used. In an embodiment, the volume of the bioreactor can be between approximately 100 mL and approximately 50,000 L. Non-limiting examples of volumes are 100 mL, 250 mL, 500 mL, 750 mL, 1 liter, 2 liters, 3 liters, 4 liters, 5 liters, 6 liters, 7 liters, 8 liters, 9 liters, 10 liters, 15 liters, 20 liters, 25 liters, 30 liters, 40 liters, 50 liters, 60 liters, 70 liters, 80 liters, 90 liters, 100 liters, 150 liters, 200 liters, 250 liters, 300 liters, 350 liters, 400 liters, 450 liters, 500 liters, 550 liters, , 600 liters, 650 liters, 700 liters, 750 liters, 800 liters, 850 liters, 900 liters, 950 liters, 1000 liters, 1500 liters, 2000 liters, 2500 liters, 3000 liters, 3500 liters, 4000 liters, 4500 liters, 5000 liters, 6000 liters, 7000 liters, 8000 liters, 9000 liters, 10,000 liters, 15,000 liters, 20,000 liters and / or 50,000 liters.Additionally, suitable reactors can be multi-use, single-use, disposable, or non-disposable and can be formed from any suitable material, including metal alloys such as stainless steel (e.g., 316L or any other suitable stainless steel) and Inconel, plastic, and / or glass.

[0067] In an embodiment, amplification suitably comprises the activation of T cell.As described herein, because T cell receptor has not yet been removed from engineered T cell line, therefore cell can still be activated.In vivo, antigen presenting cell (APC), such as dendritic cell, is as the stimulator of T cell activation by the interaction of T cell receptor (TCR) and APC major histocompatibility complex (MHC).TCR is relevant to CD3, and CD3 is a kind of T cell co-receptor, helps to activate cytotoxic T cells (for example, CD8+ original T cells) and T helper cells (for example, CD4+ original T cells).Usually, T cell activation follows dual signal model, thus needs to stimulate TCR / CD3 compound and costimulatory receptor.

[0068] Non-limiting examples of costimulatory molecules of T cells include CD28, which is a receptor for CD80 and CD86 on the APC membrane; and CD278 or ICOS (inducible T cell costimulator), which is a CD28 superfamily molecule expressed on activated T cells interacting with ICOS-L. Therefore, in some embodiments, the costimulatory molecule is CD28. In other embodiments, the costimulatory molecule is ICOS. In vivo, costimulatory signals can be provided by the B7 molecules on APC, which bind to the CD28 receptors on T cells. B7 is a peripheral transmembrane protein present on activated APCs that can interact with CD28 or CD152 surface proteins on T cells to produce costimulatory signals. Therefore, in some embodiments, the costimulatory molecule is B7.

[0069] Various activation methods are used in vitro to simulate T cell activation. In an embodiment, a T cell culture is activated with an activating agent. In another embodiment, the activating agent is an antigen presenting cell (APC). In still another embodiment, the activating agent is a dendritic cell. Dendritic cells are APCs that process antigens and present them to T cells on the cell surface. In certain embodiments, the activating agent is co-cultured with the T cell culture. Co-cultivation may require separate purification and cultivation of a second cell type, which may increase labor requirements and sources of variability. Therefore, in some embodiments, alternative activation methods are used.

[0070] In some embodiments, the activating agent is an antibody. In some embodiments, the cell culture is activated with an antibody bound to a surface (including a polymer surface, including beads). In other embodiments, the one or more antibodies are anti-CD3 and / or anti-CD28 antibodies. For example, the beads can be magnetic beads, e.g., DYNABEADS coated with anti-CD3 and anti-CD28. Anti-CD3 beads and anti-CD28 beads can appropriately provide stimulatory signals to support T cell activation. See, e.g., Riddell 1990; Trickett 2003.

[0071] In other embodiments, the cell culture is activated with a soluble antibody. In yet other embodiments, the soluble antibody is a soluble anti-CD3 antibody. OKT3 is a murine monoclonal antibody of the IgG2a isotype that targets CD3. Thus, in some embodiments, the soluble anti-CD3 antibody is OKT3. OKT3 is further described in, for example, Dudley 2003; Manger 1985; Ceuppens 1985; Van Wauwe 1980; and Norman 1995.

[0072] In certain embodiments, the co-stimulatory signal of T cell activation is provided by helper cells. Helper cells may include, for example, Fc receptors, which can cross-link CD3 antibodies with the TCR / CD3 complex on T cells. In certain embodiments, cell culture is a mixed population of peripheral blood mononuclear cells (PBMCs). PBMCs may include helper cells that can support T cell activation. For example, CD28 co-stimulatory signals may be provided by the B7 molecules present on the monocytes in PBMCs. Therefore, in certain embodiments, helper cells include monocytes or monocyte-derived cells (e.g., dendritic cells). In further embodiments, helper cells include B7, CD28 and / or ICOS. Helper cells are further described in, for example, Wolf 1994; Chai 1997; Verwilghen 1991; Schwartz 1990; Ju 2003; Baroja 1989; Austyn 1987; Tax 1983.

[0073] As described herein, activation reagents can determine the phenotype of the CAR T cells produced, thereby promoting the desired phenotype. In some embodiments, activation reagents determine T cell subsets, i.e., the ratio of CD4+ helper T cells to CD8+ cytotoxic T cells. Cytotoxic CD8+ T cells are generally responsible for killing cancer cells (i.e., anti-tumor response), infected cells (e.g., infected with viruses) or otherwise damaged cells. CD4+ T cells generally produce cytokines and help regulate immune responses, and in some cases may support T cell lysis. CD4+ cells activate APCs, and then sensitize the original CD8+ T cells to perform anti-tumor responses. Therefore, in an embodiment, the method of the present disclosure further includes producing a predefined phenotype of CAR T cells (i.e., cells promoting a desired phenotype). Predefined phenotypes can be, for example, predefined ratios of CD8+ cells to CD4+ cells. In some embodiments, the ratio of CD8+ cells to CD4+ cells in the CAR T cell population is about 1:1, about 0.25:1 or about 0.5:1. In other embodiments, the ratio of CD8+ cells to CD4+ cells in the CAR T cell population is about 2:1, about 3:1, about 4:1, or about 5:1.

[0074] In some embodiments, the T cell culture is expanded to a predefined culture size (i.e., number of cells). The predefined culture size can contain a sufficient number of cells suitable for clinical use, i.e., transfusion into a patient, research and development work, etc. In some embodiments, the clinical or therapeutic dose of T cells for administration to a patient is about 10 5 cells, about 10 6 cells, about 10 7 cells, about 10 8 cells, about 10 9 cells or about 10 10 cells. In some embodiments, the methods produce at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 clinical doses of T cells (and thus ultimately CAR T cells). In embodiments, the number of T cells produced by the methods described herein is at least about 100 million (i.e., 100 million). * 10 6 ) cells, or at least about 1 billion (i.e. 1 * 10 9 ) cells, at least about 50 billion, at least about 100 billion, at least about 250 billion, at least about 500 billion, at least about 750 billion, or at least about 1 trillion (i.e., 1 * 1012 ) cells, comprising at least about 2 trillion, at least about 3 trillion, at least about 4 trillion, at least about 5 trillion, or at least about 10 trillion T cells.

[0075] After T cell amplification, cells are suitably prepared for storage, including, for example, freezing the amplified T cell line after amplification. Methods for freezing amplified cells are known in the art and include the use of liquid nitrogen, dry ice, and may include various freeze-drying procedures. Suitably, cells are frozen at a temperature of about -80 ° C to about 0 ° C, and may include the use of cryoprotectants such as dimethyl sulfoxide (DMSO). Cells can be stored in a frozen state for weeks, months, and even years until the desired time, and then can be thawed to carry out additional processing and / or genetic modification as described herein.

[0076] In another embodiment, the present invention provides a method for producing a genetically modified T cell line, the method comprising: introducing a nucleic acid molecule encoding a CRISPR-associated nuclease under the control of a controllable promoter into the T cell line; integrating the nucleic acid molecule into the genome of the T cell line; and amplifying the T cell line. As described herein, the nucleic acid molecule encoding the CRISPR-associated nuclease is an RNA that is reverse transcribed into DNA and then integrated into the genome of the cell. As described herein, suitably, the CRISPR-associated nuclease is a Cas9 nuclease or a Cas12 nuclease.

[0077] As described herein, after expansion, the T cells can be frozen (if desired) and stored. The stored cells are then appropriately thawed before further processing.

[0078] As described herein, the method can further include inducing the expression of CRISPR related nuclease by activating a controllable promoter. In the case of an inducible promoter, such as a 4HT inducible promoter, a rapamycin inducible promoter, a hormone response element or a glutamate inducible promoter, by adding for example 4-hydroxytamoxifen, rapamycin, hormone or glutamate inducible promoter respectively. In the case of a derepressible promoter, such as the TetO sequence coupled with a CMV promoter as described herein, doxycycline is added to remove repression, and gene (engineered nuclease) is allowed to be expressed by a CMV promoter. Suitably, the nucleic acid molecule encoding Cas9 also encodes TetR repressor elements, suitably in another promoter system, such as under the control of a constitutive promoter such as hPGK promoter. As described herein, a controllable promoter can also be a Tet-on system, comprising the use of a TRE3G promoter sequence.

[0079] Figures 4A-4B An exemplary derepressible system is shown, namely the TetO system described herein. Figure 4A As shown, two TetO sequences (along with a promoter sequence) are appropriately oriented before an engineered nuclease (EN). After the tetracycline repressor protein (repressor element of the TetR-TetO sequence) binds to the TetO sequence, the promoter (e.g., CMV) is repressed. In other words, little or no transcription occurs from these promoters. Figure 4B As shown, upon binding of a binding partner of TetR, suitably doxycycline (Dox), the TetR protein changes conformation, is released from the TetO sequence, and the functional promoter (e.g., CMV) begins its normal transcription process, as it would naturally, resulting in the production of the engineered nuclease (EN).

[0080] Figure 4C Shown is an exemplary inducible vector (e.g., a lentiviral vector) that can be used to integrate an inducible engineered nuclease, in this case, the Cas9 nuclease under the control of the TET-on operating system (TRE3G), allowing expression of Cas9 in cells following induction with doxycycline. Figure 4D A more detailed vector map is shown.

[0081] Figure 4E The operation of the TET-on operating system TRE3G is shown. As shown, in the absence of doxycycline, the Tet-On 3G transactivator does not bind to the TRE3G promoter sequence. In the presence of doxycycline, the Tet-On 3G transactivator is able to bind to the TRE3G promoter sequence, which activates transcription and leads to the expression of the Cas9 nuclease (or other nucleases, if desired).

[0082] like Figure 4D As shown, the controllable systems described herein for introducing nucleases (e.g., Cas9 or Cas12) also suitably include selectable markers, such as antibiotic resistance genes (e.g., ampicillin resistance) to allow production of cells (including T cells) containing inducible nucleases that can be easily selected and enriched. Other controllable systems described herein (other inducible systems and derepressible systems) can also be used in combination with selectable markers to allow selection of cells expressing nucleases, and then enrichment is performed to provide pure cell populations with the desired nuclease (e.g., Cas9 or Cas12) integrated into the genome.

[0083] like Figure 3 As shown, in stage 3, in addition to activating the expression of the Cas9 nuclease, the guide RNA and the gene of interest are also introduced into the expanded T cell line. As described herein, the guide RNA and the gene of interest can be introduced by a transfection mechanism such as nucleofection.

[0084] Due to the introduction of this guide RNA and the gene of interest, the T cell receptor is appropriately knocked out, and the gene of interest is introduced into the genome of the T cell line. After the gene of interest is introduced, the T cells are appropriately expanded and the genetically modified T cell line is then recovered. Methods for expansion are known in the art and described herein. Methods for recovering the desired cells include various filtration methods, centrifugation, and cell separation and washing.

[0085] Suitable knockout of the T cell receptor comprises knocking out the TRAC gene (T cell receptor alpha subunit), which results in ablation of the entire T cell receptor. As described herein, various guide RNA sequences can be used to knock out the TRAC gene, and include those indicated in the examples, as well as other sequences readily determined by those of ordinary skill in the art.

[0086] As described herein, to generate chimeric antigen receptor T cells, the gene of interest suitably encodes a chimeric antigen receptor (CAR). Figure 2 As shown, gene editing by Cas9 nuclease (or Cas12 nuclease) leads to the knockout of T cell receptors and the expression of the desired CAR on T cells. Such T cells can now be administered to the desired patient population based on the desired CAR.

[0087] Integration of the desired CAR construct into the T cell suitably occurs at the site of the TRAC gene knockout, such that the CAR is under the control of the endogenous promoter of the TRAC gene.

[0088] As described herein, the ability to expand T cells to significant numbers prior to inducing Cas9 expression and then subsequently integrating the gene of interest allows for the generation of large numbers of T cells, approximately 10 9 -10 12 T cells.

[0089] In another embodiment, one or more genes can be knocked out in T cells to produce the desired modification, rather than introducing a gene of interest. For example, genes such as programmed cell death protein 1 (PD1) and / or B2M gene (β2 microglobulin) responsible for encoding serum proteins are found to be associated with major histocompatibility complex (MHC) class I heavy chain. Various methods such as antisense, siRNA, microRNA and other methods known in the art can be used to knock out such genes.

[0090] In another embodiment, the present invention provides a method for generating a chimeric antigen receptor (CAR) T cell line, the method comprising: introducing a nucleic acid molecule encoding a Cas9 nuclease under the control of a controllable promoter into a T cell line; integrating the nucleic acid molecule into the genome of the T cell line; amplifying the T cell line; inducing expression of the Cas9 nuclease by activating the controllable promoter; introducing a guide RNA and a nucleic acid encoding a chimeric antigen receptor (CAR) into the amplified T cell line; knocking out the expression of the T cell receptor and introducing the nucleic acid encoding the CAR into the genome of the T cell line; and recovering the CAR T cell line.

[0091] Described herein are examples of various controllable promoters, including inducible promoters and derepressible promoters, as well as methods for inducing expression of Cas9 nuclease by introducing molecules that induce expression or derepress derepressible promoters.

[0092] In a further embodiment, there is provided herein an allogeneic T cell line comprising a CRISPR-associated (Cas) nuclease under the control of a controllable promoter integrated into the genome of the T cell line. As described herein, the Cas nuclease is suitably a Cas9 nuclease.

[0093] As described herein, suitably, the allogeneic T cell line comprises a controllable promoter as an inducible promoter, including, for example, a 4HT inducible promoter, a rapamycin inducible promoter, a hormone response element or a glutamate inducible promoter. The controllable promoter may also be a Tet-on system.

[0094] In further embodiments, the controllable promoter can be a derepressible promoter, such as using one or more tetracycline operator sequences (TetO). In such embodiments, the T cell line further comprises a nucleic acid molecule encoding a tetracycline repressor protein.

[0095] As described, allogeneic T cells prepared according to the described examples allow for the generation of at least about 10 9 T cells, at least about 10 10 T cells, at least about 10 11 T cells, or in embodiments, at least about 10 12 T cells.

[0096] Also provided herein are methods for treating mammalian subjects, preferably human subjects, comprising administering CAR T cells prepared using allogeneic T cells as described herein, as well as CAR T cells prepared using the methods described herein. Administration to human subjects can include, for example, inhalation, injection, or intravenous administration, as well as other administration methods known in the art.

[0097] Examples

[0098] Example 1: Transduction of T cells with Cas9 inducible vectors

[0099] Three treatment regimens were investigated for transduction of T cells with an inducible Cas9 vector. The vector contained a green fluorescent protein (GFP) tag to determine transduction levels.

[0100] like Figures 5A to 5C As shown, treatment 1 (IL2) included 24-hour activation with 15 ng / mL of IL-2 and CD3 / CD28 on the day of T cell isolation. After viral transduction, expansion included only 15 ng / mL of IL-2 (IL2). Treatment 2 (IL2+CD3 / CD28) included 24-hour activation with 15 ng / mL of IL-2 and CD3 / CD28 on the day of T cell isolation. After transduction, expansion included treatment with 15 ng / mL of IL-2 and CD3 / CD28 at each culture medium change. Treatment 3 (IL2+IL) included 24-hour activation with 15 ng / mL of IL-2 and CD3 / CD28 on the day of T cell isolation. After transduction, cells were treated with 15 ng / mL of IL-2 and CD3 / CD28. Expansion was performed in the presence of only IL-2 and IL-7.

[0101] The results of transduction are provided in Figures 6A to 6B In. Figure 6A As shown, the percentage of GFP-positive cells using the CD3 / CD28+IL-2 treatment combination had the highest transduction efficiency, which was true for both the control vector (#) and the vector containing the Cas9 nuclease gene (@). IL-2+IL-7 treatment also showed good transduction ($). Figure 6B The dilution rate of the GFP-positive population is shown.

[0102] The blasticidin resistance gene in the Cas9-inducible vector is used to select cells that contain the Cas9 vector correctly inserted into the genome. Figure 7A The number of viable cells of the three treatments described in this example is shown. As shown, the IL-2+IL-7 treatment showed the most viable cells. After 12 days of expansion after selection, the cells treated with CD3 / CD28+IL-2 and IL-2+IL-7 showed a large number of viable cells ( Figure 7B ).

[0103] Exhaustion, senescence, and activation markers were measured for the three treatment regimens, and the results are shown in Figure 8 middle.

[0104] On day 11, Cas9 expression was induced and post-selected with blasticidin. Cells were induced with 1 μg / mL doxycycline for 24 hours, and protein lysates were analyzed by Western blot. Figure 9 As shown, cells grown with treatment 3 (IL-2+IL-7) showed higher Cas9 expression after induction, but cells treated with CD3 / CD28+IL-2 also showed expression of the Cas9 nuclease.

[0105] The cells were then frozen and thawed to determine whether insertion of the Cas9 vector had any effect on cell viability. Figure 10A As shown, the viability of the two treatments was almost the same before and after cryopreservation. Figure 10B Cell viability several days post-thawing is shown, indicating that for both treatments the cells were able to successfully proliferate.

[0106] Example 2: Knockout of the TRAC gene

[0107] Three sgRNA sequences were selected to investigate their ability to knock out the TRAC gene in T cells that had been transduced with the Cas9 vector. Figure 11 The three sgRNA sequences studied (SEQ ID NO: 1-3) are shown, along with the region they target in the translated TRAC gene (SEQ ID NO: 4). sgRNA sequences were transfected into T cells using the nucleofection procedure. Briefly, 1×10 T cells were cultured in 20 μL of the AMAXA P2 primary cell 4D nucleofector X kit and the EO-115 protocol at room temperature. 6 Each cell was transduced with approximately 3.3 μg of sgRNA.

[0108] Knockout experiments were performed with sgRNA TRAC#1-3 (SEQ ID NO: 1-3) and calibrated relative to CD-3. On the 1st day, Cas9 T cells (treated with 15ng / mL of CD3 / CD28 and IL-2, IL-7, and then selected with 15μg / mL of blasticidin) were thawed. On the 2nd day, Cas9 was induced with doxycycline (induced for 24 hours with 2μg / mL). On the 3rd day, cells were transduced with sgRNA TRAC#1 (SEQ ID NO: 1), TRAC#2 (SEQ ID NO: 2) and TRAC#3 (SEQ ID NO: 3) by nuclear transfection, and then amplified. On the 4th day, 7th day and 14th day, FACS analysis of CD-3 and TCR α β expression levels was performed.

[0109] Figures 12A-1 to 12B-6 Shown is TRAC knockout 4 days after nucleofection with TRAC#1-#3 sgRNA sequences. Figure 12C A compilation of the results is shown. As indicated, each of TRAC#1-TRAC#3 resulted in approximately 41-47% knockout of the TRAC gene, with TRAC#2 sgRNA showing the highest knockout (47%).

[0110] Figures 13A-1 to 13B-6 Shown is TRAC knockout 7 days after nucleofection with TRAC#1-#3 sgRNA sequences. Figure 13C A compilation of the results is shown. As indicated, each of TRAC#1-TRAC#3 resulted in approximately 66-74% knockout of the TRAC gene, with TRAC#2 sgRNA showing the highest knockout (74%).

[0111] Figures 14A-1 to 14B-6 Shown is TRAC knockout 14 days after nucleofection with TRAC#1-#3 sgRNA sequences. Figure 14C A compilation of the results is shown. As indicated, each of TRAC#1-TRAC#3 resulted in approximately 84-89% knockout of the TRAC gene, with TRAC#2 sgRNA showing the highest knockout (89%).

[0112] A compilation of the 14-day experiment is shown in FIG. 15A to FIG. 15B , thereby demonstrating efficient knockout of the TRAC gene using the methods described herein.

[0113] Example 3: Knock-in of CAR constructs

[0114] The following experiments were designed to demonstrate the ability to knock-in the desired CAR construct into T cells containing Cas9.

[0115] The CAR knock-in experiment will be performed after the knock-out experiment described above with the following additions: During nucleofection of the gRNA (directed towards the TRAC gene), a DNA template is added. This DNA template is designed to integrate into the TRAC locus using the homologous recombination mechanism when the nuclease generates a double-strand break.

[0116] The following three types of DNA templates are examined:

[0117] single-stranded DNA;

[0118] Microcircle DNA;

[0119] Linear double-stranded DNA.

[0120] To check both constructs:

[0121] conventional anti-CD19 CAR, which is assessed using FACS or functional assays;

[0122] Anti-CD19-CAR, which is fused on the cytoplasmic side to a green fluorescent protein (GFP) molecule that can be easily detected using FACS.

[0123] Additional Exemplary Embodiments

[0124] Example 1 is a method for generating a T cell line for allogeneic applications, the method comprising: introducing a nucleic acid molecule encoding an engineered nuclease under the control of a controllable promoter into the T cell line; integrating the nucleic acid molecule into the genome of the T cell line; and expanding the T cell line.

[0125] Embodiment 2 comprises the method of embodiment 1, wherein the engineered nuclease is selected from the group consisting of: a meganuclease, a zinc finger nuclease, a transcription activator-like effector-based nuclease, and a CRISPR-associated nuclease.

[0126] Embodiment 3 comprises the method of embodiment 2, wherein the CRISPR-associated nuclease is a Cas9 nuclease or a Cas12 nuclease.

[0127] Embodiment 4 comprises the method of any one of embodiments 1 to 3, wherein the controllable promoter is an inducible promoter.

[0128] Embodiment 5 comprises the method of embodiment 4, wherein the inducible promoter is a 4HT-inducible promoter, a rapamycin-inducible promoter, a hormone response element, or a glutamate-inducible promoter.

[0129] Embodiment 6 comprises the method of any one of embodiments 1 to 3, wherein the controllable promoter is a derepressible promoter.

[0130] Embodiment 7 comprises the method of embodiment 6, wherein the derepressible promoter comprises one or more tetracycline operator sequences (TetO).

[0131] Embodiment 8 comprises the method of embodiment 7, wherein the nucleic acid molecule further comprises a tetracycline repressor protein.

[0132] Embodiment 9 comprises the method of any one of embodiments 1 to 3, wherein the controllable promoter comprises a Tet-on system.

[0133] Embodiment 10 comprises the method of any one of embodiments 1 to 9, further comprising freezing the expanded T cell line after said expanding.

[0134] Embodiment 11 comprises the method of any one of embodiments 1 to 10, wherein the nucleic acid molecule is introduced into the T cell line using a lentiviral vector.

[0135] Embodiment 12 comprises the method of any one of embodiments 1 to 11, wherein the T cell line comprises at least about 10 9 T cells.

[0136] Example 13 is a method for producing a genetically modified T cell line, the method comprising: introducing a nucleic acid molecule encoding a CRISPR-associated nuclease under the control of a controllable promoter into a T cell line; integrating the nucleic acid molecule into the genome of the T cell line; amplifying the T cell line; inducing expression of the CRISPR-associated nuclease by activating the controllable promoter; introducing a guide RNA and a gene of interest into the amplified T cell line; knocking out expression of a T cell receptor and introducing the gene of interest into the genome of the T cell line; and recovering the genetically modified T cell line.

[0137] Embodiment 14 comprises the method of embodiment 13, wherein the CRISPR-associated nuclease is a Cas9 nuclease or a Cas12 nuclease.

[0138] Embodiment 15 comprises the method of embodiment 13 or embodiment 14, wherein the controllable promoter is an inducible promoter.

[0139] Embodiment 16 comprises the method of embodiment 15, wherein the inducible promoter is a 4HT-inducible promoter or a glutamate-inducible promoter.

[0140] Embodiment 17 comprises the method of embodiment 13 or embodiment 14, wherein the controllable promoter is a derepressible promoter.

[0141] Embodiment 18 comprises the method of embodiment 17, wherein the derepressible promoter comprises one or more tetracycline operator sequences (TetO).

[0142] Embodiment 19 comprises the method of embodiment 18, wherein the nucleic acid molecule further comprises a tetracycline repressor protein.

[0143] Embodiment 20 comprises the method of embodiment 18 or embodiment 19, wherein said activating said derepressible promoter comprises adding doxycycline to said T cell line.

[0144] Embodiment 21 comprises the method of embodiment 13 or 14, wherein the controllable promoter comprises a Tet-on system.

[0145] Embodiment 22 comprises the method of embodiment 21, wherein said activating said Tet-on system comprises adding doxycycline to said T cell line.

[0146] Embodiment 23 comprises the method of any one of embodiments 13-22, wherein the gene of interest encodes a chimeric antigen receptor (CAR).

[0147] Embodiment 24 comprises the method of any one of embodiments 13 to 23, further comprising freezing the T cell line after the expansion in c and thawing it before the induction in d.

[0148] Embodiment 25 comprises the method of any one of embodiments 13 to 24, wherein the genetically modified T cell line comprises at least about 10 9 T cells.

[0149] Example 26 is a method for producing a chimeric antigen receptor (CAR) T cell line, the method comprising: introducing a nucleic acid molecule encoding a Cas9 nuclease under the control of a controllable promoter into a T cell line; integrating the nucleic acid molecule into the genome of the T cell line; amplifying the T cell line; inducing expression of the Cas9 nuclease by activating the controllable promoter; introducing a guide RNA and a nucleic acid encoding a chimeric antigen receptor (CAR) into the amplified T cell line; knocking out expression of the T cell receptor and introducing the nucleic acid encoding the CAR into the genome of the T cell line; and recovering the CAR T cell line.

[0150] Embodiment 27 comprises the method of embodiment 26, wherein the controllable promoter is an inducible promoter.

[0151] Embodiment 28 comprises the method of embodiment 27, wherein the inducible promoter is a 4HT-inducible promoter or a glutamate-inducible promoter.

[0152] Embodiment 29 comprises the method of embodiment 26, wherein the controllable promoter is a derepressible promoter.

[0153] Embodiment 30 comprises the method of embodiment 29, wherein the derepressible promoter comprises one or more tetracycline operator sequences (TetO).

[0154] Embodiment 31 comprises the method of embodiment 30, wherein the nucleic acid molecule further comprises a tetracycline repressor protein.

[0155] Embodiment 32 comprises the method of embodiment 30 or embodiment 31, wherein said activating said derepressible promoter comprises adding doxycycline to said T cell line.

[0156] Embodiment 33 comprises the method of embodiment 26, wherein the controllable promoter comprises a Tet-on system.

[0157] Embodiment 34 comprises the method of embodiment 33, wherein the Tet-on system comprises adding doxycycline to the T cell line.

[0158] Embodiment 35 comprises the method of any one of embodiments 26 to 34, further comprising freezing the T cell line after the expansion in c and thawing it before the induction in d.

[0159] Embodiment 36 comprises the method of any one of embodiments 26 to 35, wherein the CAR T cell line comprises at least about 10 9 T cells.

[0160] Example 37 is an allogeneic T cell line comprising a CRISPR-associated (Cas) nuclease under the control of a controllable promoter integrated into the genome of the T cell line.

[0161] Embodiment 38 comprises the allogeneic T cell line of embodiment 37, wherein the CRISPR-associated nuclease is a Cas9 nuclease or a Cas12 nuclease.

[0162] Embodiment 39 comprises the allogeneic T cell line of embodiment 37 or embodiment 38, wherein the controllable promoter is an inducible promoter.

[0163] Embodiment 40 comprises the allogeneic T cell line of embodiment 39, wherein the inducible promoter is a 4HT-inducible promoter or a glutamate-inducible promoter.

[0164] Embodiment 41 comprises the allogeneic T cell line of embodiment 37 or embodiment 38, wherein the controllable promoter is a derepressible promoter.

[0165] Embodiment 42 comprises the allogeneic T cell line of embodiment 41, wherein the derepressible promoter comprises one or more tetracycline operator sequences (TetO).

[0166] Embodiment 43 comprises the allogeneic T cell line of embodiment 42, wherein the T cell line further comprises a nucleic acid encoding a tetracycline repressor protein.

[0167] Embodiment 44 comprises the allogeneic T cell line of embodiment 37 or embodiment 38, wherein the controllable promoter comprises a Tet-on system.

[0168] Example 45 comprises the allogeneic T cell line of any one of Examples 37 to 44, comprising at least about 10 9 T cells.

[0169] Example 46 comprises the allogeneic T cell line of Example 45, comprising at least about 10 10 T cells.

[0170] It will be apparent to those skilled in the relevant art that other suitable modifications and adaptations may be made to the methods and applications described herein without departing from the scope of any embodiment.

[0171] It should be understood that although certain embodiments have been shown and described herein, the claims are not limited to the specific forms or arrangements of parts described and illustrated. Illustrative embodiments have been disclosed in this specification, and although specific terminology is employed, it is used in a generic and descriptive sense only and not for purposes of limitation. In light of the above teachings, modifications and variations of the described embodiments are possible. Therefore, it should be understood that the described embodiments may be practiced otherwise than as specifically described.

[0172] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. An allogeneic T cell line comprising a CRISPR-associated (Cas) nuclease under the control of a controllable promoter integrated into the genome of the T cell line.

2. The allogeneic T cell line of claim 1, wherein the CRISPR-associated nuclease is a Cas9 nuclease or a Cas12 nuclease.

3. The allogeneic T cell line according to claim 1 or claim 2, wherein the controllable promoter is selected from the group consisting of an inducible promoter and a derepressible promoter.

4. The allogeneic T cell line of claim 3, wherein the controllable promoter is an inducible promoter.

5. The allogeneic T cell line of claim 4, wherein the inducible promoter is selected from the group consisting of a 4HT inducible promoter, a rapamycin inducible promoter, a hormone response element, a Tet-on system, and a glutamate inducible promoter.

6. The allogeneic T cell line of claim 5, wherein the inducible promoter is a 4HT-inducible promoter or a glutamate-inducible promoter.

7. The allogeneic T cell line of claim 3, wherein the controllable promoter is a derepressible promoter.

8. The allogeneic T cell line of claim 7, wherein the derepressible promoter comprises one or more tetracycline operator sequences (TetO).

9. The allogeneic T cell line of claim 8, wherein the T cells further comprise a nucleic acid encoding a tetracycline repressor protein.

10. The allogeneic T cell line of claim 1 or claim 2, wherein the controllable promoter comprises a Tet-on system.

11. The allogeneic T cell line of claim 10, wherein activating the Tet-on system comprises adding doxycycline to the expanded T cell line.

12. The allogeneic T cell line of claim 1, wherein the CRISPR-associated nuclease is a Cas9 nuclease, and wherein the controllable promoter comprises a Tet-on system.

13. A chimeric antigen receptor (CAR) T cell line comprising a CRISPR-associated (Cas) nuclease under the control of a controllable promoter integrated into the genome of the T cell line.

14. The CAR T cell line of claim 13, wherein the CRISPR-associated nuclease is a Cas9 nuclease or a Cas12 nuclease.

15. The CAR T cell line of claim 13 or claim 14, wherein the controllable promoter is selected from the group consisting of an inducible promoter and a derepressible promoter.

16. The CAR T cell line of claim 15, wherein the controllable promoter is an inducible promoter.

17. The CAR T cell line of claim 16, wherein the inducible promoter is selected from the group consisting of a 4HT-inducible promoter, a rapamycin-inducible promoter, a hormone response element, a Tet-on system, and a glutamate-inducible promoter.

18. The CAR T cell line of claim 17, wherein the inducible promoter is a 4HT-inducible promoter or a glutamate-inducible promoter.

19. The CAR T cell line of claim 15, wherein the controllable promoter is a derepressible promoter.

20. The CAR T cell line of claim 19, wherein the derepressible promoter comprises one or more tetracycline operator sequences (TetO).

21. The CAR T cell line of claim 20, wherein the T cells further comprise a nucleic acid encoding a tetracycline repressor protein.

22. The CAR T cell line of claim 13 or claim 14, wherein the controllable promoter comprises a Tet-on system.

23. The CAR T cell line of claim 22, wherein activating the Tet-on system comprises adding doxycycline to the expanded T cell line.

24. The CAR T cell line of claim 13, wherein the CRISPR-associated nuclease is a Cas9 nuclease, and wherein the controllable promoter comprises a Tet-on system.

Images