Engineered cells

Abstract

The present disclosure relates generally to nucleic acid constructs containing a transgene under the control of a promoter (e.g., a truncated PGK promoter), and engineered cells (e.g., engineered immune cells) including CAR T cells comprising the constructs, and uses thereof in therapeutic applications.

Description

Technical Field

[0001] This application claims priority to U.S. Provisional Application No. 63 / 594,291, filed October 30, 2023, the contents of which are hereby incorporated by reference in their entirety.

[0002] References to sequence lists This application contains a sequence list submitted electronically in XML format and hereby incorporated in its entirety by reference. The XML copy was created on October 16, 2024, named AT-059_02WO_SL.xml, and is 105,757 bytes in size.

[0003] This disclosure generally relates to engineered cells (e.g., engineered immune cells, such as chimeric antigen receptor (CAR) T cells), methods for manufacturing such engineered cells, and their use in therapeutic applications. Background Technology

[0004] Adoptive transfer of immune cells (such as T cells, including chimeric antigen receptor (CAR) modified T cells that recognize tumor-associated antigens) holds great potential in cancer treatment. However, the production of autologous CAR T cells is costly and requires a long preparation time, and the efficacy of the product varies due to the different sources of patient-specific T cells. Allogeneic CAR T cells generated from healthy donor T cells can be produced and used as off-the-shelf products, offering better product consistency and reduced manufacturing time and costs.

[0005] Most currently approved or investigational CAR T-cell therapies employ lentivirus-mediated random integration of CAR cells to engineer CAR T cells. Disadvantages of this random integration include unknown integration sites, uneven CAR copy numbers, and a potentially increased risk of genomic toxicity. Lentiviral preparation and production typically require lengthy setup times, significant resources, and sometimes inconsistent yields, all of which can increase overall manufacturing time, cost, and complexity. Therefore, there is a need for improved methods for engineering immune cells into cell-based therapies. Summary of the Invention

[0006] This disclosure provides engineered cells, such as engineered immune cells, including CAR T cells, which contain and / or express receptor peptides and / or nucleic acid inhibitors under the control of a PGK promoter (e.g., a truncated PGK promoter). In one embodiment, the receptor peptide is an antigen-specific chimeric antigen receptor (CAR), such as a CD19-specific CAR, or a CD70-binding protein, or a chimeric cytokine receptor (CCR). This document further provides engineered cells, such as engineered immune cells, comprising one or more polynucleotides. In some embodiments, the one or more polynucleotides comprise one or more coding sequences, at least two coding sequences, or two or more coding sequences encoding the receptor peptide and / or nucleic acid inhibitor.

[0007] In one aspect, the engineered cell (e.g., engineered immune cell) is a CAR T cell. In one embodiment, the CAR T cell is an autologous or allogeneic CAR T cell. In some embodiments, the CAR T cell comprises a recombinant nucleic acid sequence integrated into the constant region gene of the human T cell receptor (TCR) α chain. In one embodiment, the recombinant nucleic acid sequence comprises, in the 5' to 3' direction: (a) the 5' region of the TCR α chain constant region, (b) a truncated PGK promoter controlling the expression of the CAR nucleic acid sequence, (c) the CAR nucleic acid sequence, and (d) the 3' region of the human TCR α constant region gene. In another embodiment, the recombinant nucleic acid sequence further comprises an additional nucleic acid sequence encoding a receptor polypeptide or a nucleic acid inhibitor. In other embodiments, the receptor polypeptide is a chimeric cytokine receptor (CCR) or a CD70 binding protein. In another embodiment, the nucleic acid inhibitor is an RNA interference agent. In one embodiment, the CAR nucleic acid sequence and the additional nucleic acid sequence are linked by a P2A peptide.

[0008] In another embodiment, the truncated PGK promoter comprises a deletion of at least about 50, 60, 70, 80, or 90 nucleotides from the 5' end of a long PGK promoter having the nucleotide sequence of SEQ ID NO: 34. In another embodiment, the truncated PGK promoter is operatively linked to a recombinant nucleic acid sequence, wherein the recombinant nucleic acid sequence encodes a receptor polypeptide and / or a nucleic acid inhibitor.

[0009] In another embodiment, the truncated PGK promoter contains one or more deletions of the sequence shown in SEQ ID NO: 34. In one embodiment, the truncated PGK promoter contains one or more 5' region deletions and / or 3' region deletions.

[0010] In some embodiments, the truncated PGK promoter comprises a nucleotide sequence having about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, or about 390 nucleotides deleted from the 5' end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 35. In other embodiments, the truncated PGK promoter comprises a nucleotide sequence having about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, or about 290 nucleotides deleted from the 5' end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 36. In another embodiment, the truncated PGK promoter comprises a nucleotide sequence having about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, or about 190 nucleotides deleted from the 5' end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 37. In another embodiment, the truncated PGK promoter comprises a nucleotide sequence having about 50, about 60, about 70, about 80, about 90, or about 100 nucleotides deleted from the 5' end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 38. In one implementation, the truncated PGK promoter includes SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38.

[0011] In another embodiment, CAR T cells exhibit greater expansion and / or cytotoxicity compared to CAR T cells without a truncated PGK promoter. In another embodiment, CAR T cells exhibit less exhaustion compared to CAR T cells without a truncated PGK promoter.

[0012] In another implementation, the integrated recombinant nucleic acid sequence prevents, reduces, or impairs the expression of the TCRα constant region gene in CAR T cells.

[0013] In other embodiments, the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain. In another embodiment, the extracellular domain includes an antigen-binding domain and / or the intracellular domain includes at least one co-stimulatory domain, as described herein. In another embodiment, the intracellular domain includes at least one activating domain, such as CD3. In yet another embodiment, the activating domain is CD3 comprising CD3ζ or a variant thereof. In other embodiments, the CAR nucleic acid sequence expression binds to the following CARs: BCMA, EGFRvIII, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Liv1, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, sealing protein-18.2, Muc17, FAPα, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, FLT3, CD70, DLL3, CD52, or CD34. In another embodiment, the CAR T cells are autologous or allogeneic CAR T cells.

[0014] In another embodiment, this disclosure provides a segregated population of engineered cells, such as engineered immune cells, comprising CAR T cells as described herein.

[0015] On the other hand, this disclosure provides a method for manufacturing CAR T cells. In one embodiment, the CAR T cells are autologous or allogeneic CAR T cells. In another embodiment, the method includes introducing a recombinant nucleic acid sequence into a cell (e.g., an immune cell) comprising, in the 5' to 3' direction: (a) the 5' region of the T cell receptor (TCR) α chain constant region, (b) a truncated PGK promoter controlling the expression of the CAR nucleic acid sequence, (c) the CAR nucleic acid sequence, and (d) the 3' region of the human TCRα constant region gene. In another embodiment, the introduction step is performed under conditions sufficient to allow the recombinant nucleic acid sequence to integrate into the human T cell receptor (TCR) α chain constant region gene, thereby providing CAR T cells. In another embodiment, the integration prevents the expression of the TCRα constant region gene in the CAR T cells. In yet another embodiment, the integrated recombinant nucleic acid sequence prevents the expression of the TCRα constant region gene in the CAR T cells.

[0016] In other embodiments, the method further includes an amplification assay using CAR T cells and / or a cytotoxicity assay using CAR T cells. In one embodiment, the amplification assay is an in vitro or in vivo amplification assay, and / or the cytotoxicity assay is an in vitro or in vivo cytotoxicity assay.

[0017] In another embodiment, the method includes using a truncated PGK promoter, wherein the truncated PGK promoter comprises a deletion of at least 50 nucleotides from the 5' end of a long PGK promoter having the nucleotide sequence of SEQ ID NO: 34. In another embodiment, the truncated PGK promoter is operatively linked to a recombinant nucleic acid sequence, wherein the recombinant nucleic acid sequence encodes a receptor polypeptide and / or a nucleic acid inhibitor. In another embodiment, the truncated PGK promoter comprises one or more deletions of the sequence shown in SEQ ID NO: 34. In one embodiment, the truncated PGK promoter comprises one or more 5' region deletions and / or one or more 3' region deletions. In some embodiments, the truncated PGK promoter comprises a nucleotide sequence having about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, or about 390 nucleotides deleted from the 5' end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 35. In other embodiments, the truncated PGK promoter comprises a nucleotide sequence having about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, or about 290 nucleotides deleted from the 5' end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 36. In another embodiment, the truncated PGK promoter comprises a nucleotide sequence having about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, or about 190 nucleotides deleted from the 5' end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 37. In another embodiment, the truncated PGK promoter comprises a nucleotide sequence having about 50, about 60, about 70, about 80, about 90, or about 100 nucleotides deleted from the 5' end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 38. In one implementation, the truncated PGK promoter includes SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38.

[0018] In other embodiments, the method includes using a nucleic acid sequence encoding a CAR, wherein the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain. In another embodiment, the extracellular domain comprises an antigen-binding domain and / or wherein the intracellular domain comprises at least one co-stimulatory domain, as described herein. In another embodiment, the intracellular domain comprises at least one activating domain, such as CD3. In another embodiment, the activating domain is CD3 comprising CD3ζ or a variant thereof.

[0019] On the other hand, this disclosure provides nucleic acid constructs, nucleic acid molecules, or polynucleotides comprising a recombinant nucleic acid sequence encoding a CAR. In one embodiment, the recombinant nucleic acid sequence comprises, in the 5' to 3' direction: (a) the 5' region of the T cell receptor (TCR) α chain constant region, (b) a truncated PGK promoter controlling the expression of the CAR nucleic acid sequence, (c) the CAR nucleic acid sequence, and (d) the 3' region of the human TCR α constant region gene. In another embodiment, the recombinant nucleic acid sequence further comprises an additional nucleic acid sequence encoding a receptor polypeptide or a nucleic acid inhibitor. In one embodiment, the receptor polypeptide is a CCR or CD70 binding protein. In another embodiment, the nucleic acid inhibitor is an RNA interference agent. In yet another embodiment, the CAR nucleic acid sequence and the additional nucleic acid sequence are linked by a P2A peptide.

[0020] In another embodiment, the nucleic acid construct, nucleic acid molecule, or polynucleotide described herein comprises a truncated PGK promoter, wherein the truncated PGK promoter comprises a deletion of at least 50 nucleotides from the 5' end of a long PGK promoter having the nucleotide sequence of SEQ ID NO: 34. In another embodiment, the truncated PGK promoter is operatively linked to a recombinant nucleic acid sequence, wherein the recombinant nucleic acid sequence encodes a receptor polypeptide and / or a nucleic acid inhibitor. In another embodiment, the truncated PGK promoter comprises one or more deletions of the sequence shown in SEQ ID NO: 34. In one embodiment, the truncated PGK promoter comprises one or more 5' region deletions and / or 3' region deletions. In some embodiments, the truncated PGK promoter comprises a nucleotide sequence having about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, or about 390 nucleotides deleted from the 5' end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 35. In other embodiments, the truncated PGK promoter comprises a nucleotide sequence having about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, or about 290 nucleotides deleted from the 5' end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 36. In another embodiment, the truncated PGK promoter comprises a nucleotide sequence having about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, or about 190 nucleotides deleted from the 5' end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 37. In another embodiment, the truncated PGK promoter comprises a nucleotide sequence having about 50, about 60, about 70, about 80, about 90, or about 100 nucleotides deleted from the 5' end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 38. In one implementation, the truncated PGK promoter includes SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38.

[0021] In other embodiments, the nucleic acid constructs, nucleic acid molecules, or polynucleotides described herein comprise a nucleic acid sequence encoding a CAR, wherein the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain. In another embodiment, the extracellular domain comprises an antigen-binding domain and / or wherein the intracellular domain comprises at least one co-stimulatory domain, as described herein. In another embodiment, the intracellular domain comprises at least one activating domain, such as CD3. In another embodiment, the activating domain is CD3 comprising CD3ζ or a variant thereof.

[0022] In related aspects, this document provides vectors containing polynucleotides, and engineered cells containing said vectors or polynucleotides, such as engineered immune cells, like engineered T cells or CAR T cells. In some embodiments, the vector is an adeno-associated virus (AAV) vector or a lentiviral vector.

[0023] In another aspect, this disclosure provides CAR T cells utilizing CD70 binding protein, methods for manufacturing engineered cells (e.g., engineered immune cells), nucleic acid constructs, molecules, or polynucleotides. In some embodiments, the CD70 binding protein is a recombinant CD70 binding protein. In some embodiments, the CD70 binding protein includes an anti-CD70 antibody or an antigen-binding fragment thereof, and a transmembrane domain. In some embodiments, the anti-CD70 antibody comprises the amino acid sequence of SEQ ID NO: 16 and / or 17. In some embodiments, the anti-CD70 antibody comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the transmembrane domain includes a CD8 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the CD70 binding protein further comprises a CD3 signaling domain. In some embodiments, the CD70 binding protein does not include a co-stimulatory domain. In some embodiments, the CD70 binding protein includes a CD3 signaling domain comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the CD70-binding protein comprises the amino acid sequence of SEQ ID NO: 19 or 21, or comprises an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 19 or 21, with or without a signal peptide. In some embodiments, the coding sequence encoding the CD70-binding protein comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 20 or 22.

[0024] In another aspect, this disclosure provides CAR T cells utilizing CCR, methods for manufacturing engineered cells (e.g., engineered immune cells), nucleic acid constructs, molecules, or polynucleotides. In some embodiments, the CCR includes a thrombopoietin receptor / myeloproliferative leukemia protein receptor (TPOR / MPLR) transmembrane and JAK-binding domains and an intracellular recruitment domain. In some embodiments, the TPOR / MPLR transmembrane domain and the JAK-binding domain comprise the amino acid sequence of SEQ ID NO: 25 or 51. In some embodiments, the intracellular recruitment domain comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the CCR comprises the amino acid sequence of SEQ ID NO: 27 or 29, or an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity with SEQ ID NO: 27 or 29. In some embodiments, the coding sequence encoding the CCR comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 28 or 30.

[0025] In a further related aspect, a population of engineered cells (e.g., engineered immune cells) and a pharmaceutical composition of engineered cells or a population of engineered cells are provided. In another aspect, a method of treating a subject's disease condition is provided, comprising administering to the subject an effective amount of engineered cells, an effective amount of an engineered cell population, or an effective amount of the pharmaceutical composition described herein. In some embodiments, the subject is a human being. In some embodiments, the disease condition is cancer, including but not limited to non-Hodgkin's lymphoma, large B-cell lymphoma (LBCL), follicular lymphoma (FL), T-cell lymphoma (TCL), B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), primary central nervous system lymphoma (PCNSL), mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), and peripheral T-cell lymphoma (PTCL).

[0026] In some implementations, the disease condition is an autoimmune disease or condition, including but not limited to lupus, systemic lupus erythematosus (SLE), lupus nephritis, rheumatoid arthritis, systemic sclerosis, scleroderma, multiple sclerosis (MS), relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS), primary progressive multiple sclerosis (PPMS), neuromyelitis optica spectrum disorder (NMOSD), myasthenia gravis, Sjogren's syndrome, and myositis. In some implementations, immune cells can reduce or deplete B cells, pathogenic B cells, autoreactive B cells, pathogenic T cells, autoreactive T cells, CD70+ T cells, or allogeneic reactive T cells.

[0027] In some embodiments, the engineered cells (e.g., engineered immune cells) are autologous relative to the subject. In some embodiments, the engineered cells (e.g., engineered immune cells) are allogeneic relative to the subject. Attached Figure Description

[0028] Figures 1A to 1C The results of an in vitro long-term killing assay of anti-CD19 CAR T cells with or without co-expression of chimeric cytokine receptors (CCRs) are shown.

[0029] Figures 2A to 2B The results of in vivo cytotoxicity assays of anti-CD19CAR T cells with or without co-expression of CCR (CD19CAR / CCR) are shown.

[0030] Figure 3A Data depicted show reduced in vivo antitumor activity of anti-CD19 CAR T cells (CD19 CAR / CCR T cells) generated through site-specific integration, which co-expresses CCR, compared to CD19 CAR / CCR T cells generated via LVV transduction but expressing the same CCR and CAR. Figure 3B The study showed that CAR+ T cells generated via LVV transduction had a greater number in vivo and higher persistence compared to CAR T cells generated via site-specific integration.

[0031] Figures 4A to 4D The experimental results show the effects of different promoters driving the expression of CCR and CAR in CAR T cells generated through site-specific integration. Figure 4A The median fluorescence intensity (MFI) of CD19 CAR / CCR T cells at the end of CAR T cell production is shown. Figure 4BThe results of CAR T cell killing of wild-type Raji target cells in a continuous restimulation assay based on flow cytometry are shown. Figure 4C CARMFI during continuous restimulation assays based on flow cytometry is shown, and Figure 4D The CAR+ T cell counts during this period are shown.

[0032] Figures 5A to 5C The standard long-term killing assay was compared between CD19 CAR / CCR T cells generated by site-specific integration driven by the EFS promoter or the PGK SSI-1 promoter and CD19 CAR / CCR T cells generated by LVV transduction. Figure 5A ) or single-stimulation efficacy assay based on flow cytometry ( Figures 5B to 5C The efficacy of the drug on target Raji cells.

[0033] Figures 6A to 6B The in vivo efficacy of CD19 CAR / CCR T cells generated through site-specific integration driven by the EFS promoter, CypA300 promoter, or PGK SSI-1 promoter was compared with that of CD19 CAR / CCR T cells generated via LVV, using two different CAR T cell doses: 2x10 6 Cells ( Figure 6A ) or 4x10 6 Cells ( Figure 6B ).

[0034] Figures 7A to 7B The process of inserting transgenes (e.g., CARs) into loci through site-specific integration and homologous recombination is described.

[0035] Figures 8A to 8D Examples of different configurations of polynucleotides, nucleic acid constructs, or molecules containing transgenes and promoters are described. Detailed Implementation

[0036] Compared to autologous cell-based therapies, immune cell-based therapies starting with healthy donor cells can be manufactured on a larger scale, offering better product consistency and reduced manufacturing time and cost. To mitigate the potential risk of graft-versus-host disease (GVHD) from the use of non-HLA-matched donor cells, it is crucial to genetically modify allogeneic immune cells (such as allogeneic CAR T cells) to reduce or impair T cell receptor (TCR) αβ function or activity. Therefore, it is desirable to genetically modify allogeneic CAR T cells to reduce or impair the expression of genes involved in T cell receptor (TCR) αβ function or activity while preserving CAR function. Transgenic expression cassettes used for cell genetic modification are typically designed to both target and disrupt one or more genes and insert transgenes encoding target peptides (e.g., receptor peptides, such as chimeric antigen receptors). A key component of such cassettes is the promoter, which controls the expression of the encoded peptide. This disclosure relates to polynucleotides, nucleic acid constructs, or molecules encoding one or more receptor peptides and / or nucleic acid inhibitors (e.g., RNA interference agents), wherein the expression of one or more receptor peptides or inhibitors is controlled by a promoter derived from or obtained from the phosphoglycerate kinase (PGK1) gene (PGK). The promoter may be a truncated PGK promoter.

[0037] This document provides compositions and methods for expressing receptor peptides and / or nucleic acid inhibitors (e.g., RNA interference agents). Furthermore, uses and methods of such compositions for improving the functional activity of cells (e.g., immune cells, such as T cells) are provided. For example, these methods can be used to improve the functional activity of CAR-T cells. The methods and compositions provided herein can be used to improve the therapeutic efficacy of therapeutic cell populations (e.g., immune cell populations, including CAR T cells).

[0038] General technology Unless otherwise specified, the practice of this invention will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the scope of the art. These techniques are well explained in the literature, such as *Molecular Cloning: A Laboratory Manual, 2nd Edition* (Sambrook et al., 1989), Cold Spring Harbor Press; *Oligonucleotide Synthesis* (MJ Gait, ed., 1984); *Methods in Molecular Biology*, Humana Press; *Cell Biology: A Laboratory Notebook* (JE Cellis, ed., 1998), Academic Press; *Animal Cell Culture* (RI Freshney, ed., 1987); *Introduction to Cell and Tissue Culture* (JP Mather and PE Roberts, 1998), Plenum Press; *Cell and Tissue Culture: Laboratory Procedures* (A. Doyle, JB Griffiths, and DG Newell, ed., 1993–1998), J. Wiley and Sons; *Methods in Enzymology* (Academic Press, Inc.); and *Handbook of Experimental Immunology* (DM Weir and CC). Blackwell (ed.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos (ed., 1987); Current Protocols in Molecular Biology (FM Ausubel et al. (ed., 1987); PCR: The Polymerase Chain Reaction (Mullis et al. (ed., 1994); Current Protocols in Immunology (JE)Coligan et al., eds., 1991; Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995).

[0039] definition As used herein, the term "extracellular ligand-binding domain" refers to a polypeptide capable of binding ligands or interacting with cell surface molecules. For example, extracellular ligand-binding domains can be selected to recognize ligands used as cell surface markers on target cells associated with specific disease states.

[0040] The terms “stalk domain” or “hinge domain” are used interchangeably herein to refer to any polypeptide that acts to link a transmembrane domain to an extracellular ligand-binding domain. Specifically, the stalk domain is used to provide greater flexibility and accessibility to the extracellular ligand-binding domain.

[0041] The term "intracellular signal transduction domain" refers to the part of a protein that transduces effector signals and functional signals, guiding the cell to perform specialized functions.

[0042] "Co-stimulatory ligands" refer to molecules on antigen-presenting cells that specifically bind to homologous co-stimulatory signaling molecules on T cells, thereby providing signals. In addition to the primary signals provided by, for example, the binding of the TCR / CD3 complex to peptide-loaded MHC molecules, these signals also mediate T cell responses, including but not limited to proliferation activation and differentiation. Co-stimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin β receptor, 3 / TR6, ILT3, ILT4, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. Costimulatory ligands also include, in particular, antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0043] An "antibody" is an immunoglobulin molecule capable of specifically binding to a target (such as carbohydrates, polynucleotides, lipids, peptides, etc.) through at least one antigen recognition site located in the variable region of an immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments (such as Fab, Fab', F(ab')2, Fv), single-chain (scFv) and domain antibodies (e.g., including shark and camel antibodies), fusion proteins containing antibodies, and any other modified conformation of an immunoglobulin molecule containing an antigen recognition site. Antibodies include any class of antibodies, such as IgG, IgA, IgE, IgD, or IgM (or their subclasses), and antibodies do not necessarily belong to any particular class. Immunoglobulins can be designated into different classes based on the antibody amino acid sequence in the constant region of the antibody heavy chain. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known.

[0044] As used herein, the term “antigen-binding fragment” or “antigen-binding portion” of an antibody refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen. The antigen-binding function of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments covered within the term “antigen-binding fragment” of an antibody include Fab; Fab'; F(ab')2; the Fd fragment consisting of VH and CH1 domains; the Fv fragment consisting of the VL and VH domains of a single arm of the antibody; single-domain antibody (dAb) fragments (Ward et al., Nature 341:544-546, 1989); and isolated complementarity-determining regions (CDRs).

[0045] Antibodies, antigen-binding fragments, antibody conjugates, or peptides that “specifically bind” to a target (e.g., a protein) are terms well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit “specific binding” if its reaction or association with a particular cell or substance is more frequent, faster, longer-lasting, and / or has a greater affinity than its reaction or association with alternative cells or substances. An antibody is said to “specifically bind” to a target if its binding to a target has a greater affinity, easier binding, and / or longer-lasting binding than its binding to other substances. For example, an antibody that specifically binds to a CD19 epitope is an antibody that has a greater affinity, easier binding, and / or longer-lasting binding to that epitope compared to binding to other CD19 epitopes or non-CD19 epitopes. It should also be understood that, by reading this definition, for example, an antibody (or part or epitope) that specifically binds to a first target may or may not specifically bind to a second target. Therefore, “specific binding” does not necessarily require (but may include) exclusive binding. Usually (but not always), when we mention binding, we mean specific binding.

[0046] The “variable region” of an antibody refers to the variable region of the antibody light chain alone or in combination, or the variable region of the antibody heavy chain. As is known in the art, the variable regions of the heavy and light chains each consist of four frame regions (FRs) connected by three complementarity-determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held together tightly by the FRs and contribute to the formation of the antigen-binding site of the antibody with CDRs from the other chain. At least two techniques are used to determine CDRs: (1) techniques based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, (5th edition, 1991, National Institutes of Health, Bethesda MD)); and (2) methods based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., 1997, J. Molec. Biol. 273:927-948). As used herein, CDR may refer to a CDR defined by either method or by a combination of both.

[0047] The “CDR” in a variable domain is an amino acid residue within the variable region, which is identified according to the definitions of Kabat and Chothia, the common definition of Kabat and Chothia, the AbM definition, the contact definition, and / or the conformational definition, or any CDR identification method well known in the art. Antibody CDRs can be identified as hypervariable regions originally defined by Kabat et al. See, for example, Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington DC. The location of the CDR can also be identified as a structural loop structure originally described by Chothia et al. See, for example, Chothia et al., Nature 342:877-883, 1989. Other methods for identifying CDRs include the “AbM definition,” a compromise between Kabat and Chothia, derived using Oxford Molecular’s ​​AbM antibody modeling software (now Accelrys®); or the “contact definition” of CDRs based on observed antigen contact, as described in MacCallum et al., J. Mol. Biol., 262:732-745, 1996. In another method, referred to here as the “conformation definition” of CDRs, the position of the CDR can be identified as a residue that contributes enthalpy to antigen binding. See, for example, Makabe et al., Journal of Biological Chemistry, 283:1156-1166, 2008. Other CDR boundary definitions may not strictly follow one of the methods described above but will still overlap with at least a portion of the Kabat CDR, although they may shorten or lengthen the predicted or experimental results of antigen binding based on specific residues or groups of residues or even the entire CDR, without significantly affecting the specific residues or groups of residues or even the entire CDR. As used herein, a CDR can refer to a CDR defined by any method (including combinations of methods) known in the art. The methods used herein can utilize a CDR defined according to any of these methods. For any given embodiment containing more than one CDR, the CDR can be defined according to any of the following definitions: Kabat, Chothia, extension, AbM, contact, and / or conformation.

[0048] As used herein, a “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising that population are identical except for possible naturally occurring mutations that may be present in trace amounts. Monoclonal antibodies are highly specific (targeting a single antigenic site). Furthermore, unlike polyclonal antibody products, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. The modifier “monoclonal” indicates the characteristic of the antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibody used according to the invention can be prepared by a hybridoma method first described by Kohler and Milstein, Nature 256:495, 1975, or by a recombinant DNA method (such as the method described in U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage libraries generated using techniques described in McCafferty et al., Nature 348:552-554, 1990.

[0049] As used herein, a “humanized” antibody refers to a form of non-human (e.g., mouse) antibody that is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding sequence of the antibody) containing a minimal sequence derived from a non-human immunoglobulin. In one aspect, a humanized antibody is a human immunoglobulin (receptor antibody) in which residues from the receptor complementarity-determining region (CDR) are replaced by CDR residues from a non-human species (donor antibody) such as mouse, rat, or rabbit, possessing the desired specificity, affinity, and capability. In some cases, the Fv frame region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues not present in the receptor antibody or the introduced CDR or frame sequence, but these residues are included for further refinement and optimization of antibody performance. Typically, the humanized antibody will contain at least one and usually substantially all of two variable domains, wherein all or substantially all of the CDR regions correspond to those of the non-human immunoglobulin and all or substantially all of the FR regions are those of the human immunoglobulin common sequence. The humanized antibody may optionally also include at least a portion of an immunoglobulin constant region or structural domain (Fc), typically a portion of the immunoglobulin constant region or structural domain (Fc) of a human immunoglobulin. Antibodies having an Fc region modified as described in WO99 / 58572 are preferred. Other forms of humanized antibodies have one or more CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, or CDR H3) that are altered relative to the original antibody; these CDRs are also referred to as one or more CDRs “derived” from one or more CDRs derived from the original antibody.

[0050] As used herein, "operable linking" refers to the linking of two or more nucleic acid sequence or amino acid sequence elements. For example, a promoter nucleic acid sequence operably linked to a nucleic acid sequence encoding a polypeptide allows the promoter sequence to control the expression of the nucleic acid sequence encoding the polypeptide. The first sequence element can be operably linked to the second sequence element in a contiguous or non-contiguous manner. When two nucleic acid elements are operably linked, the coding regions remain within the same reading frame.

[0051] As used herein, “expression control sequence” refers to the nucleic acid sequence that directs nucleic acid transcription. The expression control sequence can be a promoter (such as a constitutive or inducible promoter) or an enhancer. The expression control sequence is operatively linked to the nucleic acid sequence to be transcribed.

[0052] The terms "promoter" and "promoter sequence" are used interchangeably and refer to the DNA sequence that controls the expression of a coding sequence or functional RNA. Functional RNA can be a protein-coding mRNA transcript or a non-mRNA, such as miRNA, shRNA, or other types of interfering RNA. Generally, the coding sequence is located at the 3' end of the promoter sequence. Those skilled in the art will understand that different promoters can direct gene expression in different tissues or cell types, at different developmental stages, or in response to different environmental or physiological conditions.

[0053] The terms “DNA sequence,” “nucleic acid sequence,” “nucleotide sequence,” and “polynucleotide sequence” used herein are interchangeable. Similarly, the terms “polynucleotide,” “DNA molecule,” and “nucleic acid molecule” used herein are interchangeable.

[0054] As used herein, “human antibody” means an antibody having an amino acid sequence corresponding to that of an antibody produced by a human, and / or prepared using any technique known to those skilled in the art or disclosed herein for the preparation of human antibodies. This definition of human antibody includes antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody comprising both a mouse light chain and a human heavy chain polypeptide. Human antibodies can be produced using a variety of techniques known in the art. In one embodiment, the human antibody is selected from a phage library expressing a human antibody (Vaughan et al., Nature Biotechnology, 14:309-314, 1996; Sheets et al., Proc. Natl. Acad. Sci. (USA) 95:6157-6162, 1998; Hoogenboom and Winter, J. Mol. Biol., 227:381, 1991; Marks et al., J. Mol. Biol., 222:581, 1991). Human antibodies can also be prepared by immunizing animals in which human immunoglobulin loci have been transgenic to replace endogenous loci, for example, mice in which endogenous immunoglobulin genes have been partially or completely inactivated. This method has been described in U.S. Patents 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016. Alternatively, human antibodies can be prepared by immortalizing human B lymphocytes that produce antibodies against the target antigen (such B lymphocytes can be obtained from an individual or a single-cell clone of cDNA, or can be immunized in vitro). See, for example, Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985; Boerner et al., J. Immunol., 147(1):86-95, 1991; and U.S. Patent No. 5,750,373.

[0055] The term "chimeric antibody" is intended to refer to an antibody whose variable region sequence is derived from one species and whose constant region sequence is derived from another species, such as an antibody whose variable region sequence is derived from a mouse antibody and whose constant region sequence is derived from a human antibody.

[0056] Each "monovalent antibody" molecule contains one antigen-binding site (e.g., IgG or Fab). In some cases, a monovalent antibody may have more than one antigen-binding site, but the binding sites may originate from different antigens.

[0057] A bivalent antibody contains two antigen-binding sites (e.g., IgG). In some cases, the two binding sites have the same antigen specificity. However, a bivalent antibody may be bispecific.

[0058] The antibodies of the present invention can be produced using techniques well known in the art, such as recombinant techniques, phage display techniques, synthetic techniques, or combinations of such techniques, or other techniques readily known in the art (see, for example, Jayaseena, SD, Clin. Chem., 45: 1628-50, 1999 and Fellouse, FA et al., J. MoI. Biol., 373(4):924-40, 2007).

[0059] As is known in the art, the terms "polynucleotide" or "nucleic acid," used interchangeably herein, refer to a nucleotide chain of any length and include both DNA and RNA. A nucleotide can be a deoxyribonucleotide, ribonucleotide, modified nucleotide or base and / or its analogues, or any substrate that can be incorporated into the chain by a DNA or RNA polymerase. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and their analogues. Modifications to the nucleotide structure can be conferred, if present, before or after chain assembly. The sequence of a nucleotide can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with labeled components. Other types of modifications include, for example, “caps”; substitution of one or more naturally occurring nucleotides by analogs; internucleotide modifications, such as those with non-electrolyte links (e.g., methylphosphonates, triphosphates, aminophosphates, carbamates, etc.) and those with charged links (e.g., thiophosphates, dithiophosphates, etc.); those containing overhanging moieties (e.g., proteins, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.); those with intercalating agents (e.g., acridine, psoralen, etc.); those containing chelating agents (e.g., metals, radioactive metals, boron, oxidizing metals, etc.); those containing alkylating agents; those with modified links (e.g., α-anomeric nucleic acids, etc.); and unmodified forms of one or more polynucleotides. Furthermore, any of the hydroxyl groups commonly present in sugars can be substituted (e.g., replaced by phosphonate or phosphate groups), protected by standard protecting groups, or activated to prepare additional links with other nucleotides, or can be conjugated to a solid support. The 5' and 3' OH groups can be phosphorylated or partially substituted with an amine or an organic capping group of 1 to 20 carbon atoms. Other hydroxyl groups can also be derived as standard protecting groups. The polynucleotide may also contain similar forms of ribose or deoxyribose known in the art, including, for example, 2'-O-methylribose, 2'-O-allylribose, 2'-fluororibose, or 2'-azidoribose; carbocyclic sugar analogs; α-anomeric or β-anomeric sugars; epimeric sugars such as arabinose, xylose, or lythose, pyranose, furanose, or sedoheptulose; acyclic analogs; and baseless nucleoside analogs such as methylribonucleotides. One or more phosphodiester bonds can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate ester is replaced by P(O)S (“sulfate”), P(S)S (“disulfate”), (O)NR2 (“amino ester”), P(O)R, P(O)OR', CO, or CH2 (“methylal”), wherein each R or R' is independently H or a substituted or unsubstituted alkyl group (1-20 Cs) (optionally containing an ether (-O-) bond), aryl, alkenyl, cycloalkyl, cycloalkenyl, or aromatic aldehyde. Not all links in the polynucleotide need to be identical.The preceding description applies to all polynucleotides mentioned in this article, including RNA and DNA.

[0060] As used in this article, "transfection" refers to the process by which cells absorb exogenous or heterologous RNA or DNA. When exogenous or heterologous RNA or DNA is introduced into a cell, the cell is "transfected" by that RNA or DNA. When the transfected RNA or DNA causes phenotypic changes, the cell is "transformed" by the exogenous or heterologous RNA or DNA. Transformed RNA or DNA can be integrated (covalently linked) into the chromosomal DNA that makes up the cell's genome.

[0061] As used herein, the term "homologous arm" refers to a nucleic acid sequence located at the 5' and 3' ends of a polynucleotide containing a sequence encoding a transgene of interest. Generally, homologous arms are homologous to regions in the genome containing nuclease cleavage sites located within target genes of interest, such as genes involved in the function or activity of T-cell receptor (TCR) αβ. For example, homologous arms are designed to be homologous to genomic regions flanked by nuclease cleavage sites. Homologous arms can be designed to have appropriate lengths such that they facilitate the insertion of polynucleotides into nuclease cleavage sites within target genes of interest. For example, a homologous arm may contain at least or about 50 base pairs, at least or about 75 base pairs, at least or about 100 base pairs, at least or about 125 base pairs, at least or about 150 base pairs, at least or about 175 base pairs, at least or about 200 base pairs, at least or about 300 base pairs, at least or about 400 base pairs, at least or about 500 base pairs, at least or about 600 base pairs, at least or about 700 base pairs, at least or about 800 base pairs, at least or about 900 base pairs, or at least or about 1000 base pairs.

[0062] As used in this article, "transformation" refers to the transfer of nucleic acid fragments into the genome of a host organism, thereby producing genetically stable genetic material. Host organisms containing transformed nucleic acid fragments are referred to as "transgenic," "recombinant," or "transformed" organisms.

[0063] As is known in the art, the “constant region” of an antibody refers to the constant region of the antibody light chain, alone or in combination, or the constant region of the antibody heavy chain.

[0064] As used herein, “substantially pure” means material that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or even at least 99% pure (i.e. free of contaminants).

[0065] "Host cell" includes a single cell or cell culture that can be, or has been, a recipient of a vector for integrating a polynucleotide insert. Host cells include progeny of a single host cell due to natural, accidental, or intentional mutation, and the progeny need not be identical to the original parent cell (morphologically or in complementary sequences of genomic DNA). Host cells include cells transfected in vivo with the polynucleotides of the present invention.

[0066] As is known in the art, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The "Fc region" can be a native sequence Fc region or a variant Fc region. While the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the Fc region of the human IgG heavy chain is generally defined as an extension from amino acid residue at position Cys226 or amino acid residue at position Pro230 to its C-terminus. The Fc region residues are numbered using the EU index in Kabat. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md., 1991). The Fc region of an immunoglobulin typically contains two constant regions, CH2 and CH3.

[0067] As used in the art, “Fc receptor” and “FcR” describe receptors that bind to the Fc region of an antibody. Preferred FcRs are naturally occurring human FcRs. Furthermore, preferred FcRs are receptors that bind IgG antibodies (γ receptors) and include receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splice forms of these receptors. FcγRII receptors include FcγRIIA (“activating receptor”) and FcγRIIB (“inhibiting receptor”) with similar amino acid sequences, differing primarily in their cytoplasmic domains. FcRs are reviewed in Ravetch and Kinet, Ann. Rev. Immunol., 9:457-92, 1991; Capel et al., Immunomethods, 4:25-34, 1994; and de Haas et al., J. Lab. Clin. Med., 126:330-41, 1995. "FcR" also includes the neonatal receptor FcRn, which is responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol., 117:587, 1976; and Kim et al., J. Immunol., 24:249, 1994).

[0068] As used herein with respect to antibodies, the term "competition" means that the binding of a first antibody or its antigen-binding fragment (or a portion thereof) to an epitope is sufficiently similar to the binding of a second antibody or its antigen-binding portion to the epitope, such that the binding of the first antibody to its homologous epitope in the presence of the second antibody is detectably reduced compared to the binding of the first antibody in the absence of the second antibody. Alternative scenarios in which the binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody may occur, but are not necessarily so. That is, the first antibody may inhibit the binding of the second antibody to its epitope, while the second antibody may not inhibit the binding of the first antibody to its corresponding epitope. However, when each antibody detectably inhibits the binding of another antibody to its homologous epitope or ligand, regardless of the degree of inhibition being the same, greater, or less, the antibodies are said to "cross-compete" with each other to bind to their respective epitopes. This invention encompasses competitive antibodies and cross-competitive antibodies. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or a portion thereof), based on the teachings provided herein, those skilled in the art will understand that such competing and / or cross-competing antibodies are covered by and can be used in the methods disclosed herein.

[0069] As used herein, “autologous” means that the cells, cell lines, or cell populations used to treat the patient originate from the patient.

[0070] As used in this article, "allogeneic" means that the cells or cell populations used to treat the patient did not originate from the patient, but from the donor.

[0071] As used herein, the term “endogenous” means any material that originates from or is produced within an organism, cell, tissue, or system.

[0072] As used herein, the term "exogenous" means any material introduced or generated from outside an organism, cell, tissue, or system. In some embodiments, the exogenous or recombinant sequence or protein is not a naturally occurring sequence or protein and is not endogenous or native to the cell, tissue, or organism.

[0073] As used herein, “immune cells” refers to hematopoietic cells that are functionally involved in the initiation and / or execution of innate and / or adaptive immune responses. Examples of immune cells include T cells (e.g., α / β T cells and γ / δ T cells, regulatory T (Treg) cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.

[0074] As used herein, “treatment” is a method for achieving a beneficial or desired clinical outcome. For the purposes of this disclosure, a beneficial or desired clinical outcome includes, but is not limited to, one or more of the following: reducing (or destroying) the proliferation of tumor cells or cancer cells, inhibiting the metastasis of tumor cells, shrinking or reducing the size of a tumor, alleviating a disease (e.g., cancer), reducing symptoms caused by a disease (e.g., cancer), increasing the quality of life of a patient suffering from a disease (e.g., cancer), reducing the dosage of other medications required to treat a disease (e.g., cancer), delaying the progression of a disease (e.g., cancer), curing a disease (e.g., cancer), and / or prolonging the survival of a subject suffering from a disease (e.g., cancer).

[0075] "Improvement" means that one or more symptoms are reduced or improved compared to the absence of treatment. "Improvement" also includes shortening or reducing the duration of symptoms. As used herein, an "effective dose" or "effective amount" of a drug, compound, or pharmaceutical composition is an amount sufficient to achieve any one or more beneficial or desired results. For preventative use, beneficial or desired results include eliminating or reducing the risk of disease, reducing the severity of disease, or delaying the onset of disease, including biochemical, histological, and / or behavioral symptoms of disease, complications, and intermediate pathological phenotypes that occur during disease development. For therapeutic use, beneficial or desired results include clinical outcomes such as reducing the incidence of various diseases or disorders (e.g., cancer) or improving one or more of their symptoms, reducing the dosage of other drugs required to treat the disease, enhancing the effect of another drug, and / or delaying disease progression. An effective dose may be administered in a single or multiple doses. For the purposes of this disclosure, an effective dose of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve preventative or therapeutic treatment. As understood in a clinical setting, an effective dose of a drug, compound, or pharmaceutical composition may or may not be achieved when used in combination with another drug, compound, or pharmaceutical composition. Therefore, an "effective dose" can be considered in the context of administering one or more therapeutic agents, and a single agent can be considered to be administered in an effective amount if the desired effect can or has been achieved when used in combination with one or more other agents.

[0076] As used herein, "subject" is any mammal, such as a human or a monkey. Mammals include, but are not limited to, livestock, animals, pets, primates, horses, dogs, cats, mice, and rats. In one exemplary embodiment, the subject is a human. In one exemplary embodiment, the subject is a monkey, such as a cynomolgus monkey.

[0077] As used herein, the terms “nucleic acid construct,” “recombinant construct,” or “expression cassette” are used interchangeably and refer to single-stranded or double-stranded nucleic acid molecules or polynucleotides. Such constructs are recombinant and contain combinations of different nucleic acid fragments, including but not limited to promoters and coding sequences. Generally, this combination of fragments does not exist in nature. For example, the promoter sequence of one gene can be combined with the coding sequence of another gene. Constructs can be used alone or in combination with vectors. In one embodiment, an expression cassette is one or more expression units of one or more coding sequences operatively linked to a promoter. In some embodiments, the expression of multiple coding sequences can be driven by a single promoter, and more than one coding sequence can be linked by a sequence encoding a 2A peptide (e.g., P2A or T2A). In some embodiments, the expression of multiple coding sequences is carried out via ribosome jumping. As an example, a bicistronic expression cassette allows the expression of two proteins from the same RNA transcript driven by a single promoter. In some embodiments, the expression of multiple coding sequences can be driven by multiple promoters, each operatively linked to a coding sequence.

[0078] As used herein, a "functionally expressed" gene means a gene that is expressed and that expression produces a functional genetic end product. For example, if a gene encodes a protein, then the cell functionally expresses the gene if the expression of the gene ultimately produces a protein with an appropriate function. Therefore, for example, if a gene is not transcribed, or if the expression of the gene ultimately produces untranslated or non-functional RNA (e.g., the protein is not properly folded or is not transported to its site of action (e.g., the membrane, for membrane-bound proteins)), then the gene is not functionally expressed. Functional expression can be measured directly (e.g., by measuring the gene product itself) or indirectly (e.g., by measuring the function of the gene product).

[0079] As used herein, "vector" or "recombinant vector" means a construct capable of delivering one or more genes or sequences of interest into a host cell and preferably capable of expressing said genes or sequences. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells. Vectors may include, but are not limited to, viral vectors, recombinant AAV vectors, and other vectors known to be suitable for delivering transgenes into cells (e.g., immune cells). Vectors may be based on various viruses, including but not limited to retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses (AAVs).

[0080] An "antigen-binding protein" comprises one or more antigen-binding domains. As used herein, "antigen-binding domain" means any polypeptide that binds to a specified target antigen. In some embodiments, the antigen-binding domain binds to antigens on tumor cells. In some embodiments, the antigen-binding domain binds to antigens on cells involved in hyperproliferative diseases or to viral or bacterial antigens.

[0081] Antigen-binding domains include, but are not limited to, antibody-binding regions that serve as immune functional fragments. The term "immune functional fragment" (or "fraction") in the term antigen-binding domain refers to an antigen-binding domain containing a portion of an antibody (regardless of how that portion is obtained or synthesized), which lacks at least some amino acids present in the full-length chain, but is still capable of specifically binding to a target antigen. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding domains (including intact antibodies) for binding to a given epitope.

[0082] Immunofunctional immunoglobulin fragments include, but are not limited to, scFv fragments, Fab fragments (Fab′, F(ab′)2, etc.), one or more complementarity-determining regions (“CDRs”), biantibodies (heavy chain variable domains on the same polypeptide as the light chain variable domain, linked via short peptide linkers that are too short to allow pairing between two domains on the same chain), domain antibodies, bivalent antigen-binding domains (containing two antigen-binding sites), multispecific antigen-binding domains, and single-chain antibodies. These fragments can be derived from any mammalian source, including but not limited to humans, mice, rats, camels, or rabbits. As those skilled in the art will appreciate, antigen-binding domains may include non-protein components.

[0083] The variable region typically exhibits the same overall structure as the relatively conservative frame region (FR), joined by three supervariable regions (CDRs). The CDRs from the two chains of each pair are usually aligned through the frame region, allowing them to bind to specific epitopes. From the N-terminus to the C-terminus, both the light and heavy chain variable regions typically contain the structural domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. By convention, the CDR regions in the heavy chain are usually referred to as HC CDR1, CDR2, and CDR3. The CDR regions in the light chain are usually referred to as LC CDR1, CDR2, and CDR3.

[0084] In some embodiments, the antigen-binding domain comprises one or more complementary binding regions (CDRs) present in the full-length light or heavy chain of the antibody, and in some embodiments comprises a single heavy chain and / or a portion thereof. These fragments can be generated using recombinant DNA technology, or can be generated by enzymatic or chemical cleavage of the antigen-binding domain, including the intact antibody.

[0085] In some embodiments, the antigen-binding domain is an antibody or a fragment thereof, including one or more of its complementarity-determining regions (CDRs). In some embodiments, the antigen-binding domain is a single-chain variable fragment (scFv) comprising light chain CDRs: CDR1, CDR2, and CDR3, and heavy chain CDRs: CDR1, CDR2, and CDR3.

[0086] The assignment of amino acids to each of the framework, CDR, and variable domains generally follows the following numbering scheme: Kabat numbering (see, for example, Kabat et al. in Sequences of Proteins of Immunological Interest, 5th edition, NIH Publication 91-3242, Bethesda Md. 1991); Chothia numbering (see, for example, Chothia and Lesk, (1987), J Mol Biol 196: 901-917; Al-Lazikani et al., (1997) J Mol Biol 273: 927-948; Chothia et al., (1992) J Mol Biol 227: 799-817; Tramontano et al., (1990) J Mol Biol 215(1): 175-82; and U.S. Patent No. 7,709,226); contact numbering; AbM scheme (Antibody Modeling program, Oxford). Molecular) or AHo system (Honneger and Pluckthun, J Mol Biol (2001) 309(3):657-70).

[0087] In some embodiments, the antigen-binding domain is a recombinant antigen receptor. As used herein, the term “recombinant antigen receptor” broadly refers to a non-naturally occurring surface receptor that includes an extracellular antigen-binding domain or an extracellular ligand-binding domain, a transmembrane domain, and an intracellular domain. In some embodiments, the recombinant antigen receptor is a chimeric antigen receptor (CAR). Chimeric antigen receptors (CARs) are well known in the art. A CAR is a fusion protein that includes an antigen recognition portion, a transmembrane domain, and a T-cell activation domain (see, for example, Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2): 720-724 (1993)).

[0088] In some embodiments, the intracellular domain of the recombinant antigen receptor includes a co-stimulatory domain and an ITAM-containing domain. In some embodiments, the intracellular domain of the recombinant antigen receptor contains an intracellular protein or a functional variant thereof (e.g., truncated, inserted, deleted, or substituted).

[0089] As used herein, the terms "extracellular ligand-binding domain" or "extracellular antigen-binding domain" refer to a polypeptide capable of binding a ligand or antigen. Preferably, the domain will be capable of interacting with cell surface molecules, such as ligands or surface antigens. For example, the extracellular ligand-binding or antigen-binding domain may be selected to recognize ligands used as cell surface markers (e.g., tumor-specific antigens) on target cells associated with a specific disease state. In some embodiments, the antigen-binding domain comprises an antibody or an antigen-binding fragment or antigen-binding moiety of an antibody. In some embodiments, the antigen-binding domain comprises Fv or scFv, Fab or scFab, F(ab')2 or scF(ab')2, Fd, a monobody, an affibody, a camelid antibody, a VHH antibody, a single-domain antibody, or a darpin. In some embodiments, the ligand-binding domain comprises a partner of a binding pair, such as a ligand binding to a surface receptor, or an extracellular domain of a surface receptor binding to a ligand.

[0090] The terms “stalk domain” or “hinge domain” are used interchangeably herein to refer to any oligopeptide or polypeptide that acts to link a transmembrane domain to an extracellular ligand-binding domain. Specifically, stalk or hinge domains are often used to provide greater flexibility and accessibility to the extracellular ligand-binding domain.

[0091] The term "intracellular signal transduction domain" refers to the part of a protein that transduces effector signals and functional signals, guiding the cell to perform specialized functions.

[0092] As used herein, “pharmaceuticalally acceptable carrier” or “pharmaceuticalally acceptable excipient” includes any material that, when combined with an active ingredient, allows the ingredient to remain biologically active and does not react with the subject’s immune system. Examples include, but are not limited to, any standard pharmaceutical carrier such as phosphate-buffered saline solution, water, emulsions such as oil / water emulsions, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate-buffered saline (PBS) or physiological (0.9%) saline containing such carriers. The compositions of this disclosure are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, ed. A. Gennaro, Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy 21st Ed. Mack Publishing, 2005).

[0093] When this document refers to a value or parameter “about”, it includes (and describes) embodiments involving adding or subtracting 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% from that value or parameter itself. For example, a description of “about X” includes a description of “X”. Numerical ranges include numbers that define the range.

[0094] It should be understood that wherever the implementation is described in the language "includes", similar implementations are also provided for other aspects described in the terms "comprises of" and / or "substantially constitutes of".

[0095] Where aspects or embodiments of this disclosure are described in groupings according to Markush groups or other alternatives, this disclosure covers not only the entire group listed as a whole, but also each member of the group individually, as well as all possible subgroups of the main group, and the main group that has no one or more group members. This disclosure also contemplates the explicit exclusion of one or more of any group members in the disclosed and / or claimed embodiments.

[0096] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In case of conflict, the specification, including the definitions, shall prevail. Throughout this specification and the claims, the word “comprise” or variations such as “comprises” or “comprising” shall be construed as implying inclusion of the stated integer or group of integers, but not excluding any other integer or group of integers. Unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms.

[0097] Exemplary methods and materials are described herein, but similar or equivalent methods and materials may also be used in practice or testing of this disclosure. The materials, methods, and embodiments are illustrative only and are not intended to be limiting.

[0098] This document provides engineered cells, such as engineered immune cells, including engineered T cells, that contain and / or express an antigen-specific chimeric antigen receptor (CAR) (e.g., a CD19-specific CAR) and a CD70-binding protein, and optionally contain and / or express a chimeric cytokine receptor (CCR) and / or a nucleic acid inhibitor, such as an RNA interference agent. This document also provides engineered cells, such as engineered immune cells, including engineered T cells, that contain and / or express an antigen-specific CAR (e.g., a CD19-specific CAR) and a chimeric cytokine receptor (CCR), and optionally contain and / or express a CD70-binding protein and / or a nucleic acid inhibitor (e.g., an RNA interference agent). In some embodiments, the engineered cells (e.g., engineered immune cells) are human. In other embodiments, the engineered cells (e.g., engineered immune cells) are derived from or obtained from human cells (e.g., human immune cells).

[0099] This document further provides polynucleotides comprising one or more coding sequences encoding an antigen-specific CAR (e.g., a CD19-specific CAR) and a CD70-binding protein or CCR, and / or a nucleic acid inhibitor (e.g., an RNA interference agent). In some embodiments, the antigen-specific CAR (e.g., a CD19-specific CAR) and the CD70-binding protein or CCR and / or the nucleic acid inhibitor (e.g., an RNA interference agent) are expressed by a bicistronic expression cassette and linked via a P2A or T2A self-cleaving peptide. In some aspects, antigen-specific cells (e.g., CD19-specific immune cells, such as CD19-specific CAR T cells) exhibit enhanced cytotoxicity and potency against antigen-positive and / or antigen-negative / CD70-positive cells (e.g., CD19-positive and / or CD19-negative / CD70-positive hematologic malignancies) compared to the same antigen-specific cells (e.g., CD19-specific immune cells, such as CD19-specific CAR T cells) that do not express the CD70-binding protein and / or CCR, with reduced and increased persistence and cell proliferation against host or patient allogeneic reactive immune cells. In some implementations, the expression of one or more coding sequences or bicistronic expression boxes is driven by a PGK promoter, particularly a truncated PGK promoter.

[0100] In some aspects, the polynucleotides provided herein are cloned into lentiviral vectors (LVVs) and introduced into engineered cells, such as engineered immune cells, like peripheral blood mononuclear cells (PBMCs), via lentiviral transduction. Transducing the LVV construct into PBMCs produces engineered cells with randomly integrated transgenes in the host cell genome. Alternatively, transgenes can be introduced into cells via site-specific integration (SSI) into one or more predetermined genetic loci. Another benefit of site-specific integration compared to random LVV integration is ensuring consistency of the insertion site of the transgene in the genome and reducing the number of integration events. Therefore, in some aspects, the polynucleotides provided herein are integrated into predetermined loci in the genome of engineered cells (e.g., engineered immune cells, such as CD19-specific CAR T cells). In some embodiments, the predetermined genetic locus is the T cell receptor α chain constant region (TRAC) locus. In some embodiments, the predetermined genetic locus is the CD52 locus.

[0101] Surprisingly, PGK promoters, or truncated PGK promoters as described herein (e.g., PGK SSI promoters), have been shown to provide benefits to engineered cells (e.g., engineered immune cells, including CAR T cells), such as enhanced CAR T cell expansion and / or enhanced in vitro or in vivo cytotoxicity against target tumor cells when the transgene is introduced via site-specific integration. Therefore, in some embodiments, the expression of a transgene (e.g., one or more coding sequences, such as those in a bicistronic or polycistronic expression cassette) is driven by a PGK promoter, particularly a truncated PGK promoter as described herein. In some embodiments, one or more coding sequences are present in a bicistronic or polycistronic expression cassette. In some embodiments, the transgene includes a receptor polypeptide, such as a CAR, CCR, CD70 binding protein, dominant or negative receptor, or other sequence or sequence encoding other proteins or recombinant proteins, and / or a nucleic acid inhibitor (e.g., an RNA interference agent).

[0102] In some embodiments, engineered immune cells generated through site-specific integration exhibit surprisingly improved in vivo antitumor activity compared to the EF1α short (EFS) promoter when the expression of transgenes (e.g., CD19-specific CARs) is driven by a PGK promoter (especially a truncated PGK promoter, such as the PGKSSI promoter described herein). In some embodiments, engineered immune cells generated through site-specific integration exhibit surprisingly improved in vivo antitumor activity compared to the EFS promoter when the expression of transgenes (e.g., CD19-specific CARs and CCRs) is driven by a PGK promoter (such as the PGKSSI promoter described herein). In some embodiments, engineered immune cells generated through site-specific integration exhibit surprisingly improved in vivo antitumor activity compared to the EFS promoter when the expression of transgenes (e.g., CD19-specific CARs and CD70-binding proteins) is driven by a PGK promoter (such as the PGKSSI promoter described herein). In some embodiments, the polynucleotide is integrated into, for example, the TRAC locus. In some embodiments, the polynucleotide is integrated into the CD52 locus.

[0103] 1. Nucleic acid constructs and vectors This disclosure relates to engineered cells, such as engineered immune cells, comprising one or more transgenes integrated into the genome of the engineered cell, and methods for preparing and using engineered cells. In one embodiment, the transgene is integrated into the genome, thereby disrupting a target gene. The target gene may be a gene involved in the function or activity of T cell receptor (TCR) αβ. In other embodiments, the expression of the transgene is controlled by a promoter that is not a promoter of the target gene. In another embodiment, the promoter is a non-endogenous or exogenous promoter relative to the target gene. In one embodiment, the promoter is a truncated PGK promoter. In other embodiments, the expression of the transgene is controlled by a truncated PGK promoter. In some embodiments, the engineered cell, the target gene, and / or the promoter (e.g., a truncated PGK promoter) are human. In other embodiments, the engineered cell (e.g., an engineered immune cell) is human.

[0104] In one aspect, this disclosure provides polynucleotide or nucleic acid constructs or molecules comprising one or more coding sequences or transgenes. In some embodiments, the polynucleotide, nucleic acid constructs or molecules according to the invention comprise a donor template. In another embodiment, the donor template comprises one or more transgenes containing sequences encoding receptor polypeptides and / or nucleic acid inhibitors (e.g., RNA interference agents), said sequences being controlled by a promoter. In one embodiment, the promoter may originate from another gene not directly involved in the function or activity of T cell receptor (TCR) αβ. In one embodiment, the promoter comprises a nucleic acid sequence derived from a gene, including all or part of the original promoter sequence. In one embodiment, the gene is the phosphoglycerate kinase (PGK1) gene (PGK). In another embodiment, this disclosure provides a truncated PGK promoter comprising a portion of the nucleic acid sequence of the original PGK promoter sequence derived from, obtained from, or originating from the PGK gene. In some embodiments, the PGK gene is the human PGK gene.

[0105] In one embodiment, one or more transgenes comprise a truncated PGK promoter to control the expression of one or more sequences encoding one or more peptides or agents. In other embodiments, the truncated PGK promoter comprises one or more deletions in a promoter sequence from a corresponding promoter sequence of a genomic region containing the PGK gene. Table 1 provides exemplary PGK promoter sequences and / or regions constituting a PGK promoter.

[0106] Table 1 – Promoters ( Bold, underlined text = transcription start site)

[0107] In one embodiment, the PGK promoter is derived from a genomic region containing the PGK gene. For example, the PGK promoter may be derived from a genomic region containing the PGK gene in the major assembly of Homo sapiens X chromosome GRCh38.p14 (NCBI reference sequence: NC_000023.11). In another embodiment, the PGK promoter is derived from a polynucleotide sequence corresponding to approximately 78,103,669 to 78,104,334 nucleotide positions in the genomic region containing the PGK gene in the major assembly of Homo sapiens X chromosome GRCh38.p14 (NCBI reference sequence: NC_000023.11).

[0108] In an additional aspect, the truncated PGK promoter described herein contains one or more nucleic acid sequences that bind to or are predicted to bind to one or more transcription factors. Alternatively, the truncated PGK promoter described herein may a) lack one or more nucleic acid sequences that bind to or are predicted to bind to one or more transcription factors, or b) be characterized by the lack of one or more nucleic acid sequences that bind to or are predicted to bind to one or more transcription factors. The promoter sequences described herein can be analyzed using a transcription factor binding site prediction database called JASPAR. JASPAR is an open-access database that stores an artificially compiled transcription factor (TF) binding spectrum in the form of a position frequency matrix (PFM), which summarizes the occurrence of each nucleotide at each position in an observed set of TF-DNA interactions. PFM can be used to scan any DNA sequence to predict TF binding sites. In other embodiments, the transcription factors are human transcription factors.

[0109] In one embodiment, the truncated PGK promoter comprises a nucleic acid sequence that binds to or is predicted to bind to a transcription factor selected from the group consisting of: ZNF692, ZNF257, YY1, TEAD4, TEAD1, TCF4, TCF3, TBX3, SP1, SOX18, RFX7, MYOG, MYF5, KLF9, KLF6, KLF4, KLF10, HIF1, FIGLA, DUXA, DUX4, CTCFL, BHLHE22, BHLHA15, ASCL1, and ARNT::HIF1A. In another embodiment, the transcription factor is one or more of YY1, RFX7, and HIF1. In yet another embodiment, the truncated PGK promoter comprises the nucleotide sequence of SEQ ID NO: 35, or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 35.

[0110] In another embodiment, the truncated PGK promoter is characterized by the absence of a nucleic acid sequence that binds to or is predicted to bind to a transcription factor selected from the following groups: ZNF281, ZNF148, VEZF1, TLX2, TFAP2E, TFAP2C, TFAP2B, TFAP2A, SP8, SOX15, RELB, RBPJ, PRDM1, PITX2, NKX3-2, NKX2-8, NKX2-3, NFKB1, NFATC2, MSX2, MEIS1, KLF16, KLF15 The sequence numbers are: KLF11, ISL2, HOXD4, HOXD12, HOXD11, HOXD10, HOXC9, HOXC4, HOXC12, HOXC11, HOXC10, HOXB9, HOXB7, HOXB4, HOXA9, HOXA4, HOXA1, GSX2, FOXD2, FOXC1, EN2, EGR1, EBF1, E2F8, E2F6, E2F4, E2F1, DRGX, CEBPE, CEBPD, CEBPB, BARHL2, and BARHL1. In another embodiment, the truncated PGK promoter comprises the nucleotide sequence of SEQ ID NO: 35, or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 35.

[0111] In one implementation, the truncated PGK promoter comprises a nucleic acid sequence that binds to or is predicted to bind to a transcription factor selected from the group consisting of: ZNF692, ZNF257, YY1, TEAD4, TEAD1, TCF4, TCF3, TBX3, SPI1, SP1, SOX18, SOX10, RUNX2, RFX7, PLAGL2, OSR2, OSR1, MYOG, MYF5, KLF9, KLF6, KLF4, KLF10, IKZF1, HSF4, HIF1A, FLI1, FIGLA, FEV, ETV5, ETV4, ETS2, ERG, ELK4, ELK1, ELF5, ELF1, DUXA, DUX4, CTCFL, BHLHE22, BHLHA15, ASCL1, and ARNT::HIF1A. In another embodiment, the transcription factor is one or more of YY1, RFX7, HIF1A, and ETS2. In another embodiment, the truncated PGK promoter comprises the nucleotide sequence of SEQ ID NO: 36, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 36.

[0112] In another embodiment, the truncated PGK promoter is characterized by the absence of a nucleic acid sequence that binds to or is predicted to bind to a transcription factor selected from the following groups: ZNF281, ZNF148, VEZF1, TLX2, TFAP2C, TFAP2B, TFAP2A, SP8, SOX15, RELB, RBPJ, PRDM1, PITX2, NKX3-2, NKX2-8, NKX2-3, NFKB1, NFATC2, MSX2, KLF16, KLF15, KLF11 The transcription factors are ISL2, HOXD4, HOXD12, HOXD11, HOXD10, HOXC9, HOXC4, HOXC12, HOXC11, HOXC10, HOXB9, HOXB7, HOXB4, HOXA9, HOXA4, HOXA1, GSX2, FOXD2, FOXC1, EN2, EGR1, EBF1, E2F8, E2F6, E2F4, E2F1, DRGX, CEBPE, CEBPD, CEBPB, BARHL2, and BARHL1. In another embodiment, the transcription factor is one or more of NFKB1, NFATC2, EGR1, and CEBPB. In another embodiment, the truncated PGK promoter comprises the nucleotide sequence of SEQ ID NO: 36, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 36.

[0113] In one implementation, the truncated PGK promoter contains a nucleic acid sequence that binds to or is predicted to bind to a transcription factor selected from the group consisting of: ZNF692, ZNF257, YY1, TEAD4, TEAD1, TCF4, TCF3, TBX3, STAT3, STAT1, SPI1, SPDEF, SP1, SOX18, SOX10, RUNX2, RHOXF1, RFX7, PLAGL2, OSR2, OSR1, MYOG, MYF5, KLF9, KLF6, KLF4, KLF3, KLF10, IKZF1, HSF4, HIF1A, FLI1, FIGLA, FEV, ETV5, ETV4, ETS2, ERG, ELK4, ELK1, ELF5, ELF1, DUXA, DUX4, CTCFL, CREB1, BHLHE22, BHLHA15, ASCL1, and ARNT::HIF1A. In another embodiment, the transcription factor is one or more of YY1, STAT3, STAT1, RFX7, and ETS2. In another embodiment, the truncated PGK promoter comprises the nucleotide sequence of SEQ ID NO: 37, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 37.

[0114] In another embodiment, the truncated PGK promoter is characterized by the absence of a nucleic acid sequence that binds to or is predicted to bind to a transcription factor selected from the following groups: ZNF281, ZNF148, VEZF1, TLX2, TFAP2C, TFAP2B, TFAP2A, SOX15, RELB, RBPJ, PRDM1, PITX2, NKX3-2, NKX2-8, NKX2-3, NFKB1, NFATC2, MSX2, KLF16, KLF15, ISL2. HOXD4, HOXD12, HOXD11, HOXD10, HOXC9, HOXC4, HOXC12, HOXC11, HOXC10, HOXB9, HOXB7, HOXB4, HOXA9, HOXA4, HOXA1, GSX2, FOXD2, FOXC1, EN2, EGR1, EBF1, E2F8, E2F6, E2F4, E2F1, DRGX, CEBPE, CEBPD, CEBPB, BARHL2, and BARHL1. In another embodiment, the transcription factor is one or more of NFKB1, NFATC2, EGR1, and CEBPB. In another embodiment, the truncated PGK promoter comprises the nucleotide sequence of SEQ ID NO: 37, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 37.

[0115] In one embodiment, the truncated PGK promoter includes one or more deletions, insertions, or substitutions in the PGK promoter, as shown in SEQ ID NO: 34. In some embodiments, one or more deletions in the truncated PGK promoter include one or more 5' region deletions and / or one or more 3' region deletions. In other embodiments, the 5' region deletion includes deletions from SEQ ID NO: The 5' end of 34 is missing approximately 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, and 34. 5. Approximately 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, or approximately 390 nucleotides, and related to SEQ. The nucleotide sequence of SEQ ID NO: 35 has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. In some embodiments, the truncated PGK promoter comprises, is substantially composed of, or is composed of the nucleotide sequence of SEQ ID NO: 35.

[0116] In other embodiments, the 5' region deletion includes deletions of approximately 210, approximately 211, approximately 212, approximately 213, approximately 214, approximately 215, approximately 216, approximately 217, approximately 218, approximately 219, approximately 220, approximately 221, approximately 222, approximately 223, approximately 224, approximately 225, approximately 226, approximately 227, approximately 228, approximately 229, approximately 230, approximately 231, approximately 232, approximately 233, approximately 234, approximately 235, approximately 236, approximately 237, approximately 238, approximately 239, approximately 240, approximately 241, approximately 242, approximately 243, approximately 244, approximately 245, approximately 246, approximately 247, approximately 248, approximately 249, and approximately 25 from the 5' end of SEQ ID NO: 34. 0, about 251, about 252, about 253, about 254, about 255, about 256, about 257, about 258, about 259, about 260, about 261, about 262, about 263, about 264, about 265, about 266, about 267, about 268, about 269, about 270, about 271, about 272, about 273, about 274, about 275, about 276, about 277, about 278, about 279, about 280, about 281, about 282, about 283, about 284, about 285, about 286, about 287, about 288, about 289, or about 290 nucleotides, and related to SEQ The nucleotide sequence of SEQ ID NO: 36 has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. In some embodiments, the truncated PGK promoter comprises, is substantially composed of, or is composed of the nucleotide sequence of SEQ ID NO: 36.

[0117] In other implementations, the 5' region deletion includes from SEQ ID NO: The 5' end of 34 is missing approximately 110, approximately 111, approximately 112, approximately 113, approximately 114, approximately 115, approximately 116, approximately 117, approximately 118, approximately 119, approximately 120, approximately 121, approximately 122, approximately 123, approximately 124, approximately 125, approximately 126, approximately 127, approximately 128, approximately 129, approximately 130, approximately 131, approximately 132, approximately 133, approximately 134, approximately 135, approximately 136, approximately 137, approximately 138, approximately 139, approximately 140, approximately 141, approximately 142, approximately 143, approximately 144, approximately 145, approximately 146, approximately 147, approximately 148, approximately 149, approximately 150, approximately 151, approximately 152, approximately 153, approximately 154, approximately 15 5. Approximately 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or approximately 200 nucleotides, and related to SEQ. The nucleotide sequence of SEQ ID NO: 37 has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. In some embodiments, the truncated PGK promoter comprises, is substantially composed of, or is composed of the nucleotide sequence of SEQ ID NO: 37.

[0118] In other embodiments, the 5' region deletion includes the deletion of about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, or about 100 nucleotides from the 5' end of SEQ ID NO: The nucleotide sequence of SEQ ID NO: 38 has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. In some embodiments, the truncated PGK promoter comprises, is substantially composed of, or is composed of the nucleotide sequence of SEQ ID NO: 38.

[0119] In some embodiments, the truncated PGK promoter is substantially composed of or constitutes a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the nucleotide sequence of SEQ ID NO: 35, 36, 37, or 38.

[0120] On the other hand, the polynucleotide, nucleic acid constructs, or molecules described herein include a donor template. The donor template may contain one or more transgenic or coding sequences encoding one or more receptor peptides and / or one or more nucleic acid inhibitors, such as RNA interference agents, as described herein. In other embodiments, the donor template includes a promoter, such as a truncated PGK promoter, to control the expression of one or more transgenic or coding sequences.

[0121] In other embodiments, the polynucleotide, nucleic acid construct, or molecule comprises a donor template, which can be integrated into the transgene via homologous recombination. In one embodiment, the donor template comprises a transgene having a sequence encoding a target molecule (e.g., a receptor peptide). The target molecule, such as the receptor peptide, can be an antigen-binding molecule. In another embodiment, the antigen-binding molecule can be part of a chimeric antigen receptor (CAR), or the antigen-binding molecule is a CAR. In one embodiment, the receptor peptide is a CAR that includes an antigen-binding domain. In other embodiments, the template comprises a sequence encoding a chimeric cytokine receptor (CCR) or CD70-binding protein. In one embodiment, the sequence encoding the target molecule is controlled by a PGK promoter (e.g., a truncated PGK promoter).

[0122] In other embodiments, the polynucleotide, nucleic acid construct, or molecule further comprises one or more homologous sequences that are homologous to a portion of the target nucleic acid sequence in the target gene. In one embodiment, the target gene is a gene involved in the function or activity of T cell receptor (TCR) αβ. In another embodiment, the gene is a constant domain of the TCRα gene (TRAC gene). In yet another embodiment, the target is one or more of the components of the TCR, HLA, TCRα, and TCRβ. In other embodiments, the target gene is a human gene.

[0123] In one embodiment, the target gene is a gene expressed by host immune cells, which, upon administration to a subject, participate in a potential immune response against engineered cells (e.g., engineered immune cells, such as CAR T cells). In other embodiments, the host immune cells are human. Clinical data suggest that temporarily depleting host immune cells at the start of CAR T cell therapy is advantageous. Temporary depletion can be achieved by administering an agent that targets and depletes host immune cells expressing the target gene. In one embodiment, cells (e.g., immune cells) need to be genetically modified to reduce or impair the expression of the target gene. In one embodiment, the target gene is CD52. A binder targeting CD52 (e.g., an anti-CD52 antibody) is administered to the subject before, during, or after administration of engineered cells (e.g., engineered immune cells) to the subject in need. In another embodiment, the binder depletes host immune cells expressing CD52 but does not bind to engineered cells with reduced or impaired CD52 expression. In yet another embodiment, the binder does not deplete engineered cells with reduced or impaired CD52 expression. In one embodiment, the anti-CD52 antibody is alemtuzumab (SEQ ID NO:67-74).

[0124] In some implementations, polynucleotides, nucleic acid constructs, or molecules are used to generate engineered cells, such as immune cells, resistant to one or more chemotherapeutic agents. The chemotherapeutic agents can be, for example, purine nucleotide analogs (PNAs), thereby adapting the immune cells for cancer treatment in combination with adoptive immunotherapy and chemotherapy. Exemplary PNAs include, for example, clofarabine, fludarabine, cyclophosphamide, and cytarabine, alone or in combination. PNAs are metabolized by deoxycytidine kinase (dCK) to monophosphate, diphosphate, and triphosphate PNAs. Their triphosphate forms compete with ATP for DNA synthesis, act as pro-apoptotic agents, and are potent inhibitors of ribonucleotide reductase (RNR), which is involved in trinucleotide production.

[0125] In another embodiment, the target gene is deoxycytidine kinase (dCK). Certain PNAs can lymphode host immune cells. DCK-deficient cells are known to be resistant to therapies such as lymphodepletion therapy. In one embodiment, the target gene is dCK. A lymphodepletion compound, such as PNA, is administered to a subject before, during, or after administration of engineered cells (e.g., engineered immune cells) to the subject in need. In another embodiment, the lymphodepletion compound depletes host immune cells expressing dCK, but does not deplete engineered cells whose dCK expression is reduced or impaired.

[0126] In another embodiment, the target gene is the glucocorticoid receptor (GR), such as human GR. Certain glucocorticoids, such as dexamethasone, are known to have immunosuppressive effects on host immune cells. In one embodiment, the target gene is GR. A glucocorticoid (e.g., dexamethasone) is administered to a subject before, during, or after the administration of engineered cells (e.g., engineered immune cells) to a subject in need. In another embodiment, the glucocorticoid suppresses the immune response of host immune cells expressing GR, but does not suppress engineered cells where GR expression is reduced or impaired.

[0127] In another embodiment, the target gene is a checkpoint inhibitor. Certain immune receptors, such as PD-1, PDL1, and CTLA-4, are involved in the suppression of host immune cells. In one embodiment, the target gene is a checkpoint inhibitor. A conjugate targeting one or more checkpoint inhibitors (e.g., anti-PD-1, anti-PDL1, or anti-CTLA4 antibody) is administered to the subject before, during, or after administration of engineered cells (e.g., engineered immune cells) to the subject in need. In another embodiment, the conjugate depletes host immune cells expressing the checkpoint inhibitor but does not bind to engineered cells with reduced or impaired checkpoint inhibitor expression. In another embodiment, the conjugate does not deplete engineered cells with reduced or impaired checkpoint inhibitor expression. In another embodiment, the target gene is one or more of PD-1, PDL1, and CTLA-4. In other embodiments, the target gene is a human gene.

[0128] On the other hand, target genes are molecules involved in the rejection and / or recognition of engineered cells (e.g., engineered immune cells as described herein) by host immune cells. In one embodiment, the target gene is a gene involved in the presentation of various peptides, such as the TAP2 component of antigen-processing-associated transporters (TAPs). The primary pathway for MHC class I molecules to load peptides is TAP-dependent: peptides generated by the proteasome (or IFN-γ-inducible immunoproteasome) are transported to the endoplasmic reticulum (ER) via TAP and then loaded onto MHC class I molecules. Compared to the significant reduction (10-100-fold) of surface β2 microglobulin (β2m) in β2m KO cells, knockout of TAP2 resulted in only a slight reduction in surface β2m (a 2-fold reduction after selecting KO cells) (see PCT / US2022 / 14393). Figure 4A (The patent mentioned herein is incorporated herein by reference in its entirety.) In another embodiment, the target gene is TAP2 and / or β2m. In other embodiments, the target gene is a human gene.

[0129] In another embodiment, the target gene is a gene involved in regulating the transcription of HLA-I and HLA-II molecules. HLA-I and HLA-II molecules are tightly regulated at the transcriptional level by similar key cis-regulatory elements: W / S, X1, X2, and Y-box motifs. The regulatory factor X (RFX) heterologous complex contains RFX5, RFXAP, and RFXANK and binds to the X1 box. The X2 box is occupied by CREB / ATF1 family transcription factors, and the Y box is bound by the NF-Y protein. Furthermore, nucleotide-binding domains and two members of the leucine-rich repeat receptor (NLR) family, NLRC5 and CIITA, are essential for the formation of the HLA enhancer complex, thereby promoting the transcription of HLA-I and HLA-II. In one embodiment, the target gene is one or more of TAP2, NLRC5, β2m, CIITA, RFX5, RFXAP, and RFXANK. In other embodiments, the target gene is a human gene.

[0130] In another embodiment, the target gene is a cell surface receptor involved in immune cell adhesion and activation at the immune synapse. Certain cell surface molecules (including but not limited to CD58 and CD2, CD48 and CD2 and / or ICAM-1 and LFA-1) interact to provide appropriate cell adhesion and immune cell activation (Dustin, ML The immunologicalsynapse, Cancer Immunol Res. 2014 Nov;2(11):1023-33). Furthermore, the loss of CD58 is associated with tumor cell resistance to T cell-mediated killing and immune escape (Frangieh, CJ et al. Multimodalpooled Perturb-CITE-seq screens in patient models define mechanisms of cancer immune evasion. Nat Genet. Mar 2021;53(3):332-341, e-book March 1, 2021; Challa-Malladi, M. et al., Combined genetic inactivation of β2-Microglobulin and CD58 reveals frequent escape from immune recognition in diffuse large B cell lymphoma. Cancer Cell. Dec 13, 2011;20(6):728-40, e-book December 1, 2011). In one embodiment, the target gene is one or more of CD48, CD58, and ICAM-1. In other embodiments, the target gene is a human gene.

[0131] In another aspect of this disclosure, the target gene of the engineered cell may be one or more genes encoding one or more molecules that function in one or more cellular pathways involving the rejection of host or recipient immune cells that respond to T-cell and NK-cell epitope determinants on the surface of an allogeneic cell product different from the host. In one embodiment, the target gene is one or more genes encoding: i) cell surface receptors known to function in immune cell adhesion and immune synapse activation (e.g., one or more of CD48, CD58, and ICAM-1); and / or ii) transcription factors or regulators of HLA-I and HLA-II molecules (e.g., RFX5, NLRC5, CIITA, RFXAP, and RFXANK). The methods described herein for generating engineered cells with reduced or eliminated expression of one or more genes may focus solely on genes encoding molecules that function at the immune synapse, but may also be supplemented by the reduction or elimination of expression of genes encoding molecules that have a major function as transcription factors for HLA-I and / or HLA-II molecules.

[0132] In another embodiment, the target gene is CD70, such as human CD70. CD70 is expressed on the surface of immune cells (e.g., T cells, especially activated T cells), and CD70-binding proteins can cause lysis of such CD70-expressing immune cells (see PCT / US2022 / 33598). Figure 1A (The patent mentioned herein is incorporated herein by reference in its entirety.) In some embodiments, the donor template comprises a homologous arm targeting CD70 and a transgene encoding a CD70-binding protein. In another embodiment, the engineered cells (e.g., engineered immune cells) have reduced or impaired CD70 expression and express the CD70-binding protein. Upon administration of the engineered cells (e.g., engineered immune cells) to a subject in need, the CD70-binding protein causes lysis of the host immune cells expressing CD70, but does not cause lysis of other engineered cells whose CD70 expression is reduced or impaired.

[0133] In other embodiments, the polynucleotide, nucleic acid construct, or molecule includes a donor template that can be integrated into a double-strand break site in the host cell genome via homologous recombination. In one embodiment, the donor template includes one or more, at least two, or more coding sequences or transgenes. In one embodiment, the expression of one or more, at least two, or more coding sequences or transgenes is controlled by a PGK promoter (e.g., a truncated PGK promoter).

[0134] In other embodiments, the polynucleotide, nucleic acid construct, or molecule further comprises one or more homologous sequences that are homologous to a portion of the integration site in the engineered immune cell genome (for site-specific integration). In one embodiment, the integration site is a gene (target site or target gene) involved in the function or activity of T cell receptor (TCR) αβ. In another embodiment, the gene is the TCRα constant region (TRAC) gene. In yet another embodiment, the integration site is located in the TRAC (a component of TCR), HLA, TCRα, TCRβ, β2-microglobulin (“β2m”), CD52, GR, deoxycytidine kinase (DCK), PD-1, and CTLA-4 genes.

[0135] In other embodiments, the polynucleotide, nucleic acid construct, or molecule includes a 5' homologous arm having a sequence homologous to a 5' sequence of a double-strand break in the target nucleic acid sequence located in the target gene. In another embodiment, the polynucleotide, nucleic acid construct, or molecule further includes a 3' homologous arm having a sequence homologous to a 3' sequence of a double-strand break in the target nucleic acid sequence located in the target gene. When the polynucleotide, nucleic acid construct, or molecule is delivered to a cell and after the target nucleic acid sequence is cleaved, homologous recombination occurs between the target nucleic acid sequence and one or more homologous sequences of the polynucleotide, nucleic acid construct, or molecule.

[0136] Nucleotide sequence homology exists in the upstream and downstream flanking regions of double-strand break sites and genome integration sites, and the nucleic acid sequence to be introduced (e.g., one or more transgenes) is located between two homologous arms. In some embodiments, in the context of site-specific integration, the terms 5' homologous arm and 3' homologous arm, as well as the terms upstream and downstream flanking regions, are used to refer to the orientation of the target site or target gene for genome integration.

[0137] Nucleotide sequence homology exists in the upstream and downstream flanking regions of double-strand break sites and genome integration sites, and the nucleic acid sequence to be introduced (e.g., one or more transgenes) is located between two homologous arms. In some embodiments, in the context of site-specific integration, the terms 5' homologous arm and 3' homologous arm, as well as the terms upstream and downstream flanking regions, are used to refer to the orientation of the target site or target gene for genome integration.

[0138] In some embodiments, double-strand breaks can be generated by rare cleavage endonucleases, such as, but not limited to, zinc finger endonucleases, TALE endonucleases, or CRISPR. Polynucleotides, nucleic acid constructs, or molecules can be integrated into the desired integration site of the genome via homologous recombination between the 5' and 3' homologous arms and between homologous sequences at the integration site. In some embodiments, 5' and 3' homologous sequences are provided in Table 3, for example, SEQ ID NO: 42-45.

[0139] In one embodiment, the polynucleotide, nucleic acid construct, or molecule comprises a donor template including a 5' homologous arm, a 3' homologous arm, a transgene, and a promoter, such as a truncated PGK promoter. In other embodiments, the polynucleotide, nucleic acid construct, or molecule comprises a 5' homologous arm, a first sequence encoding a first receptor polypeptide, a second sequence encoding a second receptor polypeptide, a 3' homologous arm, and a promoter, such as a truncated PGK promoter.

[0140] In some embodiments, the orientation of one or more coding sequences or transgenes is the same as or opposite to the orientation of the target site for genome integration. In some embodiments, the polynucleotide, nucleic acid construct, or molecule includes a donor template comprising a 5' homologous arm, a promoter, one or more coding sequences or transgenes, and a 3' homologous arm. In some embodiments, the promoter comprises a truncated PGK promoter, such as the PGKSSI-1, PGK SSI-2, or PGK SSI-3 promoters as described herein.

[0141] Figures 8A to 8D Examples of different configurations of polynucleotides, nucleic acid constructs, or molecules containing transgenes and promoters are described. Figure 8A (Left figure) depicts a polynucleotide, nucleic acid construct or molecule containing a donor template having a first homologous arm (HA1), a PGK promoter (e.g., a truncated PGK promoter), a receptor polypeptide sequence, and a second homologous arm (HA2). Figure 8A (Right figure) depicts a polynucleotide, nucleic acid construct or molecule having a donor template comprising a first homologous arm (HA1), a PGK promoter (e.g., a truncated PGK promoter), a first receptor polypeptide sequence, an adapter sequence, a second receptor polypeptide sequence, and a second homologous arm (HA2). Figure 8B The polynucleotide, nucleic acid construct or molecule is described having a donor template comprising a first homologous arm (HA1), a PGK promoter (e.g., a truncated PGK promoter), a CAR sequence (including sequences encoding scFv, CD8 / hinge region, 4-1BB costimulatory region and CD3ζ region), and a second homologous arm (HA2). Figure 8CThe polynucleotide, nucleic acid construct or molecule is described having a donor template comprising a first homologous arm (HA1), a PGK promoter (e.g., a truncated PGK promoter), a chimeric cytokine receptor sequence, a linker sequence, a CAR sequence (including sequences encoding scFv, CD8 / hinge region, 4-1BB co-stimulatory region and CD3ζ region), and a second homologous arm (HA2).

[0142] In one aspect, this disclosure provides a variety of polynucleotide, nucleic acid constructs or molecules that target different genes of interest in the same cell. In one embodiment, a first nucleic acid construct (or polynucleotide or molecule) comprises a donor template having a homologous arm of a first target gene, a PGK promoter (e.g., a truncated PGK promoter), and a first receptor polypeptide sequence, while a second nucleic acid construct (or polynucleotide or molecule) comprises a homologous arm of a second target gene, a promoter that is not a truncated PGK promoter, and a second receptor polypeptide sequence. Both the first and second nucleic acid constructs (or polynucleotides or molecules) can be used to modify the first and second target genes in the same cell. Figure 8D (Left figure) depicts a first nucleic acid construct (or polynucleotide or molecule) having a donor template containing a first transgene, the first transgene having a homologous arm that targets and modifies a first gene. The first nucleic acid construct (or polynucleotide or molecule) has a first donor template containing a first homologous arm (HA1), a PGK promoter (e.g., a truncated PGK promoter), a first receptor polypeptide sequence, and a second homologous arm (HA2). Figure 8D (Right figure) depicts a second nucleic acid construct (or polynucleotide or molecule) having a second donor template containing a second transgene, the second transgene having a homologous arm targeting a second gene for modification. The second nucleic acid construct (or polynucleotide or molecule) includes a third homologous arm (HA3), a promoter that is not a truncated PGK promoter, a second receptor polypeptide sequence, and a fourth homologous arm (HA4). In one embodiment, the promoter that is not a truncated PGK promoter includes an EFS promoter or an EFL promoter.

[0143] In one aspect, the nucleic acid construct (or polynucleotide or molecule) comprises a donor template having a promoter (e.g., a truncated PGK promoter) positioned such that the promoter can initiate transcription in the same or opposite direction as the target gene of interest, which is controlled by an endogenous promoter. In one embodiment, the promoter is located between the transgene and the 3' homologous arm, allowing transcription to proceed from the end of the 3' homologous arm to the end of the 5' homologous arm. In other embodiments, the promoter initiates transcription in the opposite direction to the transcription direction normally occurring with the endogenous promoter of the target gene of interest. This promoter-transgene configuration is advantageous because it allows the promoter (e.g., the PGK promoter) to initiate the transcription of the transgene without any interference from the endogenous promoter.

[0144] Figures 7A to 7B The process of inserting transgenes (such as CAR) into genes of interest is described. Figure 7A This describes the insertion of a CAR into the TRAC locus via site-specific integration and homologous recombination (TRAC knock-in or TRAC KI). The P2A peptide is derived from the *Thosea asigna* virus. As shown by the large dashed arrow in the figure above, transcription of the TRAC endogenous promoter typically proceeds in one direction. After CAR insertion into the TRAC locus, transcription of the CAR-associated promoter proceeds in the opposite direction, as shown by the smaller solid arrow in the figure below. Figure 7B This describes the insertion of a CAR into the CD52 locus via site-specific integration and homologous recombination (CD52 knock-in or CD52 KI). As shown by the large dashed arrow in the figure above, transcription of the CD52 endogenous promoter typically proceeds in one direction. After CAR insertion into the CD52 locus, transcription of the CAR-related promoter proceeds in the opposite direction, as shown by the smaller solid arrow in the figure below. Figure 7A and Figure 7B The illustrations use adeno-associated virus 6 (AAV6) as an example to illustrate site-specific integration of CARs under non-endogenous or exogenous promoters, relative to endogenous TRAC or CD52 promoters. LHA and RHA: left and right homologous arms, respectively. In one embodiment, the inserted sequence encoding the CAR is controlled by a PGK promoter (e.g., a truncated PGK promoter).

[0145] Figure 7AThe process of inserting a transgene with a promoter into a TRAC gene is described, such that transcription of the transgene can be initiated in the opposite direction to that of the endogenous TRAC promoter. Alternatively, the promoter is located between the transgene and the 5' homologous arm, such that transcription can be initiated from the end of the 5' homologous arm to the end of the 3' homologous arm. In other embodiments, the promoter initiates transcription in the same direction as the transcription of the target gene of interest by the endogenous promoter. In one embodiment, the non-endogenous promoter is a PGK promoter, for example, a truncated PGK promoter.

[0146] In one embodiment, the polynucleotide, nucleic acid construct, or molecule comprises a donor template comprising, in the 5' to 3' direction: (a) a 5' homologous arm, a transgene, a truncated PGK promoter, and a 3' homologous arm; or (b) a 5' homologous arm, a truncated PGK promoter, a transgene, and a 3' homologous arm. In other embodiments, the polynucleotide, nucleic acid construct, or molecule has a donor template comprising, in the 5' to 3' direction: (a) a 5' homologous arm, a first sequence encoding a first receptor polypeptide, a second sequence encoding a second receptor polypeptide, a truncated PGK promoter, and a 3' homologous arm; or (b) a 5' homologous arm, a truncated PGK promoter, a first sequence encoding a first receptor polypeptide, a second sequence encoding a second receptor polypeptide, and a 3' homologous arm.

[0147] In another embodiment, the polynucleotide, nucleic acid construct, or molecule includes a donor template having a transgene having a first and a second sequence with the same orientation (opposite to each other). For example, the first and second sequences may be arranged in a 5' to 3' or 3' to 5' orientation relative to a homologous arm in the nucleic acid construct (or polynucleotide or molecule). Furthermore, the first and second sequences may be arranged in a 5' to 3' or 3' to 5' orientation relative to a promoter in the nucleic acid construct (or polynucleotide or molecule) such that a promoter (e.g., a truncated PGK promoter) can initiate transcription in a 5' to 3' orientation. The first sequence may be located at a 5' or 3' position relative to the second sequence. In one embodiment, the first sequence has a 5' to 3' orientation, while the second sequence has a 3' to 5' orientation.

[0148] In some embodiments, stoichiometric co-expression of the first and second receptor peptides is achieved using a linker sequence between the two sequences. The linker sequence may encode a P2A peptide from the *Melilothorax glabra* virus, as further described below.

[0149] In one aspect, the polynucleotide, nucleic acid construct, or molecule has a donor template containing a nucleic acid repressive sequence, such as a nucleic acid inhibitor, or an RNA interference agent containing one or more RNA interference sequences. The donor template may have the nucleic acid repressive sequence alone or in combination with a sequence encoding a receptor polypeptide. Upon introduction into cells, the RNA interference sequence reduces the expression level of the target gene relative to comparable cells that do not have said sequence. This reduction in expression level may be expression knockout or knockdown. Different knockdown methods may be suitable, such as employing various RNA-based technologies (e.g., short hairpin RNA (shRNA), antisense RNA, microRNA (miRNA), small (or short) interfering RNA (siRNA); See For example, Van Hoeck et al., Biomaterials, Vol. 286, July 2022, 121510, ISSN 0142-9612; Lam et al. people [Mol. Ther.-Nucleic Acids 4:e252 (2015), doi:10.1038 / mtna.2015.23; Sridharan and Gogtay, Brit. J. Clin. Pharmacol. 82: 659-72 (2016), and U.S. Patent No. 9,556,433 to Krause et al., all of which are incorporated herein by reference in their entirety.] In another embodiment, the RNA interference agent is a microRNA-adapted shRNA, wherein the miRNA scaffold contains one or more shRNA sequences. RNA-based agents can be delivered to cells (e.g., immune cells) to knock out one or more genes. In one embodiment, the RNA-based agent can be configured to target one or more genes or be targeted by one or more genes. In one embodiment, the length of the RNA interference sequence can be between about 15 and about 30 nucleotides. In another embodiment, a nucleic acid inhibitor (e.g., an RNA interference agent containing one or more RNA interference sequences) reduces the expression of genes involved in the function or activity of T cell receptor (TCR) αβ. In other embodiments, the gene is selected from TRAC (a component of TCR), HLA, TCRα, TCRβ, β2-microglobulin (“β2m”), CD52, GR, deoxycytidine kinase (DCK), PD-1, and CTLA-4.

[0150] In one aspect, the polynucleotide, nucleic acid construct, or molecule includes a donor template having a homologous arm, an RNA interference sequence, and a promoter, such as a truncated PGK promoter. In one embodiment, the donor template further includes a receptor polypeptide sequence. In another embodiment, the polynucleotide, nucleic acid construct, or molecule has a donor template comprising, in a 5' to 3' orientation: (a) a 5' homologous arm, a truncated PGK promoter, an RNA interference sequence, and a 3' homologous arm, or (b) a 5' homologous arm, an RNA interference sequence, a truncated PGK promoter, and a 3' homologous arm. In yet another embodiment, the donor template includes a first RNA interference sequence and a second RNA interference sequence with the same orientation (opposite to each other). For example, the first and second RNA interference sequences may be arranged in a 5' to 3' orientation or a 3' to 5' orientation relative to the homologous arm in the nucleic acid construct (or polynucleotide or molecule). Furthermore, the first and second RNA interference sequences can be arranged in the nucleic acid construct (or polynucleotide or molecule) in a 5' to 3' or 3' to 5' orientation relative to the promoter, such that the promoter (e.g., a truncated PGK promoter) can initiate transcription in a 5' to 3' orientation. The first RNA interference sequence may be located at a 5' or 3' position relative to the second RNA interference sequence. In one embodiment, the first RNA interference sequence has a 5' to 3' orientation, while the second RNA interference sequence has a 3' to 5' orientation.

[0151] On the other hand, polynucleotides, nucleic acid constructs, or molecules are provided as part of one or more recombinant vectors (e.g., virus-based vectors) suitable for introduction into one or more cells (e.g., immune cells) to modify a target gene of interest. In some embodiments, the vector comprises a donor template containing homologous arm sequences homologous to the target gene sequence. In one embodiment, the target gene is a gene involved in the function or activity of T cell receptor (TCR) αβ. In another embodiment, the gene is a TCRα gene constant domain (TRAC gene). In another embodiment, the recombinant virus-based vector is adenovirus-associated virus (AAV). In one embodiment, the AAV vector contains AAV serotype sequences, such as viral inverted terminal repeat (ITR) sequences. Polynucleotides, nucleic acid constructs, or molecules may contain one or more AAV serotype sequences, including but not limited to AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-DJ, or AAV-DJ / 8 serotype sequences. It is possible to generate recombinant AAV vectors incorporating nucleic acid sequences containing transgenes, and to produce recombinant AAV particles to infect cells (e.g., immune cells) in order to modify target genes of interest.

[0152] In some embodiments, the vector comprises a donor template, which further comprises a transgene. In another embodiment, the transgene comprises a polynucleotide encoding a receptor polypeptide and / or an RNA interference sequence. In some embodiments, the polynucleotide encoding the receptor polypeptide and / or the RNA interference sequence is present in the expression vector for stable expression in cells. In some embodiments, the polynucleotide is present in a viral vector for stable expression in cells. In some embodiments, the viral vector may be, for example, an AAV vector.

[0153] In one aspect, this disclosure provides expression vectors containing polynucleotides, nucleic acid constructs, or molecules, as described herein. In one embodiment, the expression vector comprises a polynucleotide, nucleic acid construct, or molecule having a donor template comprising a 5' homologous arm, a transgene, a promoter (e.g., a truncated PGK promoter), and a 3' homologous arm. The donor template in the vector may comprise, in the 5' to 3' direction: (a) a 5' homologous arm, a transgene, a truncated PGK promoter, and a 3' homologous arm; or (b) a 5' homologous arm, a truncated PGK promoter, a transgene, and a 3' homologous arm. In other embodiments, the donor template in the vector comprises, in the 5' to 3' direction: (a) a 5' homologous arm, a first sequence encoding a first receptor polypeptide (or a first nucleic acid inhibitor or a second nucleic acid inhibitor), a second sequence encoding a second receptor polypeptide (or a first nucleic acid inhibitor or a second nucleic acid inhibitor), a truncated PGK promoter, and a 3' homologous arm; or (b) a 5' homologous arm, a truncated PGK promoter, a first sequence encoding a first receptor polypeptide (or a first nucleic acid inhibitor or a second nucleic acid inhibitor), a second sequence encoding a second receptor polypeptide (or a first nucleic acid inhibitor or a second nucleic acid inhibitor), and a 3' homologous arm.

[0154] On the other hand, this disclosure provides a method for preparing any nucleic acid construct or molecule, expression vector, or polynucleotide comprising the nucleic acid sequence described herein. This disclosure also covers polynucleotides complementary to any such sequence. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA, or synthetic) or RNA molecules. RNA molecules include HnRNA molecules containing introns and corresponding one-to-one with DNA molecules, and mRNA molecules without introns. The polynucleotides of this disclosure may, but do not necessarily, contain additional coding or non-coding sequences, and the polynucleotides may, but do not necessarily, be linked to other molecules and / or supporting materials.

[0155] On the other hand, this disclosure provides variants of the nucleic acid sequences described herein. A nucleic acid variant of a given polynucleotide may contain a deletion and / or insertion at at least one nucleotide position in the given polynucleotide. In one embodiment, a variant of a given polynucleotide is configured such that the open reading frame of the variant is preserved. The variant preferably has at least about 70% identity with the polynucleotide sequence described herein, more preferably at least about 80% identity, more preferably at least about 90% identity, and most preferably at least about 95% identity.

[0156] Two polynucleotide or polypeptide sequences are said to be "identical" if the nucleotide or amino acid sequences are identical when best aligned as described below. Typically, comparisons between two sequences are performed by comparing sequences within a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 20 consecutive positions, typically 30 to about 75 positions or 40 to about 50 positions, within which a sequence can be compared to a reference sequence having the same number of consecutive positions, provided that the two sequences have been best aligned.

[0157] Using the Megalign program in the Lasergene Bioinformatics Software Suite (DNASTAR, Inc., Madison, Wl), with default parameters, optimal sequence alignment can be performed for comparison. This procedure embodies several alignment schemes described in the following references: Dayhoff, MO, 1978, A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, MO (edited) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC, Vol. 5, Supplement 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes, pp. 626-645; Methods in Enzymology, Vol. 183, Academic Press, Inc., San Diego, CA; Higgins, DG and Sharp, PM, 1989, CABIOS 5:151-153; Myers, EW and Muller W., 1988, CABIOS 4:11-17; Robinson, ED, 1971, Comb. Theor. 11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath, PHA and Sokal, RR, 1973, Numerical Taxonomythe Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, WJ and Lipman, DJ, 1983, Proc. Natl. Acad. Sci. USA80:726-730.

[0158] Preferably, the "sequence identity percentage" is determined by comparing two best-aligned sequences within a comparison window of at least 20 positions, wherein a portion of the polynucleotide or polypeptide sequence in the comparison window may contain 20% or less, typically 5% to 15% or 10% to 12%, of additions or deletions (i.e., vacancies) compared to a reference sequence (excluding additions or deletions) for best-alignment of the two sequences. The percentage is calculated as follows: the number of positions in the two sequences where the same nucleic acid residues or amino acid bases are present is determined to produce the number of matching positions; the number of matching positions is divided by the total number of positions in the reference sequence (i.e., the window size); and the result is multiplied by 100 to produce the sequence identity percentage.

[0159] Variants can also, or alternatively, be substantially homologous to the natural gene or a portion thereof or its complementary sequence. Such polynucleotide variants are capable of hybridizing with naturally occurring DNA sequences encoding natural antibodies (or complementary sequences) under moderately stringent conditions.

[0160] Suitable “moderately stringent conditions” include: prewashing in a solution of 5X SSC, 0.5% SDS, and 1.0 mM EDTA (pH 8.0); hybridization overnight at 50-65°C with 5X SSC; followed by washing twice at 65°C with 2X, 0.5X, and 0.2X SSC containing 0.1% SDS, for 20 minutes each time.

[0161] The “highly stringent conditions” or “highly stringent conditions” used in this article are: (1) washing with low ionic strength and high temperature, for example, 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; (2) using denaturing agents such as formamide during hybridization, for example, 50% (v / v) formamide, 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer (pH 6.5) containing 750 mM sodium chloride and 75 mM sodium citrate at 42°C; or (3) using salmon sperm DNA treated with 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, and sonication at 42°C. The sample was washed with 0.2×SSC (sodium chloride / sodium citrate) at 42°C, followed by washing with 50% formamide at 55°C, and then a high-rigidity wash with 0.1×SSC containing EDTA at 55°C. Skilled technicians will know how to adjust the temperature, ionic strength, etc., as needed to accommodate factors such as probe length.

[0162] Those skilled in the art will understand that, due to the degeneracy of the genetic code, there are many nucleotide sequences that encode polypeptides as described herein. Some of these polynucleotides have very low homology to the nucleotide sequences of any natural gene. However, this disclosure specifically considers polynucleotides that differ due to codon usage. Furthermore, alleles of genes containing the polynucleotide sequences provided herein are within the scope of this disclosure. An allele is an endogenous gene altered by one or more mutations (such as nucleotide deletions, additions, and / or substitutions). The resulting mRNA and protein structure or function may (but not necessarily) be altered. Alleles can be identified using standard techniques such as hybridization, amplification, and / or database sequence comparison.

[0163] The polynucleotides disclosed herein can be obtained using chemical synthesis, recombination methods, or PCR. Chemical polynucleotide synthesis methods are well known in the art and need not be described in detail herein. Those skilled in the art can use the sequences provided herein and commercial DNA synthesizers to generate the desired DNA sequences.

[0164] For the preparation of polynucleotides using recombinant methods, a polynucleotide containing the desired sequence can be inserted into a suitable vector, and the vector can then be introduced into a suitable host cell for replication and amplification, as discussed further herein. Polynucleotides can be inserted into host cells by any means known in the art. Cells are transformed by introducing exogenous polynucleotides through direct uptake, endocytosis, transfection, F-crossing, or electroporation. Once introduced, the exogenous polynucleotide can be maintained intracellularly as a non-integrating vector (such as a plasmid) or integrated into the host cell genome. The amplified polynucleotide can then be isolated from the host cell using methods well-known in the art. See, for example, Sambrook et al., 1989.

[0165] Alternatively, PCR allows for the replication of DNA sequences. PCR technology is well known in the art and is described, for example, in U.S. Patent Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202, and PCR: The Polymerase Chain Reaction, edited by Mullis et al., Birkauswer Press, Boston, 1994.

[0166] Therefore, this article provides a polynucleotide comprising a promoter and at least a first coding sequence and a second coding sequence, wherein the first coding sequence or the second coding sequence encodes an antigen-specific CAR (e.g., a CD19-specific CAR).

[0167] RNA can be obtained by using isolated DNA in a suitable vector and inserting it into a suitable host cell. When the cell replicates and transcribes DNA into RNA, RNA can be isolated using methods well known to those skilled in the art, such as those described above, as in Sambrook et al., 1989.

[0168] Suitable cloning vectors can be constructed according to standard techniques or selected from a large number of cloning vectors available in the art. While the chosen cloning vector may vary depending on the intended host cell, useful cloning vectors typically possess self-replicating capabilities, may have a single target against a specific restriction endonuclease, and / or carry genes that can be used to select clones containing said vectors. Suitable examples include plasmids and bacterial viruses such as pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial suppliers such as BioRad, Stragene, and Invitrogen.

[0169] Expression vectors are typically replicable polynucleotide constructs containing polynucleotides according to this disclosure. This means that the expression vector must be able to replicate in the host cell either as a free organism or as part of the chromosomal DNA as a whole. Suitable expression vectors include, but are not limited to, plasmids, viral vectors (including adenoviruses, adeno-associated viruses, and retroviruses), granules, and the expression vectors disclosed in PCT Publication WO 87 / 04462. Vector components typically include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; and suitable transcriptional control elements (such as promoters, enhancers, and terminators). For expression (i.e., translation), one or more translational control elements are also typically required, such as a ribosome binding site, a translation initiation site, and a stop codon.

[0170] Vectors containing the polynucleotide of interest can be introduced into host cells by a variety of appropriate means, including electroporation; transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-glucan, or other substances; microbolite bombardment; liposome transfection; and infection (e.g., where the vector is an infectious pathogen, such as vaccinia virus). The choice of vector or polynucleotide for introduction generally depends on the characteristics of the host cell.

[0171] Polynucleotides encoding receptor polypeptides (e.g., one or more of CAR, CCR, or CD70 binding proteins) and / or nucleic acid inhibitors (e.g., RNA interference agents) disclosed herein may be present in expression cassettes or expression vectors (e.g., plasmids for introduction into bacterial host cells, or viral vectors (such as baculovirus vectors) for transfection of insect host cells, or plasmids or viral vectors (e.g., lentiviruses) for transfection of mammalian host cells). In some embodiments, the polynucleotide or vector may contain a nucleic acid sequence encoding a ribosomal jumping sequence, such as, but not limited to, a sequence encoding a 2A peptide. The 2A peptide has been identified in the foot-and-mouth disease virus subgroup of piconenucleoviruses, which causes the ribosome to “jump” from one codon to the next without forming a peptide bond between the two amino acids encoded by the two codons (see (Donnelly and Elliott 2001; Atkins, Wills et al. 2007; Doronina, Wu et al. 2008)). A “codon” refers to three nucleotides on mRNA (or on the sense strand of a DNA molecule) that are translated by the ribosome into one amino acid residue. Therefore, two polypeptides can be synthesized from a single, consecutive open reading frame within mRNA, with the polypeptides separated by a 2A oligopeptide sequence within the frame. This type of ribosome jumping mechanism is well known in the art and is known to be used by several vectors to express several proteins encoded by a single messenger RNA.

[0172] This document further provides polynucleotides comprising one or more coding sequences encoding a CAR and a CD70-binding protein or a CCR. In some embodiments, the CAR and CD70-binding protein or CCR is expressed by a bicistronic expression cassette and linked via a P2A or T2A peptide. In some embodiments, expression of one or more coding sequences or bicistronic expression cassettes is driven by a PGK promoter, particularly a truncated PGK promoter.

[0173] To guide transmembrane polypeptides into the secretory pathway of host cells, in some embodiments, a secretion signal sequence (also known as a leader sequence, prepro sequence, or pre-sequence) is provided in a polynucleotide sequence or vector sequence. The secretion signal sequence is operatively linked to the transmembrane nucleic acid sequence, i.e., the two sequences are joined at the correct reading frame and positioned to guide the newly synthesized polypeptide into the secretory pathway of the host cell. The secretion signal sequence is typically located at the 5' of the nucleic acid sequence encoding the polypeptide of interest, although some secretion signal sequences may be located at other positions within the nucleic acid sequence of interest (see, for example, Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830). Those skilled in the art will recognize that considerable sequence variation may exist in these polynucleotide molecules due to the degeneracy of the genetic code. In some embodiments, the nucleic acid sequences of this disclosure are codon-optimized for expression in mammalian cells, preferably in human cells. Codon optimization refers to the process of swapping a sequence of interest in a codon that is typically rare in highly expressed genes of a given species with a codon that is typically frequent in highly expressed genes of that species, and that encodes the same amino acid as the swapped codon.

[0174] The polynucleotide may contain a native sequence (e.g., an endogenous sequence or endogenous promoter sequence encoding an antibody or a portion thereof) or a variant of such a sequence. Polynucleotide variants contain one or more substitutions, additions, deletions, and / or insertions such that the activity of the encoded polypeptide is not reduced relative to the native molecule. As described herein, the effect on the immunoreactivity of the encoded polypeptide can generally be assessed. The variant preferably has at least about 70% identity with the polynucleotide sequence, more preferably, at least about 80% identity, even more preferably, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, and most preferably, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or about 99% identity.

[0175] 2. Receptor peptides In one aspect, this disclosure provides polynucleotides, nucleic acid constructs or molecules having a sequence encoding a receptor polypeptide, engineered cells (e.g., engineered immune cells) expressing the receptor polypeptide, and methods for preparing and using the nucleic acid construct (or polynucleotide or molecule) and the engineered cells. In one embodiment, the engineered cells are human.

[0176] In one embodiment, one or more receptor peptides include, but are not limited to, antigen-binding fragments or portions, chimeric antigen receptors (CARs), chimeric cytokine receptors (CCRs), and CD70-binding proteins. Different CCRs include, but are not limited to, inducible CCRs and constitutively active CCRs, as described in WO19 / 169290, WO20 / 180694, WO20 / 180664, and WO21 / 041806, each of which is incorporated herein by reference in its entirety. CD70-binding proteins are described in PCT / US2022 / 033598, which is also incorporated herein by reference in its entirety.

[0177] As used herein, chimeric antigen receptors (CARs) are proteins that specifically recognize target antigens (e.g., target antigens on cancer cells). Upon binding to a target antigen, a CAR can activate immune cells to attack and destroy cells carrying that antigen (e.g., cancer cells). CARs can also be incorporated with co-stimulatory or signal transduction domains to increase their potency. See Krause et al., J. Exp. Med., Vol. 188, No. 4, 1998 (619–626); Finney et al. people , Journal of Immunology 1998, 161: 2791–2797; Song et al. people , Blood 119:696-706 (2012); Kalos et al. people , Sci. Transl. Med. 3:95 (2011); Porter et al. people , N. Engl. J. Med. 365:725-33 (2011); and Gross et al. people , Annu. Rev. Pharmacol. Toxicol. 56:59–83 (2016); U.S. Patent Nos. 7,741,465 and 6,319,494.

[0178] The chimeric antigen receptor described herein comprises an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain includes an antigen-binding domain that specifically binds to a target. In some embodiments, the CAR is a CD19-specific CAR comprising, from the N-terminus to the C-terminus, the following elements: a signal sequence, a CD19 antigen-binding domain (e.g., anti-CD19scFv), a hinge region, a transmembrane region, and one or more consecutive signal transduction domains. In some embodiments, the CD19-specific CAR disclosed herein comprises, from the N-terminus to the C-terminus, the following elements: a CD8α signal sequence, the CD19 scFv described herein, a CD8α hinge domain and a transmembrane domain, a 4-1BB cytoplasmic signal transduction domain, and a CD3ζ cytoplasmic signal transduction domain. Exemplary sequences are shown in Table 3.

[0179] In some implementations, the antigen-specific CAR further includes a safety switch and / or one or more monoclonal antibody-specific epitopes.

[0180] a. Antigen-binding domain As discussed above, the CAR described herein includes an antigen-binding domain. As used herein, "antigen-binding domain" means any polypeptide that binds to a specified target antigen. In some embodiments, the antigen-binding domain binds to antigens on tumor cells. In some embodiments, the antigen-binding domain binds to antigens on cells involved in hyperproliferative diseases.

[0181] In some embodiments, the antigen-binding domain comprises a variable heavy chain, a variable light chain, and / or one or more CDRs as described herein. In some embodiments, the antigen-binding domain is a single-chain variable fragment (scFv) comprising light chains CDRs CDR1, CDR2, and CDR3, and heavy chains CDRs CDR1, CDR2, and CDR3.

[0182] An antigen-binding domain is said to be "selective" when it binds more tightly or has a higher affinity to one target than to another.

[0183] In some implementations, the antigen-binding domain specifically binds to BCMA, MUC16 (also known as CA125), EGFR, EGFRvIII, MUC1, Flt-3, WT-1, CD20, CD23, CD30, CD38, CD70, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, MHC-NY-ESO1, HER2 (ERBB2), CAIX (carbonic anhydrase IX), LIV1, ADAM10, CHRNA2, LeY, NKG2D, CS1, CD44v6, ROR1, CD19, sealing protein-18.2 (sealing protein-18A2 or sealing protein-18 isotype 2), PSCA, DLL3 (δ-like protein 3, Drosophila δ homolog 3, δ3), Mud 7 (Mucin17, Muc3, Muc3), FAPα (fibroblast activator protein α), and Ly6G6D. (Lymphocyte antigen 6 complex locus proteins G6d, c6orf23, G6D, MEGT1, NG25), PSMA, MSLN, or RNF43 (E3 ubiquitin ligase RNF43, RING finger protein 43). For example, the following patents disclose CARs and / or antibodies targeting antigens: BCMA: WO201616630, WO2020150339, WO2019196713, WO2016014565, WO2017025038 (each of which is incorporated herein by reference in its entirety); MUC16: US9,169,328, WO2016149368, WO2020023888 (each of which is incorporated herein by reference in its entirety); EGFRvIII: WO2017125830, WO2016016341 (each of which is incorporated herein by reference in its entirety); Flt3: WO2018222935, WO2020010284, WO2017173410 (Each of these patents is incorporated herein by reference in its entirety); CD20: WO2018145649, WO2020010235, WO2020123691 (Each of these patents is incorporated herein by reference in its entirety); CD38: WO2017025323 (Each of these patents is incorporated herein by reference in its entirety); CD70: WO2019152742, WO2018152181 (Each of these patents is incorporated herein by reference in its entirety); CD33: WO2016014576 (Each of these patents is incorporated herein by reference in its entirety); CD133: WO2018072025 (Each of these patents is incorporated herein by reference in its entirety); CS1: WO2019030240 (Each of these patents is incorporated herein by reference in its entirety);ROR1: WO2016115559 (the patent is incorporated herein by reference in its entirety); CD19: WO2002077029, US11,077,144 (each patent is incorporated herein by reference in its entirety); Sealing Protein: WO2018006882, WO2021008463 (each patent is incorporated herein by reference in its entirety); DLL3: WO2020180591 (the patent is incorporated herein by reference in its entirety); WT1: US20160152725A1, US7622119B2 (each patent is incorporated herein by reference in its entirety); CD23: US6011138A, CN1568198A (each patent is incorporated herein by reference in its entirety); CD30: US10815301B2, US10808035B2 (Each of the aforementioned patents is incorporated herein by reference in its entirety); PRAME: US20180148503A1, WO2020186204A1 (Each of the aforementioned patents is incorporated herein by reference in its entirety); LIV1: US20200231699A1 (Each of the aforementioned patent is incorporated herein by reference in its entirety); NKG2D: WO2021179353A1, US20210269501A1 (Each of the aforementioned patents is incorporated herein by reference in its entirety); FAPα: US20200246383A1, US20210115102A1 (Each of the aforementioned patents is incorporated herein by reference in its entirety); PSMA: US20210277141A1, WO2020108646A1 (Each of the aforementioned patents is incorporated herein by reference in its entirety); and MSLN: CN109680002A, CN109628492A (Each of the aforementioned patents is incorporated herein by reference in its entirety).

[0184] In some implementations, the cancer antigen is selected from the group consisting of: carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CDS, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, antigens of cytomegalovirus (CMV)-infected cells (e.g., cell surface antigens), epithelial glycoprotein (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine protein kinase erb-B2,3,4, folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor, ganglioside G2 (GD2), ganglioside G3 (GD3), and human epidermal growth factor receptor 2. (HER-2), human telomerase reverse transcriptase (hTERT), interleukin-13 receptor subunit α-2 (IL-13Ra2), κ-light chain, kinase insertion domain receptor (KDR), Lewis A (CA19.9), LI cell adhesion molecule (LICAM), melanoma antigen family A, 1 (MAGE-AI), mucin 16 (Muc-16), mucin 1 (Muc-1), mesothelin (MSLN), NKG2D ligand, cancer-testis antigen NY-ESO-1, carcinoembryonic antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), and Wilms tumor protein (WT-1).

[0185] Variants of the antigen-binding domain (e.g., variants of CDR, VH, and / or VL) are also within the scope of this disclosure, for example, variant light chains and / or variant heavy chains, each having at least 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-99%, or greater than 99% identity with the amino acid sequence of the antigen-binding domain. In some cases, such molecules comprise at least one heavy chain and one light chain, while in others, the variant forms contain two variant light chains and two variant heavy chains (or sub-parts thereof). Those skilled in the art will be able to identify suitable variants of the antigen-binding domain as shown herein using well-known techniques. In some embodiments, those skilled in the art can identify suitable regions in the molecule that can be altered without destroying activity by targeting regions considered unimportant to activity.

[0186] In some embodiments, the polypeptide structure of the antigen-binding domain is based on an antibody, including but not limited to monoclonal antibodies, bispecific antibodies, microantibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimics"), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), and fragments thereof. In some embodiments, the antigen-binding domain comprises or is composed of an avimer.

[0187] In some implementations, the antigen-binding domain is scFv.

[0188] In some implementations, antigen-selective CARs contain a leader sequence or signal peptide.

[0189] In some embodiments, the CAR comprises a leader peptide or a signal peptide; in some embodiments, the leader peptide comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the amino acid sequence MALVTALLLPLALLLHAARP (SEQ ID NO: 1). In some embodiments, the leader (signal) peptide comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the leader (signal) peptide is encoded by a nucleic acid sequence comprising the following: ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGCCGCACGCCCG (SEQ ID NO: 48).

[0190] In other embodiments, this disclosure relates to isolated polynucleotides encoding any of the antigen-binding domains described herein. In some embodiments, this disclosure relates to isolated polynucleotides encoding CARs. Vectors comprising polynucleotides and methods for their preparation are also provided herein.

[0191] In other embodiments, this disclosure relates to isolated polynucleotides encoding any of the antigen-binding domains described herein. In some embodiments, this disclosure relates to isolated polynucleotides encoding CARs. Vectors comprising polynucleotides and methods for their preparation are also provided herein.

[0192] In some embodiments, CAR-immune cells (e.g., CAR-T cells) that can form components of an engineered population of immune cells (derived from donor cells of a donor cell population as described herein) generated by practicing the methods of this disclosure contain a polynucleotide encoding a safety switch polypeptide (e.g., RQR8). See, for example, WO2013153391A, which is hereby incorporated by reference in its entirety. In CAR-immune cells (e.g., CAR-T cells) containing said polynucleotide, the safety switch polypeptide may be expressed on the surface of the CAR-immune cell (e.g., CAR-T cell).

[0193] b. Hinge domain The extracellular domain of the CAR disclosed herein may include a "hinge" domain (or hinge region). This term generally refers to any polypeptide that acts to link a transmembrane domain in the CAR to an extracellular antigen-binding domain in the CAR. Specifically, the hinge domain can be used to provide greater flexibility and accessibility to the extracellular antigen-binding domain.

[0194] The hinge domain may contain up to 300 amino acids, with 10 to 100 amino acids in some embodiments and 25 to 50 amino acids in others. The hinge domain may be derived from all or part of a naturally occurring molecule, such as all or part of an extracellular region derived from CD8, CD4, CD28, 4-1BB, or IgG (particularly the hinge region of IgG; it will be understood that the hinge region may contain some or all of members of the immunoglobulin family, such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or fragments thereof), or from all or part of a constant region of the antibody heavy chain. Alternatively, the hinge domain may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or it may be a fully synthetic hinge sequence. In some embodiments, the hinge domain is a portion of the human CD8α chain (e.g., NP_001139345.1). In other embodiments, the hinge and transmembrane domain comprise a portion of the human CD8α chain. In some embodiments, the hinge domain of the CAR described herein comprises a subsequence of CD8α, IgG1, IgG4, PD-1, or FcγRIIIα, particularly a hinge region of any one of CD8α, IgG1, IgG4, PD-1, or FcγRIIIα. In some embodiments, the hinge domain comprises a human CD8α hinge, a human IgG1 hinge, a human IgG4 hinge, a human PD-1 hinge, or a human FcγRIIIα hinge. In some embodiments, the CAR disclosed herein comprises scFv, a human CD8α hinge and a transmembrane domain, a CD3ζ signaling domain, and a 4-1BB signaling domain. Table 3 provides amino acid sequences of exemplary hinge domains as provided herein. In some embodiments, the hinge in the CAR of this disclosure is a CD8 hinge comprising the amino acid sequence TTTAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 9). In other embodiments, the hinge in the CAR of this disclosure is a CD28 hinge containing the amino acid sequence IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 93).

[0195] c. Transmembrane domain The CAR disclosed herein is designed to have a transmembrane domain fused to the extracellular domain of the CAR. It can similarly fuse to the intracellular domain of the CAR. In some cases, the transmembrane domain may be selectively modified or modified by amino acid substitution to prevent such domains from binding to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex. In some embodiments, short linkers may form links between any and some of the extracellular domain, transmembrane domain, and intracellular domain of the CAR.

[0196] The transmembrane domain of the CAR disclosed herein has the ability to: (a) be expressed on the surface of immune cells, such as, for example but not limited to, lymphocytes, such as T helper (T) cells. h Cellular, cytotoxic T (T) c ) cells, T regulation (T reg (a) cells or natural killer (NK) cells, and / or (b) interact with extracellular antigen-binding domains and intracellular signal transduction domains to guide the cellular response of immune cells against target cells.

[0197] The transmembrane domain can be derived from a natural or synthetic source. In the case of a natural source, the domain can originate from any membrane-binding or transmembrane protein.

[0198] The transmembrane region specifically used in this disclosure may be derived from (including or correspond to) CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death receptor-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC Class 1 molecules, TNF receptor protein, immunoglobulins, cytokine receptors, integrins, signal transduction lymphocyte activating molecules (SLAM protein), activating NK cell receptors, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile protein), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, ligands that specifically bind to CD83, or any combination thereof.

[0199] As a non-limiting example, the transmembrane domain may be derived from or a subset of the following: T-cell receptors, such as α, β, γ, or δ; polypeptides constituting the CD3 complex; IL-2 receptor p55 (α chain), p75 (β chain), or γ chain; subunit chains of Fc receptors, particularly Fcγ receptor III; or CD proteins. Alternatively, the transmembrane domain may be synthetic and may primarily contain hydrophobic residues, such as leucine and valine. In some embodiments, the transmembrane domain is derived from the human CD8α chain (e.g., NP_001139345.1).

[0200] In some embodiments, the transmembrane domain in the CAR of this disclosure is a CD8α transmembrane domain. In some embodiments, the transmembrane domain in the CAR of this disclosure is a CD8α transmembrane domain containing the amino acid sequence IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 97). In some embodiments, the hinge domain and transmembrane domain in the CAR of this disclosure are a CD8α hinge domain and a CD8α transmembrane domain, respectively.

[0201] In some embodiments, the transmembrane domain of the CAR of this disclosure is a CD28 transmembrane domain. In some embodiments, the transmembrane domain of the CAR of this disclosure is a CD28 transmembrane domain containing the amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 56). In some embodiments, the hinge domain and transmembrane domain of the CAR of this disclosure are a CD28 hinge domain and a CD28 transmembrane domain. In other embodiments, the CD28 hinge and CD28 transmembrane domain contain the amino acid sequence IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 94). In some embodiments, the CAR of this disclosure contains a CD8 hinge and a CD28 transmembrane domain. In other embodiments, the CD8 hinge and CD28 transmembrane domain contain the amino acid sequence TTTAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 95).

[0202] d. Intracellular domains The intracellular (cytoplasmic) domain of the CAR disclosed herein can provide activation of at least one of the normal effector functions of an immune cell containing the CAR, such as signal 1 / activation and / or signal 2 / co-stimulation. For example, T cell effector functions can refer to cytolytic activity or helper activity, including cytokine secretion.

[0203] In some embodiments, the activating intracellular signal transduction domains used in CARs can be, for example, but not limited to, cytoplasmic sequences of T-cell receptors and helper receptors that work together to initiate signal transduction upon antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences having the same functional capabilities.

[0204] It will be understood that suitable (e.g., activating) intracellular domains include, but are not limited to, signal transduction domains derived from (or corresponding to) the following: CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death receptor-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC Class 1 molecules, TNF receptor protein, immunoglobulins, cytokine receptors, integrins, signal transduction lymphocyte activating molecules (SLAM protein), activating NK cell receptors, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile protein), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, ligands that specifically bind to CD83, or any combination thereof.

[0205] In addition to the aforementioned activating domains, the intracellular domains of the CAR disclosed herein may also incorporate co-stimulatory signal transduction domains (which are interchangeably referred to herein as co-stimulatory molecules) to enhance their potency. Besides the primary signal provided by the stimulatory molecules as described herein, the co-stimulatory domains can provide signaling.

[0206] As used herein, "co-stimulatory molecules" refer to homologous binding partners on immune cells (e.g., T cells) that specifically bind to co-stimulatory ligands, thereby mediating cellular co-stimulatory responses such as, but not limited to, proliferation. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors. Examples of co-stimulatory molecules include CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0207] It will be understood that suitable costimulatory domains within the scope of this disclosure may be derived from (or correspond to), for example, CD28, OX40, 4-1BB / CD137, CD2, CD3 (α, β, δ, ε, γ, ζ), CD4, CD5, CD7, CD9, CD16, CD22, CD27, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1 (CD11a / CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC Class I molecules, TNFR, integrins, signal transduction lymphocyte activating molecules, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1-1d, IT GAE, CD103, ITGAL, CD1-1a, LFA-1, ITGAM, CD1-1b, ITGAX, CD1-1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile protein), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83 ligands, or fragments or combinations thereof. It will be understood that other co-stimulatory molecules or fragments thereof not listed above are within the scope of this disclosure.

[0208] In some embodiments, the intracellular / cytoplasmic domain of the CAR may be designed to include a 4-1BB / CD137 domain, either alone or in combination with one or more other desired intracellular domains useful in the context of the CAR disclosed herein. The complete native amino acid sequence of 4-1BB / CD137 is described in NCBI reference sequence: NP_001552.2. The complete native 4-1BB / CD137 nucleic acid sequence is described in NCBI reference sequence: NM_001561.5.

[0209] In some implementations, the intracellular / cytoplasmic domain of the CAR may be designed to include a CD28 domain, either alone or in combination with one or more other desired intracellular domains useful in the context of the CAR disclosed herein. The complete native amino acid sequence of CD28 is described in NCBI reference sequence: NP_006130.1. The complete native CD28 nucleic acid sequence is described in NCBI reference sequence: NM_006139.1.

[0210] In some embodiments, the intracellular / cytoplasmic domain of the CAR may be designed to include a CD3ζ domain, either alone or in combination with any other desired intracellular domain useful in the context of the CAR disclosed herein.

[0211] For example, the intracellular domain of a CAR may include a portion of the CD3ζ chain and a portion of a co-stimulatory signaling molecule. The intracellular signaling sequences within the intracellular signaling portion of the CAR disclosed herein may be linked to each other in a random or designated order. In some embodiments, the intracellular domain is designed to include an activation domain of CD3ζ and a signaling domain of CD28. In some embodiments, the intracellular domain is designed to include an activation domain of CD3ζ and a 4-1BB signaling domain.

[0212] In some embodiments, the 4-1BB (intracellular domain) comprises the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 12). In some embodiments, the 4-1BB (intracellular domain) is encoded by the following nucleic acid sequence: AAGGCGCGGCAGGAAGAAGCTCCTCTACATTTTTAAGCAGCCTTTTATGAGGCCCGTACAGACAACACAGGAGGAAGATGGCTGTAGCTGCAGATTTCCCGAGGAGGAGGAAGGTGGGTGCGAGCTG (SEQ ID NO: 57).

[0213] In some implementations, the intracellular domain in the CAR is designed to include a portion of CD28 and CD3ζ, wherein the intracellular CD28 contains the amino acid sequence of SEQ ID NO: 13 and is encoded by the nucleic acid sequence described in SEQ ID NO: 58.

[0214] In some embodiments, the CD3ζ amino acid sequence may include SEQ ID NO: 11, and the nucleic acid sequence encoding the CD3ζ amino acid sequence may include SEQ ID NO: 59: AGGGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCAACTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGCGCAGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAGGAGGGTCTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAGGGACTCAGCACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAGG (SEQ ID NO: 59).

[0215] In some embodiments, the intracellular signaling domain of the CAR of this disclosure includes a domain of a co-stimulatory molecule. In some embodiments, the intracellular signaling domain of the CAR of this disclosure includes a portion of a co-stimulatory molecule selected from the group consisting of fragments of 4-1BB (GenBank: AAA53133.) and CD28 (NP_006130.1).

[0216] In some embodiments, the intracellular signal transduction domain of the CAR of this disclosure comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, 95%, 97%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the intracellular signal transduction domain of the CAR of this disclosure comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, 95%, 97%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 13.

[0217] In an exemplary embodiment, the CAR of this disclosure comprises, from the N-terminus to the C-terminus: a (cleavable) CD8α signaling sequence, an anti-CD19 scFv, a CD8α hinge and transmembrane region, a 4-1BB cytoplasmic (co-stimulatory) signaling domain, and a CD3ζ cytoplasmic signaling domain.

[0218] In some respects, this article provides engineered immune cells that contain and / or express CD19-specific CARs, as well as CD70-binding proteins and / or chimeric cytokine receptors (CCRs).

[0219] e. CD-70 binding protein In a related respect, this disclosure provides a CD70-binding protein as described herein, wherein the CD70-binding protein comprises an extracellular ligand or antigen-binding domain (or a CD70-binding domain) that binds to CD70 and a transmembrane domain. The CD70-binding protein may comprise zero to one or more intracellular signaling domains, as described herein. In some embodiments, the CD70-binding protein is a non-naturally occurring or recombinant CD70-binding protein.

[0220] In some embodiments, the CD70 binding protein provided herein comprises an extracellular domain that binds to CD70 (e.g., an anti-CD70 single-chain variable fragment (scFv)) and a transmembrane domain. In some embodiments, the CD70 binding protein or CD70 CAR provided herein comprises an extracellular ligand-binding domain (e.g., scFv), a transmembrane domain, and an intracellular signaling domain. In some embodiments, the anti-CD70 scFv comprises the amino acid sequence of SEQ ID NO: 16, 17, or 18. In some embodiments, the anti-CD70 scFv comprises the amino acid sequence of SEQ ID NO: 18.

[0221] In some embodiments, the CD70-binding protein comprises one or more intracellular signaling domains selected from the group consisting of: CD3ζ signaling domain, CD3δ signaling domain, CD3γ signaling domain, CD3ε signaling domain, CD28 signaling domain, CD2 signaling domain, OX40 signaling domain, and 4-1BB signaling domain, or variants thereof. In some embodiments, the intracellular signaling domain comprises an amino acid sequence of one or more of SEQ ID NO: 11, 12, 13, 82, 83, 84, 85, 86, 87, 88, 89, 90, or 91. In some embodiments, the intracellular signaling domain comprises an amino acid sequence of one or more of SEQ ID NO: 11, 88, 89, or 90. In some embodiments, the CD70-binding protein comprises a CD3ζ signaling domain or a variant thereof and does not comprise a co-stimulatory domain. In some embodiments, the CD70-binding protein comprises a 4-1BB signaling domain or a variant thereof and does not comprise a CD3 signaling domain. In some embodiments, the CD70-binding protein comprises a 4-1BB signaling domain and a CD3ζ signaling domain. In some embodiments, the CD70-binding protein does not comprise an intracellular signaling domain. Different intracellular signaling domains or combinations thereof can confer different signaling strengths, which can promote T cell proliferation, potency, survival, persistence, and / or resistance to host immune cell rejection. This document describes CD70-binding proteins comprising zero to one or more intracellular signaling domains. In some embodiments, the CD70-binding protein comprises the amino acid sequences of SEQ ID NO: 18, SEQ ID NO: 10, and SEQ ID NO: 11, and an amino acid sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 19 or 21. In some embodiments, the CD70-binding protein comprises an amino acid sequence with one or more conserved amino acid substitutions having the amino acid sequence of SEQ ID NO: 19 or 21.

[0222] CD70 is expressed on T cells, particularly activated T cells, including host allogeneic reactive T cells. In some embodiments, engineered immune cells containing the CD70-binding protein described herein can exhibit varying levels of persistence and / or resistance to host immune cell rejection and can be adapted for in vivo lymphocyte depletion upon administration to a patient. In some embodiments, engineered immune cells containing the CD70-binding protein described herein can inhibit the proliferation and / or activity of host immune cells to varying degrees upon administration to a patient, which can allow for fine tuning of the depth of in vivo lymphocyte depletion. For example, engineered immune cells containing the CD70-binding protein, exhibiting prolonged expansion and / or inhibition of host immune cell proliferation or activity in MLR assays, can be used to prolong lymphocyte depletion. In contrast, when less thorough or incomplete lymphocyte depletion is required, engineered immune cells containing the CD70-binding protein, which exhibit less prolonged expansion and / or inhibition of host immune cells in the same or similar assays, can be used.

[0223] In some respects, engineered cells (e.g., engineered immune cells, such as CAR T cells) containing and / or expressing receptor-specific CAR and CD70-binding proteins, as described herein, can target both receptor-positive and receptor-negative / CD70-positive tumor cells. Receptor-specific CAR T cells co-expressing CD70-binding proteins advantageously prevent receptor antigen escape from tumor cells. Receptor-specific CAR T cells co-expressing CD70-binding proteins and / or chimeric cytokine receptors (CCRs) exhibit further enhanced activity.

[0224] f. Chimeric cytokine receptor (CCR) The CCR disclosed herein includes a transmembrane domain. The transmembrane domain of this disclosure contains sequences that enable the two monomers to constitutively dimerize, thereby allowing constitutive JAK activation on the intracellular portion and constitutive recruitment and phosphorylation of STAT, for example, on the cytoplasmic region of the receptor.

[0225] The transmembrane domain is located at the N-terminus and is coupled to the intracellular / cytoplasmic domain at the C-terminus. In some embodiments, coupling is optionally achieved via a linker.

[0226] The transmembrane domains used herein can be inserted into the cell membrane expressing the transmembrane domain. In some embodiments, the CCR of this disclosure comprises a transmembrane domain spanning the cell membrane, and an extracellular and / or intracellular portion. In some embodiments, the CCR of this disclosure comprises a transmembrane domain spanning the cell membrane, and an extracellular ligand-binding domain and / or an intracellular signal transduction domain. In some embodiments, the CCR of this disclosure comprises a transmembrane domain spanning the cell membrane, and further comprises an intracellular domain, and does not comprise an extracellular ligand-binding domain. In some embodiments, the CCR comprises the amino acid sequence of SEQ ID NO: 27 or 29.

[0227] In some embodiments, the transmembrane domains of this disclosure are engineered (synthetic) and do not resemble any naturally occurring transmembrane domains; for example, they are non-natural.

[0228] In other embodiments, the transmembrane domains of this disclosure are derived from or obtained from naturally occurring receptors.

[0229] In some embodiments, the transmembrane domain and / or JAK-binding / activating domain (or simply JAK-binding domain) of this disclosure are derived from one or more of the following receptors: erythropoietin receptor (EpoR), interleukin-6 signal transducer (GP130 or IL6ST), prolactin receptor (PrlR), growth hormone receptor (GHR), granulocyte colony-stimulating factor receptor (GCSFR), and thrombopoietin receptor / myeloproliferative leukemia protein receptor (TPOR / MPLR). When derived from naturally occurring receptors, the entire receptor or the entire transmembrane sequence of the receptor may not be necessary to achieve constitutive activation and constitutive JAK binding / activation at the intracellular portion. In some embodiments, the transmembrane domain of this disclosure is derived from a truncated version of the naturally occurring TPOR / MPLR (myeloproliferative leukemia protein) receptor, as shown in SEQ ID NO: 51. Therefore, fragments of naturally occurring receptors can be utilized. Furthermore, certain mutations can be introduced into the transmembrane domain of a naturally occurring receptor to further modulate downstream signaling. In some embodiments, the transmembrane domains of this disclosure are derived from TPOR / MPLR and have H499L / S505N / W515K substitutions as shown in SEQ ID NO: 25.

[0230] The CCR of this disclosure includes a cytoplasmic recruitment domain. The recruitment domain may be a STAT recruitment domain, an AP1 recruitment domain, a Myc / Max recruitment domain, or an NFkB recruitment domain. In some embodiments, the recruitment domain is a signal transducer and transcription activator (STAT) recruitment (STAT activation) domain, for example, derived from a receptor tail (cell tail) or a cytokine receptor tail. These intracellular recruitment domains of the CCR of this disclosure allow signal transduction to propagate in immune cells containing CARs and chimeric cytokine receptors (e.g., CAR-T cells with chimeric cytokine receptors of this disclosure). Cytokine signaling propagates through the STAT recruitment domain, thereby achieving cytokine-based immune enhancement of the cell. In some embodiments, the immune enhancement is homeostatic, for example, the signal transduction leads to an increase in CAR-carrying immune cells. In some embodiments, the immune enhancement is inflammatory, for example, the signal transduction leads to an increase in the potency of CAR-carrying immune cells. In some embodiments, the immune enhancement can prevent exhaustion, for example, the signal transduction can maintain the long-term function of CAR-carrying immune cells.

[0231] In some embodiments, the recruitment domain of this disclosure is synthetic and does not resemble any naturally occurring receptor fragment. In some embodiments, immune enhancement can prevent depletion; for example, signaling can maintain the long-term function of CAR-carrying immune cells. In some embodiments, the Stat recruitment domain of this disclosure is synthetic and does not resemble any naturally occurring receptor fragment.

[0232] In other embodiments, the Stat recruitment domain of this disclosure is derived from the cytoplasmic tail of a naturally occurring receptor, such as a naturally occurring cytokine receptor. These cytoplasmic tails of naturally occurring receptors may be regions downstream of the JAK-binding domain of a transmembrane domain of the receptor. The Stat recruitment domain of a chimeric cytokine receptor includes at least one STAT recruitment domain derived from at least one receptor. In some embodiments, the Stat recruitment domain includes at least one STAT1 recruitment domain. In some embodiments, the Stat recruitment domain includes at least one STAT2 recruitment domain. In some embodiments, the Stat recruitment domain includes at least one STAT3 recruitment domain. In some embodiments, the Stat recruitment domain includes at least one STAT4 recruitment domain. In some embodiments, the Stat recruitment domain includes at least one STAT5 recruitment domain. In some embodiments, the Stat recruitment domain includes at least one STAT6 recruitment domain. In some embodiments, the Stat recruitment domain includes at least one STAT7 recruitment domain.

[0233] In some implementations, the naturally occurring receptor from which the Stat recruitment domain originates is not a cytokine receptor.

[0234] In some embodiments, the naturally occurring receptor from which the Stat recruitment domain is derived is a cytokine receptor. Exemplary cytokine receptors that enhance cytokine signaling in T-cell immunity include, but are not limited to, IL-2, IL-7, IL-15, and IL-21 receptors. In alternative embodiments, the receptor from which the Stat recruitment domain is derived is not a cytokine receptor. By selecting the Stat recruitment domain of the CCR, the receptor can be redirected to the selected signal transduction pathway. In some embodiments, the recruitment domain comprises the amino acid sequence of SEQ ID NO: 26.

[0235] In some embodiments, the CCR of this disclosure comprises a recruitment domain linked to the C-terminus of a transmembrane / JAK2 binding domain, with or without a linker. In some embodiments, the linker comprises one or more amino acid residues. In some embodiments, the CCR comprises the amino acid sequence of SEQ ID NO: 27 or 29.

[0236] In some embodiments, the CCR of this disclosure comprises an extracellular ligand-binding domain. In some embodiments, the extracellular ligand-binding domain binds to an immunosuppressive molecule, such as PDL1. In some embodiments, the extracellular ligand-binding domain of the CCR comprises an antibody that binds to PDL-1. In some embodiments, the extracellular ligand-binding domain of the CCR comprises the extracellular domain of PD-1 (PD-1 CCR). In some embodiments, the binding of the PD-1 extracellular domain of the PD-1 CCR to PDL-1 can convert an inhibitory signal of PDL-1 into a stimulatory signal via signal transduction through an intracellular cytoplasmic recruitment domain. In some embodiments, the PD-1 CCR comprises a TPOR / MPLR transmembrane / JAK binding domain comprising the amino acid sequence of SEQ ID NO: 50, and an intracellular recruitment domain of SEQ ID NO: 26. In some embodiments, the PD-1 extracellular domain comprises the wild-type PD-1 extracellular domain and comprises the amino acid sequence of SEQ ID NO: 52.

[0237] In some embodiments, the PD-1 extracellular domain contains a mutation targeting the wild-type PD-1 extracellular domain sequence. In some embodiments, the PD-1 extracellular domain sequence is a high-affinity PD-1 extracellular domain. In some embodiments, the PD-1 extracellular domain sequence is a high-affinity PD-1 extracellular domain and contains the amino acid sequence of SEQ ID NO: 53. In some embodiments, the PD-1 extracellular domain contains one or more tandem repeat sequences of a high-affinity PD-1 extracellular domain.

[0238] g. Dominant negative receptors On the other hand, this document provides a dominant PD-1 receptor comprising a PD-1 extracellular domain and a transmembrane domain, but without a functional intracellular signaling domain. In some embodiments, the PD-1 extracellular domain comprises a wild-type PD-1 extracellular domain. In some embodiments, the PD-1 extracellular domain comprises a mutant PD-1 extracellular domain. In some embodiments, the PD-1 extracellular domain comprises a high-affinity PD-1 extracellular domain. In some embodiments, the PD-1 extracellular domain comprises the amino acid sequence of SEQ ID NO: 53.

[0239] In other embodiments, the transmembrane domain comprises a PD-1 transmembrane domain. In some embodiments, the transmembrane domain comprises a CD8a transmembrane domain. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NOS: 10, 50, or 60. Conserved amino acid substitutions of the peptides disclosed herein are considered.

[0240] In some embodiments, the PD-1 dominant-negative receptor comprises the amino acid sequence of SEQ ID NO: 52 or 53. In some embodiments, the PD-1 dominant-negative receptor comprises the amino acid sequence of SEQ ID NO: 52 or 53 and has at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 55. In some embodiments, the PD-1 dominant-negative receptor comprises an amino acid sequence having one or more conserved amino acid substitutions of the amino acid sequence of SEQ ID NO: 54 or 55. In some embodiments, the PD-1 dominant-negative receptor comprises the amino acid sequence of SEQ ID NO: 54 or 55.

[0241] This disclosure covers modifications of the peptides disclosed herein, such as CARs, CCRs, CD70-binding proteins, dominant and negative receptors, etc., that do not significantly affect their properties, and variants that enhance or reduce activity and / or affinity as needed. Peptide modification is common practice in the art and need not be described in detail herein. Examples of modified peptides include peptides with conserved substitutions of amino acid residues, deletions or additions of one or more amino acids, said modifications that do not significantly and harmfully alter functional activity, or said modifications that mature (enhance) the peptide's affinity for its ligands, or the use of chemical analogs.

[0242] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions of peptides ranging in length from one residue to one hundred or more residues, as well as intra-sequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue or antibodies fused to an epitope tag. Other insertional variants of antibody molecules include fusions of enzymes or peptides with the N-terminus or C-terminus of the antibody, which increase the antibody's half-life in blood circulation.

[0243] Substitutional variants remove at least one amino acid residue from the antigen-binding domain and insert a different residue at its position. In some embodiments, the substitutional mutagenic site of interest includes the hypervariable region / CDR, but FR alterations are also considered. Conservative substitutions are shown under the heading “Conservative Substitutions” in Table 2. If such substitutions result in a change in biological activity, more substantial changes (named “Exemplary Substitutions” in Table 2, or further described below with respect to amino acid categories) can be introduced and products screened.

[0244] Table 2: Amino Acid Substitutions

[0245] In some aspects, this disclosure provides immune cells or engineered cells, such as engineered immune cells, comprising polynucleotides including a first coding sequence and a second coding sequence, wherein the first or second coding sequence encodes an anti-CD19 CAR. In some embodiments, the engineered immune cells further comprise additional polynucleotides including one or more coding sequences.

[0246] In some embodiments, one or more coding sequences include a coding sequence encoding a dominant-negative PD-1 receptor. In some embodiments, the dominant-negative PD-1 receptor comprises a wild-type PD-1 extracellular domain. In some embodiments, the dominant-negative PD-1 receptor comprises a high-affinity PD-1 extracellular domain. In some embodiments, the dominant PD-1 receptor does not comprise a functional intracellular signaling domain. In some embodiments, the dominant-negative PD-1 receptor comprises the amino acid sequence SE ID NO:54.

[0247] In some embodiments, the additional polynucleotide comprises one or more coding sequences. In some embodiments, the one or more coding sequences encode a CCR. In some embodiments, the CCR comprises the amino acid sequence of SEQ ID NO: 27 or 29. In some embodiments, the one or more coding sequences encode a CD70 binding protein. In some embodiments, the CD70 binding protein comprises the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 21. In some embodiments, the one or more coding sequences encode a PD-1 CCR. In some embodiments, the PD-1 CCR comprises a PD-1 extracellular domain comprising the amino acid sequence of SEQ ID NO: 54 or SEQ ID NO: 55; a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 50; and an intracellular signal transduction domain comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the one or more coding sequences encode a PD-1 dominant-negative receptor. In some embodiments, the PD-1 dominant-negative receptor comprises the amino acid sequence of SEQ ID NO: 54 or SEQ ID NO: 55.

[0248] In some embodiments, the additional polynucleotide from 5' to 3' contains a promoter operatively linked to both the third and fourth coding sequences. In some embodiments, the third coding sequence encodes a CCR or CD70-binding protein. In some embodiments, the fourth coding sequence encodes a PD-1 CCR or a PD-1 dominant / negative receptor. In some embodiments, the third coding sequence encodes a PD-1 CCR or a PD-1 dominant / negative receptor. In some embodiments, the fourth coding sequence encodes a CCR or CD70-binding protein.

[0249] In other respects, this article provides engineered immune cells, such as CAR T cells expressing CD19-specific CAR and CD70 binding proteins and / or CCRs. In some embodiments, engineered immune cells, such as CD19 CAR T cells, further express PD-1 dominant-negative receptors.

[0250] In some embodiments, the engineered cell (e.g., engineered immune cell) comprises a polynucleotide or integrated nucleic acid sequence comprising, from 5' to 3', a first coding sequence and a second coding sequence driven by a truncated PGK promoter, wherein the first coding sequence encodes an antigen-specific CAR (e.g., a CD19-specific CAR) and the second coding sequence encodes a CD70-binding protein (as described herein), or wherein the first coding sequence encodes a CD70-binding protein and the second coding sequence encodes an antigen-specific CAR (e.g., a CD19-specific CAR) (as described herein), or wherein the first coding sequence encodes an antigen-specific CAR (e.g., a CD19-specific CAR) and the second coding sequence encodes a nucleic acid inhibitor (e.g., an RNA interference agent) (as described herein), or wherein the first coding sequence encodes a CD70-binding protein and the second coding sequence encodes a nucleic acid inhibitor (e.g., an RNA interference agent) (as described herein).

[0251] In some embodiments, the engineered cells (e.g., engineered immune cells) further comprise additional polynucleotide or additional integrated nucleic acid sequences containing one or more coding sequences, wherein said one or more coding sequences encode a CCR and optionally encode a PD-1 dominant / negative receptor or a nucleic acid inhibitor, such as an RNA interference agent, as described herein. In some embodiments, the additional polynucleotide or additional integrated nucleic acid sequence comprises a third coding sequence and a fourth coding sequence from 5' to 3', wherein the third coding sequence encodes a CCR and the fourth coding sequence encodes a PD-1 dominant / negative receptor or a nucleic acid inhibitor, such as an RNA interference agent, as described herein, or wherein the third coding sequence encodes a PD-1 dominant / negative receptor and the fourth coding sequence encodes a CCR or a nucleic acid inhibitor, such as an RNA interference agent, as described herein.

[0252] In some embodiments, the engineered cell (e.g., engineered immune cell) comprises a polynucleotide or integrated nucleic acid sequence comprising, from 5' to 3', a first coding sequence and a second coding sequence driven by a truncated PGK promoter, wherein the first coding sequence encodes a CCR and the second coding sequence encodes an antigen-specific CAR (e.g., a CD19-specific CAR) (as described herein), or wherein the first coding sequence encodes an antigen-specific CAR (e.g., a CD19-specific CAR) and a fourth coding sequence encodes a CCR (as described herein). In some embodiments, the engineered cell (e.g., engineered immune cell) further comprises additional polynucleotides or additional integrated sequences comprising one or more coding sequences, wherein said one or more coding sequences encode a CD70-binding protein and optionally encode a PD-1 dominant / negative receptor, as described herein. In some embodiments, the additional polynucleotide from 5' to 3' comprises a third coding sequence and a fourth coding sequence, wherein the third coding sequence encodes a CD70-binding protein and the fourth coding sequence encodes a PD-1 dominant-negative receptor, as described herein; or wherein the third coding sequence encodes a PD-1 dominant-negative receptor and the fourth coding sequence encodes a CD70-binding protein, as described herein.

[0253] Also provided are populations of engineered immune cells, such as CAR T cells. In some embodiments, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the engineered immune cell population expresses the CD19 CAR described herein. In some embodiments, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the engineered immune cell population expresses the CD19 CAR and CD70 binding protein described herein. In some embodiments, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the engineered immune cell population expresses the CD19 CAR, CD70 binding protein, and CCR described herein. In some embodiments, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the engineered immune cell population expresses the CD19 CAR, CD70 binding protein, and CCR described herein.

[0254] 3. Engineered cells This document provides engineered cells (e.g., engineered immune cells) that express the receptor peptides and / or nucleic acid inhibitors disclosed herein. For the subject receiving the engineered cells, the engineered cells may be allogeneic or autologous.

[0255] In some embodiments, the engineered cells are engineered immune cells selected from: T cells (e.g., inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes (Tregs), helper T lymphocytes, tumor-infiltrating lymphocytes (TILs)), natural killer (NK) cells, natural killer T cells (NKTs), TCR-expressing cells, dendritic cells, killer dendritic cells, mast cells, or B cells. In some embodiments, the engineered immune cells may be derived from a group consisting of CD4+ T lymphocytes and CD8+ T lymphocytes. In some exemplary embodiments, the engineered immune cells are T cells. In some exemplary embodiments, the engineered immune cells are γδ T cells. In some exemplary embodiments, the engineered immune cells are macrophages. In some exemplary embodiments, the engineered immune cells are natural killer (NK) cells.

[0256] In some implementations, engineered immune cells may be derived from, for example, but not limited to, stem cells. Stem cells may be adult stem cells, non-human embryonic stem cells (more specifically, non-human stem cells), umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells (iPSCs), totipotent stem cells, or hematopoietic stem cells.

[0257] In some embodiments, the cells are obtained or prepared from peripheral blood. In some embodiments, the cells are obtained or prepared from peripheral blood mononuclear cells (PBMCs). In some embodiments, the cells are obtained or prepared from bone marrow. In some embodiments, the cells are obtained or prepared from umbilical cord blood. In some embodiments, the cells are human cells.

[0258] In some implementations, cells are transfected or transduced using polynucleotides, nucleic acid constructs, or nucleic acid vectors via a method selected from the group consisting of: electroporation, acoustic pore effect, gene gun method (e.g., gene gun), lipid transfection, polymer transfection, nanoparticles, viral transfection or transduction (e.g., retrovirus, lentivirus, AAV), or polymeric complexes.

[0259] In some embodiments, engineered cells (e.g., engineered immune cells) expressing the antigen-specific CAR (e.g., CD19-specific CAR), CD70-binding protein, and / or CCR of this disclosure at their cell surface membrane contain greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of stem cell memory cells and central memory cells. In some embodiments, the antigen-specific CAR of this disclosure is expressed at its cell surface membrane. Engineered cells (e.g., CD19-specific CARs), CD70-binding proteins, and / or CCRs (e.g., engineered immune cells) comprise approximately 10% to approximately 100%, approximately 10% to approximately 90%, approximately 10% to approximately 80%, approximately 10% to approximately 70%, approximately 10% to approximately 60%, approximately 10% to approximately 50%, approximately 10% to approximately 40%, approximately 10% to approximately 30%, approximately 10% to approximately 20%, approximately 15% to approximately 100%, approximately 15% to approximately 90%, approximately 15% to approximately 80%, approximately 15% to approximately 70%, approximately 15% to approximately 60%, approximately 15% to approximately 50%, approximately 15% to approximately 40%, approximately 15% to approximately 30%, approximately 20% to approximately 100%, approximately 20% to approximately 90%, approximately 20% to approximately 80%, approximately 20% to approximately 70%, approximately 20% to approximately 60%, approximately 20% to approximately 50%, approximately 20% to approximately 40%, approximately 20% to approximately 30%, and approximately 30% to approximately 100%. Approximately 30% to approximately 90%, approximately 30% to approximately 80%, approximately 30% to approximately 70%, approximately 30% to approximately 60%, approximately 30% to approximately 50%, approximately 30% to approximately 40%, approximately 40% to approximately 100%, approximately 40% to approximately 90%, approximately 40% to approximately 80%, approximately 40% to approximately 70%, approximately 40% to approximately 60%, approximately 40% to approximately 50%, approximately 50% to approximately 100%, approximately 50% to approximately 90%, approximately 50% to approximately 80%, approximately 50% to The percentages of stem cell memory cells and central memory cells are approximately 70%, approximately 50% to approximately 60%, approximately 60% to approximately 100%, approximately 60% to approximately 90%, approximately 60% to approximately 80%, approximately 60% to approximately 70%, approximately 70% to approximately 90%, approximately 70% to approximately 80%, approximately 80% to approximately 100%, approximately 80% to approximately 90%, approximately 90% to approximately 100%, approximately 25% to approximately 50%, approximately 75% to approximately 100%, or approximately 50% to approximately 75%.

[0260] In some embodiments, the cells are immune cells, wherein the immune cells are inflammatory T lymphocytes expressing an antigen-specific CAR (e.g., a CD19-specific CAR) as described herein. In some embodiments, the immune cells are cytotoxic T lymphocytes expressing any of the antigen-specific CARs described herein. In some embodiments, the immune cells are regulatory T lymphocytes expressing any of the antigen-specific CARs described herein. In some embodiments, the immune cells are helper T lymphocytes expressing any of the CARs described herein.

[0261] Prior to amplification and genetic modification, cell sources can be obtained from the subject using a variety of non-limiting methods. Cells can be obtained from many non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, any number of T cell lines available and known to those skilled in the art can be used. In some embodiments, cells can be derived from a healthy donor or from a patient, such as a patient diagnosed with cancer or an infection. In some embodiments, cells can be part of a mixed population of cells exhibiting different phenotypic characteristics.

[0262] On the other hand, this disclosure provides engineered cells (e.g., engineered immune cells) containing transgenes expressed under the control of a PGK promoter (such as a truncated PGK promoter). In one embodiment, the engineered cells contain a recombinant nucleic acid sequence encoding a receptor polypeptide (e.g., CAR, CCR, CD70 binding protein) or a nucleic acid inhibitor, as described herein.

[0263] In some embodiments, the engineered cells according to this disclosure (e.g., engineered immune cells) may contain one or more disrupted or inactivated genes. In some embodiments, the engineered cells according to this disclosure contain one disrupted or inactivated gene selected from the group consisting of CD52, GR, DCK, PDL1, PD-1, CTLA-4, LAG3, TIM3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, 2B4, HLA, TCRα, and TCRβ, and / or express CAR, multi-chain CAR, and / or pTα transgenes. In some embodiments, the isolated cells contain a polynucleotide encoding a polypeptide comprising a multi-chain CAR. In some embodiments, the isolated cells according to this disclosure contain two disrupted or inactivated genes selected from the group consisting of: CD52 and GR, CD52 and TCRα, CD52 and TCRβ, GR and TCRα, GR and TCRβ, TCRα and TCRβ, PD-1 and TCRα, PD-1 and TCRβ, CTLA-4 and TCRα, CTLA-4 and TCRβ, LAG3 and TCRα, LAG3 and TCRβ, TIM3 and TCRα, Tim3 and TCRβ, BTLA and TCRα, BTLA and TCRβ, BY55 and TCRα, BY55 and TCRβ, TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 and TCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ, 2B4 and TCRα, 2B4 and TCRβ, and / or expressing CAR, multi-strand CAR, and pTα transgenes. In some embodiments, the method includes destroying or inactivating one or more genes by introducing a nuclease capable of selectively inactivating the gene through selective DNA cleavage into the cell. In some embodiments, the nuclease may be, for example, a zinc finger nuclease (ZFN), a megaTAL nuclease, a mega-nuclease, a transcription activator-like effector nuclease (TALE nuclease / TALEN), or a CRISPR (e.g., Cas9, Cas12a, or Cas12b) nuclease.

[0264] In another embodiment, the recombinant nucleic acid sequence is integrated into a first site in the cell genome, such that the receptor polypeptide is expressed by the cell at the cell surface and / or the nucleic acid inhibitor is expressed intracellularly (e.g., an RNA interference sequence is expressed intracellularly), and wherein the integration of the recombinant nucleic acid into the first site reduces or prevents the expression of the target gene. In one embodiment, the integration reduces or prevents the expression of the target gene at the cell surface.

[0265] In other embodiments, integration reduces or prevents the expression of the functional T-cell receptor (TCR) complex at the cell surface. In some embodiments, nucleic acid sequences encoding receptor peptides and / or nucleic acid inhibitors are integrated into a single site within the cell genome.

[0266] In some embodiments, the nucleic acid sequence encoding the receptor polypeptide and / or nucleic acid inhibitor is integrated into two sites within the cellular genome. In some embodiments, the first site is an exon of a gene encoding a TCR complex protein. In other embodiments, the first site is an exon of a gene encoding a protein involved in the function or activity of the T cell receptor (TCR) αβ. In another embodiment, the target gene is a gene involved in the function or activity of the T cell receptor (TCR) αβ. In one embodiment, the first site is located in an exon selected from the following genes: TRAC (a component of TCR), HLA, TCRα, TCRβ, β2-microglobulin (“β2m”), CD52, GR, deoxycytidine kinase (DCK), PD-1, and CTLA-4. In another embodiment, the target gene is selected from TRAC (a component of TCR), HLA, TCRα, TCRβ, β2-microglobulin (“β2m”), CD52, GR, deoxycytidine kinase (DCK), PD-1, and CTLA-4.

[0267] This disclosure relates to an engineered cell, such as an engineered immune cell, comprising a transgene integrated into a target gene of the cell (e.g., an immune cell), wherein the expression of the transgene is controlled by a promoter that is not a promoter of the target gene, and methods for preparing and using such engineered cells. In another embodiment, the promoter is a non-endogenous or non-exogenous promoter relative to the target gene. In one embodiment, the promoter is a truncated PGK promoter. In other embodiments, the expression of the transgene is controlled by a truncated PGK promoter.

[0268] In one embodiment, the engineered cell comprises a polynucleotide, nucleic acid construct, or molecule containing a donor template for integration into the genome and subsequent expression by the cell, wherein the donor template contains a transgene. In another embodiment, the transgene comprises a recombinant nucleic acid sequence encoding a receptor polypeptide (e.g., CAR, CCR, CD70 binding protein) or a nucleic acid inhibitor, as described herein. In yet another embodiment, the transgene comprises one or more sequences encoding one or more receptor polypeptides and / or one or more nucleic acid inhibitors, as described herein.

[0269] In one aspect, this disclosure provides genetically modified immune cells for immunotherapy. The invention also provides genetically modified immune cells comprising a transgene having a nucleic acid sequence encoding a naturally occurring or non-natural polypeptide, such as a recombinant or chimeric polypeptide, said transgene being inserted into a gene involved in the function or activity of T-cell receptor (TCR) αβ. In one embodiment, the encoded polypeptide is a chimeric antigen receptor (CAR), a chimeric cytokine receptor (CCR), or a CD70-binding protein. In another embodiment, the nucleic acid sequence of the transgene encodes a nucleic acid inhibitor.

[0270] In some embodiments, the transgene includes a promoter that is exogenous relative to the gene to which the transgene is targeted. The promoter may originate from another gene that is not directly involved in the function or activity of T-cell receptor (TCR) αβ. In one embodiment, the promoter includes a nucleic acid sequence derived from the gene, comprising all or part of the original promoter sequence. In one embodiment, the gene is the phosphoglycerate kinase (PGK1) gene (PGK). In another embodiment, this disclosure provides a truncated PGK promoter that includes a portion of the nucleic acid sequence of the original PGK promoter sequence derived from, obtained from, or originating from the PGK gene.

[0271] In other embodiments, immune cells modified with a transgene containing a truncated PGK promoter-controlled CAR nucleic acid sequence exhibit improved amplification and / or cytotoxicity compared to immune cells modified with a transgene containing a CAR nucleic acid sequence controlled by an untruncated PGK promoter. In one embodiment, the CAR nucleic acid sequence is inserted into a gene involved in the function or activity of T cell receptor (TCR) αβ. In another embodiment, the gene is selected from TRAC (a component of TCR), HLA, TCRα, TCRβ, β2-microglobulin (“β2m”), CD52, GR, deoxycytidine kinase (DCK), PD-1, and CTLA-4. In another embodiment, the target gene is selected from TRAC (a component of TCR), HLA, TCRα, TCRβ, β2-microglobulin (“β2m”), CD52, GR, deoxycytidine kinase (DCK), PD-1, and CTLA-4.

[0272] In some embodiments, TCRs are rendered nonfunctional in cells according to this disclosure by disrupting or inactivating the TCRα gene and / or one or more TCRβ genes. Modified cells capable of proliferating independently of the TCRα signaling pathway are covered within the scope of this disclosure. In some embodiments, a method is provided for obtaining modified cells derived from an individual, wherein said cells are capable of proliferating independently of the major histocompatibility complex (MHC) signaling pathway. Modified cells readily obtainable by this method that can proliferate independently of the MHC signaling pathway are covered within the scope of this disclosure. The modified cells disclosed herein can be used for patients in need of treatment against host-vG rejection and graft-versus-host disease (GvHD); therefore, the scope of this disclosure includes methods for treating patients in need of treatment against host-vG rejection and graft-versus-host disease (GvHD) comprising treating the patient by administering to the patient an effective amount of modified cells containing disrupted or inactivated TCRα and / or TCRβ genes.

[0273] This document provides antigen-specific CAR-T cells, such as CD19-specific CAR-T cells, which contain a destroyed or inactivated dCK gene. In some embodiments, dCK knockout cells are prepared by transfecting T cells with a polynucleotide encoding a specific TAL nuclease targeting the dCK gene, for example, by transfecting T cells with mRNA encoding a specific TAL nuclease via electroporation. dCK knockout antigen-specific CAR-T cells (e.g., CD19-specific CAR-T cells) are resistant to PNAs, including, for example, clofarabine and / or fludarabine, and retain T-cell cytotoxicity against cells expressing antigens (e.g., cells expressing CD19).

[0274] In some embodiments, the isolated cells or cell lines of this disclosure may contain pTα or a functional variant thereof. In some embodiments, the isolated cells or cell lines may be further genetically modified by disrupting or inactivating the TCRα gene.

[0275] This disclosure also provides engineered immune cells comprising any of the CAR polynucleotides described herein. In some embodiments, the CAR can be introduced into immune cells as a transgene via a plasmid vector. In some embodiments, the plasmid vector may also contain, for example, a selection marker that provides identification and / or selection of cells receiving the vector.

[0276] On the other hand, receptor peptides (e.g., CAR, CCR, or CD70-binding peptides) can be synthesized in situ within the cell after the polynucleotide encoding the receptor peptide is introduced into the cell. Alternatively, the receptor peptide can be generated extracellularly and then introduced into the cell. Methods for introducing polynucleotide or nucleic acid constructs into cells are known in the art. In some embodiments, stable transformation methods (e.g., using lentiviral vectors) can be used to integrate polynucleotide or nucleic acid constructs into the cellular genome. In other embodiments, transient transformation methods can be used to transiently express polynucleotide or nucleic acid constructs, as well as polynucleotide or nucleic acid constructs not integrated into the cellular genome. In other embodiments, virus-mediated methods can be used. Polynucleotide or nucleic acid constructs can be introduced into cells by any suitable means, such as, for example, recombinant viral vectors (e.g., retroviruses, adenoviruses), liposomes, etc. Transient transformation methods include, for example, but not limited to, microinjection, electroporation, or particle bombardment. Polynucleotide or nucleic acid constructs can be included in a vector (e.g., a plasmid vector or viral vector).

[0277] In other implementations, the modified immune cells are used in immunotherapy.

[0278] Engineered immune cells can be genetically engineered to express one or more transgenes. These transgenes can be introduced into the genome of immune cells via various methods, including retroviral or lentiviral transduction. Transgenes delivered to cells in retroviral or lentiviral vectors can randomly integrate into the cell's genome after viral transduction. Random integration of transgenes can negatively impact the activity of engineered cells. Therefore, in some embodiments, one or more transgenes are introduced into engineered immune cells via site-specific integration.

[0279] In some embodiments, engineered immune cells express higher levels of one or more transgenes introduced via site-specific integration compared to cells introduced via lentiviral transduction, for example. In some embodiments, engineered immune cells exhibit improved activity of one or more transgenes introduced via site-specific integration compared to cells introduced via lentiviral transduction, for example. In some embodiments, engineered immune cells show fewer genomic translocations when one or more transgenes are introduced via site-specific integration compared to cells introduced via lentiviral transduction, for example. In some embodiments, one or more transgenes are introduced via site-specific integration into the CD52 locus. In some embodiments, the immune cells contain additional genetic modifications, including but not limited to knockout or knockdown of endogenous genes, enhancement of endogenous genes, and integration of exogenous genes. In some embodiments, the engineered immune cells include transgenes integrated into the CD52 locus and further include one or more genetic modifications at the TRAC locus. In some embodiments, one or more genetic modifications at the TRAC locus include integrating a transgene at the TRAC locus.

[0280] In some embodiments, the engineered cells (e.g., engineered immune cells) contain a polynucleotide integrated into the TRAC gene. In some embodiments, the engineered cells further contain an additional polynucleotide comprising a promoter operatively linked to one or more coding sequences, wherein said one or more coding sequences express a CCR, CD70 binding protein, or nucleic acid inhibitor, as described herein. In some embodiments, the one or more coding sequences of the additional polynucleotide further express a PD-1 dominant / negative receptor. In some embodiments, the additional polynucleotide further comprises a 5' homologous arm and a 3' homologous arm, wherein each of said 5' homologous arm and said 3' homologous arm is at least 100 nucleotides in length. In some embodiments, each of said 5' homologous arm and 3' homologous arm comprises a nucleotide sequence homologous to the nucleotide sequence of the CD52 gene. In some embodiments, the promoter comprises the EFS promoter described herein or a truncated PGK promoter. In some embodiments, the additional polynucleotide is integrated into the CD52 gene. In some implementations, the immune cells are T cells, NK cells, NK-T cells, tumor-infiltrating lymphocytes (TILs), or T cells or NK cells derived from iPSCs.

[0281] In related aspects, engineered cells (e.g., engineered immune cells) are provided that comprise a recombinant polynucleotide sequence integrated into the human TRAC gene, wherein the recombinant polynucleotide sequence comprises the 5' region of the human TRAC gene, a polynucleotide as described herein, and the 3' region of the human TRAC gene, wherein the integrated recombinant polynucleotide sequence prevents the expression of the human TRAC gene. In some embodiments, the engineered cell further comprises an additional recombinant polynucleotide sequence integrated into the human CD52 gene, wherein the additional recombinant polynucleotide sequence comprises the 5' region of the human CD52 gene, one or more coding sequences, and the 3' region of the human CD52 gene.

[0282] This document also provides cell lines obtained from transformed immune cells (e.g., T cells) according to any of the methods described above. This document also provides modified cells resistant to immunosuppressive therapy. In some embodiments, the isolated cells according to this disclosure contain a polynucleotide encoding a CAR.

[0283] The immune cells of this disclosure can be activated and expanded using well-known methods before or after genetic modification of the immune cells. Typically, the engineered immune cells of this disclosure can be expanded, for example, by contacting agents that stimulate the CD3 TCR complex and co-stimulatory molecules on the surface of T cells to generate T cell activation signals. For example, chemicals such as the calcium ion carrier A23187, phorbol 12-myristate 13-acetate (PMA), or mitogen lectins (such as phytohemagglutinin (PHA)) can be used to generate T cell activation signals.

[0284] In some embodiments, it can be achieved by contacting, for example, an anti-CD3 antibody (such as an OKT3 antibody) or its antigen-binding fragment, or an anti-CD2 antibody immobilized on a surface, or by contacting a protein kinase C activator (e.g., ， Bryostatin is contacted with a calcium ion carrier to stimulate cell populations, such as T cells, in vitro. Ligands binding to helper molecules on the surface of T cells are used to co-stimulate these molecules. For example, under conditions suitable for stimulating T cell proliferation, T cell populations can be contacted with anti-CD3 antibodies (e.g., OKT3 antibodies) and anti-CD28 antibodies. The anti-CD3 and anti-CD28 antibodies can be set on beads (such as plastic or magnetic beads), plates, or other substrates. Suitable conditions for T cell culture include appropriate culture media (e.g., [missing information - likely related to a specific culture medium]). ， Minimal essential culture medium or RPMI Media 1640 or X-vivo 15 (Lonza) may contain factors required for proliferation and survival, including serum (e.g., serum). ，Fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGFβ, and TNF, or any other cell growth additives known to the craftsman. Other additives for cell growth include, but are not limited to, surfactants, plasma salts, and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. Culture media may include RPMI 1640, A1M-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, which contain added amino acids, sodium pyruvate, and vitamins, and may be serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined group of hormones, and / or a sufficient amount of cytokines (e.g., IL-7 and / or IL-15) to promote T cell growth and expansion. Antibiotics, such as penicillin and streptomycin, are contained only in the experimental culture and not in the cell culture to be infused into the subject. The target cells are maintained under conditions necessary to support growth, such as a suitable temperature (e.g., 37°C) and atmosphere (e.g., air with 5% CO2). T cells exposed to different stimulation times may exhibit different characteristics. In some embodiments, the cells of this disclosure can be expanded by co-culturing with tissues or cells. The cells can also be expanded in vivo, for example, by expanding in the blood of a subject after the cells have been administered to the subject.

[0285] 4. Methods for generating engineered cells A variety of known techniques can be used in the preparation of polynucleotides, peptides, vectors, antigen-binding domains, engineered cells (e.g., engineered immune cells), compositions, etc., according to this disclosure. Cells can be obtained from a subject (e.g., a patient) or from a healthy donor prior to the in vitro manipulation or genetic modification of the immune cells described herein.

[0286] In one aspect, engineered cells are derived from suitable source materials, such as immune cells. In some embodiments, immune cells include T cells. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the art (such as FICOLL™ isolation).

[0287] Cells can be obtained from an individual's circulating blood via apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, the cells collected via apheresis can be washed to remove the plasma fraction and placed in a suitable buffer or medium for subsequent processing.

[0288] In some embodiments, T cells are isolated from PBMCs by lysing red blood cells and depleting monocytes (e.g., using centrifugation via a PERCOLL™ gradient). Specific T cell subsets (e.g., CD28+, CD4+, CDS+, CD45RA-, CD45RO+, CDS+, CD62-, CD95-, CD95+, IL2Rβ+, IL2Rβ-, CCR7+, CCR7-, CDL-, CD62L+, and combinations thereof) can be further isolated using positive or negative selection techniques known in the art. For example, T cell subsets are CD45RA+, CD95-, IL-2Rβ-, CCR7+, and CD62L+. For example, T cell subsets are CD45RA+, CD95+, IL-2Rβ+, CCR7+, and CD62L+. For example, T cell subsets are CD45RO+, CD95+, IL-2Rβ+, CCR7+, and CD62L+. For example, T cell subsets include CD45RO+, CD95+, IL-2Rβ+, CCR7-, and CD62L-. For example, T cell subsets include CD45RA+, CD95+, IL-2Rβ+, CCR7-, and CD62L-. For example, T cell populations can be enriched by negative selection by conjugating antibodies against surface markers specific to negatively selected cells. One method used herein is cell sorting and / or selection or flow cytometry via negative magnetic immunoadsorption, using a mixture of monoclonal antibodies against cell surface markers present on negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody mixture typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting can also be used to isolate cell populations of interest for use in this disclosure.

[0289] PBMCs can be directly used for genetic modification with immune cells (such as CARs or TCRs) as described herein. In some embodiments, after PBMC isolation, before or after genetic modification and / or expansion, T lymphocytes can be further isolated, and both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subsets.

[0290] In some embodiments, CD8+ cells are further sorted into naive, stem cell memory, central memory, and effector cells by identifying characteristic cell surface antigens associated with each of these types of CD8+ cells. In some embodiments, the expression of phenotypic markers for central memory T cells includes CD45RO, CD62L, CCR7, CD28, CD3, and CD127, and they are negative for granzyme B. In some embodiments, stem cell memory T cells are CD45RO-, CD62L+, and CD8+ T cells. In some embodiments, central memory T cells are CD45RO+, CD62L+, and CD8+ T cells. In some embodiments, effector T cells are negative for CD62L, CCR7, CD28, and CD127, and positive for granzyme B and perforin.

[0291] In some implementations, CD4+ T cells are further sorted into subsets. For example, CD4+ T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations with characteristic cell surface antigens.

[0292] On the other hand, engineered cells (e.g., engineered immune cells) are stem cell-derived cells, such as stem cell-derived immune cells. In some embodiments, immune cells may be derived from stem cells, such as progenitor cells, bone marrow stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells, and mesenchymal stem cells. iPS cells and other types of stem cells may be cultured immortalized cell lines or isolated directly from a patient. Various methods for isolating, developing, and / or culturing stem cells are known in the art and may be used in the practice of this invention.

[0293] In some embodiments, the cells (e.g., immune cells) are induced pluripotent stem cells (iPSCs) derived from reprogrammed cells (such as T cells). In some embodiments, the source material can be induced pluripotent stem cells (iPSCs) derived from T cells or non-T cells. The source material can alternatively be B cells, or any other cells derived from peripheral blood mononuclear cell isolates, hematopoietic progenitor cells, hematopoietic stem cells, mesenchymal stem cells, adipose stem cells, or any other somatic cell type.

[0294] This article provides methods for preparing engineered cells (e.g., engineered immune cells) for immunotherapy. In some embodiments, the methods include obtaining cells from a donor (e.g., donor immune cells), introducing a receptor polypeptide sequence or nucleic acid inhibitor (e.g., an RNA interference agent) (as described herein) under the control of a PGK promoter (e.g., a truncated PGK promoter) into the donor cells, and expanding the cells.

[0295] This disclosure provides methods for generating populations of engineered cells (e.g., engineered immune cells) comprising one or more engineered cells expressing one or more receptor peptides (e.g., CAR, CCR, or CD70 binding proteins) and / or nucleic acid inhibitors (e.g., RNA interference agents). In one embodiment, the transgene comprises a sequence encoding such a receptor peptide or nucleic acid inhibitor (e.g., an RNA interference agent), which can be introduced into the cell using a targeting method such that the transgene is inserted at a specific location in a target gene of interest. In one embodiment, the targeting method includes using a nuclease (such as a rare cleavage nuclease) that can cleave at a specific site within the target gene, thereby allowing the insertion of a transgene-containing polynucleotide, nucleic acid construct, or molecule at the specific cleavage site. In one embodiment, the expression of the receptor peptide and / or nucleic acid inhibitor is controlled by a PGK promoter (e.g., a truncated PGK promoter).

[0296] Prior to engineering the cells, cell sources can be obtained from the subject using a variety of non-limiting methods. Cells can be obtained from many non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, any number of T cell lines available and known to those skilled in the art can be used. In some embodiments, the cells can be derived from a healthy donor. In some embodiments, the cells can be part of a mixed population of cells exhibiting different phenotypic characteristics.

[0297] In one aspect, the present invention provides a method for preparing genetically modified immune cells, said genetically modified immune cells comprising one or more genetic modifications to one or more target genes involved in the function or activity of T cell receptor (TCR) αβ. In one embodiment, the one or more target genes are TRAC and / or CD52. The genetic modification is performed such that the expression and / or activity of one or more target genes is disrupted or inactivated. By disrupting or inactivating the target genes, the aim is to prevent the target genes from being expressed in the form of functional proteins and to impair the activity of the gene products.

[0298] In some embodiments, the method includes inactivating one or more target genes by introducing a rare-cutting endonuclease into cells, the rare-cutting endonuclease being capable of selectively inactivating the target gene by introducing a specific double-strand break at a target sequence within the target gene. In other embodiments, the method includes inactivating or reducing the expression level of one or more genes by introducing a rare-cutting endonuclease capable of selectively inactivating genes through selective DNA cleavage into cells. In some embodiments, the rare-cutting endonuclease may be, for example, a transcription activator-like effector nuclease (TALE nuclease or TALEN), a megaTAL endonuclease, a zinc finger endonuclease, a mega-nuclease, or a CRISPR endonuclease. In one embodiment, the rare-cutting endonuclease is TALEN. In another embodiment, the CRISPR endonuclease is a Cas9 endonuclease or a Cas12 endonuclease, including Cas12a and Cas12b.

[0299] In one aspect, target gene inactivation is achieved via the use of CRISPR endonucleases. CRISPR endonucleases are clustered regularly spaced short palindromic repeat (CRISPR)-associated endonucleases, such as Cas9 or Cas12. These endonucleases associate with guide RNA (gRNA), which guides the endonuclease to cleave at a specific site by hybridization to the target region. In this case, one or more gRNA molecules can be designed to hybridize to one or more specific sites within one or more target genes to guide cleavage by at least one CRISPR endonuclease. Exemplary gRNAs targeting the TRAC and CD52 genes are provided in Table 3, SEQ ID NO: 46 and 47, respectively.

[0300] On the other hand, rare-cutting endonucleases can be introduced into cells before, during, or after genetic modification of cells, such as immune cells. In one embodiment, a polynucleotide encoding a rare-cutting endonuclease is introduced into the cell by a suitable method, said method including but not limited to transfection, liposome transfection, transduction, electroporation, acoustic pore effect, gene gun method (e.g., gene gun), lipid transfection, polymer transfection, nanoparticles, viral transfection or transduction (e.g., retrovirus, lentivirus, AAV), or polymeric complex.

[0301] In some embodiments, the polynucleotide encoding a rare-cutting endonuclease according to this disclosure can be, for example, mRNA introduced directly into cells via electroporation. In some embodiments, cell pulse technology can be used to temporarily permeate living cells to deliver substances into the cells. Parameters can be adjusted to determine conditions for achieving high transfection efficiency with minimal mortality. In some embodiments, the rare-cutting endonuclease is a TALE nuclease.

[0302] This document also provides methods for transfecting cells (such as immune cells, e.g., T cells). In some embodiments, the method includes: contacting the cells with nucleic acids (e.g., RNA) and applying agile pulse sequences to the cells, said agile pulse sequences comprising: (a) electrical pulses with a voltage range of about 2250 to 3000 V per centimeter; (b) pulse widths of 0.1 ms; (c) pulse intervals of about 0.2 to 10 ms between the electrical pulses of steps (a) and (b); (d) electrical pulses with a voltage range of about 2250 to 3000 V per centimeter, pulse widths of about 100 ms, and pulse intervals of about 100 ms between the electrical pulse of step (b) and the first electrical pulse of step (c); and (e) four electrical pulses with a voltage of about 325 V, pulse widths of about 0.2 ms, and pulse intervals of 2 ms between each of the four electrical pulses.

[0303] In some implementations, the method of transfecting cells (such as immune cells, e.g., T cells) includes contacting the cells with RNA and applying a fast pulse sequence to the cells, said fast pulse sequence comprising: (a) an electrical pulse with a voltage of approximately 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900, or 3000 V per centimeter; (b) a pulse width of 0.1 ms; (c) a pulse interval between the electrical pulses in steps (a) and (b) of approximately 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ms; and (d) a voltage range of approximately 2250 to 3000 V per centimeter. V, for example, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900 or 3000 V per centimeter, with a pulse width of 100 ms and a pulse interval of 100 ms between the electrical pulse in step (b) and the first electrical pulse in step (c); and (e) 4 electrical pulses with a voltage of about 325 V, a pulse width of about 0.2 ms, and a pulse interval of about 2 ms between each of the four electrical pulses. This application discloses any values ​​included within the above range. The electroporation medium can be any suitable medium known in the art. In some embodiments, the conductivity of the electroporation medium ranges from about 0.01 to about 1.0 millisiemens.

[0304] In other embodiments, the rare cleavage endonuclease is specific for recognizing nucleic acid sequences within the target gene. In another embodiment, the endonuclease is expressed in the cell upon introduction and generates double-strand breaks or cleavage sites at the nucleic acid recognition sequences within the target gene. The presence of double-strand breaks or cleavage sites allows for site-specific integration of the transgene via a recombinant vector, as further described herein.

[0305] Engineered cells, such as engineered immune cells, can be genetically engineered to express one or more transgenes. Transgenes can be introduced into the cell genome via various methods, including retroviral or lentiviral transduction. Transgenes delivered into cells in retroviral or lentiviral vectors can randomly integrate into the cell's genome after viral transduction. Random integration of transgenes can negatively impact the activity of engineered cells. Therefore, in some embodiments, one or more transgenes are introduced into engineered immune cells via site-specific integration.

[0306] On the other hand, site-specific integration methods for preparing genetically modified cells (e.g., genetically modified immune cells) include the use of homology-directed repair (HDR). In one embodiment, HDR includes homology recombination (HR). In another embodiment, the method includes using a recombinant vector configured to use HDR (e.g., HR) to facilitate transgene integration into cells (e.g., immune cells). In another embodiment, the method includes using a polynucleotide containing one or more sequences homologous to a sequence of a target gene. In another embodiment, the method does not include the use of non-homologous end joining (NHEJ), and / or is not performed without using a polynucleotide containing one or more sequences homologous to a sequence of a target gene.

[0307] In one aspect, the site-specific integration method includes introducing a recombinant viral vector (e.g., an AAV vector) into a population of cells (e.g., immune cells), wherein the recombinant viral vector targets a double-strand break or cleavage site in a target sequence of a target gene. The introduction of the recombinant viral vector into the cells can be performed before, simultaneously with, or after the introduction of a rare cleavage endonuclease into the cells, as further described herein. In one embodiment, the method includes providing a recombinant viral vector comprising a donor template. The donor template comprises a transgene encoding a receptor polypeptide or a nucleic acid inhibitor. In other embodiments, the donor template further comprises flanking homologous arms. In another embodiment, the flanking homologous arms comprise a 5' homologous arm and a 3' homologous arm. In another embodiment, the 5' homologous arm comprises a sequence homologous to a sequence 5' upstream of the double-strand break or cleavage site in the target sequence, and the 3' homologous arm comprises a sequence homologous to a sequence 3' downstream of the double-strand break or cleavage site in the target sequence. In another embodiment, the donor template is integrated into the genome of a population of cells (e.g., an immune cell population) at the double-strand break or cleavage site. In another embodiment, the method includes culturing cells under conditions sufficient to allow transgene integration after the introduction of the recombinant viral vector. In one embodiment, the expression of the receptor peptide and / or nucleic acid inhibitor is controlled by a PGK promoter (e.g., a truncated PGK promoter).

[0308] In some embodiments, the method includes introducing a donor template containing a transgene into cells (e.g., immune cells) and amplifying the cells. In some embodiments, this disclosure relates to a method for engineering cells (e.g., immune cells) comprising the steps of: providing cells (e.g., immune cells), and i) expressing at least one receptor polypeptide at the cell surface, and / or ii) expressing a nucleic acid inhibitor (such as an RNA interference agent) intracellularly. In some embodiments, the method includes: introducing at least one polynucleotide encoding a receptor polypeptide and / or at least one nucleic acid inhibitor (e.g., an RNA interference agent) into the cells (e.g., by transfecting the cells with it), and expressing said at least one polynucleotide and / or said at least one nucleic acid inhibitor in the cells. In one embodiment, the expression of the receptor polypeptide and / or the nucleic acid inhibitor is controlled by a PGK promoter (e.g., a truncated PGK promoter).

[0309] In some embodiments, cells generated using SSI methods (e.g., engineered immune cells) express higher levels of one or more introduced transgenes compared to cells introduced via non-site-specific integration (SSI) methods (e.g., retroviral or lentiviral transduction). In some embodiments, engineered cells generated via SSI methods (e.g., engineered immune cells) exhibit improved activity of one or more introduced transgenes compared to cells introduced via non-SSI methods (e.g., retroviral or lentiviral transduction). In some embodiments, engineered cells generated using SSI methods (e.g., engineered immune cells) exhibit fewer genomic translocations of one or more transgenes compared to cells introduced via non-SSI methods (e.g., retroviral or lentiviral transduction).

[0310] In some embodiments, one or more transgenes are introduced into a target locus via site-specific integration. In some embodiments, engineered cells (e.g., engineered immune cells) contain additional genetic modifications, including but not limited to knocking out or knocking down endogenous genes, enhancing endogenous genes, and integrating exogenous genes. In some embodiments, engineered immunotherapy includes a transgene integrated into a first genetic locus and further includes one or more genetic modifications at a second genetic locus. In some embodiments, one or more genetic modifications at the first and / or second genetic locus include integrating a transgene into the first and / or second genetic locus. In one embodiment, the expression of the coding sequence of the transgene (e.g., the coding sequence of a receptor peptide and / or a nucleic acid inhibitor) is controlled by a PGK promoter (e.g., a truncated PGK promoter).

[0311] In some embodiments, the method includes the step of introducing a polynucleotide, nucleic acid construct, or molecule into a cell, wherein the polynucleotide, nucleic acid construct, or molecule contains a polynucleotide template for homologous recombination. In other embodiments, the polynucleotide template contains a sequence homologous to at least a portion of a nucleic acid sequence in a target gene, such that homologous recombination occurs between the target gene nucleic acid sequence and the polynucleotide template. In some embodiments, the polynucleotide template contains a 5' homologous arm homologous to a 5' sequence of a double-strand break in the target sequence of the target gene, a sequence encoding a transgene, and a 3' homologous arm homologous to a 3' sequence of a double-strand break in the target sequence of the target gene. After the target nucleic acid sequence is cleaved, a homologous recombination event occurs between the target nucleic acid sequence and the polynucleotide template.

[0312] 5. Treatment methods Engineered cells obtained by the methods described herein, such as engineered immune cells (e.g., CAR T cells), or cell lines derived from such engineered cells, can be used as pharmaceutical agents or for the preparation of pharmaceutical agents. In some embodiments, such pharmaceutical agents can be used to treat conditions, diseases, or disorders. This disclosure includes methods for treating or preventing disorders in a subject associated with an antigen or adverse and / or elevated levels of an antigen, as described herein. In one embodiment, the method includes administering an effective amount of at least one immune cell, comprising a CAR containing an antigen-binding domain specific to the antigen, as disclosed herein, to a patient in need.

[0313] In some embodiments, engineered cells according to this disclosure, such as engineered immune cells (e.g., CAR T cells), or cell lines derived from such engineered cells, can be used to manufacture agents for treating diseases or conditions in subjects in need.

[0314] Methods for treating diseases or conditions, including cancer and autoimmune diseases, are provided. In some embodiments, this disclosure relates to generating a T-cell-mediated immune response in a subject, including administering an effective amount of the engineered immune cells of this application to the subject. In some embodiments, the T-cell-mediated immune response targets one or more target cells. In some embodiments, the engineered immune cells comprise a chimeric antigen receptor (CAR). The CAR-containing immune cells of this disclosure can be used to treat diseases or conditions, such as CD19-related diseases or conditions and / or CD70-related diseases or conditions. In some embodiments, the disease or disorder can be an autoimmune disease, including but not limited to lupus, systemic lupus erythematosus (SLE), lupus nephritis, rheumatoid arthritis, systemic sclerosis, scleroderma, and myositis. In some embodiments, the target cells are tumor cells. In some aspects, this disclosure includes a method for treating or preventing malignant tumors, the method comprising administering an effective amount of at least one isolated antigen-binding domain described herein to a subject in need. In some aspects, this disclosure includes a method for treating or preventing malignant tumors, the method comprising administering an effective amount of immune cells to a subject in need, wherein the immune cells contain at least one chimeric antigen receptor, and a CD70 binding protein and / or CCR as described herein. In some embodiments, the method for treating or preventing malignant tumors includes administering an effective amount of a population of immune cells to a subject in need, wherein the immune cells contain at least one chimeric antigen receptor, and a CD70 binding protein and / or CCR as described herein. The CAR-containing immune cells of this disclosure can be used to treat malignant tumors associated with an antigen (e.g., CD19) or malignant tumors involving abnormal expression of an antigen (e.g., CD19). In some embodiments, the CAR-containing immune cells of this disclosure can be used to treat malignant tumors such as non-Hodgkin lymphoma (NHL), refractory and / or relapsed NHL, large B-cell lymphoma (LBCL), follicular lymphoma (FL), T-cell lymphoma (TCL), B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), primary central nervous system lymphoma (PCNSL), mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), and peripheral T-cell lymphoma (PTCL). In exemplary embodiments, the CAR-containing immune cells of this disclosure, such as anti-CD19 CAR-T cells, are used to treat LBCL.

[0315] A method for reducing tumor size in a subject is also provided, the method comprising administering engineered cells of the present disclosure to the subject, wherein the cells contain a chimeric antigen receptor containing an antigen-binding domain, such as a CD19 antigen-binding domain, and binding to an antigen on the tumor, such as a CD19 antigen.

[0316] In some embodiments, the subject has a solid tumor or a hematologic malignancy such as lymphoma or leukemia. In some embodiments, engineered cells are delivered to a tumor bed, such as a tumor bed in small cell lung cancer. In some embodiments, the cancer is present in the subject's bone marrow. In some embodiments, the engineered cells are autologous immune cells, such as autologous T cells. In some embodiments, the engineered cells are allogeneic immune cells, such as allogeneic T cells. In some embodiments, the engineered cells are heterologous immune cells, such as heterologous T cells. In some embodiments, the engineered cells are transfected or transduced in vitro. As used herein, the term "in vitro cell" refers to any cell cultured in vitro.

[0317] In some embodiments, cells containing chimeric antigen receptors that include antigen-binding domains (e.g., CD19 antigen-binding domains) exhibit enhanced cytotoxicity and potency against pathological cells associated with autoimmune indications, such as CD19-positive pathological cells associated with autoimmune indications.

[0318] "Effective dose" is any amount that provides the expected or beneficial outcome when used alone or in combination with another agent. For a therapeutic agent (such as engineered CAR T cells), "therapeutic effective dose," "effective dose," or "therapeutic effective dose" refers to any amount that, when used alone or in combination with another therapeutic agent, protects a subject from disease onset or promotes disease remission (manifested as a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom resolution, or prevention of injury or disability caused by the disease). The ability of a therapeutic agent to promote disease remission can be assessed using a variety of methods known to skilled practitioners (e.g., physicians or clinicians), such as assessment in human subjects during clinical trials, assessment in animal model systems predicting efficacy in humans, or assessment by measuring the activity of the agent in an in vitro assay.

[0319] The terms “patient” and “subject” are used interchangeably and include human and non-human animal subjects, as well as people with a formally diagnosed condition, people with an undiagnosed condition, people receiving medical care, people at risk of developing a condition, etc.

[0320] The term "treatment" includes therapeutic treatment, preventative treatment, and applications that reduce the risk of a subject developing a certain condition or other risk factors. Treatment does not necessarily require a complete cure for the condition and encompasses implementation plans that alleviate symptoms or potential risk factors. The term "prevention" does not require a 100% elimination of the likelihood of the event. Rather, it indicates a reduced likelihood of the event occurring in the presence of a compound, immune cell, therapeutic agent, or method.

[0321] The desired therapeutic total number of cells in the composition includes at least two cell types (e.g., at least one CD8+ T cell and at least one CD4+ T cell, or two CD8+ T cells, or two CD4+ T cells), or more typically greater than 10. 2 Cells, and at most 10 6 One, up to 10 8 One or 10 9 One cell, and can be 10 10 Or 10 12 One or more cells. The number of cells will depend on the intended use of the composition and the type of cells included in the composition. A cell density greater than 10 is typically desired. 6 10 cells / ml and usually greater than 10 7 10 cells / ml, usually 10 8 Cells / ml or greater. Clinically relevant numbers of immune cells can be allocated to multiple infusions, accumulating to 10 or more. 5 10 6 10 7 10 8 10 9 10 10 10 11 Or 10 12 Cells. In some aspects of this disclosure, particularly because all infused cells will be redirected to a specific target antigen (e.g., CD19), a lower number of cells can be administered, in 10 6 / kg (10 6 -10 11 CAR therapy can be administered multiple times at doses within these ranges. For patients undergoing therapy, the cells can be autologous, allogeneic, or xenogeneic.

[0322] In some implementations, the therapeutically effective dose of CAR T cells is approximately 1 x 10⁻⁶. 5 Cells / kg, approximately 2 x 10 5 Cells / kg, approximately 3 x 10 5 1 cell / kg, approximately 4 x 10 5 Cells / kg, approximately 5 x 10 5 cells / kg, approximately 6 x 10 5 1 cell / kg, approximately 7 x 10 5 Cells / kg, approximately 8 x 10 5 Cells / kg, approximately 9 x 10 5 cells / kg, 2 x 10 6 Cells / kg, approximately 3 x 10 61 cell / kg, approximately 4 x 10 6 Cells / kg, approximately 5 x 10 6 cells / kg, approximately 6 x 10 6 1 cell / kg, approximately 7 x 10 6 Cells / kg, approximately 8 x 10 6 Cells / kg, approximately 9 x 10 6 cells / kg, approximately 1 x 10 7 Cells / kg, approximately 2 x 10 7 Cells / kg, approximately 3 x 10 7 1 cell / kg, approximately 4 x 10 7 Cells / kg, approximately 5 x 10 7 cells / kg, approximately 6 x 10 7 1 cell / kg, approximately 7 x 10 7 Cells / kg, approximately 8 x 10 7 1 cell / kg or approximately 9 x 10 7 Cells / kg

[0323] In some implementations, the target dose range for CAR+ / CAR-T+ cells is approximately 1 × 10⁻⁶. 6 To approximately 1×10 10 cells / kg, for example, about 1×10⁻⁶ 6 cells / kg, approximately 1×10 7 cells / kg, approximately 1×10 8 cells / kg, approximately 1×10 9 Cells / kg or approximately 1×10⁻⁶ 10 Cells / kg. It should be understood that doses higher or lower than this range may be suitable for some subjects, and healthcare providers may determine the appropriate dose level as needed. Furthermore, multiple doses of cells may be provided according to this disclosure.

[0324] In some aspects, this disclosure includes a pharmaceutical composition comprising at least one antigen-specific CAR as described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises an additional active agent.

[0325] The CAR-expressing cell populations disclosed herein can be administered alone or as pharmaceutical compositions in combination with diluents and / or other components (such as IL-2 or other cytokines or cell populations). The pharmaceutical compositions of this disclosure may comprise CAR-expressing cell populations (such as T cells) as described herein, said cell populations in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of this disclosure can be formulated for intravenous administration.

[0326] Pharmaceutical compositions (solutions, suspensions, etc.) may include one or more of the following: sterile diluents, such as water for injection, saline solutions (preferably physiological saline, Ringer's solution, isotonic sodium chloride), non-volatile oils (such as synthetic monoglycerides or diglycerides) that can be used as solvents or suspension media, polyethylene glycol, glycerol, propylene glycol, or other solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates; and agents for adjusting tonicity, such as sodium chloride or dextran. Parenteral preparations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. For therapeutic applications, injectable pharmaceutical compositions are preferably sterile.

[0327] In some embodiments, when administered to a patient, engineered immune cells expressing any of the antigen-specific CARs described herein (e.g., CD19-specific CARs) at their cell surface can reduce, kill, or lyse the patient's endogenous antigen-expressing cells, such as CD19-expressing cells. In one embodiment, the percentage reduction or lysis of antigen-expressing endogenous cells (e.g., CD19-expressing endogenous cells) or antigen-expressing cell lines (e.g., CD19-expressing cell lines) by engineered immune cells expressing any of the antigen-specific CARs described herein (e.g., CD19-specific CARs) is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In one embodiment, the percentage reduction or lysis of endogenous cells expressing the antigen (e.g., endogenous cells expressing CD19) or cell lines expressing the antigen (e.g., cell lines expressing CD19) by engineered immune cells expressing any of the antigen-specific CARs described herein (e.g., CD19-specific CARs) is about 5% to about 95%, about 10% to about 95%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 25% to about 75%, or about 25% to about 60%. In one embodiment, the endogenous antigen-expressing cells (e.g., CD19-expressing cells) are endogenous antigen-expressing bone marrow cells, such as endogenous CD19-expressing bone marrow cells.

[0328] In one embodiment, the assays disclosed herein can be used to measure the percentage reduction or lysis of target cells (e.g., cell lines expressing antigens such as CD19) by engineered immune cells expressing the antigen-specific CAR (e.g., CD19-specific CAR) disclosed herein at their cell surface membranes.

[0329] The method may further include administering one or more chemotherapeutic agents to the patient prior to the administration of the engineered cells provided herein. In some embodiments, the chemotherapeutic agent is a lymphodepleting (preconditioning) chemotherapeutic agent. For example, a method of preconditioning a patient requiring T-cell therapy includes administering a specific beneficial dose of cyclophosphamide (200 mg / m²) to the patient. 2 / day up to 2000 mg / m 2 / day, approximately 100 mg / m 2 / day to approximately 2000 mg / m 2 / day; for example, approximately 100 mg / m² 2 / day, approximately 200 mg / m 2 / day, approximately 300mg / m 2 / day, approximately 400 mg / m 2 / day, approximately 500 mg / m 2 / day, approximately 600 mg / m 2 / day, approximately 700 mg / m 2 / day, approximately 800 mg / m 2 / day, approximately 900 mg / m 2 / day, approximately 1000 mg / m 2 / day, approximately 1500 mg / m 2 / day or approximately 2000 mg / m 2 ( / day) and a specific dose of fludarabine (20 mg / m²) 2 / day up to 900 mg / m 2 / day, approximately 10 mg / m² 2 / day to approximately 900 mg / m 2 / day; for example, approximately 10mg / m² 2 / day, approximately 20 mg / m 2 / day, approximately 30 mg / m 2 / day, approximately 40 mg / m 2 / day, approximately 40 mg / m 2 / day, approximately 50 mg / m 2 / day, approximately 60 mg / m 2 / day, approximately 70 mg / m 2 / day, approximately 80 mg / m 2 / day, approximately 90 mg / m 2 / day, approximately 100 mg / m 2 / day, approximately 500mg / m 2 / day or approximately 900 mg / m 2 / day). An exemplary dosing regimen involves treating a patient and includes administering approximately 300 mg / m² to the patient daily. 2 / day of cyclophosphamide and approximately 30 mg / m 2 Fludarabine per day, or cyclophosphamide administered to the patient before or after fludarabine administration for three days, followed by a therapeutically effective dose of engineered T cells.

[0330] In some embodiments, particularly when the engineered cells provided herein have been genetically edited to eliminate or minimize the surface expression of CD52, lymphocyte depletion further includes administration of an anti-CD52 antibody, such as alemtuzumab. In some embodiments, the CD52 antibody is administered at a dose of about 1-20 mg / day IV, such as about 13 mg / day IV, such as about 20 mg / day IV, such as about 30 mg / day IV, for 1, 2, 3 days or longer. The antibody may be administered in combination with other elements of the lymphocyte depletion regimen (e.g., cyclophosphamide and / or fludarabine), before or after the use of these components. Exemplary anti-CD52 antibody sequences, such as SEQ ID NO: 67-74, are provided in Table 3.

[0331] In other implementations, the antigen-binding domain, transducing (or other engineered) cells, and chemotherapeutic agents are each administered in an amount effective in treating the subject's disease or ailment.

[0332] In some embodiments, the compositions disclosed herein comprising CAR-expressing immune effector cells can be administered in combination with any number of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; and ethyleneimine and methylmelamine derivatives, including altretamine, triethylenemelamine, and triethylenylphosphamide. ephosphoramide, triethylenethiophosphoramide, and trimethylomelamine; nitrogen mustards, such as chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, and mechlorethamine hydrochloride. Oxygen hydrochloride, melphalan, novombhichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitroureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine;Antibiotics, such as aclacinomysin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin C, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-leucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, and mycophenolic acid. Folic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and fluorouracil (5-FU); folic acid analogues, etc. Examples include denopterin, methotrexate, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; and pyrimidine analogs such as ancitabine, azacitidine, 6-azouridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, fluxuridine, and 5-FU.Androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenergics, such as aminoglutethimide, mitotane, and trilostane; folic acid supplements, such as frolinic acid; aceglatone; aldophosphamide glycoside; and aminolevulinic acid. acid); amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; mitoguazone; mitoxantrone; mopidamnol; nitraerine; pentostatin; phenamet; pirarubicin; podophyllinic acid acid); 2-ethylhydrazine; procarbazine; PSK®; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2”-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactalol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa;Taxanes, such as paclitaxel (TAXOL™, Bristol-Myers Squibb) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer); chloranmbucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C. C); mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RF S2000; difluoromethylornithine (DMFO); retinic acid derivatives such as Targretin™ (bexarotene), Panretin™ (alitretinoin); ONTAK™ (denileukin diftitox); esperamicins; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. This definition also includes anti-hormonal agents that modulate or inhibit the effects of hormones on tumors, such as anti-estrogens, including, for example, tamoxifen, raloxifene, aromatase inhibitors 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, raloxifene hydrochloride, LY117018, onapristone, and toremifene (Fareston); and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing. Chemotherapy agents, including but not limited to the CHOP regimen, may also be administered in combination where appropriate, namely cyclophosphamide (Cytoxan®), doxorubicin (hydroxydoxorubicin), vincristine (Oncovin®), and prednisone.

[0333] In some embodiments, the chemotherapeutic agent is administered concurrently with or within one week of the engineered cells, peptides, or nucleic acids. In other embodiments, the chemotherapeutic agent is administered approximately 1–7 days, approximately 1 to approximately 4 weeks, approximately 1 week to approximately 1 month, approximately 1 week to approximately 2 months, approximately 1 week to approximately 3 months, approximately 1 week to approximately 6 months, approximately 1 week to approximately 9 months, or approximately 1 week to approximately 12 months after the administration of the engineered cells, peptides, or nucleic acids. In other embodiments, the chemotherapeutic agent is administered at least 1 month prior to the administration of the cells, peptides, or nucleic acids. In some embodiments, the method further includes administering two or more chemotherapeutic agents.

[0334] A variety of additional therapeutic agents can be used in combination with the compositions described herein. For example, potentially useful additional therapeutic agents include PD-1 inhibitors such as nivolumab (Opdivo®), pembrolizumab (Keytruda®), pembrolizumab, pidilizumab, and atezolizumab.

[0335] Additional therapeutic agents suitable for use in combination with this disclosure include, but are not limited to: ibrutinib (Imbruvica®), ofatumumab (Arzerra®), rituximab (Rituxan®), bevacizumab (Avastin®), trastuzumab (Herceptin®), trastuzumab emtansine (KADCYLA®), imatinib (Gleevec®), cetuximab (Erbitux®), and panitumumab.Vectibix®, catumaxomab, ibritumomab, oftatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, and more. Latinib, Axitinib, Masitinib, Pazopanib, Sunitinib, Sorafenib, Toceranib, Lestaurtinib, Axitinib, Cedilanib, Lenvatinib, Nintedanib, Pazopanib, Regorafenib nib), semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib Bosutinib, Letatinib, Ruxolitinib, Pacritinib, Cobimetinib, Selumetinib, Trametinib, Binimetinib, Alectinib, Ceritinib, Crizotinib, Aflibercept, Adipotide, Denileukin Diftitox, mTOR inhibitors such as Everolimus and Temsirolimus, Hedgehog inhibitorsInhibitors such as sodegib and vismodegib, and CDK inhibitors such as the CDK inhibitor palbociclib.

[0336] In some embodiments, compositions comprising CAR-containing immune cells may be administered in conjunction with a treatment regimen to prevent or reduce cytokine release syndrome (CRS) or neurotoxicity. Treatment regimens for preventing CRS or neurotoxicity may include lenzilumab, tocilizumab, atrial natriuretic peptide (ANP), anakinra, and iNOS inhibitors (e.g., L-NIL or 1400W). In other embodiments, compositions comprising CAR-containing immune cells may be administered in conjunction with an anti-inflammatory agent. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDs) (including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide), anti-TNF drugs, cyclophosphamide, and mycophenolate mofetil. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, COX-2 inhibitors, and sialic acid derivatives. Exemplary analgesics include acetaminophen, oxycodone, tramadol, or propoxyphene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules targeting cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors such as TNF antagonists (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®), and infliximab (REMICADE®), chemokine inhibitors, and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies and recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold preparations (oral (auronoxine) and intramuscular), and minocycline.

[0337] In some embodiments, the compositions described herein are administered in combination with cytokines. Examples of cytokines include lymphokines, monokines, and conventional polypeptide hormones. Among the cytokines are: growth hormones, such as human growth hormone, N-methionylhuman growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; pro-relaxin; glycoprotein hormones, such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatocyte growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental prolactin; Müllerian inhibitory substances; mouse gonadotropin-related peptides; and inhibitory peptides. Inhibitors; activators; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor (NGF), such as NGF-β; platelet-derived growth factor; transforming growth factor (TGF), such as TGF-α and TGF-β; insulin-like growth factor-I and insulin-like growth factor-II; erythropoietin (EPO); bone-inducing factor; interferons, such as interferon-α, interferon-β, and interferon-γ; colony-stimulating factors (CSF), such as macrophage-CSF. (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, IL-21; tumor necrosis factor, such as TNF-α or TNF-β; and other polypeptide factors, including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins derived from natural sources or recombinant cell cultures, as well as bioactive equivalents of naturally occurring sequence cytokines.

[0338] 6. Reagent kits and products This application provides a kit comprising any of the CD19 CAR-containing immune cells described herein, and pharmaceutical compositions thereof. In one embodiment of the kit, engineered CAR cells (e.g., CAR T cells) are frozen in a suitable culture medium, such as CryoStor® CS10, CryoStor® CS2, or CryoStor® CS5 (BioLife Solutions).

[0339] In some exemplary embodiments, the kits disclosed herein include T cells containing allogeneic CD19 CAR for administering lymphocyte depletion and CAR-T regimens to subjects.

[0340] This application also provides articles or kits comprising any of the therapeutic compositions described herein. Examples of articles include vials (e.g., sealed vials).

[0341] All references cited in this article, including patents, patent applications, papers, textbooks, and the references cited therein, are hereby included in their entirety by reference, if they have not already been cited.

[0342] Table 3

[0343] Example Experimental methods To prepare lentiviruses encoding the desired constructs, HEK293T cells were seeded at 450,000 cells / mL in 2 mL of DMEM (Gibco) supplemented with 10% FBS (Hyclone) in each well of a 6-well plate the day before transfection. On the day of transfection, lentiviruses were prepared as follows: 1.5 μg psPAX2 lentiviral packaging vector, 0.5 μg pMD2G, and 0.5 μg appropriate transfer CAR vector were mixed together in 250 μL Opti-MEM (Gibco) in each well of a 6-well plate (“DNA mixture”). 10 μL of Lipofectamine 2000 (Invitrogen) in the 250 μL Opti-MEM was incubated at room temperature for 5 minutes, and then added to the DNA mixture. The mixture was incubated at room temperature for 20 minutes, and a total volume of 500 μL was slowly added to the well walls containing HEK293T cells. One day after transfection, the culture medium for HEK293T cells in each well of a 6-well plate was replaced with 2 mL of T cell transduction medium per well, i.e., X-Vivo-15 medium supplemented with 10% FBS. Two days after transfection, the lentiviral supernatant from HEK293T cells was harvested and passed through a 0.45 μm filter (EMD Millipore) to remove cell debris. The supernatant was then concentrated 25-fold using a Lenti-X concentrator (Takara Bio) according to the manufacturer's instructions, aliquoted, and rapidly frozen. Lentiviral titers were determined by thawing aliquots of frozen lentivirus, performing 4-fold serial dilutions, and then performing limiting dilution titrations on JurkaT cells (clone E6-1; ATCC). On day 0, purified T cells were activated in Grex-24 plates (Wilson Wolf, catalog number 80192M) in X-Vivo-15 medium (Lonza) supplemented with 100 IU / mL human IL-2 (Miltenyi Biotec), 10% FBS (Hyclone), and human T TransAct (Miltenyi Biotec, catalog number 130-111-160, 1:100 dilution). On day 2, T cells were resuspended at 500,000 cells / mL in T cell transduction medium and transduced in Grex-24 plates at MOI=5 with the appropriate lentiviral stock solution and 100 IU / mL human IL-2. On day 5 after transduction, cells were harvested and washed to remove residual IL-2. They were then resuspended in T-cell expansion medium, namely X-Vivo-15 supplemented with 5% human AB serum (Gemini Bio), and each sample was divided into two parts, one part receiving 100 IU / mL human IL-2 according to the standard protocol, and the other part receiving a lower concentration of 25 IU / mL human IL-2.Cells were expanded into larger G-Rex containers (Wilson Wolf) as needed using T-cell expansion medium and appropriate concentrations of human IL-2. The absolute number of T cells in each sample was counted on days 5, 9, and 14, and transduction efficiency was determined by detecting the percentage of T cells binding to the FITC-conjugated v5-tagged monoclonal antibody (Thermo Fisher) using flow cytometry. The CAR-T cell products were cryopreserved on day 14 or 15 and thawed as needed for further assays.

[0344] For cells generated via AAV6 site-specific integration, TransAct was removed by centrifugation two days after T cell activation. Cells were then washed with PBS and electroporated using an AMAXA 4D nuclear transfection electroporator at 1 x 10⁻⁶ cells per cell. 6 Cells were electroporated with 0.5 μg of TALEN® mRNA per arm of nuclease. Following the gene editing step, recombinant AAV6 containing one or more transgenes was added to T cells at an MOI of 10,000–15,000. After electroporation, cells were incubated overnight at 30°C. Cells were then returned to 37°C and the above T cell culture conditions. On day 14 post-activation, TCRα / β depletion was performed using the EasySep™ Human TCRα / β Depletion Kit (STEMCELL Technologies) according to the manufacturer's instructions. On day 15 or 16 post-activation, T cells were cryopreserved in 90% FBS / 10% DMSO using a rate-controlled freezer and stored in liquid nitrogen vapor phase.

[0345] AAV-mediated site-specific integration can also be performed using CRISPR endonucleases, such as Cas12i endonuclease, or CRISPR reagents. Pan-T cell donors were thawed and activated for 2 days with GMP-grade TransAct and IL-2. Prior to electroporation, Cas12i nuclease and TRAC-targeting guide RNA (gRNA) were incubated together at room temperature to form a ribonucleoprotein complex (RNP). T cells were then mixed with the RNP and electroporated. Immediately afterward, cells were incubated overnight with AAV containing one or more transgenes at a specified MOI and then transferred to GREX plates in complete T cell culture medium supplemented with IL-2. Cells were subsequently expanded for 12 days and then frozen. Flow cytometry analysis was performed on days 4, 7, and 11 post-electroporation. The transgene cassette was expected to insert into the target sequence GACCCTGCC within exon 1 of TRAC.

[0346] Standard Long-Term Kill Assay (LTKA). CAR T cells were co-cultured with Raji cells expressing luciferase-GFP at an effector-to-target cell ratio of 8:1. Every 2–3 days, half of the cells were passaged into fresh Raji cells. The remaining half of the cells were then used to determine target cell killing using Bright-glo reagent (Promega).

[0347] Continuous restimulation assays based on flow cytometry were performed in 24-well G-rex® (Wilson Wolf, catalog number 80192M) using 5 x 10⁻⁶ cells. 5 CAR T cells and 5x10 5 Raji luciferase-GFP cells were co-cultured in RPMI 1640 + 10% FBS medium (Hyclone). A small aliquot of the co-cultured CAR T cells and target cells was stained with antibodies using 123counteBeads™ counting beads (Thermo Fisher Scientific, catalog number 01-1234-42) to determine the baseline phenotype and cell count at the start of the assay. Every 3–4 days, 50% of the supernatant was removed from the G-rex® container, and the remaining cell suspension was thoroughly mixed. A small aliquot was then used for phenotypic analysis and cell counting, as described above. After determining the cell count, fresh Raji cells were added back to each well to restore the E:T ratio to 1:1. Continuous restimulation was repeated until CAR T cells no longer killed Raji target cells.

[0348] Single-stimulation FACS assays are similar to continuous-stimulation assays based on flow cytometry. CAR T cells and Raji cells are co-cultured in 24-well G-rex® cells at an E:T ratio of 1:2 or 1:4, instead of a 1:1 E:T ratio. Every 3–4 days, 50% of the supernatant is removed from the G-rex® cells, and the remaining cell suspension is thoroughly mixed. A small aliquot is taken for phenotypic analysis and cell counting, as described above. Fresh Raji cells are not added back; only fresh culture medium is added back. Time points are collected every 3–4 days until Raji cells are completely eliminated by CAR T cells, or until Raji cells outnumber T cells.

[0349] Sensitized T-mixed lymphocyte reaction (MLR) human PBMCs (host) are sensitized to irradiated, unedited T cells derived from donors used to create the aforementioned gene-edited transplanted T cells to promote the expansion of allogeneic reactive T cell clones. Briefly, transplanted PBMCs are irradiated with 30 Gy and co-cultured with host PBMCs at a 1:1 ratio in R10 + 20 IU / mL IL-2 + 10 ng / mL IL-7 + 10 ng / mL IL-15 (Miltenyi, catalog number 130-095-765) for 4 days. The medium is then replaced with cytokine-free R10, and the cells are cultured for another 3 days. Subsequently, pan-T cells are isolated using MACS negative selection (Miltenyi, Human Pan-T Cell Isolation Kit, catalog number 130-096-535) as recommended by the manufacturer. In 96-well plates, 20,000 gene-edited transplanted T cells were seeded together with 20,000 sensitized host T cells and cultured for 2 days at 37°C and 5% CO2 in R10 + 20 IU / mL IL-2. The viability of the transplanted T cells was determined by flow cytometry using gated absolute counts of live TCRαβ-CAR+ T cells.

[0350] Example 1: Generation of CAR T cells expressing anti-CD19 CAR from lentiviral vector Based on the anti-CD19 scFv, CD3z signaling domain, and 4-1BB co-stimulatory domain of SEQ ID NO: 4, constructs for expressing second-generation anti-CD19 CAR were designed and cloned into lentiviral vectors (LVV). CAR T cells generated from donor PBMCs either expressed CD19 CAR alone or expressed a chimeric cytokine receptor (CCR) and CD19 CAR (CD19 CAR / CCR) from a bicistronic expression cassette linked via a cleavable P2A peptide. The CCR contained a TPOR transmembrane / JAK binding and activation domain and an IL2R intracellular signaling domain, as illustrated in SEQ ID NO: 27. Gene expression in all LVV constructs was driven by the elongation factor 1α (EF-1α) promoter. CAR T cells were generated from donor PBMCs activated by LVV transduction. The cytotoxic activity of CAR T cells with different constructs from three different donors against CD19-positive Raji and Daudi cells was evaluated by a long-term killing assay (LTKA) in vitro. like Figures 1A to 1C As shown, in most cases, co-expression of CCR enhances the cytotoxicity of CD19 CAR T cells.

[0351] In an orthotopic tumor model established by injecting Raji cells into NSG mice, the in vivo cytotoxicity of CAR T cells engineered with a selected construct was evaluated. Briefly, Raji cells expressing luciferase were intravenously injected into mice. CAR T cells were injected 4 days after tumor cell transplantation. Tumor burden was measured using an IVIS Spectrum instrument. Figures 2A to 2B As shown, CD19 CAR T cells co-expressing CCR in 3x10 6 At a dose level of CAR T cells, tumor cells in animal models were effectively reduced.

[0352] Example 2: Site-specific integration of CAR T cells expressing anti-CD19 CAR LVV constructs were transduced into PBMCs to generate engineered cells with randomly integrated transgenes in the host cell genome. Transgenes can be introduced into cells via site-specific integration (SSI) into predetermined genetic loci to ensure consistency of insertion sites in the genome and limit the number of integration events. Site-specific integration using vectors such as adeno-associated virus (AAV) can also accommodate one or more transgenes, which can be larger than those accommodated by lentiviral vectors, while maintaining high transduction efficiency. CD19 CAR / CCR constructs cloned into AAV vectors were inserted into the TRAC locus via homologous recombination, and the expression of CCR and CD19 CAR was driven by a short EF1α promoter (EFS). The resulting CAR T cells were evaluated for in vivo cytotoxicity in a Raji orthotopic tumor model in NSG mice and compared with CAR T cells expressing the same CCR and CD19 CAR generated via LVV transduction (N=8 mice / group, 2.5 x 10⁻⁶ cells / year). 6 CAR + (cells / mouse).

[0353] Figure 3A The results showed, unexpectedly, that CAR T cells generated through site-specific integration exhibited significantly lower tumor control in vivo compared to CAR T cells transduced via LVV. CAR T cells derived from different donors reproduced these results. Consistent with poor in vivo cytotoxicity, CAR T cells generated through site-specific integration failed to expand during the experimental period and did not persist in treated mice, in contrast to CAR T cells transduced via LVV. Figure 3B ).exist Figures 5A to 5CSimilar in vitro results were also observed, in which CAR T cells generated by site-specific integration (SSI) driven by the EFS promoter (SSI EFS CD19 / CCR) showed lower cytotoxicity in standard long-term killing assays and expanded less than CAR T cells generated by LVV transduction.

[0354] Example 3: An improved anti-CD19 CAR construct targeting site-specific integration To enhance the in vitro and in vivo antitumor activity of our SSI-engineered CAR T cells, we tested a range of different promoters to drive CCR and CAR expression. All SSI constructs were designed to integrate into the T cell receptor α constant region (…). TRAC The CD19CAR gene locus was used. Surface expression of CD19CAR was measured by flow cytometry using anti-idiotype (anti-ID) antibodies (ACROBiosystems) to measure the mean fluorescence intensity (MFI) of CAR and to detect the antitumor activity of CAR T cells. Several heterologous promoters were tested, including three derived from human phosphoglycerate kinase 1 (…). PGK1 The promoters of the gene were named PGK-SSI-1, PGK-SSI-2, and PGK-SSI-3. SSI-engineered CAR T cells with constructs containing the endogenous promoter (Endo) of SEQ ID NO: 33 were also tested. Figures 4A to 4B ).

[0355] CAR MFI is measured at the end of manufacturing. It is derived from human ubiquitin B (…). UBB The CAR construct driven by the promoter (UBB300) exhibited the highest CAR expression at the end of manufacturing. Figure 4A The three PGK SSI promoters, as well as the human elongation factor 1-α1 short (EFS) promoter and the human cyclophilin A (CypA300) promoter, all produced similar CAR expression at the end of manufacturing. Figure 4A Next, in a flow cytometry-based continuous restimulation cytotoxicity assay, CAR T cells were analyzed against the CD19+ tumor cell line Raji at an effector cell to target cell (E:T) ratio of 1:1. Surprisingly, the PGK SSI promoter construct exhibited superior antitumor activity compared to promoters used in all other assays. Figure 4B ).

[0356] To understand the mechanisms underlying the differences in antitumor efficacy, we monitored CAR expression (MFI) during consecutive restimulation assays. We noted that although the CAR MFI was similar between the EFS and PGK SSI promoters at the end of the process ( Figure 4AHowever, the promoters regulated CAR expression differently after exposure to CD19+ tumor cells. On day 3 after target exposure, all promoters increased CAR expression, with the EFS and UBB promoters achieving the highest expression levels. Figure 4C By day 17, all promoters had downregulated CAR expression; however, the EFS and UBB promoters maintained significantly higher expression levels for the remainder of the assay compared to the PGK SSI promoter. Figure 4C Surprisingly, this sustained high level of CAR expression after day 17 was associated with CAR T cell expansion. Figure 4D ) and antitumor activity ( Figure 4B The expression was negatively correlated with CAR expression. In contrast, the PGK SSI promoter maintained low levels of CAR expression from day 17 onwards, but showed the highest peak amplification compared to other constructs. Figure 4D ) and optimal antitumor activity ( Figure 4B Despite the reluctance to be limited to specific mechanisms, the results indicate that the EFS and UBB promoters lead to long-term high CAR expression, which may result in CAR T cell exhaustion and dysfunction; while the PGK SSI promoter leads to low CAR expression after target exposure, thereby maintaining CAR T cell function and resulting in better CAR T cell expansion and better anti-tumor activity.

[0357] Based on these results, we then tested the PGK SSI promoter (e.g., PGK SSI-1) and compared it with the EFS promoter in CAR T cells generated by site-specific integration and CAR T cells generated by LVV transduction. Figure 5A The results showed that, in standard LTKA, when tested against wild-type Raji cells at an E:T ratio of 4:1, CD19 CAR T cells co-expressing CCR under the PGK SSI-1 promoter (but not the EFS promoter) performed comparably to LVV cells expressing the same CD19 CAR and CCR. When tested against wild-type Raji cells at an E:T ratio of 1:2 in a single-stimulation FACS-based power assay, the PGK SSI-1-driven SSI construct outperformed not only the EFS construct but also LVV-transduced CAR T cells, as evidenced by the number of remaining target cells (…). Figure 5B ) and the expansion of CAR T+ cells ( Figure 5C This has been confirmed in all of them. Figures 5A to 5CAll results shown were obtained using CAR T cells derived from cells from the same donor. In another LTKA experiment, CAR T cells utilizing the PGK SSI-1 promoter were found to outperform CAR T cells utilizing an optimized endogenous promoter (SEQ ID NO: 96) (data not shown).

[0358] The results of the in vitro killing assay were confirmed in vivo in the Raji orthotopic tumor NSG mouse model. In the animal tumor model, CD19 CAR T cells driven by the PGK SSI-1 promoter were superior to LVV-transduced CAR T cells and other constructs driven by the Cyp300A and EFS promoters at two different cell doses. Figures 6A to 6B ).

[0359] While the disclosed teachings have been described with reference to various applications, methods, kits, and compositions, it should be understood that various changes and modifications can be made without departing from the teachings herein and the inventions claimed below. The above embodiments are intended to better illustrate the disclosed teachings and are not intended to limit the scope of the teachings presented herein. Although the teachings herein are described through these exemplary embodiments, those skilled in the art will readily understand that many variations and modifications can be made to these exemplary embodiments without requiring extensive experimentation. All such variations and modifications are within the scope of the existing teachings.

Claims

1. A chimeric antigen receptor (CAR) T cell comprising: A recombinant nucleic acid sequence integrated into the constant region of the human T cell receptor (TCR) α chain gene, wherein the recombinant nucleic acid sequence comprises, in the 5' to 3' direction: (a) The 5' region of the constant region of the TCRα chain. (b) A truncated PGK promoter that controls the expression of the CAR nucleic acid sequence. (c) The CAR nucleic acid sequence, and (d) The 3' region of the human TCRα constant region gene.

2. The CAR T cells of claim 1, compared with CAR T cells that do not contain a truncated PGK promoter, exhibit greater expansion and / or cytotoxicity.

3. The CAR T cells of claim 1, which exhibit lower exhaustion compared to CAR T cells that do not contain a truncated PGK promoter.

4. The CAR T cell of claim 1 or 2, wherein the integrated recombinant nucleic acid sequence prevents the expression of the TCRα constant region gene in the CAR T cell.

5. The CAR T cell of claim 1, wherein the recombinant nucleic acid sequence further comprises an additional nucleic acid sequence encoding a receptor polypeptide or a nucleic acid inhibitor.

6. The CAR T cell of claim 5, wherein the receptor polypeptide is a chimeric cytokine receptor (CCR) or CD70 binding protein.

7. The CAR T cell of claim 5, wherein the nucleic acid inhibitor is an RNA interference agent.

8. The CAR T cell of claim 5, wherein the CAR nucleic acid sequence and the additional nucleic acid sequence are linked by a P2A peptide.

9. The CAR T cell of any one of claims 1 to 4, wherein the truncated PGK promoter comprises one or more deletions of the sequence shown in SEQ ID NO:

34.

10. The CAR T cell of any one of claims 1 to 4, wherein the truncated PGK promoter comprises one or more 5' region deletions and / or 3' region deletions.

11. The CAR T cell of claim 9 or 10, wherein the truncated PGK promoter comprises a nucleotide sequence: (a) The nucleotide sequence comprises a deletion of about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380 or about 390 nucleotides from the 5' end of SEQ ID NO: 34, and has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 35; (b) The nucleotide sequence comprises a deletion of about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280 or about 290 nucleotides from the 5' end of SEQ ID NO: 34, and has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 36; (c) The nucleotide sequence comprises a deletion of about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, or about 190 nucleotides from the 5' end of SEQ ID NO: 34, and has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 37; or (d) The nucleotide sequence comprises about 50, about 60, about 70, about 80, about 90 or about 100 nucleotides deleted from the 5' end of SEQ ID NO: 34, and has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO:

38.

12. The CAR T cell of claim 9 or 10, wherein the truncated PGK promoter comprises SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:

38.

13. The CAR T cell of any one of claims 1 to 10, wherein the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain.

14. The CAR T cell of claim 13, wherein the extracellular domain comprises an antigen-binding domain and / or wherein the intracellular domain comprises at least one co-stimulatory domain.

15. The CAR T cell of claim 14, wherein the co-stimulatory domain is a signal transduction region comprising: CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death receptor-1 (PD-1), inducible T cell co-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1 (CD11a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC. Class I molecules, TNF receptor protein, immunoglobulins, cytokine receptors, integrins, signal transduction lymphocyte activating molecules (SLAM protein), activating NK cell receptors, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile protein), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, ligands that specifically bind to CD83, or any combination thereof.

16. The CAR T cell of claim 13 or 14, wherein the intracellular domain comprises at least one activating domain.

17. The CAR T cell of claim 16, wherein the activating domain comprises CD3.

18. The CAR T cell of claim 17, wherein the CD3 comprises CD3ζ.

19. CAR T cells according to any one of claims 1 to 18, wherein the CAR nucleic acid sequence expresses a CAR that binds to: BCMA, EGFRvIII, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Liv1, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, sealing protein-18.2, Muc17, FAPα, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, FLT3, CD70, DLL3, CD52, or CD34.

20. The CAR T cell according to any one of claims 1 to 19, wherein the CAR T cell is an autologous or allogeneic CAR T cell.

21. A method for manufacturing CAR T cells, comprising introducing a recombinant nucleic acid sequence into the cell, said recombinant nucleic acid sequence comprising, in the 5' to 3' direction: (a) The 5' region of the T cell receptor (TCR) α chain constant region (b) A truncated PGK promoter that controls the expression of the CAR nucleic acid sequence. (c) The CAR nucleic acid sequence, and (d) The 3' region of the human TCRα constant region gene. The introduction is performed under conditions sufficient to allow the recombinant nucleic acid sequence to integrate into the constant region gene of the human T cell receptor (TCR) α chain, thereby providing CAR T cells.

22. The method of claim 21, wherein the integration causes the expression of the TCRα constant region gene in the CAR T cells to be blocked.

23. The method of claim 21, wherein the integrated recombinant nucleic acid sequence prevents the expression of the TCRα constant region gene in the CAR T cells.

24. The method of claim 21, further comprising using the CAR T cells for amplification assay.

25. The method of claim 21, further comprising performing a cytotoxicity assay using the CAR T cells.

26. The method of claim 23, wherein the amplification assay is an in vitro or in vivo amplification assay, and / or wherein the cytotoxicity assay is an in vitro or in vivo cytotoxicity assay.

27. The method of any one of claims 21 to 26, wherein the truncated PGK promoter comprises one or more deletions of the wild-type promoter as shown in SEQ ID NO:

34.

28. The method of claim 27, wherein the one or more defects include 5' region defects and / or 3' region defects.

29. The method of claim 28, wherein the truncated PGK promoter comprises a nucleotide sequence: (a) The nucleotide sequence comprises a deletion of about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380 or about 390 nucleotides from the 5' end of SEQ ID NO: 34, and has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 35; (b) The nucleotide sequence comprises a deletion of about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280 or about 290 nucleotides from the 5' end of SEQ ID NO: 34, and has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 36; (c) The nucleotide sequence comprises a deletion of about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, or about 190 nucleotides from the 5' end of SEQ ID NO: 34, and has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 37; or (d) The nucleotide sequence comprises about 50, about 60, about 70, about 80, about 90 or about 100 nucleotides deleted from the 5' end of SEQ ID NO: 34, and has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO:

38.

30. The method of claim 28 or 29, wherein the truncated PGK promoter comprises a sequence selected from the group consisting of SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO:

38.

31. The method of any one of claims 21 to 30, wherein the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain.

32. The method of claim 31, wherein the extracellular domain comprises an antigen-binding domain.

33. The method of claim 31 or 32, wherein the intracellular domain comprises at least one co-stimulatory domain.

34. The method of claim 33, wherein the co-stimulatory domain is a signal transduction region comprising: CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death receptor-1 (PD-1), inducible T cell co-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1 (CD11a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC class I molecules, TNF receptor protein, immunoglobulin, cytokine receptor, integrin, signal transduction lymphocyte activating molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile protein), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, ligands that specifically bind to CD83, or any combination thereof.

35. The method of claim 31 or 33, wherein the intracellular domain comprises at least one activating domain.

36. The method of claim 35, wherein the activating domain comprises CD3.

37. The method of claim 36, wherein the CD3 comprises CD3ζ.

38. The method of any one of claims 20 to 37, wherein the CAR nucleic acid sequence expresses a CAR that binds to: BCMA, EGFRvIII, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Liv1, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, sealing protein-18.2, Muc17, FAPα, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, FLT3, CD70, DLL3, CD52, or CD34.

39. The method of any one of claims 20 to 38, wherein the CAR T cells are autologous or allogeneic CAR T cells.

40. An isolated population of CAR T cells comprising CAR T cells according to any one of claims 1 to 20.

41. A nucleic acid molecule comprising a recombinant nucleic acid sequence encoding a CAR, wherein the recombinant nucleic acid sequence comprises, in the 5' to 3' direction: (a) The 5' region of the T cell receptor (TCR) α chain constant region (b) A truncated PGK promoter that controls the expression of the CAR nucleic acid sequence. (c) The CAR nucleic acid sequence, and (d) The 3' region of the human TCRα constant region gene.

42. The nucleic acid molecule of claim 41, wherein the recombinant nucleic acid sequence further comprises an additional nucleic acid sequence encoding a receptor polypeptide or a nucleic acid inhibitor.

43. The nucleic acid molecule of claim 42, wherein the receptor polypeptide is a CCR or CD70 binding protein.

44. The nucleic acid molecule of claim 42, wherein the nucleic acid inhibitor is an RNA interference agent.

45. The nucleic acid molecule of claim 42, wherein the CAR nucleic acid sequence and the additional nucleic acid sequence are linked by a P2A peptide.

46. ​​The nucleic acid molecule of any one of claims 41 to 45, wherein the truncated PGK promoter comprises one or more deletions of the sequence shown in SEQ ID NO:

34.

47. The nucleic acid molecule of any one of claims 41 to 45, wherein the truncated PGK promoter comprises one or more 5' region deletions and / or 3' region deletions.

48. The nucleic acid molecule of claim 46 or 47, wherein the truncated PGK promoter comprises a nucleotide sequence: (a) The nucleotide sequence comprises a deletion of about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380 or about 390 nucleotides from the 5' end of SEQ ID NO: 34, and has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 35; (b) The nucleotide sequence comprises a deletion of about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280 or about 290 nucleotides from the 5' end of SEQ ID NO: 34, and has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 36; (c) The nucleotide sequence comprises a deletion of about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, or about 190 nucleotides from the 5' end of SEQ ID NO: 34, and has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 37; or (d) The nucleotide sequence comprises about 50, about 60, about 70, about 80, about 90 or about 100 nucleotides deleted from the 5' end of SEQ ID NO: 34, and has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO:

38.

49. The nucleic acid molecule of claim 41, wherein the truncated PGK promoter comprises SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 or SEQ ID NO:

38.

50. The nucleic acid molecule of any one of claims 41 to 49, wherein the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain.

51. The nucleic acid molecule of claim 50, wherein the extracellular domain comprises an antigen-binding domain and / or wherein the intracellular domain comprises at least one co-stimulatory domain.

52. The nucleic acid molecule of claim 51, wherein the co-stimulatory domain is a signal transduction region comprising: CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death receptor-1 (PD-1), inducible T cell co-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1 (CD11a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC. Class I molecules, TNF receptor protein, immunoglobulins, cytokine receptors, integrins, signal transduction lymphocyte activating molecules (SLAM protein), activating NK cell receptors, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile protein), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, ligands that specifically bind to CD83, or any combination thereof.

53. The nucleic acid molecule of claim 51 or 52, wherein the intracellular domain comprises at least one activating domain.

54. The nucleic acid molecule of claim 53, wherein the activating domain comprises CD3.

55. The nucleic acid molecule of claim 54, wherein the CD3 comprises CD3ζ.

56. The nucleic acid molecule of any one of claims 41 to 55, wherein the CAR nucleic acid sequence expresses a CAR that binds to: BCMA, EGFRvIII, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Liv1, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, sealing protein-18.2, Muc17, FAPα, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, FLT3, CD70, DLL3, CD52, or CD34.

57. A vector comprising the nucleic acid according to any one of claims 41 to 56.

58. A cell comprising any one of claims 41 to 56.

59. A cell comprising the carrier of claim 57.

60. The cell of claim 59, wherein the cell is an immune cell selected from the group consisting of: T cells, dendritic cells, killer dendritic cells, mast cells, natural killer (NK) cells, macrophages, monocytes, B cells, and immune cells derived from stem cells.

61. The cell according to claim 58 or 59, wherein the cell is an immune cell.

62. The cell according to claim 60 or 61, wherein the immune cell is an autologous immune cell or an allogeneic immune cell.

63. An isolated cell population comprising CAR T cells according to any one of claims 1 to 20 or cells according to any one of claims 58 to 62.

64. A pharmaceutical composition comprising CAR T cells according to any one of claims 1 to 20, cells according to any one of claims 58 to 62, or the isolated CAR T cell population according to claim 63.

65. A kit comprising CAR T cells according to any one of claims 1 to 20, cells according to claims 43 to 47, an isolated CAR T cell population according to claim 63, or a pharmaceutical composition according to claim 64.

66. A method of treating cancer in a subject in need, the method comprising administering to the subject a therapeutically effective amount of any one of claims 1 to 20 CAR T cells, any one of claims 58 to 62 cells, an isolated cell population as described in claim 63, or a pharmaceutical composition as described in claim 64.

67. The method of claim 66, wherein the cancer includes a solid tumor.

68. The method of claim 66, wherein the cancer comprises a liquid tumor.

69. The CAR T cell according to any one of claims 1 to 20, the cell according to any one of claims 58 to 62, or the nucleic acid molecule according to any one of claims 41 to 56, wherein the truncated PGK promoter comprises a nucleic acid sequence that binds to or is predicted to bind to a transcription factor selected from the group consisting of: a) YY1, RFX7, and HIF1; b) YY1, RFX7, HIF1A, and ETS2; or c) YY1, STAT3, STAT1, RFX7 and ETS2.

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