Immune cells expressing mutant interleukin-15

Abstract

This disclosure provides methods and constructs for improving the persistence or function of immune cells. In particular, the methods or constructs include a mutant interleukin-15 (IL-15) transgene under the control of a constitutive or inductive promoter to enhance the function of immune cells. The mutant IL-15 is modified to focus signaling to cells expressing IL-15Rα and / or signaling complexes containing IL-15Rα. More specifically, this disclosure provides methods and constructs for enhancing the function of immune cells by enhancing immune cell proliferation, reducing antigen-independent interferon-gamma (IFNγ) release, and suppressing tumor growth and unregulated immune cell proliferation.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 499,467, filed May 1, 2023, and U.S. Provisional Patent Application No. 63 / 502,875, filed May 17, 2023, the contents of both of which are hereby incorporated by reference in their entirety as if fully set forth herein.

[0002] Reference to Sequence Listing The sequence listing associated with this application is provided in XML format instead of a paper copy and is hereby incorporated by reference into this specification. The name of the file containing the sequence listing is 3401142.xml. The file is 346,334 bytes, was created on April 29, 2024, and was electronically submitted via the Patent Center.

[0003] The present disclosure provides methods and artificial expression constructs for improving the persistence or function of immune cells. In particular, the methods or artificial expression constructs include a mutant interleukin - 15 (IL - 15) transgene under the control of a constitutive or inducible expression control sequence to enhance the function of immune cells (e.g., recombinant receptor - expressing T cells).

Background Art

[0004] According to the World Health Organization, cancer is a major cause of death worldwide and was involved in nearly 10 million deaths in 2020.

[0005] For many years, the selected treatments for cancer have been surgery, chemotherapy, and / or radiotherapy. In recent years, more targeted therapies have emerged to specifically target cancer cells by identifying and exploiting specific molecular and / or immunophenotypic changes found mainly in those cells. For example, many cancer cells preferentially express specific markers on their cell surface, and these markers have provided targets for antibody - based therapeutic agents.

[0006] Meaningful progress has been made in genetically engineering cells of the immune system to target and kill unwanted cell types, such as cancer cells. Many of these immune cells are T cells that have been genetically engineered to express recombinant receptors, such as chimeric antigen receptors (CARs). A CAR is a protein that contains several different subcomponents that enable the genetically modified T cell to recognize and kill the target cell type. The subcomponents include at least an extracellular component and an intracellular component that are expressed as a single protein or assembled into a functional unit. The extracellular component includes a binding domain that binds to a marker (e.g., antigen) that preferentially exists on the surface of the unwanted cell. When the binding domain binds to such a marker, the intracellular component signals the T cell to destroy the bound cell. A CAR can also include a transmembrane domain that can link the extracellular component to the intracellular component.

[0007] Other subcomponents that can increase the function of a CAR can also be used. For example, a spacer provides additional conformational mobility to the CAR, often increasing the ability of the binding domain that binds to the targeted cell marker and leading to an enhanced cytolytic effect. The appropriate length of the spacer within a particular CAR can depend on a number of factors, including how close or far the target marker is positioned from the surface of the membrane of the unwanted cell.

[0008] CAR T cells have had substantial success in treating various cancers, but challenges remain. For example, CAR T cells can have suboptimal proliferation, functional anergy or exhaustion, impaired memory differentiation, and / or limited persistence that limits their ability to create or maintain remission in patients. Thus, strategies to improve the effectiveness of CAR T cells are needed, particularly for scenarios involving long-term antigen exposure that can lead to functional anergy and exhaustion. Genetic enhancement of CAR T cell efficacy may improve anti-cancer efficacy, but it carries the risk of adverse effects, such as unregulated cell growth. Intermittent administration of IL-15 to non-human primates is well tolerated and promotes memory T cell proliferation, while continuously delivered high doses of IL-15 are toxic (Berger et al, Blood 114(12):2417). [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Berger et al.Blood 114(12):2417 [Overview of the project]

[0010] This disclosure provides regulated IL-15 secretion for enhancing the efficacy of immune cells without driving autonomous immune cell growth or severe toxicity. This disclosure generally relates, in part, to methods and artificial expression constructs comprising mutant interleukin-15 (IL-15) transgenes under the control of constitutive or inducible expression regulatory sequences for enhancing the function of immune cells (e.g., recombinant receptor-expressing T cells). The mutant IL-15 is modified to focus signaling to cells expressing IL-15Rα and / or signaling complexes containing IL-15Rα. In certain embodiments, the mutant IL-15 is modified to restrict signaling to cells expressing IL-15Rα and / or signaling complexes containing IL-15Rα. In certain embodiments, the mutant IL-15 has a lower affinity for, or is unable to bind to, a complex containing the common gamma receptor without IL2Rβ and IL-15Rα compared to a complex containing the common gamma receptor with IL2Rβ and IL-15Rα. In various embodiments, mutant IL-15 has lower affinity for the common gamma receptor and / or atypical binding to IL2Rβ compared to wild-type IL-15. In various embodiments, mutant IL-15 is unable to bind to the common gamma receptor. More specifically, the disclosure provides methods or constructs for enhancing the function of immune cells by enhancing immune cell proliferation, reducing antigen-independent interferon-gamma (IFNγ) release, and suppressing tumor growth and unregulated immune cell proliferation. Methods for improving the persistence (or function) of adoptive cell therapy (ACT) and for treating subjects in need thereof are also provided.

[0011] In certain embodiments, the artificial expression construct comprises mutant IL-15 under the control of a constitutive or inducible expression regulatory sequence. In certain embodiments, mutant IL-15 preferentially binds to an IL-15 receptor complex containing IL15Rα. In certain embodiments, mutant IL-15 comprises a D-to-S mutation at position 8 compared to wild-type IL-15. In certain embodiments, mutant IL-15 comprises the sequence shown in SEQ ID NO: 9. In certain embodiments, mutant IL-15 is encoded by the sequence shown in SEQ ID NO: 11. In certain embodiments, the inducible synthetic promoter comprises the sequence shown in SEQ ID NO: 15. In certain embodiments, the artificial expression construct further comprises a recombinant receptor or exogenous lymphocyte receptor (e.g., T cell receptor; TCR) under the control of a constitutive expression regulatory sequence. In certain embodiments, the recombinant receptor comprises a chimeric antigen receptor (CAR), an engineered T cell receptor (eTCR), a dimerizing agent-regulated immunoreceptor complex (DARIC), or a hybrid thereof. In certain embodiments, the CAR comprises an anti-CD33 CAR. In certain embodiments, the CAR includes an anti-CLL1 CAR. In certain embodiments, the DARIC includes an anti-CD33 DARIC (DARIC33). In certain embodiments, the constitutive expression regulatory sequence includes the MNDU3 promoter or the EF1a promoter. In certain embodiments, the EF1a promoter includes the first intron of the human EF1a gene. In certain embodiments, the EF1a promoter lacks the first intron of the human EF1a gene. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 shows a schematic diagram of the design of an anti-CD33 dimerizer-modulated immunoreceptor complex (DARIC33) containing a constitutively expressed soluble or membrane-bound IL-15 variant. [Figure 2] Figure 2 shows the growth dynamics between control cells and T cells transduced using IL-15-containing lentivirus. [Figure 3A] Figures 3A and 3B show anti-CD33 expression and median fluorescence intensity (MFI) on T cells as measured by flow cytometry. [Figure 3B] Figures 3A and 3B show anti-CD33 expression and median fluorescence intensity (MFI) on T cells as measured by flow cytometry. [Figure 4A] Figures 4A and 4B show the percentage positive expression of FKBP-rapamycin binding (FRB) and MFI on T cells as measured by flow cytometry. [Figure 4B] Figures 4A and 4B show the percentage positive expression of FKBP-rapamycin binding (FRB) and MFI on T cells as measured by flow cytometry. [Figure 5] Figure 5 shows the virus copy number (VCN) for the given conditions. [Figure 6] Figure 6 shows the CD4 / CD8 staining by flow cytometry under the specified conditions. [Figure 7] Figure 7 shows the phenotype of T cells transduced using the DARIC33 / IL15 construct or control. [Figure 8] Figure 8 shows the median CD54 fluorescence intensity as measured by flow cytometry on T cells transduced using the indicated construct. [Figure 9] Figure 9 shows IFNγ secretion from untransduced T cells or T cells transduced using the shown construct, without the presence of target cells. [Figure 10] Figure 10 shows IFNγ secretion from untransduced T cells or T cells transduced using the indicated DARIC33 construct and co-cultured with the acute myeloid leukemia cell line MV-4-11. [Figure 11] Figure 11 shows IFNγ secretion from untransduced T cells or T cells transduced using the shown construct and co-cultured with CD33-expressing A549 cells (A549-CD33). [Figure 12] Figure 12 shows IFNγ secretion from untransduced T cells or T cells transduced using the indicated construct and co-cultured with B cell maturation antigen (BCMA)-expressing A549 cells (A549-BCMA). [Figure 13] Figure 13 shows A549-CD33 spheroid killing by transduced T cells using the shown construct, without the presence of AP21967. [Figure 14] Figure 14 shows A549-CD33 spheroid killing by transduced T cells using the shown construct, accompanied by AP21967. [Figure 15] Figure 15 shows A549-BCMA spheroid killing by transduced T cells using the shown construct, without the presence of AP21967. [Figure 16] Figure 16 shows A549-BCMA spheroid killing by transduced T cells using the shown construct, accompanied by AP21967. [Figure 17] Figure 17 shows a schematic diagram of DARIC33 designs for MND promoter-driven DARIC33 constructs, including soluble or membrane-bound IL-15 variants with adjustable promoter (iSynPro) expression in both forward and reverse orientations. [Figure 18A] Figures 18A-18C show sIL15 secretion by transduced T cells using the indicated constructs, in or out of the presence of non-transduced T cells or A549 tumor cells overexpressing BCMA or CD33. [Figure 18B] Figures 18A-18C show sIL15 secretion by transduced T cells using the indicated constructs, in or out of the presence of non-transduced T cells or A549 tumor cells overexpressing BCMA or CD33. [Figure 18C] Figures 18A-18C show sIL15 secretion by transduced T cells using the indicated constructs, in or out of the presence of non-transduced T cells or A549 tumor cells overexpressing BCMA or CD33. [Figure 19A] Figures 19A and 19B show IFNγ secretion by transduced T cells using the indicated constructs, in or out of the presence of non-transduced T cells or A549 tumor cells overexpressing BCMA or CD33. [Figure 19B]Figures 19A and 19B show IFNγ secretion by transduced T cells using the indicated constructs, in or out of the presence of non-transduced T cells or A549 tumor cells overexpressing BCMA or CD33. [Figure 20A] Figures 20A and 20B show IFNγ and IL-2 secretion by transduced T cells using the indicated constructs in the presence of untransduced T cells or HL60 or CD33low OCI-AML tumor cells ± rapamycin. [Figure 20B] Figures 20A and 20B show IFNγ and IL-2 secretion by transduced T cells using the indicated constructs in the presence of untransduced T cells or HL60 or CD33low OCI-AML tumor cells ± rapamycin. [Figure 20C] Figure 20C shows IL-15 secretion by transduced T cells using the indicated constructs in the absence or presence of HL60 tumor cells ± rapamycin, or in the absence of or presence of HL60 tumor cells. [Figure 21A] Figures 21A-21C show the proliferation of untransduced T cells or transduced T cells cultured in the presence of CD33+MV4-11 cells and rapamycin in different cytokine-containing media, using the constructs shown. [Figure 21B] Figures 21A-21C show the proliferation of untransduced T cells or transduced T cells cultured in the presence of CD33+MV4-11 cells and rapamycin in different cytokine-containing media, using the constructs shown. [Figure 21C] Figures 21A-21C show the proliferation of untransduced T cells or transduced T cells cultured in the presence of CD33+MV4-11 cells and rapamycin in different cytokine-containing media, using the constructs shown. [Figure 22] Figure 22 shows in vivo tumor growth in an NSG mouse model in which CD33+ MV4-11 tumor cells expressing firefly luciferase were engrafted and transduced T cells (10 × 10⁶) using the shown construct were administered without the presence of rapamycin. [Figure 23] Figure 23 shows in vivo tumor growth in an NSG mouse model in which CD33+ MV4-11 tumor cells expressing firefly luciferase were engrafted and administered with transduced T cells (10 × 10⁶) using the shown construct, along with present rapamycin. [Figure 24] Figure 24 shows in vivo tumor growth in an NSG mouse model in which CD33+ MV4-11 tumor cells expressing firefly luciferase were engrafted and administered with transduced T cells (3 × 10⁶) using the shown construct, along with present rapamycin. [Figure 25A] Figures 25A and 25B show schematic diagrams of constructs and cells containing a controllable promoter (iSynPro)-expressing soluble IL-15.D8S variant and eTCR. [Figure 25B] Figures 25A and 25B show schematic diagrams of constructs and cells containing a controllable promoter (iSynPro)-expressing soluble IL-15.D8S variant and eTCR. [Figure 26A] Figures 26A–26D show in vivo tumor growth in an NSG mouse model in which tumor cells expressing firefly luciferase were engrafted and transduced using the shown constructs, with or without present rapamycin, were administered. [Figure 26B] Figures 26A–26D show in vivo tumor growth in an NSG mouse model in which tumor cells expressing firefly luciferase were engrafted and transduced using the shown constructs, with or without present rapamycin, were administered. [Figure 26C] Figures 26A–26D show in vivo tumor growth in an NSG mouse model in which tumor cells expressing firefly luciferase were engrafted and transduced using the shown constructs, with or without present rapamycin, were administered. [Figure 26D]Figures 26A–26D show in vivo tumor growth in an NSG mouse model in which tumor cells expressing firefly luciferase were engrafted and transduced using the shown constructs, with or without present rapamycin, were administered. [Figure 27] Figure 27 shows the survival curves of NSG mice that were administered T cells transduced using the indicated construct, with or without present rapamycin, after engraftment using tumor cells. [Modes for carrying out the invention]

[0013] A brief explanation of array identifiers Sequence IDs 1-5 show the amino acid sequences of exemplary anti-CD33 dimerizer-modulated immunoreceptor complex (DARIC33) signaling, targeting, and fusion polypeptides.

[0014] Sequence ID 6 shows the amino acid sequence of an exemplary membrane-bound IL15 construct having a CD8α transmembrane domain.

[0015] Sequence ID 7 shows the amino acid sequence of an exemplary membrane-bound IL15 construct having an AMN transmembrane domain.

[0016] Sequence ID 8 shows the amino acid sequence for wild-type IL15.

[0017] Sequence ID 9 shows the amino acid sequence for the mutant IL15, which has a D-to-S mutation at position 8.

[0018] Sequence ID 10 shows the polynucleotide sequence for wild-type IL15.

[0019] Sequence ID 11 shows the polynucleotide sequence for mutant IL15, which has a D-to-S mutation at position 8.

[0020] Sequence ID 12 shows the polynucleotide sequence for an exemplary MNDU3 promoter.

[0021] Sequence ID 13 shows the polynucleotide sequence for an exemplary EF1α promoter.

[0022] Sequence ID 14 shows the polynucleotide sequence for an exemplary CMV promoter.

[0023] Sequence ID 15 shows the polynucleotide sequence for an exemplary adjustable promoter (iSynPro).

[0024] Sequence IDs 16–19 show polynucleotide sequences for exemplary iSynPro promoters operably ligated in forward and reverse orientation to polynucleotides encoding wild-type IL15 or mutant IL15.D8S.

[0025] Sequence IDs 20-40 show the amino acid sequences of the components of DARIC.

[0026] Sequence ID 41 shows the amino acid sequence of treated wild-type IL15.

[0027] Sequence ID 42 shows the amino acid sequence of the treated mutant IL15.

[0028] Sequence ID 43 shows the polynucleotide sequence for treated wild-type IL15.

[0029] Sequence ID 44 shows the polynucleotide sequence for the treated mutant IL15.

[0030] Sequence IDs 45-83 show exemplary iSynPro promoters.

[0031] Sequence ID 84 shows the amino acid sequence for the smallest CD4 hinge.

[0032] Sequence ID 85 shows the amino acid sequence for the CD3ε domain.

[0033] Sequence IDs 86-89 show the amino acid sequences of illustrative FRB and FKBP12 polypeptides.

[0034] Sequence ID 90 shows an illustrative amino acid sequence of the CD4 transmembrane domain.

[0035] Sequence IDs 91 and 92 show the amino acid sequences of illustrative cleaved intracellular CD4 polypeptides.

[0036] Sequence IDs 93-95 show the CDR amino acid sequences of exemplary anti-CD33 VHH conjugates.

[0037] Sequence ID 96 shows the amino acid sequence of an exemplary anti-CD33 VHH conjugate.

[0038] Sequence IDs 97-99 show the CDR amino acid sequences of exemplary anti-CLL1 VHH conjugates.

[0039] Sequence ID 100 shows the amino acid sequence of an exemplary anti-CLL1 VHH conjugate.

[0040] Sequence IDs 101 and 102 show the amino acid sequences of illustrative signal sequences.

[0041] Sequence ID 103 shows the amino acid sequence of an exemplary engineered T cell receptor (eTCR) signaling component.

[0042] Sequence ID 104 shows the amino acid sequence of an exemplary eTCR-targeting component.

[0043] Sequence ID 105 shows the amino acid sequence of an exemplary eTCR fusion polypeptide.

[0044] Sequence ID 106 represents the consensus Kozak sequence.

[0045] Sequence ID 107 shows the polynucleotide sequence for the IL2 minimal promoter.

[0046] Sequence ID 108 shows the amino acid sequence for the CD28 transmembrane domain.

[0047] Sequence ID 109 shows the amino acid sequence for the CD8α transmembrane domain.

[0048] Sequence ID 110 shows the amino acid sequence for the CD3z signaling domain.

[0049] Sequence ID 111 shows the amino acid sequence for the 4-1BB signaling domain.

[0050] In the sequence described above, X represents any amino acid, if present, or the absence of any amino acid. (Modes for carrying out the invention)

[0051] A. Overview According to the World Health Organization, cancer is a leading cause of death worldwide, accounting for nearly 10 million deaths in 2020.

[0052] For many years, the preferred treatments for cancer have been surgery, chemotherapy, and / or radiation therapy. In recent years, more targeted therapies have emerged to specifically target cancer cells, primarily by identifying and utilizing specific molecular and / or immunophenotypic changes observed in those cells. For example, many cancer cells preferentially express certain markers on their cell surface, and these markers have provided targets for antibody-based therapeutics.

[0053] Significant advances have been made in genetically engineering cells of the immune system to target and kill undesirable cell types, such as cancer cells. Many of these immune cells are T cells genetically engineered to express recombinant receptors, such as chimeric antigen receptors (CARs). A CAR is a protein containing several distinct subcomponents that enable genetically modified T cells to recognize and kill target cell types. The subcomponents include at least an extracellular and an intracellular component, which are expressed as a single protein or assembled into a functional unit. The extracellular component includes a binding domain that preferentially binds to markers (e.g., antigens) on the surface of undesirable cells. When the binding domain binds to such a marker, the intracellular component signals the T cell to destroy the binding cell. In addition, a CAR may include a transmembrane domain that can link the extracellular component to the intracellular component.

[0054] Other subcomponents that can enhance the function of CARs can also be used. For example, spacers provide CARs with additional steric mobility, often increasing the ability of binding domains to targeted cell markers, leading to an enhanced cytolytic effect. The appropriate length of a spacer within a particular CAR can depend on a number of factors, including how close or far the target marker is located from the surface of the undesirable cell membrane.

[0055] While CAR T cells have achieved substantial success in treating various cancers, challenges remain. For example, CAR T cells may exhibit suboptimal proliferation, functional anergy or depletion, impaired memory differentiation, and / or limited persistence that restricts their ability to create or maintain remission in patients. Thus, strategies to improve the efficacy of CAR T cells are needed, particularly in scenarios involving prolonged antigen exposure that can lead to functional anergy and depletion.

[0056] Genetic enhancement of CAR T cell efficacy may improve anticancer efficacy, but it carries the risk of adverse effects, such as unregulated cell growth. Intermittent administration of IL-15 to non-human primates is well tolerated and promotes memory T cell proliferation, while continuously delivered high doses of IL-15 are toxic (Berger et al, Blood 114(12):2417). Thus, this disclosure provides regulated IL-15 secretion for enhancing the efficacy of immune cells without driving autonomous immune cell growth or severe toxicity.

[0057] This disclosure provides methods and artificial expression constructs comprising a mutant interleukin-15 (IL-15) transgene under the control of constitutive or inducible expression regulatory sequences for enhancing the function of immune cells (e.g., recombinant receptor-expressing T cells). The mutant IL-15 is modified to focus signaling to cells expressing IL-15Rα and / or signaling complexes containing IL-15Rα. In certain embodiments, the mutant IL-15 is modified to restrict signaling to cells expressing IL-15Rα and / or signaling complexes containing IL-15Rα. In certain embodiments, the mutant IL-15 has a lower affinity for, or is unable to bind to, a complex containing IL2Rβ and the common gamma receptor without IL-15Rα, compared to a complex containing IL2Rβ and the common gamma receptor with IL-15Rα. In various embodiments, the mutant IL-15 has a lower affinity for the common gamma receptor and / or atypical binding to IL2Rβ compared to wild-type IL-15. In various embodiments, mutant IL-15 is unable to bind to the common gamma receptor. More specifically, the disclosure provides methods or constructs for enhancing the function of immune cells by enhancing immune cell proliferation, reducing antigen-independent interferon-gamma (IFNγ) release, and suppressing tumor growth and unregulated immune cell proliferation. Methods for improving the persistence (or function) of adoptive cell therapy (ACT) and for treating subjects in need thereof are also provided.

[0058] In certain embodiments, the cell or artificial expression construct comprises mutant IL-15 under the control of a constitutive or inducible expression regulatory sequence. In certain embodiments, mutant IL-15 preferentially binds to an IL-15 receptor complex containing IL15Rα. In certain embodiments, mutant IL-15 comprises a D-to-S mutation at position 8 compared to treated wild-type IL-15 (SEQ ID NO: 41) or untreated wild-type IL-15 (SEQ ID NO: 8). In certain embodiments, mutant IL-15 comprises the sequence shown in SEQ ID NO: 9. In certain embodiments, mutant IL-15 comprises the sequence shown in SEQ ID NO: 42. In certain embodiments, mutant IL-15 is encoded by the sequence shown in SEQ ID NO: 11. In certain embodiments, mutant IL-15 is encoded by the sequence shown in SEQ ID NO: 44. In certain embodiments, the inducible synthetic promoter comprises the sequence shown in SEQ ID NO: 15. In certain embodiments, the artificial expression construct further comprises a recombinant receptor or exogenous lymphocyte receptor under the control of a constitutive expression regulatory sequence. In certain embodiments, the recombinant receptor includes a chimeric antigen receptor (CAR), an engineered T cell receptor (eTCR), a dimerizing agent-regulated immunoreceptor complex (DARIC), or a hybrid thereof. In certain embodiments, the exogenous lymphocyte receptor includes a T cell receptor or B cell receptor introduced into genetically engineered cells. In certain embodiments, the recombinant receptor includes an anti-CD33 recombinant receptor. In certain embodiments, the recombinant receptor includes an anti-CLL1 recombinant receptor. In certain embodiments, the recombinant receptor includes a CAR. In certain embodiments, the recombinant receptor includes a DARIC. In certain embodiments, the constitutive expression regulatory sequence includes an MNDU3 promoter or an EF1a promoter. In certain embodiments, the EF1a promoter includes the first intron of the human EF1a gene. In certain embodiments, the EF1a promoter lacks the first intron of the human EF1a gene.

[0059] Recombinant (i.e., manipulated) DNA, peptide and oligonucleotide synthesis, immunoassays, tissue culture, transformation (e.g., electroporation, lipofection), enzymatic reactions, purification, and related techniques and procedures may be commonly carried out as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology, and immunology, as cited and discussed throughout this specification. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford Univ. Press USA, 1985); Current Protocols in Immunology (Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M.Shevach、Warren Strober 2001 John Wiley & Sons、NY、NY);Real-Time PCR:Current Technology and Applications,Edited by Julie Logan,Kirstin Edwards and Nick Saunders,2009,Caister Academic Press,Norfolk,UK;Anand,Techniques for the Analysis of Complex Genomes,(Academic Press,New York,1992);Guthrie and Fink,Guide to Yeast Genetics and Molecular Biology (Academic Press,New York,1991);Oligonucleotide Synthesis (N.Gait,Ed.,1984);Nucleic Acid The Hybridization (B.Hames & S.Higgins,Eds.,1985);Transcription and Translation(B.Hames & S.Higgins,Eds.,1984);Animal Cell Culture(R.Freshney,Ed.,1986);Perbal,A Practical Guide to Molecular Cloning(1984);Next-Generation Genome Sequencing(Janitz,2008 Wiley-VCH);PCR Protocols(Methods in Molecular Biology)(Park,Ed.,3rd Edition,2010 Humana Press);Immobilized Cells And Enzymes(IRL Press,1986);the treatise,Methods In Enzymology(Academic Press,Inc.,N.Y.);Gene Transfer Vectors For Mammalian Cells(J.H.Miller and M.P.Calos eds.See also: 1987, Cold Spring Harbor Laboratory; Harlow and Lane, Antibodies (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (DMWeir and CC Blackwell, eds., 1986); Roitt, Essential Immunology, 6th Edition (Blackwell Scientific Publications, Oxford, 1988); Current Protocols in Immunology (QEColigan, AMKruisbeek, DHMargulies, EMShevach and W.Strober, eds., 1991); Annual Review of Immunology; and research articles in journals such as Advances in Immunology.

[0060] B. Definition Before providing further details of this disclosure, it may be helpful to provide definitions of certain terms used herein.

[0061] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention pertains. Similar or equivalent methods and materials may be used in the implementation or verification of specific embodiments, but preferred embodiments of compositions, methods, and materials are described herein. For the purposes of this disclosure, the following terms are defined below:

[0062] The articles "a," "an," and "the" are used herein to refer to one or more of the grammatical objects of the articles (i.e., at least one, or one or more). For example, "an element" means one element, or one or more elements.

[0063] The use of options (e.g., "or") should be understood to mean one of those options, both of them, or any combination thereof.

[0064] "And / or" should be understood to mean either one of the options, or both of those options.

[0065] In one embodiment, the range, for example, 1 to 5, refers to each numerical value that is included within that range. For example, in one non-restrictive, purely illustrative embodiment, the range "1-5" is equivalent to the expressions 1, 2, 3, 4, 5; or 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0; or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0. The enumeration of value ranges in this specification is intended merely as a concise way of referring individually to each separate value that falls within that range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually enumerated herein. All methods described herein may be carried out in any suitable order unless otherwise indicated herein, or unless otherwise clearly contradicted by the context. Any and all examples or exemplary language (e.g., "etc.") provided herein are intended merely to better illustrate the invention and do not impose any limitation on the scope of the invention as otherwise claimed. No language herein should be construed as indicating any unclaimed element essential to the practice of the invention.

[0066] "Substantially" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, "substantially the same" means an effect that is approximately the same as a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length, for example, a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that produces a physiological effect.

[0067] Throughout this specification, any reference to “an embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment,” or any combination thereof, means that the particular features, structures, or characteristics described in relation to an embodiment are included in at least one embodiment. Thus, the occurrence of the aforementioned terms in various parts of this specification does not necessarily all refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Also, it is understood that the positive enumeration of features in an embodiment serves as a basis for excluding those features in a particular embodiment.

[0068] Additional definitions are provided throughout this disclosure.

[0069] C. Mutant IL-15 polypeptide. In certain embodiments, mutant IL-15 preferentially binds to IL-15 receptor complexes containing IL-15Rα compared to IL-15 receptor complexes without IL-15Rα. In certain embodiments, mutant IL-15 contains D to S mutations compared to wild-type IL-15. In certain embodiments, wild-type IL-15 includes the sequence shown in SEQ ID NO: 8 and / or is encoded by the sequence shown in SEQ ID NO: 10. In certain embodiments, wild-type IL-15 includes the sequence shown in SEQ ID NO: 41 and / or is encoded by the sequence shown in SEQ ID NO: 43. In certain embodiments, mutant IL-15 includes the sequence shown in SEQ ID NO: 9, or a sequence having at least 90%, 95%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 9. In certain embodiments, mutant IL-15 includes the sequence shown in SEQ ID NO: 42, or a sequence having at least 90%, 95%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 42. In certain embodiments, mutant IL-15 is encoded by the sequence shown in SEQ ID NO: 11, or a sequence having at least 90%, 95%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 11. In certain embodiments, mutant IL-15 is encoded by the sequence shown in SEQ ID NO: 44, or a sequence having at least 90%, 95%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 44. In this specification, mutant IL-15 is also referred to as variant IL-15.

[0070] D. Recombinant receptor. In certain embodiments, the recombinant receptor is or comprises a binding domain that binds to a target antigen, wherein the recombinant receptor is expressed by a cell after artificial introduction of the nucleic acid encoding the recombinant receptor into the cell. The recombinant receptor may be, for example, a CAR, an engineered T cell receptor (eTCR), a dimerizing agent-regulated immunoreceptor complex (DARIC), or a hybrid thereof.

[0071] As previously described, CARs include several distinct subcomponents that enable genetically modified cells to recognize and kill unwanted cells, such as cancer cells. The subcomponents include at least extracellular and intracellular components. The extracellular component includes a binding domain that specifically binds to a target antigen preferentially present on the cell surface or its region. When the binding domain binds to such an antigen, the intracellular component activates the cell (e.g., an immune effector cell) to destroy the binding cell. In addition, CARs include transmembrane domains that directly or indirectly link the extracellular component to the intracellular component, and other subcomponents that can enhance the function of the CAR. For example, the inclusion of spacer regions and / or one or more linker sequences allows the CAR to have additional steric mobility and often can increase the ability of the binding domain to bind to the target antigen.

[0072] Many of the considerations associated with CARs also apply to eTCRs. The eTCRs disclosed herein include binding domains that bind to a target antigen (e.g., scFv) linked to the Cα and / or Cβ chains of the TCR. The TCR is typically a heterodimer fusion protein containing α and β chains. Each chain contains a variable region (Vα and Vβ) and a constant region (Cα and Cβ). In certain embodiments, the eTCR does not include the native TCR variable region, but does include the native TCR constant region. In certain embodiments, the eTCR includes scFv as the variable region of either the α or β chain. In certain embodiments, the eTCR includes scFv as the variable region of both the α and β chains. In certain embodiments, the eTCR comprises a Cα and / or Cβ chain sequence that is 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%, at least 99.5%, or 100% identical to the amino acid sequence of a known or identified TCR Cα or Cβ.

[0073] In certain embodiments, the TCR or eTCR lacks substantial intracellular domains. Rather, the TCR or eTCR associates with (or is associated with) CD3 dimers to activate downstream signaling mechanisms. CD3 dimers can be constructed from CD3ε, CD3γ, CD3δ, and CD3ζ chains.

[0074] For specific examples of TCRs that may be used in the context of this disclosure, see, for example, WO2018 / 129270;WO2017 / 112944;WO2011 / 039507;US8,008,438;US2016 / 0083449;US2015 / 0246959;Sromnes, et al. (2015) Cancer Cell 28(5):638-652;Kobayashi, et al. (2013) Nature Medicine 19:1542-1546);Varela-Rohena, et al. (2008) Nature Medicine. 14(12):1390-1395); and Robbins et al. (2008) The Journal of Immunology 180(9):6116-6131.

[0075] In addition, in certain embodiments, the TCRs and eTCRs described herein include amino acid substitutions that improve expression, stability, and / or functional avidity. In various embodiments, the TCRs and eTCRs include a minimally mouse-modified TCRα chain and a minimally mouse-modified TCRβ chain. In various embodiments, the TCRα chain transmembrane domain includes one or more hydrophobic amino acid substitutions. See, for example, WO2021 / 195503. In certain embodiments, the eTCR includes a modified TCR. In certain embodiments, the eTCR includes a minimally mouse-modified TCR.

[0076] Similarly, considerations associated with CARs also apply to DARICs. This disclosure provides methods and compositions for polymerizing components of a DARIC so that the DARIC is primed for signal transduction. In certain embodiments, the DARIC comprises a first fusion protein (or first portion of a recombinant receptor) containing a first polymerizing domain, and a second fusion protein (or second portion of a recombinant receptor) containing a second polymerizing domain and intracellular components, wherein a dimerizing agent binds to the first and second polymerizing domains so that the first and second fusion proteins polymerize to form a DARIC ready for activation (i.e., primed for signal transduction).

[0077] In certain embodiments, “primed for signaling,” “priming for signaling,” “to prime for signaling,” and similar phrases (e.g., “priming DARIC for signaling”) refer to the reconfiguration of the components of DARIC so that the fusion protein containing the binding domain and the fusion protein containing the intracellular component are functionally coupled, allowing activation or downstream signaling to occur within the engineered cell upon binding of the target antigen. In some places herein, DARIC is referred to as activated or active when it is primed for signaling. In some embodiments, DARIC does not contain a binding domain. In some embodiments, DARIC without a binding domain contains an intracellular component on each of the first and second fusion proteins, in which signaling occurs upon multimerization.

[0078] In certain embodiments, DARIC may comprise a dimer, trimer, or higher-order multimer formed by at least two different proteins, comprising at least one protein having a target-specific binding domain, and / or one protein having an intracellular component, such as an intracellular signaling domain, a costimulatory domain, or a coreceptor domain. DARIC is primed for signal transduction when the dimerizing agent brings together at least two of the protein and associated proteins. In certain embodiments, DARIC comprises at least an intracellular component that enables or transmits intracellular signals. In other embodiments, DARIC comprises a binding domain.

[0079] In certain embodiments, DARIC comprises a targeting component and a signaling component. In certain embodiments, the targeting component is a fusion protein comprising at least a binding domain and a multimerizing domain. The targeting component may also comprise a linker, a spacer, and / or a transmembrane domain. In certain embodiments, the signaling component is a fusion protein comprising at least an intracellular component (e.g., an effector domain, a costimulatory domain) and a multimerizing domain. The signaling component may also comprise a linker, a spacer, and / or a transmembrane domain.

[0080] For further information regarding DARIC or similar recombinant receptors that may be used in the context of this disclosure, see, for example, WO2023 / 108158, WO2023 / 196996A2, WO2023 / 196997A2, and WO2020 / 227474A1.

[0081] In certain embodiments, recombinant receptors include an extracellular component containing a binding domain, an intracellular component containing a signaling domain, and a transmembrane domain. Each of these subcomponents is not mutually exclusive and may have some overlap. For example, the transmembrane domain may extend into either or both the extracellular and intracellular compartments. The intracellular and / or extracellular components may also extend into the transmembrane domain. Each of these subcomponents is described in more detail in the following section subheadings.

[0082] D1. Joint Domain

[0083] A "binding domain" refers to a protein, polypeptide, oligopeptide, peptide, or other molecule that has the ability to specifically recognize and bind to a target (e.g., CD19, CD20, CD33, CLL1, and / or other target antigens).

[0084] Useful binding domains in this disclosure include those known in the art, those described herein, or those generated by various methods known in the art (see, e.g., U.S. Patents 6,291,161 and 6,291,158). For example, binding domains can be identified by screening a Fab phage library for Fab fragments that specifically bind to a target of interest (see Hoet et al., Nat. Biotechnol. 23:344, 2005). In addition, anti-target antibodies having target-specific binding domains of interest can be generated using conventional strategies for hybridoma generation, such as using target antigens as immunogens in convenient systems (e.g., mice, HuMAb mice®, TC mice®, KM-mouse®, llamas, sheep, chickens, rats, hamsters, rabbits, etc.).

[0085] Further sources of binding domains include target-specific antibody-variable domains from various species, including humans, rodents, birds, and sheep (these can be formatted as antibodies, single-chain variable fragments (scFv), single-chain antibodies (sdAb), fragment antigen-binding regions (Fab), or soluble heavy-chain variable (VH) domains, single-chain single-domain antibodies (VHH), or domain antibodies). Additional sources of binding domains include other species, such as camelids (camels, dromedaries, or llamas (Ghahroudi et al., FEBS Letters 414:521, 1997; Vincke et al., J. Biol. Chem. 284:3273, 2009; and Hamers-Casterman et al., Nature 363:446, 1993; and Nguyen et al., J. Mol. Biol. 275:413, 1998), nurse sharks (Roux et al., Proc. Nat'l. Acad. Sci. (USA) 95:11804, 1998), spotted ratfish (Nguyen et al., Immunogenetics 54:39, 2002), or lampreys (Herrin et al.) This includes the variable domains of antibodies from al., Proc. Nat'l. Acad. Sci. (USA) 105:2040, 2008 and Alder et al., Nature Immunol. 9:319, 2008. These antibodies can clearly form an antigen-binding region using only the heavy chain variable region; that is, these functional antibodies are heavy chain-only homodimers (referred to as "heavy chain antibodies") (Jespers et al., Nat. Biotechnol. 22:1161, 2004; Cortez-Retamozo et al., Cancer Res. 64:2853, 2004; Baral et al., Nature Med. 12:580, 2006, and Barthelemy et al., J. Biol. Chem. 283:3639, 2008).

[0086] Other alternative sources of target-specific binding domains include sequences encoding random peptide libraries or sequences encoding manipulated diversity of amino acids in loop regions of alternative non-antibody backbones, e.g., fibrinogen domains (see Weisel et al. (1985) Science 230:1388), Kunitz domains (see U.S. Patent No. 6,423,498), ankyrin repeat proteins (also known as DARPins; Binz et al., J. Mol. Biol. 332:489, 2003 and Binz et al., Nat. Biotechnol. 22:575, 2004), and fibronectin-binding domains (also known as adnectin or monobodies; Richards et al., J. Mol. Biol. 326:1475, 2003; Parker et al., Protein Eng.Des.Sel.18:435,2005 and Hackel et al.,J.Mol.Biol.381:1238,2008), cysteine ​​knot miniprotein (Vita et al.,Proc.Nat'l.Acad.Sci.(USA)92:6404,1995;Martin et al.,Nat.Biotechnol.21:71,2002 and Huang et al.,Structure 13:755,2005), tetratricopeptide repeat domain (Main et al.,Structure 11:497,2003 and Cortajarena et al.,ACS Chem.Biol.3:161,2008), leucine-rich repeat domain (Stumpp et al. al., J.Mol.Biol.332:471,2003), anticarin (Skerra, FEBS J.275:2677,2008), lipocalin domain (e.g., PCT publication number WO2006 / 095164, see Beste et al., Proc.Nat'l.Acad.Sci.(USA)96:1898,1999 and Schonfeld et al., Proc.Nat'l.Acad.Sci.(USA)106:8198,2009), armadillo repeat proteins (ArmRPs; Varadamsetty et al., J.Mol.Biol.424:68,2012), diabody (Manzke et al., Int. J. Cancer 82:700,1999), repebody (Lee et al., Proc. Nat'l. Acad. Sci. USA 109:3299,2012), minibody (Hu et al., Cancer Res. 56:3055,1996), cyclotide (Craik et al., J. Mol. Biol. 294:1327,1999), V-like domain (e.g., see US Patent Application Publication No. 2007 / 0065431), C-type lectin domain (Zelensky and Gready, FEBS J. 272:6179,2005; Beavil et al.) mAb. 2 Or Fcab (trademark) (e.g., see PCT publication numbers WO2007 / 098934; WO2006 / 072620), or similar (Nord et al., Protein Eng. 8:601, 1995; Nord et al., Nat. Biotechnol. 15:772, 1997; Nord et al., Eur. J. Biochem. 268:4269, 2001; and Binz et al. (2005) Nat. Biotechnol. 23:1257, 2005), etc.

[0087] In further embodiments, the binding domain is specific to a target that is an antigen associated with cancer (e.g., solid malignancies, hematological malignancies), inflammatory diseases, autoimmune diseases, or graft-versus-host diseases. Exemplary target antigens include alpha-folate receptor (FRα), α vβ6 integrin, ADGRE2, BACE2, B-cell maturation antigen (BCMA), B7-H3 (CD276), B7-H4, B7-H6, CA19.9, carbonic anhydrase IX (CAIX), CCR1, CD7, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, CD244, carcinoembryonic antigen (CEA), C-type lectin-like molecule-1 (CLL-1), CD2 subset 1 (CS-1), CLDN6, cMET, chondroitin sulfate proteoglycan 4 (CSPG4), CLDN18.2. Cutaneous T-cell lymphoma-associated antigen 1 (CTAGE1), DLL3, epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), EGFR806, epidermal glycoprotein 2 (EGP2), epidermal glycoprotein 40 (EGP40), EPHB2, ERBB4, epidermal cell adhesion molecule (EPCAM), ephrin type A receptor 2 (EPHA2), fibroblast-activating protein (FAP), Fc receptor-like 5 (FCRL5), Fetal Acetylcholinesterase Receptor (AchR), FLT3, FN, FN-EDB, FRBeta, Ganglioside G2 (GD2), Ganglioside G3 (GD3), Glypican-3 (GPC3), EGFR family including ErbB2 (HER2), HER2p95, EGFRv3, IL-10Rα, IL-13Rα2, Copper, Cancer / Testicular Antigen 2 (LAGE-1A), K-Ras, K-Ras G12C, K-Ras G12D, K-Ras G12V, lambda, Lewis-Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, LY6G6GD, melanoma antigen 1 recognized by T cells (MelanA or MART1), mesothelin (MSLN), MMP10, MUC1, MUC16, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), nerve cell adhesion molecule (NCAM), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan This includes receptor 1 (ROR1), synovial sarcoma, X-section point 2 (SSX2), survivor, tumor-associated glycoprotein 72 (TAG72), transmembrane activator and CAML-interacting factor (TACI), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), TIM3, trophoblast glycoprotein (TPBG), UL16-binding protein (ULBP) 1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and vascular endothelial growth factor receptor 2 (VEGFR2). Additional exemplary binding domains can be found in WO2023196996A2, WO2023196997A2, and WO2017172981A2.

[0088] In some embodiments, one or more antigen-binding domains bind to CD19, CD20, CD22, CD33, CD79A, CD79B, B7H3, Muc16, Her2, EGFR, FN-EDB, CLDN18.2, DLL3, FLT3, CLL1, CD123, or BCMA. In some embodiments, one or more antigen-binding domains bind to CD33, CLL1, CD19, CD20, CD22, CD79A, CD79B, or BCMA. In some embodiments, one or more antigen-binding domains bind to CD33 and / or CLL1. In certain embodiments, the binding domain binds to CD33. In certain embodiments, the binding domain binds to CLL1. In certain embodiments, the binding domain binds to CD33 and CLL1. In certain embodiments, the binding domain is an anti-CD33 VHH antibody, an anti-CD33 scFv, or an anti-CD33 sdAb. In certain embodiments, the binding domain is an anti-CLL1 VHH antibody, an anti-CLL1 scFv, or an anti-CLL1 sdAb.

[0089] D2. Intracellular components.

[0090] The intracellular components of recombinant receptors include one or more intracellular signaling domains, co-stimulatory domains, or co-receptor domains that transmit or enable intracellular signals. In certain embodiments, the intracellular components generate signals that promote immunoeffector function in recombinant receptor-modified cells. In certain embodiments, the intracellular components generate stimulatory and / or co-stimulatory signals based on ligand binding. Examples of immunoeffector function include cytolytic activity and helper activity, and cytokine secretion. Intracellular component signals can also lead to proliferation, activation, differentiation, and similar processes in immune cells.

[0091] The intracellular effector domain of a recombinant receptor is involved in the activation of the cell on which the recombinant receptor is expressed. “Effector domain” thus means any portion of the intracellular domain sufficient to transduce an activation signal. When receiving a suitable signal, the effector domain can directly or indirectly stimulate a biological or physiological response in the cell. In certain embodiments, the effector domain is part of a protein or protein complex that receives a signal when bound, or it binds directly to a target molecule, thereby inducing a signal from the effector domain. The effector domain may directly stimulate a cellular response if it contains one or more signaling domains or motifs, such as an immunoreceptor tyrosine-based activation motif (ITAM). In other embodiments, the effector domain indirectly stimulates a cellular response by associating with one or more other proteins that directly stimulate a cellular response, such as a co-stimulatory domain.

[0092] The effector domain can provide activation of at least one function of the modified cell upon binding to a cellular marker expressed by cancer cells. Activation of the modified cell may include differentiation, proliferation, and / or activation, or one or more other effector functions. In certain embodiments, the effector domain may include an intracellular signaling component, including a T cell receptor, and a costimulatory domain, which may include a cytoplasmic sequence from a co-receptor or costimulatory molecule.

[0093] An effector domain may contain one, two, three, or more intracellular signaling components (e.g., receptor signaling domains, cytoplasmic signaling sequences), a co-stimulatory domain, or a combination thereof. An exemplary effector domain includes signaling and stimulatory domains selected from: 4-1BB (CD137), CARD11, CD3γ, CD3δ, CD3ε, CD3ζ, CD27, CD28, CD79A, CD79B, DAP10, FcRα, FcRβ (FcεR1b), FcRγ, Fyn, HVEM (LIGHTR), ICOS, LAG3, LAT, Lck, LRP, NKG2D, NOTCH1, pTα, PTCH2, OX40, ROR2, Ryk, SLAMF1, Slp76, TCRα, TCRβ, TRIM, Wnt, Zap70, or any combination thereof.In certain embodiments, exemplary effector domains include signaling domains and co-stimulatory domains selected from: CD86, FcγRIIa, DAP12, CD30, CD40, PD-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligands that specifically bind to CD83, CDS, ICAM-1, GITR, BAFFR, SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD 11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1( CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), PSGL1, CD100( SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LT BR, GADS, PAG / Cbp, NKp44, NKp30, NKp46, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9. In certain embodiments, the effector domain includes a CD3ζ signaling domain. In certain embodiments, the CD3ζ signaling domain includes the sequence shown in Sequence ID No. 110.

[0094] The intracellular signaling component sequences that act in the mode of stimulation may include iTAMs. Examples of iTAMs containing primary cytoplasmic signaling sequences include those derived from CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD66d, CD79a, CD79b, and common FcRγ (FCER1G), FcγRlla, FcRβ (FcεRib), DAP10, and DAP12. In certain embodiments, variants of CD3ζ retain at least one, two, three, or all of the ITAM regions.

[0095] In certain embodiments, the effector domain comprises a cytoplasmic portion that associates with a cytoplasmic signaling protein, wherein the cytoplasmic signaling protein is a lymphocyte receptor or its signaling domain, a protein comprising multiple ITAMs, a co-stimulatory domain, or any combination thereof.

[0096] Examples of additional intracellular signaling components include the cytoplasmic sequence of the CD3ζ chain and / or co-receptors that act cooperatively to initiate signaling after binding domain engagement. In certain embodiments, the intracellular component may include more than just the cytoplasmic portion of the sequence. For example, the intracellular component may include the transmembrane domain or a portion thereof of the same molecule.

[0097] Co-stimulatory domains are domains whose activation may be required for an efficient lymphocyte response to cell marker binding. Some molecules are interchangeable as intracellular signaling components or co-stimulatory domains. Examples of co-stimulatory domains include ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. For example, CD27 co-stimulation has been demonstrated to enhance the proliferation, effector function, and survival of immune effector cells in vitro, as well as to increase the persistence and anticancer activity of human T cells in vivo (Song et al. Blood. 2012;119(3):696-706). Further examples of such co-stimulatory domain molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, ITGAM, and CDl This includes lb, ITGAX, CDllc, ITGBl, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, CEACAM1, CRTAM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, and CD19a. In certain embodiments, the co-stimulatory domain includes a 4-1BB signaling domain. In certain embodiments, the 4-1BB signaling domain includes the sequence shown in Sequence ID No. 111.

[0098] Intracellular components also include Wnt signaling pathways (e.g., LRP, Ryk, or ROR2), NOTCH signaling pathways (e.g., NOTCH1, NOTCH2, NOTCH3, or NOTCH4), Hedgehog signaling pathways (e.g., PTCH or SMO), receptor tyrosine kinases (RTKs) (e.g., epidermal growth factor (EGF) receptor family, fibroblast growth factor (FGF) receptor family, hepatocyte growth factor (HGF) receptor family, insulin receptor (IR) family, platelet-derived growth factor (PDGF) receptor family, vascular endothelial growth factor (VEGF) receptor family, tropomycin receptor kinase (Trk) receptor family, ephrin (Eph) receptor family, AXL receptor family, leukocyte tyrosine kinase (LTK) receptor family) This may include one or more proteins of the following types: Lee, tyrosine kinase 1 (TIE) receptor family with immunoglobulin-like and EGF-like domains; receptor tyrosine kinase-like orphan (ROR) receptor family; discoidin domain (DDR) receptor family; reconstitution during transfection (RET) receptor family; tyrosine protein kinase-like (PTK7) receptor family; receptor tyrosine kinase (RYK) receptor family; or muscle-specific kinase (MuSK) receptor family; G protein-coupled receptors; GPCRs (Frizzled or Smoothened); serine / threonine kinase receptors (BMPR or TGFR); or cytokine receptors (IL1R, IL2R, IL7R, or IL15R).

[0099] In certain embodiments, the intracellular component includes a 4-1BB signaling domain, a CD3ζ signaling domain, a CD3ε signaling domain, or a CD4 signaling domain. In certain embodiments, the intracellular component includes a 4-1BB signaling domain. In certain embodiments, the intracellular component includes a CD3ζ signaling domain. In certain embodiments, the intracellular component includes a CD3ε signaling domain. In certain embodiments, the intracellular component includes a CD4 signaling domain. In certain embodiments, the CD4 signaling domain is a cleaved intracellular polypeptide.

[0100] D3. Transmembrane domain.

[0101] Recombinant receptors can be designed to include a transmembrane domain. The transmembrane domain can anchor the recombinant receptor to the cell membrane. The transmembrane domain may include one or more additional amino acids adjacent to the transmembrane region, for example, one or more amino acids associated with the extracellular region of the protein from which the transmembrane originates (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) and / or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein originates (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more). In certain embodiments, the transmembrane domain may originate from the same protein from which the intracellular component signaling domain, co-stimulatory domain, hinge domain, or co-receptor originates. In certain embodiments, the transmembrane domain does not originate from the same protein from which any other domain of the recombinant receptor originates. In certain embodiments, the transmembrane domain may be selected or modified by amino acid substitution to avoid binding to or minimize interaction with other domains in the recombinant receptor.

[0102] In certain embodiments, the transmembrane domain has a three-dimensional structure that is thermodynamically stable within the cell membrane and is generally in the range of 15 to 30 amino acids in length. The structure of the transmembrane domain may include an alpha-helix, beta-barrel, beta-sheet, beta-helix, or any combination thereof.

[0103] The transmembrane domain may be derived from either a natural source or / or a synthetic source. If the source is natural, the transmembrane domain may be derived from any membrane-bound protein or transmembrane protein. The transmembrane domain may include at least the transmembrane region of the α, β, or ζ chain of the T cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22; CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In certain embodiments, the transmembrane domains are, for example, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2Rβ, IL2Rγ, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, ITGAM, CDl lb, ITGAX, CDl It may include at least the transmembrane region of lc, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, or NKG2C. In certain embodiments, various human hinges may also be used, including human Ig (immunoglobulin) hinges (e.g., IgG4 hinge, IgD hinge), GS linkers (e.g., GS linkers described herein), KIR2DS2 hinges, or CD8a hinges.In certain embodiments, the recombinant receptor includes a CD28 transmembrane domain, a CD4 transmembrane domain, or a CD8α transmembrane domain. In certain embodiments, the CD28 transmembrane domain includes the sequence shown in SEQ ID NO: 108. In certain embodiments, the CD4 transmembrane domain includes the sequence shown in SEQ ID NO: 90. In certain embodiments, the CD8α transmembrane domain includes the sequence shown in SEQ ID NO: 109.

[0104] In certain embodiments, the transmembrane domain may primarily consist of hydrophobic residues, such as leucine and valine. In certain embodiments, the transmembrane domain may consist of a triplet of phenylalanine, tryptophan, and valine found at each end of the transmembrane domain. In certain embodiments, the CD28, CD4, or CD8 hinge is juxtaposed with the transmembrane domain on the extracellular side.

[0105] D4. Linkers and spacers.

[0106] A linker within a recombinant receptor can be any part of the recombinant receptor that functions to connect two subcomponents or domains of the recombinant receptor. In certain embodiments, the linker can provide mobility between different components of the recombinant receptor. A linker in relation to ligating the VH and VL of the antibody-derived binding domain of scFv is described above. The linker may also include a spacer region and junctional amino acids.

[0107] A spacer region is a type of linker region used to create appropriate distance and / or mobility from other linked components.

[0108] In certain embodiments, the length of the spacer region can be customized for individual purposes. For example, the spacer region can be customized for individual cell markers on target cells to optimize cell recognition and destruction after recombinant receptor binding. In certain examples, the spacer may be of a length that provides increased responsiveness of recombinant receptor-expressing cells after antigen binding compared to the absence of the spacer. In certain embodiments, the spacer region length may be selected based on the location of the cell marker epitope, the affinity of the binding domain for the epitope, and / or the ability of recombinant receptor-modified cells to destroy target cells ex vivo and / or in vivo in response to cell marker recognition. The spacer region can also enable high expression levels in recombinant receptor-modified cells. In certain embodiments, the extracellular spacer region of the recombinant receptor is located between the transmembrane domain and the extracellular binding domain.

[0109] Exemplary spacers include spacers having 10-250 amino acids, 10-200 amino acids, 10-150 amino acids, 10-100 amino acids, 10-50 amino acids, or 10-25 amino acids. In certain embodiments, the spacer region has 12 amino acids, 20 amino acids, 21 amino acids, 26 amino acids, 27 amino acids, 45 amino acids, or 50 amino acids. In certain embodiments, the long spacers are greater than 119 amino acids, the intermediate spacers are 13-119 amino acids, and the short spacers are 10-12 amino acids.

[0110] In certain embodiments, the spacer region includes an immunoglobulin hinge region. The immunoglobulin hinge region may be a wild-type immunoglobulin hinge region or a modified wild-type immunoglobulin hinge region. In certain embodiments, the immunoglobulin hinge region is a human immunoglobulin hinge region. The immunoglobulin hinge region may be an IgG, IgA, IgD, IgE, or IgM hinge region. The IgG hinge region may be an IgG1, IgG2, IgG3, or IgG4 hinge region. In certain embodiments, the spacer region may include all or part of a hinge region sequence from IgG1, IgG2, IgG3, IgG4, or IgD, either alone, or in combination with all or part of a CH2 region; all or part of a CH3 region; or all or part of a CH2 region and all or part of a CH3 region. The "wild-type immunoglobulin hinge region" refers to naturally occurring upper and intermediate hinge amino acid sequences found in the heavy chain of antibodies that are inserted between and connect the CH1 and CH2 domains (for IgG, IgA, and IgD), or between the CH1 and CH3 domains (for IgE and IgM), and connect them.

[0111] Exemplary spacers include the IgG4 hinge alone, the IgG4 hinge linked to the CH2 domain and the CH3 domain, or the IgG4 hinge linked to the CH3 domain. The hinge region may be modified to avoid undesirable structural interactions, such as dimerization with an unintended partner. Other examples of hinge regions that may be used in recombinant receptors described herein include hinge regions present in the extracellular regions of type 1 membrane proteins, such as CD8α, CD4, CD28, and CD7, which may be wild-type or variants thereof.

[0112] In certain embodiments, the spacer region includes a hinge region of the type II C-lectin interdomain (stalk) region or a differentiation cluster (CD) molecule stalk region. The "stalk region" of a type II C-lectin or CD molecule refers to the extracellular domain portion of the type II C-lectin or CD molecule, located between the C-lectin-like domain (CTLD; e.g., similar to the CTLD of natural killer cell receptors) and the hydrophobic portion (transmembrane domain). For example, the extracellular domain of human CD94 (GenBank acceptance number AAC50291.1) corresponds to amino acid residues 34-179, while the CTLD corresponds to amino acid residues 61-176. Therefore, the stalk region of the human CD94 molecule contains amino acid residues 34-60, which are located between the hydrophobic portion (transmembrane domain) and the CTLD (see Boyington et al., Immunity 10:15, 1999; for other descriptions of the stalk region, also see Beavil et al., Proc. Nat'l. Acad. Sci. USA 89:153, 1992; and Figdor et al., Nat. Rev. Immunol. 2:11, 2002). These type II C-lectins or CD molecules may also have junctional amino acids between the stalk region and the transmembrane domain or CTLD. In another example, the 233-amino acid human NKG2A protein (UniProt ID P26715.1) has a hydrophobic region (transmembrane domain) in the range of amino acids 71–93 and an extracellular domain in the range of amino acids 94–233. The CTLD contains amino acids 119–231, and the stalk region contains amino acids 99–116, which may be flanked by additional junctional amino acids. Other type II C-lectin molecules or CD molecules, as well as their extracellular ligand-binding domains, stalk regions, and CTLDs, are known in the art (e.g., GenBank acceptance numbers NP 001993.2; AAH07037.1; NP 001773.1; AAL65234.1; CAA04925.1; for the sequences and descriptions of human CD23, CD69, CD72, NKG2A, and NKG2D, respectively).

[0113] Linkers can be mobile, rigid, or semi-rigid, depending on the desired function of the linker. Linkers can contain junctional amino acids. For example, in certain embodiments, the linker provides mobility and room for conformational transfer between different components of a recombinant receptor. Commonly used mobile linkers include Gly-Ser linkers. In certain embodiments, the linker sequence is a set of glycine repeats and serine repeats, for example (Gly x Ser y ) n This includes 1 to 10 repeats of the formula, where x and y are independent integers from 0 to 10, but both x and y are not 0, provided that n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). Specific examples include GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, G4S, 2xG4S, 3xG4S, 4xG4S, 5xG4S, and any combination thereof, where "G4" indicates four Gly amino acids. Linkers can be used to connect the components of scFv, such as the linker described in Whitlow et al. (Protein Eng. 6(8):989-95, 1993).

[0114] D5. Multimerization domain.

[0115] A "multimerizing domain" refers to a molecule that preferentially interacts with or associates with another molecule, either directly or via a dimerizing agent, where the interaction of different multimerizing domains substantially contributes to or efficiently facilitates multimerization (i.e., the formation of dimers, trimers, or polysegmented complexes, which may be homodimers, heterodimers, homotrimers, heterotrimers, homomultimers, or heteromultimers).

[0116] In certain embodiments, the multimerizing domains associate using a dimerizing agent. In certain embodiments, the dimerizing agent is rapamycin or an analogue thereof. For example, the first and second multimerizing domains are a pair selected from the FK506-binding protein (FKBP) multimerizing domain and the FKBP-rapamycin-binding (FRB) multimerizing domain, or variants thereof. The FRB domain is a polypeptide region (protein "domain") capable of forming a ternary complex with the FKBP protein and rapamycin or its rapalogue. FRB domains are present in numerous naturally occurring proteins, including mTOR proteins from human and other species (also referred to in the literature as FRAP, RAPT1, or RAFT); yeast proteins containing Tor1 and Tor2; and Candida FRAP homologs. Information regarding the nucleotide sequences, cloning, and other embodiments of these proteins is publicly known in the art. For example, the accession number for human mTOR is GenBank accession number L34075.1 (Brown et al., Nature 369:756, 1994).

[0117] In certain embodiments, the first and second multimerization domains are localized extracellularly when the first and second portions of the recombinant receptor are expressed. In certain embodiments, the first and second multimerization domains are localized intracellularly when the first and second portions of the recombinant receptor are expressed.

[0118] In certain embodiments, the “FKBP-rapamycin-binding (FRB) multimerization domain” refers to the FRB polypeptide. The FRB domain for use in recombinant receptors of this disclosure generally comprises at least 85 to 100 amino acid residues. In certain embodiments, the FRB amino acid sequence for use in recombinant receptors of this disclosure comprises a 93-amino acid sequence from Ile-2021 to Lys-2113, and a T2098L mutation (T82L is an equivalent position in the 93-amino acid FRB polypeptide), with a reference to GenBank acceptance number L34075.1. The FRB domain for use in recombinant receptors of this disclosure is capable of binding to a complex of the FKBP protein bound to rapamycin or its analogues of this disclosure. In certain embodiments, the peptide sequence of the FRB domain includes (a) a naturally occurring peptide sequence extending at least the indicated 93-amino acid region of human mTOR or the corresponding region of a homologous protein; (b) a variant of the naturally occurring FRB, in which up to 10 amino acids, or 1 to 5 amino acids, or 1 to 3 amino acids, or in some embodiments, only one amino acid, is deleted, inserted, or substituted from the naturally occurring peptide; or (c) a peptide encoded by a nucleic acid molecule capable of selectively hybridizing to a DNA molecule encoding the naturally occurring FRB domain, or by a DNA sequence that, apart from the degeneracy of the genetic code, would be capable of selectively hybridizing to a DNA molecule encoding the naturally occurring FRB domain. In certain embodiments, the FRB polypeptide binds to the FKBP polypeptide via a crosslinking factor, thereby forming a triple complex.

[0119] Certain embodiments utilize the FRB sequence shown in SEQ ID NO: 86, and certain embodiments utilize the sequence shown in SEQ ID NO: 87.

[0120] In certain embodiments, the “FK506-binding protein (FKBP) multimerization domain” refers to the FKBP polypeptide. FKBP is a cytoplasmic receptor for macrolides, such as FK506, FK520, and rapamycin, and is highly conserved across species. For the purposes of this disclosure, FKBP is a protein or protein domain capable of binding to rapamycin or its analogues and further forming a ternary complex with an FRB-containing protein or recombinant receptor. The FKBP domain may also be referred to as the “rapamycin-binding domain.” Information regarding the nucleotide sequences, cloning, and other embodiments of various FKBP species is publicly known in the art (e.g., see Staendart et al., Nature 346:671, 1990 (human FKBP12); Kay, Biochem. J. 314:361, 1996). Homologous FKBP proteins in other mammalian species, in yeast, and in other organisms are also publicly known in the art and may be used in the recombinant receptors disclosed herein. The size of the FKBP domain for use in this disclosure will vary depending on which FKBP protein is used. The FKBP domain of the recombinant receptor in this disclosure may bind to rapamycin or its analogues and be involved in a ternary complex with FRB-containing proteins (as can be determined by any direct or indirect means for detecting such binding).

[0121] The peptide sequences of the FKBP domain of the FKBP recombinant receptor of this disclosure include (a) a naturally occurring FKBP peptide sequence preferably derived from human FKBP12 protein (GenBank acceptance number AAA58476.1), or a peptide sequence derived therefrom from another human FKBP, from mouse or other mammalian FKBP, or from certain other animals, yeast or fungal FKBP; (b) a variant of the naturally occurring FKBP sequence, wherein up to 10 amino acids, or 1 to 5 amino acids, or 1 to 3 amino acids, or in some embodiments, only 1 amino acid, are deleted, inserted, or substituted from the naturally occurring peptide; or (c) a peptide sequence encoded by a nucleic acid molecule capable of selectively hybridizing to a DNA molecule encoding naturally occurring FKBP, or by a DNA sequence that would be capable of selectively hybridizing to a DNA molecule encoding naturally occurring FKBP, except for genetic coding degeneracy. In certain embodiments, the FKBP polypeptide is an FKBP12 polypeptide, or an FKBP12 polypeptide containing the F36V mutation. In certain embodiments, the FKBP polypeptide intended herein binds to the FRB polypeptide via a crosslinking factor, thereby forming a ternary complex.

[0122] In certain embodiments, FKBP includes the sequence shown in sequence number 88. In certain embodiments, FKBP includes the sequence shown in sequence number 89.

[0123] A “crosslinking factor” refers to a molecule that associates with two or more multimerizing domains and is positioned between them. In certain embodiments, the multimerizing domains substantially contribute to or efficiently promote the formation of the polypeptide complex only in the presence of the crosslinking factor. In certain embodiments, the multimerizing domains do not contribute to or efficiently promote the formation of the polypeptide complex in the absence of the crosslinking factor. Exemplary examples of crosslinking factors suitable for use in the specific embodiments contemplated herein include AP21967, rapamycin (sirolimus) or its rapalogue, coumamycin or its derivatives, gibberellin or its derivatives, abscisic acid (ABA) or its derivatives, methotrexate or its derivatives, cyclosporine A or its derivatives, FKCsA or its derivatives, synthetic ligand (SLF) for trimethoprim (Tmp)-FKBP or its derivatives, or any combination thereof.

[0124] Other multimerized domain pairs include FKBP and calcineurin, FKBP and cyclophyllin, FKBP and bacterial DHFR, calcineurin and cyclophyllin, PYL1 and ABI1, or GIB1 and GAI, or variants thereof.

[0125] In certain embodiments, the first polymerizing domain is the FRB polymerizing domain, and the second polymerizing domain is the FKBP polymerizing domain. In certain embodiments, the dimerizing agent / crosslinking factor is rapamycin and / or its analogues.

[0126] In certain embodiments, the first and second polymerizing domains are either the same or different.

[0127] A "dimerizing agent" refers to any molecule capable of binding to the first and second polymerizing domains, thereby combining the two polymerizing domains and any constituent elements, and thus attaching to the polymerizing domains.

[0128] In certain embodiments, the dimerizing agent is rapamycin (marketed under the trade name Rapamune® (Amgen, Thousand Oaks, California) and also known as sirolimus). Rapamycin analogs (rapalogs) may also be used. Exemplary rapamycin analogs include those disclosed in U.S. Patent No. 6,649,595, which describes various rapalog structures. In certain embodiments, the dimerizing agent is a rapalog with substantially reduced immunosuppressive effect compared to rapamycin. "Substantially reduced immunosuppressive effect" means a rapalog having at least less than 0.1 to 0.005 times the immunosuppressive effect observed or predicted for equimolar amounts of rapamycin, as measured clinically or in suitable in vitro (e.g., inhibition of T cell proliferation) or in vivo surrogate for human immunosuppressive activity. Alternatively, "substantially reduced immunosuppressive effect" means the EC observed for rapamycin in the same assay. 50 EC in such in vitro assays is at least 10 to 250 times larger than the value. 50This refers to rapalogs that have a value. Other exemplary rapalogs include everolimus, novolimus, pimecrolimus, ridafololimus, tacrolimus, temsirolimus, umilolimus, zotarolimus, limitaside (AP1903), AP20187 (other names: 2,2'-[[2-[(dimethylamino)methyl]-1,3-propanediyl]bis[imino(2-oxo-2,1-ethanediyl)oxy-3,1-phenylene[(1R)-3-(3,4-dimethoxyphenyl)propyridene]]] ester; (2S,2'S)-1-[(2S)-1-oxo-2-(3,4,5-trimethoxyphenyl)butyl]-2-piperidinecarboxylic acid It contains B / B homodimerizing agent, AP21967 (other name: C16-(S)-7-methylindolerapamycin; C16-AiRap), and BPC015.

[0129] In certain embodiments, the dimerizing agent includes rapamycin (sirolimus) or its rapalogue, coumamycin or its derivatives, gibberellin or its derivatives, abscisic acid (ABA) or its derivatives, methotrexate or its derivatives, cyclosporine A or its derivatives, FKCsA or its derivatives, synthetic ligand (SLF) for trimethoprim (Tmp)-FKBP or its derivatives, or any combination thereof.

[0130] E. Polynucleotides A "constitutive regulatory sequence" refers to a promoter, enhancer, or promoter / enhancer that enables continuous or sequential transcription of a manipulably linked sequence. A constitutive regulatory sequence may be a "ubiquitous" promoter, enhancer, or promoter / enhancer that enables expression in a wide range of cell and tissue types, or it may be a "cell-specific," "cell type-specific," "cell line-specific," or "tissue-specific" promoter, enhancer, or promoter / enhancer that enables expression in a limited range of cell and tissue types, respectively.

[0131] A "promoter" refers to a recognition site of a polynucleotide (DNA or RNA) to which RNA polymerase binds. RNA polymerase initiates and transcribes the polynucleotide operably ligated to the promoter. In certain embodiments, in mammalian cells, the operational promoter includes an AT-rich region located 25–30 bases upstream from the transcription initiation site, and / or another sequence, the CNCAAT region, found 70–80 bases upstream from the transcription initiation, where N can be any nucleotide.

[0132] An "enhancer" refers to a segment of DNA containing a sequence capable of providing enhanced transcription, and in some examples, can function independently of their orientation relative to other regulatory sequences. Enhancers can function cooperatively or additively with promoters and / or other enhancer elements. A "promoter / enhancer" refers to a segment of DNA containing a sequence capable of providing both promoter and enhancer functions.

[0133] Exemplary ubiquitous expression regulatory sequences suitable for use in specific embodiments include, but are not limited to, the cytomegalovirus (CMV) pre-early promoter, viral Simianvirus 40 (SV40) (e.g., early or late), Moroni mouse leukemia virus (MoMLV) LTR promoter, Rous sarcoma virus (RSV) LTR, herpes simplex virus (HSV) (thymidine kinase) promoter, vaccinia virus-derived H5, P7.5 and P11 promoters, and extensions. Growth factor 1 alpha (EF1a) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde triphosphate dehydrogenase (GAPDH), eukaryotic cell translation initiation factor 4A1 (EIF4A1), heat shock 70kDa protein 5 (HSPA5), heat shock protein 90kDa beta, member 1 (HSP90B1), heat shock protein 70kDa (HSP70), β-kinesin (β-KIN), human ROSA It includes 26 gene loci (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter, β-actin promoter and myeloproliferative sarcoma virus enhancer, negative regulatory region deletion type, and dl587rev primer binding site substitution (MND) U3 promoter (Haas et al. Journal of Virology. 2003;77(17):9439-9450).

[0134] In one embodiment, the artificial expression construct includes an MNDU3 promoter. In one embodiment, the vector includes an MNDU3 promoter.

[0135] In one embodiment, the artificial expression construct includes an EF1a promoter containing the first intron of the human EF1a gene. In one embodiment, the vector includes an EF1a promoter containing the first intron of the human EF1a gene.

[0136] In one embodiment, the artificial expression construct includes an EF1a promoter lacking the first intron of the human EF1a gene. In one embodiment, the vector includes an EF1a promoter lacking the first intron of the human EF1a gene.

[0137] "Conditional expression" may refer to any type of conditional expression, but is not limited to, inducible expression; repressive expression; or expression in cells or tissues having a particular physiological, biological, or disease state. This definition is not intended to exclude cell-type or tissue-specific expression. Certain embodiments provide conditional expression of a polynucleotide of interest, for example, where expression is controlled by subjecting cells, tissues, organisms, etc., to a process or condition that causes the expression of the polynucleotide or an increase or decrease in the expression of the polynucleotide encoded by the polynucleotide of interest.

[0138] In some alternatives, an inducible synthetic promoter is used, which comprises a first sequence encoding a transcription factor response element and a second sequence encoding a promoter sequence, and optionally, the inducible synthetic promoter comprises one or more of SEQ ID NOs. 45-83. In some alternatives, the inducible synthetic promoter is inducible by chimeric antigen receptor activation. In some alternatives, the inducible synthetic promoter is inducible by binding of the chimeric antigen receptor to a ligand. In some alternatives, the inducible synthetic promoter is inducible by interaction with CD3 / CD28. In some alternatives, CD3 / CD28 is conjugated on beads. In some alternatives, the inducible synthetic promoter is inducible by a chemical substance. In some alternatives, the chemical substance is PMA or ronomycin. In some alternatives, the promoter sequence comprises an IL2 minimal promoter sequence. In certain embodiments, the IL2 minimal promoter sequence comprises the sequence shown in SEQ ID NO. 107. In some alternatives, the first sequence in the inducible synthetic promoter comprises the sequence shown in any one of SEQ ID NOs. 45-83. In some alternatives, the inducible synthetic promoter includes a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the sequence shown in any one of SEQ ID NOs. 45-83, or a sequence having sequence identity within the range of any two of the aforementioned percentages. In some alternatives, the transcription factor response element includes E2F1, EGR1, HIF1A, NFAT, LEF1, SP1, PU.1, NFKB, JUN, FOS, and / or STAT4. In certain embodiments, the inducible synthetic promoter includes the sequence shown in any one of SEQ ID NOs. 45-83 and the IL2 minimal promoter. In certain embodiments, the inducible synthetic promoter includes the sequence shown in SEQ ID NO. 15.

[0139] Additional examples of inductive promoters / systems include, but are not limited to, steroid-inductive promoters, e.g., promoters for genes encoding glucocorticoid receptors or estrogen receptors (inducible by treatment with the corresponding hormone), metallothionein promoters (inducible by treatment with various heavy metals), MX-1 promoters (inducible by interferon), the "GeneSwitch" mifepristone-regulated system (Sirin et al., 2003, Gene, 323:67), cuminate-inducible gene switches (WO2002 / 088346), and tetracycline-dependent regulatory systems.

[0140] The term “operatably linked” refers to juxtaposition, where the described components are in a relationship that enables them to function in their intended manner. In one embodiment, “operatably linked” refers to a functional linkage between a nucleic acid expression regulatory sequence (e.g., a promoter and / or enhancer) and a second polynucleotide sequence, e.g., the polynucleotide of interest, where the expression regulatory sequence directs the transcription of the nucleic acid corresponding to the second sequence.

[0141] F. Genetically modified cells In various embodiments, cells are modified to contain and / or express polypeptides and / or polynucleotides as intended herein. In certain embodiments, cells are for use in the treatment of diseases or disorders (e.g., cancer or autoimmune diseases or disorders). Cells may be non-genetically modified to express one or more polypeptides as intended herein, or in certain preferred embodiments, cells may be genetically modified to express one or more polypeptides as intended herein. "Genetically engineered" or "genetically modified" refers to the addition of extra genetic material in the form of DNA or RNA to the total genetic material in a cell. "Genetically modified cell," "modified cell," and "non-natural" are used interchangeably in certain embodiments.

[0142] In certain embodiments, the artificial expression constructs envisioned herein are introduced and expressed in cells (e.g., lymphocytes or immune effector cells) to improve the efficacy, function, and / or persistence of the cells. In certain embodiments, one or more artificial expression constructs are introduced and expressed in cells redirected to target cells by co-expressing recombinant receptors or exogenous lymphocyte receptors.

[0143] In some embodiments, persistence is increased compared to similar cells or cell populations expressing exogenous wild-type IL-15. In some embodiments, the improved function includes reduced antigen-independent IFNγ release compared to similar cells or cell populations expressing exogenous wild-type IL-15. In some embodiments, the improved function includes increased proliferation compared to similar cells or cell populations expressing exogenous wild-type IL-15.

[0144] "Immune effector cells" are any cells of the immune system that have one or more effector functions (e.g., cytotoxic cell-killing activity, cytokine secretion, antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) induction). The illustrative immune effector cells contemplated herein include, but are not limited to, T lymphocytes, cytotoxic T cells (CTLs; CD8+ T cells), TILs, and helper T cells (HTLs; CD4+ T cells). In certain embodiments, the cells include αβ T cells. In certain embodiments, the cells include γδ T cells. In one embodiment, the immune effector cells include natural killer (NK) cells. In one embodiment, the immune effector cells include natural killer T (NKT) cells. In certain embodiments, immune cells and immune effector cells are used interchangeably.

[0145] Immune effector cells can be self / autologous ("self") or non-self ("non-self," e.g., allogeneic, syngeneic, or heterogeneous). "Self" refers to cells from the same subject. "Allogeneic" refers to cells of the same species but genetically different from the cells being compared. "Syngeneic" refers to cells of a different subject but genetically identical to the cells being compared. "Heterogeneous" refers to cells of a different species to the cells being compared. In a preferred embodiment, the cells are human autoimmune effector cells.

[0146] Appropriate exemplary immunoeffector cells for introducing the artificial expression constructs envisioned herein include T lymphocytes. “T cells” or “T lymphocytes” are recognized in the Art and include thymocytes, immature T lymphocytes, mature T lymphocytes, quiescent T lymphocytes, or activated T lymphocytes. T cells may be T helper (Th) cells, e.g., T helper 1 (Th1) or T helper 2 (Th2) cells. T cells may be helper T cells (HTLs; CD4+ T cells), CD4+ T cells, cytotoxic T cells (CTLs; CD8+ T cells), CD4+CD8+ T cells, CD4-CD8-T cells, or any other subset of T cells. Other exemplary populations of T cells suitable for use in specific embodiments include naive T cells and memory T cells. For example, suitable populations of T cells for use in a particular embodiment include naive T cells (TN), T memory stem cells (TSCM), central memory T cells (TCM), effector memory T cells (TEM), and effector T cells (TEFF).

[0147] As will be understood by those skilled in the art, other cells may also be used as immune effector cells expressing artificial expression constructs and / or variant IL15 polypeptides as intended herein. In certain embodiments, immune effector cells also include NK cells, NKT cells, neutrophils, and macrophages. Immune effector cells also include effector cell precursors, in which such precursor cells can be induced to differentiate into immune effector cells in vivo or in vitro. Thus, in certain embodiments, immune effector cells include immune effector cell precursors, such as hematopoietic stem cells (HSCs) contained within a CD34+ population of cells derived from umbilical cord blood, bone marrow, or mobilized peripheral blood, which differentiate into mature immune effector cells upon administration to a subject, or which can be induced in vitro to differentiate into mature immune effector cells.

[0148] "CD34+ cells" refer to cells that express the CD34 protein on their cell surface. "CD34" often refers to a cell surface glycoprotein (e.g., sialomucin protein) that acts as a cell-cell adhesion molecule and is involved in T cell entry into lymph nodes. The CD34+ cell population includes hematopoietic stem cells (HSCs), which differentiate upon administration to a patient and contribute to the entire hematopoietic system, including T cells, NK cells, NKT cells, neutrophils, and monocyte / macrophage lineages.

[0149] Methods for producing immunoeffector cells expressing and / or mutant IL15 polypeptides, including the artificial expression constructs contemplated herein, are provided in certain embodiments. In one embodiment, the method comprises transfecting or transducing immunoeffector cells isolated from an organism so that the immunoeffector cells express one or more of the artificial expression constructs contemplated herein. In one embodiment, the method comprises transfecting or transducing immunoeffector cells isolated from an organism so that the immunoeffector cells express i) mutant IL15 polypeptide and ii) recombinant receptor or exogenous lymphocyte receptor. In certain embodiments, immunoeffector cells are isolated from an organism and genetically modified without further in vitro manipulation. Such cells can then be directly re-administered to an organism. In further embodiments, immunoeffector cells are first activated and stimulated to proliferate in vitro before genetic modification. In this regard, immunoeffector cells can be cultured before and / or after genetic modification.

[0150] In certain embodiments, the cells are human cells. In certain embodiments, the cell source is obtained from the subject prior to the in vitro manipulation or genetic modification of the immune effector cells described herein. In certain embodiments, the modified immune effector cells include T cells. In certain embodiments, the immune effector cells are genetically modified in vivo.

[0151] T cells can be obtained from many sources, including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from the site of infection, ascites, pleural fluid, splenic tissue, and tumors. In certain embodiments, T cells can be obtained from units of blood collected from a subject using any number of techniques known to those skilled in the art, such as sedimentation, e.g., FICOLL® (Cytiva Sweden AB, Sweden) isolation.

[0152] In other embodiments, isolated or purified populations of T cells are used. In some embodiments, after isolation of peripheral blood mononuclear cells (PBMCs), both cytotoxic T lymphocytes and helper T lymphocytes can be sorted into naive T cell subgroups, memory T cell subgroups, and effector T cell subgroups, either before or after activation, proliferation, and / or genetic modification.

[0153] In one embodiment, the isolated or purified population of T cells expresses one or more markers, including, but not limited to, CD3+, CD4+, CD8+, or a combination thereof.

[0154] In certain embodiments, T cells are isolated from an organism and first activated and stimulated and grown in vitro before being modified to include an artificial expression construct and / or to express a mutant IL15 polypeptide as intended herein.

[0155] To achieve a sufficient therapeutic dose of the T cell composition, the T cells are often subjected to one or more rounds of stimulation, activation, and / or proliferation. In certain embodiments, T cells can generally be activated and proliferated using the methods described in, for example, U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; and 6,867,041, each of which is incorporated herein by reference in whole. In certain embodiments, T cells are activated and proliferated for 6, 12, 18, or 24 hours, optionally in combination with recombinant receptors or exogenous lymphocyte receptors, prior to the introduction of vectors or polynucleotides encoding the artificial expression constructs intended herein.

[0156] In one embodiment, T cells are activated at the same time they are modified.

[0157] In various embodiments, a method for generating immune effector cells includes activating a population of cells including T cells and proliferating this population of T cells. T cell activation can be achieved by providing a primary stimulatory signal through the T cell TCR / CD3 complex and by providing a secondary co-stimulatory signal through accessory molecules, such as CD28.

[0158] The TCR / CD3 complex may be stimulated by contacting T cells with a suitable CD3-binding substance, such as a CD3 ligand or an anti-CD3 monoclonal antibody. Exemplary examples of CD3 antibodies include, but are not limited to, OKT3, G19-4, BC3, and 64.1.

[0159] In addition to the primary stimulatory signal provided via the TCR / CD3 complex, secondary co-stimulatory signals are required to induce a T cell response. In certain embodiments, CD28-binding agents can be used to provide these co-stimulatory signals. Exemplary examples of CD28-binding agents include, but are not limited to, natural CD28 ligands, e.g., natural ligands for CD28 (e.g., members of the B7 family of proteins, e.g., B7-1 (CD80) and B7-2 (CD86)); as well as anti-CD28 monoclonal antibodies or fragments thereof capable of crosslinking the CD28 molecule, e.g., monoclonal antibodies 9.3, B-T3, XR-CD28, KOLT-2, 15E8, 248.23.2, and EX5.3D10.

[0160] In one embodiment, a molecule that provides a primary stimulus signal, such as a molecule that provides stimulation through a TCR / CD3 complex, and a co-stimulatory molecule are conjugated to the same surface.

[0161] In certain embodiments, the conjugated agent providing the stimulatory and co-stimulatory signals is localized on the cell surface. This can be achieved by transfecting or transducing cells with a nucleic acid encoding the conjugated agent in a form appropriate for its expression on the cell surface, or by conjugating the conjugated agent to the cell surface.

[0162] In another embodiment, molecules that provide a primary stimulus signal, such as molecules that provide stimulation through the TCR / CD3 complex, and co-stimulatory molecules are presented on antigen-presenting cells.

[0163] In one embodiment, a molecule that provides a primary stimulus signal, such as a molecule that provides stimulation through a TCR / CD3 complex, and a co-stimulatory molecule are presented on separate surfaces.

[0164] In certain embodiments, one of the conjugating agents providing the stimulatory signal and the co-stimulatory signal is soluble (provided in solution), and the other agent is provided on one or more surfaces. In certain embodiments, both conjugating agents providing the stimulatory signal and the co-stimulatory signal are provided in a soluble form (provided in solution). In various embodiments, the method for producing T cells as intended herein includes activating T cells with a soluble anti-CD3 antibody and / or a soluble anti-CD28 antibody, or fragments thereof. In various embodiments, the method for producing T cells as intended herein includes activating T cells with a surface-bound anti-CD3 antibody and / or a surface-bound anti-CD28 antibody, or fragments thereof. In various embodiments, the method for producing T cells as intended herein includes activating T cells with a bead-bound anti-CD3 antibody and / or a bead-bound anti-CD28 antibody, or fragments thereof.

[0165] In one embodiment, proliferating immune cells (e.g., T cells) activated by the method intended herein further includes culturing a population of cells containing immune cells for a period of several hours (3 hours) to 7 to 28 days, or any integer number of hours in between. In another embodiment, the immune cell composition may be cultured for 14 days. In a particular embodiment, the immune cells are cultured for 21 days. In yet another embodiment, the immune cell composition is cultured for 2 to 3 days. Several cycles of stimulation / activation / proliferation may also be desired.

[0166] In certain embodiments, suitable conditions for culturing immune cells (e.g., T cells) include, but are not limited to, a suitable culture medium (e.g., Minimal Essential Media or RPMI Media 1640 or X-vivo 15 (Lonza)) and one or more factors necessary for proliferation and survival, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, IL-21, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α, or any other additives known to those skilled in the art that are suitable for cell growth.

[0167] Further illustrative examples of cell culture media include, but are not limited to, RPMI 1640, Clicks, AIM-V, DMEM, MEM, a-MEM, IMDM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with appropriate amounts of serum (or plasma) or a defined set of hormones and / or sufficient amounts of cytokines for the growth and proliferation of immune cells.

[0168] Antibiotics, such as penicillin and streptomycin, are present only in the experimental culture and not in the cell culture into which the target cells are injected. The target cells are maintained under conditions necessary to support growth, such as a suitable temperature (e.g., 37°C) and air (e.g., air with 5% CO2 added).

[0169] In certain embodiments, PBMCs or isolated immune cells are brought into contact with stimulating and co-stimulating agents, such as anti-CD3 antibodies and anti-CD28 antibodies typically attached to beads or other surfaces, in a culture medium containing suitable cytokines, such as IL-2, IL-7, and / or IL-15.

[0170] In other embodiments, artificial antigen-presenting cells (aAPCs) are produced by manipulating K562 cells, U937 cells, 721.221 cells, T2 cells, and C1R cells to have stable expression and secretion of various costimulatory molecules and cytokines. In specific embodiments, K32 aAPCs or U32 aAPCs are used and directed to present one or more antibody-based stimulating molecules on the surface of the aAPC cells. Populations of T cells can be proliferated by aAPCs expressing various costimulatory molecules, including, but not limited to, CD137L (4-1BBL), CD134L (OX40L), and / or CD80 or CD86. Ultimately, aAPCs provide an efficient platform for proliferating genetically modified immune cells and maintaining CD28 expression on CD8 T cells. The aAPCs provided in WO03 / 057171 and US2003 / 0147869 are incorporated herein by reference in their entirety.

[0171] In certain embodiments, an expression construct or polynucleotide encoding mutant IL15 is introduced into a population of immune cells. In certain embodiments, an expression construct or polynucleotide encoding mutant IL15 is introduced into a population of immune cells expressing a recombinant receptor or an exogenous lymphocyte receptor. In certain embodiments, i) mutant IL15 and ii) an expression construct or polynucleotide encoding a recombinant receptor or an exogenous lymphocyte receptor are introduced into a population of immune cells. The polynucleotide may be introduced into T cells by microinjection, transfection, lipofection, heat shock, electroporation, transduction, gene gun, microinjection, DEAE-dextran-mediated transduction, and similar methods.

[0172] A “vector” refers to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The nucleic acid to be transferred is typically ligated into the vector nucleic acid molecule, for example, by insertion into it. The vector may contain a sequence directed towards autonomous replication in a cell, or a sequence sufficient to enable integration into host cell DNA. In certain embodiments, a non-viral vector is used to deliver one or more polynucleotides intended herein to immune cells.

[0173] Exemplary examples of nonviral vectors include, but are not limited to, mRNA, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes. Other nonviral vectors are discussed above.

[0174] Exemplary methods for nonviral delivery of polynucleotides intended in certain embodiments include, but are not limited to, electroporation, sonoporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, nanoparticles, polycation or lipid:nucleic acid complexes, naked DNA, artificial virions, DEAE-dextran-mediated transfer, gene guns, and heat shock.

[0175] Exemplary examples of nonviral / polynucleotide delivery systems suitable for use in specific embodiments contemplated in particular embodiments include, but are not limited to, systems provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc. Lipofection reagents are commercially available (e.g., Transfectam® and Lipofectin®). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides are described in the literature. See, for example, Liu et al. (2003) Gene Therapy. 10:180-187; and Balazs et al. (2011) Journal of Drug Delivery. 2011:1-12. Antibody-targeted delivery, bacterial-inducible delivery, and non-biological nanocell system delivery are also anticipated in particular embodiments.

[0176] In various embodiments, a polynucleotide is an mRNA introduced into a cell to transiently express a desired polypeptide. "Transient" refers to the expression of a non-integrated transgene over a period of several hours, days, or weeks, where the duration of expression is shorter than the duration of polynucleotide expression, if it is integrated into the genome in the cell or contained within a stable plasmid replicon.

[0177] In certain embodiments, a viral vector is used to deliver one or more polynucleotides intended herein to immune cells (e.g., T cells).

[0178] Exemplary examples of viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40).

[0179] In one embodiment, polynucleotides are introduced into immune cells by AAV transduction. In another embodiment, polynucleotides are introduced into immune cells by retroviral transduction. In another embodiment, polynucleotides are introduced into immune cells by lentiviral transduction. In another embodiment, polynucleotides are introduced into immune cells by adenovirus transduction. In another embodiment, polynucleotides are introduced into immune cells by herpes simplex virus transduction. In another embodiment, polynucleotides are introduced into immune cells by vaccinia virus transduction.

[0180] Additional illustrative examples of expression vectors include, but are not limited to, the pClneo vector (Promega) for expression in mammalian cells; and pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentiviral-mediated gene transfer and expression in mammalian cells. In certain embodiments, the coding sequences of polypeptides disclosed herein may be ligated into such expression vectors for polypeptide expression in mammalian cells.

[0181] In certain embodiments, the vector is an episomal vector or a vector maintained outside of chromosomes. "Episome" refers to a vector that can replicate without integration into the host's chromosomal DNA and without gradual loss from dividing host cells, and also means that the vector replicates outside of chromosomes or episomalally.

[0182] The “regulatory elements” or “regulatory sequences” present in an artificial expression vector are the vector’s untranslated regions—the origin of replication, selection cassette, promoter, enhancer, translation initiation signal (Shine Dalgarno sequence or Kozak sequence), introns, polyadenylated sequences, and 5’ and 3’ untranslated regions—which interact with host cellular proteins to perform transcription and translation. Such elements can vary in their intensity and specificity. Depending on the vector system and host used, any number of appropriate transcription and translation elements, ubiquitous promoters, and inducible promoters may be included.

[0183] In certain embodiments, vectors include, but are not limited to, expression vectors and viral vectors, and include exogenous, endogenous, or heterologous regulatory sequences, such as promoters and / or enhancers. “Endogenous” regulatory sequences are sequences that are naturally linked to a given gene in the genome. “Exogenous” regulatory sequences are those that are positioned alongside a gene using genetic engineering (i.e., molecular biological techniques) so that the transcription of that gene is directed by the linked enhancer / promoter. “Hexlogous” regulatory sequences are exogenous sequences that come from a different species than the cell being genetically engineered.

[0184] Conditional expression can also be achieved by using site-specific DNA recombinases. According to certain embodiments, the vector contains at least one (typically two) sites for recombination mediated by site-specific recombinases. The “recombinase” or “site-specific recombinase” includes excisable or integrated proteins, enzymes, cofactors or related proteins involved in a recombination reaction involving one or more recombination sites (e.g., 2, 3, 4, 5, 7, 10, 12, 15, 20, 30, 50, etc.), which may be wild-type proteins (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)), or mutants, derivatives (e.g., fusion proteins containing recombinant protein sequences or fragments thereof), fragments, and variants thereof. Exemplary examples of recombinases suitable for use in specific embodiments include, but are not limited to, Cre, Int, IHF, Xis, Flop, Fis, Hin, Gin, ΦC31, Cin, Tn3 resolverase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCE1, and ParA.

[0185] A vector may contain one or more recombination sites for any of a wide variety of site-specific recombinases. It should be understood that the target site for the site-specific recombinase is added to any site required for integration into the vector, e.g., a retroviral vector or a lentiviral vector. “Recombinant sequence,” “recombinant site,” or “site-specific recombination site” refers to a specific nucleic acid sequence that a recombinase recognizes and binds to.

[0186] For example, one recombination site for Cre recombinase is loxP, a 34-base pair sequence containing two 13-base pair inverse repeats (which act as recombinase binding sites) adjacent to an 8-base pair core sequence (see Figure 1 in Sauer, B., Current Opinion in Biotechnology 5:521-527 (1994)). Other exemplary loxP sites include, but are not limited to, lox511 (Hoess et al., 1996; Bethke and Sauer, 1997), lox5171 (Lee and Saito, 1998), lox2272 (Lee and Saito, 1998), m2 (Langer et al., 2002), lox71 (Albert et al., 1995), and lox66 (Albert et al., 1995).

[0187] Appropriate recognition sites for FLP recombinase are not limited to, but include: FRT (McLeod, et al., 1996), F1, F2, F3 (Schlake and Bode, 1994), F4, F5 (Schlake and Bode, 1994), FRT(LE) (Senecoff et al., 1988), and FRT(RE) (Senecoff et al., 1988).

[0188] Other examples of recognition sequences are the attB, attP, attL, and attR sequences, which are recognized by the recombinase enzyme λ-integrase, e.g., phi-c31. The φC31 SSR mediates recombination only between the heterotype sites attB (34 bp long) and attP (39 bp long) (Groth et al., 2000). attB and att are named after the attachment sites for phage integrase on bacterial and phage genomes, respectively, and both contain incomplete reverse repeats that can be bound by the φC31 aposidimer (Groth et al., 2000). The product sites, attL and attR, are further effectively inactive to φC31-mediated recombination (Belteki et al., 2003), making the reaction irreversible. To catalyze insertion, attB-carrying DNA has been found to be more readily inserted into genomic attP sites than attP sites into genomic attB sites (Thyagarajan et al., 2001; Belteki et al., 2003). Thus, by a typical strategy, homologous recombination positions the attP-carrying "docking site" in a designated locus, which then partners with the attB-carrying input sequence for insertion.

[0189] An "internal ribosome entry site" or "IRES" refers to an element that facilitates direct internal ribosome entry into the start codon of a cistron (protein-coding region), such as ATG, leading to cap-independent gene translation. See, for example, Jackson et al., 1990. Trends Biochem Sci 15(12):477-83) and Jackson and Kaminski. 1995. RNA 1(10):985-1000. In certain embodiments, the vector contains one or more polynucleotides of interest encoding one or more polypeptides. In certain embodiments, to achieve efficient translation of each of the polypeptides, the polynucleotide sequence may be separated by one or more IRES sequences or polynucleotide sequences encoding self-cleaving polypeptides. In one embodiment, the IRES used in the polynucleotides contemplated herein is an EMCV IRES.

[0190] In certain embodiments, the artificial expression construct may include a polynucleotide encoding a self-cleaving polypeptide. Exemplary self-cleaving polypeptides include 2A peptides from porcine rhinitis virus-1 (P2A), Thosea asigna virus (T2A), equine rhinitis A virus (E2A), foot-and-mouth disease virus (F2A), or their variants. Further exemplary nucleic acid and amino acid sequences of 2A peptides are shown, for example, in Kim et al. (PLOS One 6:e18556 (2011)). In certain embodiments, cells are genetically modified to contain the self-cleaving polypeptide. In certain embodiments, the sequence encoding the self-cleaving polypeptide lies between the sequence encoding the extracellular component of the recombinant receptor and the sequence encoding the intracellular component of the recombinant receptor. In certain embodiments, the sequence encoding the self-cleaving polypeptide lies between the sequence encoding mutant IL-15 and the sequence encoding the extracellular component of the recombinant receptor. In certain embodiments, the sequence encoding the self-cleaving polypeptide lies between the sequence encoding mutant IL-15 and the sequence encoding the intracellular component of the recombinant receptor.

[0191] A "Kozak sequence" refers to a short nucleotide sequence that greatly promotes the initial binding of mRNA to the small subunit of the ribosome, thereby increasing translation. The consensus Kozak sequence is shown in Sequence ID No. 106, where R is purine (A or G) (Kozak, 1986. Cell. 44(2):283-92, and Kozak, 1987. Nucleic Acids Res. 15(20):8125-48). In certain embodiments, the vector contains a polynucleotide having the consensus Kozak sequence and encoding the desired polypeptide.

[0192] Elements that direct the efficient termination and polyadenylation of exogenous nucleic acid transcripts increase exogenous gene expression. Transcription termination signals are generally found downstream of polyadenylation signals. In certain embodiments, the vector contains a polyadenylation sequence 3' of the polynucleotide encoding the polypeptide to be expressed. The "polyA site" or "polyA sequence" represents a DNA sequence directed by RNA polymerase II to both terminate and polyadenylate the nascent RNA transcript. The polyadenylation sequence can promote mRNA stability by adding a polyA tail to the 3' end of the coding sequence, thus contributing to increased translation efficiency. Cleavage and polyadenylation are directed by the poly(A) sequence in the RNA. For mammalian premRNA, the core poly(A) sequence has two recognition elements adjacent to the cleavage polyadenylation site. Typically, a nearly invariant AAUAAA hexamer is located 20-50 nucleotides upstream of the more variable element, which is rich in U or GU residues. The cleavage of the nascent transcript occurs between these two elements and is coupled with the addition of adenosine up to 250 to the 5' cleavage product. In certain embodiments, the core poly(A) sequence is an ideal poly(A) sequence (e.g., AATAAA, ATTAAA, AGTAAA). In certain embodiments, the poly(A) sequence is the SV40 poly(A) sequence, the bovine growth hormone poly(A) sequence (BGHpA), the rabbit β-globin poly(A) sequence (rβgpA), their variants, or another suitable exogenous or endogenous poly(A) sequence known in the art.

[0193] In some embodiments, polynucleotides, or cells containing polynucleotides, utilize suicide genes and include inducible suicide genes to reduce the risk of direct toxicity and / or uncontrolled amplification. In certain embodiments, the suicide genes are not immunogenic to the host or cells containing the polynucleotides. Certain examples of suicide genes that may be used are caspase-9, caspase-8, or cytosine deaminase. Caspase-9 can be activated using a specific chemical inducer of dimerization (CID).

[0194] Other control elements may include tag cassettes, transduction markers, or selection cassettes.

[0195] Tag cassettes and transduction markers can be used to activate, promote the growth of, detect, concentrate, isolate, track, deplete, and / or remove genetically modified cells in vitro, in vivo, and / or ex vivo. A “tag cassette” refers to a unique synthetic peptide sequence that is attached to, fused to, or part of an expression molecule (e.g., recombinant receptor or chemokine receptor) to which a congenerally binding molecule (e.g., ligand, antibody, or other binding partner) can bind, where the binding properties can be used to activate tagged proteins and / or cells expressing tagged proteins, promote their growth, detect, concentrate, isolate, track, deplete, and / or remove them. Transduction markers can serve the same purpose, but are derived from naturally occurring molecules and are often expressed using skipping elements (or self-cleaving polypeptides) that separate the transduction marker from the rest of the expression molecule.

[0196] Exemplary tags include His tag, Flag tag, Xpress tag, Avi tag, calmodulin-binding peptide (CBP) tag, polyglutamic acid tag, HA tag, Myc tag, Strep tag (which refers to the original STREP® tag, STREP® tag II (IBA Institut fur Bioanalytik, Germany); see, for example, US7,981,632), Softag1, Softag3, and V5. See Figure 6 for exemplary sequences.

[0197] Conjugate-binding molecules that specifically bind to the tag sequences disclosed herein are commercially available. For example, His-tagged antibodies are commercially available from suppliers including Life Technologies, Pierce Antibodies, and GenScript. Flag-tagged antibodies are commercially available from suppliers including Pierce Antibodies, GenScript, and Sigma-Aldrich. Xpress-tagged antibodies are commercially available from suppliers including Pierce Antibodies, Life Technologies, and GenScript. Avi-tagged antibodies are commercially available from suppliers including Pierce Antibodies, IsBio, and Genecopoeia. Calmodulin-tagged antibodies are commercially available from suppliers including Santa Cruz Biotechnology, Abcam, and Pierce Antibodies. HA-tagged antibodies are commercially available from suppliers including Pierce Antibodies, Cell Signal, and Abcam. Myc-tagged antibodies are commercially available from suppliers including Santa Cruz Biotechnology, Abcam, and Cell Signal. Strep-tagged antibodies are commercially available from suppliers including Abcam, Iba, and Qiagen.

[0198] In certain embodiments, the transduction marker may include any cell surface presentation marker that can be detected using an antibody that binds to the marker and enables sorting of cells having the marker. In certain embodiments, the transduction marker may be truncated low-affinity nerve growth receptor (LNGFRF) and one-step selection using streptavidin conjugate magnetic beads (Matheson et al. (2014) PloS one 9(10):e111437), or truncated human epidermal growth factor receptor (EGFR) (tEGFR or EGFRt; see Wang et al., Blood 118:1255, 2011), truncated CD19 (tCD19 or CD19t; see Budde et al., Blood 122:1660, 2013); truncated HER2 protein (Her2tG); human CD34 ECD; and / or CD34 antigen (see Fehse et al., Mol. Therapy 1(5 Pt 1); 448-456, 2000) and CD20 antigen (see Philip et al., Blood It may include a magnetically sortable marker streptavidin-binding peptide (SBP) presented on the cell surface by RQR8 combined with a target epitope from 124:1277-1278. In certain embodiments, cells are genetically modified to express EGFRt.

[0199] Transduction markers may include any suitable fluorescent protein, including blue fluorescent protein (e.g., BFP, eBFP, eBFP2); cyan fluorescent protein (e.g., eCFP, cerulean, CyPet); green fluorescent protein (e.g., GFP-2, tagGFP, turboGFP, eGFP); orange fluorescent protein (e.g., mOrange, mKO, Kusabira-Orange); red fluorescent protein (e.g., mKate, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express); yellow fluorescent protein (e.g., YFP, eYFP, citrin, Venus); and any other suitable fluorescent protein, such as firefly luciferase.

[0200] In certain embodiments, the selection cassette provides for positive or negative selection of a desired cell population. Negative selection is where some cell types are removed and the cell type of interest remains. Positive selection involves targeting the desired cell population in order to retain only the desired cells.

[0201] The selection cassette can encode a protein that (a) confers resistance to antibiotics or other toxins, (b) complements an auxotrophic defect, or (c) supplies an essential nutrient not available from complex media, such as a gene encoding D-alanine racemase for Bacillus. Any number of selection systems can be used to recover transformed cells. In certain embodiments, positive selection cassettes include genes for resistance to neomycin, hygromycin, ampicillin, puromycin, phleomycin, zeomycin, blasticidin, or biomycin. In certain embodiments, the selection cassette includes the DHFR (dihydrofolate reductase) gene or the DHFR double mutant (DHFRdm) gene that provides resistance to methotrexate (MTX), O 6 the MGMT P140K gene involved in resistance to BG / BCNU, the HPRT (hypoxanthine phosphoribosyltransferase) gene involved in transformation of specific bases present in HAT selection medium (aminopterin, hypoxanthine, thymidine), or other genes for detoxification of some drugs. In certain embodiments, the selection agents include neomycin, hygromycin, puromycin, phleomycin, zeomycin, blasticidin, biomycin, ampicillin, O 6 BG / BCNU, MTX, tetracycline, aminopterin, hypoxanthine, thymidine kinase, DHFR, Gln synthetase, or ADA.

[0202] In certain embodiments, the selection cassette includes DHFRdm. In certain embodiments, the method does not require a selection cassette to obtain a highly purified cell population.

[0203] In certain embodiments, the negative selection cassette contains genes for transformation into toxic substances for cells expressing the substrate present in the culture medium. These molecules include diphtheria toxin (DTA) detoxification genes (Yagi et al., Anal Biochem. 214(1):77-86, 1993; Yanagawa et al., Transgenic Res. 8(3):215-221, 1999), ganciclovir, or thymidine kinase genes for herpesvirus (HSV TK) susceptible to the presence of FIAU. The HPRT gene may also be used as a negative selection by adding 6-thioguanine (6TG) to the medium, as well as for all positive and negative selections, polyA transcription termination sequences from different origins, the most classic ones derived from SV40 polyA, or eukaryotic polyA genes (e.g., bovine growth hormone, rabbit β-globin).

[0204] Viral vectors containing polynucleotides as envisioned in certain embodiments can be delivered in vivo by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial infusion) or topical application to individual patients, typically as described below. Alternatively, the vector can be delivered ex vivo to cells, such as cells explanted from individual patients (e.g., mobilized peripheral blood, lymphocytes, bone marrow aspirate, tissue biopsy, etc.) or hematopoietic stem cells from a universal donor, followed by re-implantation of these cells into the patient.

[0205] G. Compositions and Formulations In certain embodiments, genetically modified cells can be collected from culture medium, washed, and concentrated in a carrier in a therapeutically effective amount to prepare a formulation. In certain embodiments, pharmaceutically acceptable carrier solutions are well known to those skilled in the art, and are similar to the development of appropriate dosing and therapeutic regimens for using the specific compositions and formulations described herein in various therapeutic regimens, including, for example, intestinal and parenteral administration, intravascular, intravenous, intraarterial, intraosseous, intraventricular, intracerebral, intracranial, intrathecal, subarachnoid, and intramedullary administration and formulation. It will be understood by those skilled in the art that certain embodiments contemplated herein may include other formulations, for example, those well known in the art of pharmaceuticals, and formulations described in, for example, Remington: The Science and Practice of Pharmacy, volume I and volume II, 22nd Edition, Edited by Loyd V. Allen Jr., Philadelphia, PA: Pharmaceutical Press; 2012, which is incorporated herein by reference in its entirety.

[0206] Exemplary carriers include physiological saline, buffered physiological saline, physiological saline, water, Hanks' solution, Ringer's solution, Normosol-R (Abbott Labs), PLASMA-LYTE A® (registered trademark) (Baxter Laboratories, Inc., Morton Grove, Illinois), and combinations thereof.

[0207] In certain embodiments, the carrier may be supplemented with human serum albumin (HSA) or other human serum components or fetal bovine serum. In certain embodiments, the carrier for infusion comprises 5% HSA or buffered saline with dextrose. Additional isotonic agents include trivalent or higher sugar alcohols, such as polyhydric sugar alcohols including glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.

[0208] The carrier may include buffers such as citrate buffers, succinate buffers, tartarate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactic acid buffers, acetate buffers, phosphate buffers, histidine buffers, and / or trimethylamine salts.

[0209] Stabilizers refer to a broad category of excipients that help prevent cell adhesion to container walls, ranging in function from fillers to additives. Typical stabilizers include: polyhydric sugar alcohols; amino acids, e.g., arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, e.g., lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myo-inititol, galactitol, glycerol, and cyclitol, e.g., inositol; PEG; amino acid polymers; sulfur-containing reducing agents, e.g., urea, glutathione, thio This may include: citric acid, sodium thioglycolate, thioglycerol, alpha-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (i.e., <10 residues); proteins, such as HSA, bovine serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; monosaccharides, such as xylose, mannose, fructose, and glucose; disaccharides, such as lactose, maltose, and sucrose; trisaccharides, such as raffinose; and polysaccharides, such as dextran.

[0210] Where necessary or beneficial, the formulation may contain a local anesthetic, such as lidocaine, to reduce pain at the injection site.

[0211] Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halide, hexamethonium chloride, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0212] The therapeutically effective amount of cells in the formulation is 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10 More than a cell, or 10 11 It can surpass that.

[0213] In the formulations disclosed herein, cells are generally in volumes of liter or less, 500 ml or less, 250 ml or less, or 100 ml or less. Therefore, the density of administered cells is typically 10 4 cells / ml, 10 7 cells / ml, or 10 8 Larger than cells / ml.

[0214] In certain embodiments, the formulation may contain at least one genetically modified cell type (e.g., modified T cells, NK cells, or stem cells). The formulation may contain different types of genetically modified cells (e.g., combinations of T cells, NK cells, and / or stem cells).

[0215] Different types of genetically modified cells or cell subsets (e.g., modified T cells, NK cells, and / or stem cells) may be provided in different ratios, such as 1:1:1, 2:1:1, 1:2:1, 1:1:2, 5:1:1, 1:5:1, 1:1:5, 10:1:1, 1:10:1, 1:1:10, 2:2:1, 1:2:2, 2:1:2, 5:5:1, 1:5:5, 5:1:5, 10:10:1, 1:10:10, 10:1:10, etc. These ratios may also be applied to the number of cells expressing the same or different expression molecules (e.g., mutant IL-15 and / or recombinant receptors). If only two cell types are combined, or only two combinations of expressed molecular components are included in the formulation, the ratio may include any two combinations that can be created from the three number of combinations provided above. In embodiments, the combined cell population is tested for efficacy and / or cell proliferation in vitro, in vivo, and / or ex vivo, and a cell ratio that provides cell efficacy and / or proliferation is selected. Certain embodiments include genetically modified cells expressing mutant IL-15.

[0216] The cell-based formulations disclosed herein can be prepared, for example, for administration by injection, infusion, perfusion, or lavage. The formulations can be further formulated for intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, subarachnoid, intratumoral, intramuscular, intravesical, bone marrow, and / or subcutaneous injection.

[0217] Targeted vectors and / or nanoparticles can also be used to genetically modify immune cells in vivo or ex vivo. Vectors that can be used to deliver artificial expression constructs (encoding mutant IL-15 and / or recombinant receptors) to cells are described elsewhere in this specification, and numerous vectors are known in the art.

[0218] Exemplary cell-targeted nanoparticles include cell-targeting ligands (e.g., CD3, CD4, CD8, CD34) on the surface of the nanoparticles, where the cell-targeting ligands result in selective uptake of the nanoparticles by a selected cell type. The nanoparticles then deliver genetically modified components resulting in the expression of mutant IL-15 and / or recombinant receptors.

[0219] Exemplary nanoparticles include liposomes (microscopic vesicles containing at least one concentric lipid bilayer surrounding an aqueous core), liposome nanoparticles (liposome structures used to encapsulate other smaller nanoparticles within their core), and lipid nanoparticles (liposome-like structures lacking the continuous lipid bilayer characteristic of liposomes). Other polymer-based nanoparticles can also be used, as can porous nanoparticles constructed from any material capable of forming a porous network. Exemplary materials include metals, transition metals, and metalloids (e.g., lithium, magnesium, zinc, aluminum, and silica).

[0220] For in vivo delivery and cell uptake, nanoparticles may have a neutral or electrostatic coating and a size of 130 nm or less. The dimensions of the nanoparticles can be determined, for example, using conventional techniques such as dynamic light scattering and / or electron microscopy. In certain embodiments, the nanoparticles may be those described in WO2014153114, WO2017181110, and WO201822672.

[0221] The therapeutically effective dose of the vector and / or nanoparticles in the formulation may be in the range of 0.1–5 μg / kg or 0.5–1 μg / kg. In other examples, doses may include 1 μg / kg, 30 μg / kg, 90 μg / kg, 150 μg / kg, 500 μg / kg, 750 μg / kg, 0.1–5 mg / kg, or 0.5–1 mg / kg. In other examples, doses may include 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg, or more.

[0222] Methods for administering vector compositions and / or nanoparticles as envisioned in a particular embodiment include any method that is effective in producing modified immunoeffector cells.

[0223] H. How to use The methods disclosed herein include treating subjects (humans, non-human primates, veterinary animals (dogs, cats, reptiles, birds, etc.), livestock (horses, cattle, goats, pigs, chickens, etc.) and research animals (monkeys, rats, mice, fish, etc.)) using the formulations disclosed herein. Treating a subject includes delivering a therapeutically effective dose. A therapeutically effective dose includes an effective dose, a prophylactic dose, and / or a dose that provides a therapeutic dose.

[0224] An "effective dose" is the amount of formulation required to produce a desired physiological change in a subject. For example, an effective dose may provide an immunogenic anticancer effect or an anti-infective effect. Effective doses are often administered for research purposes. The effective doses disclosed herein may produce a statistically significant effect in animal models or in vitro assays related to the evaluation of the development or progression of cancer or infection. Immunogenic formulations may be provided in an effective dose, in which the effective dose stimulates an immune response.

[0225] "Prophylactic treatment" includes treatment administered to subjects who do not present with signs or symptoms of cancer or infection, or who present only with early signs or symptoms of cancer or infection, and the treatment is administered for the purpose of reducing or decreasing the risk of further development of cancer or infection. Thus, prophylactic treatment functions as a prophylactic treatment against targeted antigen-expressing cancer or infection. In certain embodiments, prophylactic treatment reduces, delays, or prevents the development of metastasis from a primary cancerous tumor site. In certain embodiments, prophylactic treatment reduces, delays, or prevents infection from bacteria, viruses, fungi, parasites, or arthropods.

[0226] "Therapeutic treatment" includes treatments administered to subjects presenting with symptoms or signs of cancer or infection, and is administered to subjects for the purpose of reducing or eliminating those signs or symptoms of cancer or infection. Therapeutic treatments can reduce, control or eliminate the presence or activity of cancer or infection, and / or reduce, control or eliminate the side effects of cancer or infection.

[0227] The functions as an effective dose, prophylactic treatment, or therapeutic treatment are not mutually exclusive, and in certain embodiments, the administered dose may achieve a treatment level greater than 1.

[0228] In certain embodiments, the therapeutically effective dose provides an anticancer effect. The anticancer effect includes a reduction in the number of cancer cells, a reduction in the number of metastases, a reduction in tumor volume, an increase in life expectancy, induced chemosensitivity or radiosensitivity in cancer cells, inhibited angiogenesis near cancer cells, inhibited cancer cell proliferation, inhibited tumor growth, prevention or reduction of metastasis, extended life expectancy, reduced cancer-related pain, and / or reduced relapse or recurrence of cancer after treatment. In certain embodiments, cancer is a hematological malignancy. In certain embodiments, cancer is a solid tumor or tumor.

[0229] A tumor is a swelling or lesion formed by the abnormal growth of cells (called neoplastic cells or tumor cells). Tumor cells are abnormal cells that grow through rapid, unregulated cell proliferation and continue to grow after the stimulus that initiated their growth has ceased. Tumors exhibit a partial or complete lack of structural, tissue, and functional coordination with normal tissue and usually form a different mass of tissue, which can be benign, premalignant, or malignant.

[0230] Examples of hematological malignancies that can be treated using the methods and constructs disclosed herein include leukemia, lymphoma, or multiple myeloma. In certain embodiments, leukemia includes acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloid monocytic leukemia (CMML), or myeloproliferative neoplasm (MPN). In certain embodiments, leukemia includes AML. In certain embodiments, lymphoma includes non-Hodgkin lymphoma and Hodgkin lymphoma. In certain embodiments, multiple myeloma includes light chain myeloma, nonsecretory myeloma, solitary plasmacytoma, extramedullary plasmacytoma, monoclonal gamma disease of unknown significance (MGUS), smoldering multiple myeloma (SMM), immunoglobulin D (IgD) myeloma, or immunoglobulin E (IgE) myeloma.

[0231] Examples of solid tumors that can be treated using the methods and constructs disclosed herein include lung cancer, squamous cell carcinoma, colorectal cancer, pancreatic cancer, breast cancer, thyroid cancer, bladder cancer, cervical cancer, esophageal cancer, ovarian cancer, gastric cancer, endometrial cancer, or brain cancer. In certain embodiments, lung cancer is non-small cell lung cancer. In certain embodiments, brain cancer includes glioma, glioblastoma, or oligodendroglioma.

[0232] Regarding administration, the therapeutically effective dose (also referred to as dose in this specification) can be initially estimated based on results from in vitro assays and / or animal model studies. Using such information, a more accurate useful dose in the subject of interest can be determined. The actual dosage administered to a particular subject can be determined by a physician, veterinarian, or researcher, taking into account parameters such as the target, body weight, severity of the condition, type of cancer or infection, stage of cancer or infection, previous or concurrent therapeutic interventions, the subject's idiopathic disease, and physical and physiological factors including the route of administration.

[0233] The therapeutically effective amount of cell-based formulation is 10 4 ~10 9 cells / kg body weight, or 10 3 ~10 11It can include cells / kg body weight. The therapeutically effective dose to administer is 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10 More than a cell, or 10 11 It can exceed, or include.

[0234] The therapeutically effective dose of the vector and / or nanoparticles in the formulation may be in the range of 0.1–5 μg / kg or 0.5–1 μg / kg. In other examples, doses may include 1 μg / kg, 30 μg / kg, 90 μg / kg, 150 μg / kg, 500 μg / kg, 750 μg / kg, 0.1–5 mg / kg, or 0.5–1 mg / kg. In other examples, doses may include 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg, or more.

[0235] The therapeutically effective dose can be achieved by administering a single dose or multiple doses over the course of a treatment regimen (e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, or annually). In specific embodiments, the treatment protocol may be directed by a clinical trial protocol or an FDA-approved treatment protocol.

[0236] The therapeutically effective dose can be administered, for example, by injection, infusion, perfusion, or lavage. Routes of administration may include intravesical, intravenous, intradermal, intraarterial, intraparenchymal, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, vaginal, intrarectal, local, subarachnoid, intratumoral, intramuscular, or subcutaneous administration.

[0237] In certain embodiments, the formulations and / or compositions are administered to the patient in combination with any number of relevant therapeutic modalities (e.g., before, concurrently, or after). In certain embodiments, the cells may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunosuppressants such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludarivine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation.

[0238] In some embodiments, the chemotherapeutic agent is administered simultaneously with or within one week after the administration of the engineered cells or artificial expression construct. In other embodiments, the chemotherapeutic agent is administered 1 to 4 weeks, or 1 week to 1 month, 1 week to 2 months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months, or 1 week to 12 months after the administration of the engineered cells or nucleic acid. In other embodiments, the chemotherapeutic agent is administered at least one month before the administration of the cells or nucleic acid. In some embodiments, the method further includes administering two or more chemotherapeutic agents.

[0239] In additional embodiments, the formulations and / or compositions disclosed herein may be administered with an anti-inflammatory agent. The anti-inflammatory agent or drug includes steroids, glucocorticoids, and nonsteroidal anti-inflammatory drugs (NSAIDS).

[0240] In certain embodiments, the formulations and / or compositions described herein are administered in combination with cytokines. "Cytokine" refers to a protein released by one cell population and acting as an intercellular mediator on another cell. Examples of cytokines are lymphokines, monokines, and conventional polypeptide hormones. Among cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; 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 lactogen; mullerian inhibitor substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor (NGF) such as NGF-beta; platelet growth factor; transforming growth factor (TGF) such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II; erythropoietin (EPO); osteogenic factor; interferons such as interferon-alpha, beta, and -gamma; colony-stimulating factors (CSF) such as macrophage-CSF (M-CSF); granulocyte macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL) such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, tumor necrosis factors such as TNF-alpha or TNF-beta; and other polypeptide factors including LIF and kit ligand (KL). Cytokines include proteins from natural sources or from recombinant cell culture, and biologically active equivalents of native sequence cytokines.

Table 1-1

Table 1-2

[0241] While the embodiments described above are illustrated in some detail by illustration and examples for the purpose of clarity of understanding, it will be readily apparent to those skilled in the art that certain changes and modifications can be made thereto in light of the teachings intended herein without departing from the spirit or scope of the appended claims. The following embodiments are provided for illustrative purposes only and not for limiting purposes. Those skilled in the art will readily recognize a variety of non-material parameters that can be changed or modified to produce essentially similar results.

[0242] K. Experimental Examples Example 1. Generation and characterization of engineered T cells expressing cytokines. Lentiviral vectors comprising constructs containing at least a recombinant receptor and / or a polynucleotide encoding an IL15 polypeptide were designed, constructed, and validated. In certain lentiviral vectors, the recombinant receptor is an anti-CD33 dimerizer-modulated immunoreceptor complex (DARIC33) containing anti-CD33 VHH and signaling components. Anti-CD33 VHH DARIC (DARIC33), containing a lentiviral vector, was constructed including a constitutive MNDU3 promoter operably linked to a polynucleotide encoding the following: DARIC signaling components (CD8α-signal peptide, FRB variant (T82L), CD8α transmembrane domain, intracellular 4-1BB costimulatory domain, and CD3 zeta signaling domain); P2A sequence; and DARIC targeting components (Igκ signaling peptide, CD33-specific VHH binding domain (cameloid or humanized), G4S linker, FKBP12 domain, and CD4-derived transmembrane domain with cleavage intracellular domain. See, for example, SEQ ID NOs: 1-5). Some lentiviral vectors also contained polynucleotides encoding an IL15 polypeptide operably linked to a polynucleotide encoding a recombinant receptor via polynucleotides encoding one or more viral 2A self-cleaving polypeptides (e.g., P2A). See Figure 1.

[0243] T cells from three donors were transduced using lentiviral vectors (LVVs) encoding either DARIC33 alone or DARIC33 with various IL15 variants. Transduced T cells were grown for 10 days with similar growth kinetics between control cells and T cells transduced with IL15-containing lentiviruses (Figure 2). Expression of both DARIC33 targeting components (Figures 3A and 3B) and DARIC33 signaling components (Figures 4A and 4B) was similar across all LVVs, with the exception of T cells transduced with soluble wild-type (WT) IL15, which exhibited reduced expression of DARIC33 components. Viral copy numbers were within acceptable ranges for all DARIC33 variants (Figure 5). Inclusion of either soluble IL15 or membrane-bound IL15 had minimal effect on the CD4:CD8 ratio (Figure 6). Transduced T cells using an IL15-containing vector showed a slight decrease in the central memory T cell compartment, as determined by CD62L staining (Figure 7). CD54 expression is a marker of sustained signaling in LVV transduced T cells, and increased CD54 expression was observed in WT, but not sIL-15 variant (D8S mutation), compared to control DARIC33 cells (Figure 8).

[0244] Example 2. IL15-expressing DARIC33 T cells are functionally active. Transduced T cells were co-cultured with either the medium alone or with endogenous CD33+ tumor cell lines MV4-11 and A549, which were engineered to overexpress CD33. Cytokine secretion was quantified by Meso Scale Discovery (MSD®, Merck Sharp & Dohme Corp., Leway, N.J.) analysis of the culture supernatant. Minimal IFNγ production was observed when untransduced or anti-CD33 dimerizer-regulated immune receptor complex (DARIC33) T cells were cultured in the absence of tumor. Expression of DARIC33 with soluble wild-type (WT) IL15 resulted in significant IFNγ production in the absence of tumor. Neither the D8S sIL15 mutant (IL-15 with D8S mutation) nor the membrane-fixed IL15 variant showed high levels of IFNγ production in the absence of tumor. Dimerization of the DARIC33 signaling complex with rapamycin or the non-immunosuppressive rapalog AP21967 did not affect IL15 secretion from either construct (Figure 9).

[0245] To analyze the functional capacity of IL15-expressing T cells, DARIC33 T cells were cultured with CD33+ MV4-11 tumor cells for 24 hours in a 1:1 E:T ratio, either in the presence or absence of rapamycin or AP21967 rapalog. All constructs exhibited inducible IFNγ production in response to the tumor co-culture, with similar levels of IFNγ secretion for all DARIC33 variants (Figure 10). Minimal cytokine production was detected in the non-transduced control. Similarly, DARIC33-targeted T cells secreted equivalent amounts of IFNγ to A549 cell lines engineered to express CD33 (Figure 11). However, when co-cultured with a control A549 cell line engineered to overexpress B cell maturation antigen (BCMA), T cells engineered to express DARIC33 in combination with WT soluble IL15 secreted high levels of IFNγ (Figure 12).

[0246] To evaluate DARIC33 cytotoxicity, parental and IL15-expressing DARIC33 T cells were co-cultured with A549 cells engineered to express a fluorescent reporter gene (NLR) and either CD33 or BCMA as negative controls. Co-culture of DARIC33 cells with A549-CD33 spheroids in the absence of dimerized drugs resulted in some cytotoxicity, particularly in T cells expressing soluble WT IL-15 variants (Figure 13). Addition of AP21967 produced rapid and similar tumor cell killing for all DARIC33 variants (Figure 14). Similar to the above findings, co-culture of DARIC33 variants with control A549-BCMA tumor cell lines resulted in minimal cell death in the presence or absence of AP21967 (Figures 15 and 16). T cells expressing WT soluble IL15 showed antigen-independent cytotoxicity.

[0247] Example 3. Generation and Characterization of Lentiviral Vectors. Lentiviral vectors were designed, constructed, and validated, comprising constructs containing polynucleotides comprising at least a polynucleotide encoding a recombinant receptor (e.g., DARIC33) and / or an inducible / adjustable promoter (e.g., iSynPro) operably linked to a polynucleotide encoding sIL15 or sIL15.D8S polypeptide. As shown in Figure 17, the iSynPro-IL15 polypeptide was constructed either forward-oriented or reverse-oriented relative to the polynucleotide encoding the recombinant receptor.

[0248] T cells were activated, transduced, and proliferated as described in Example 1. After a 10-day proliferation protocol, transduced T cells were cultured with NLR+ A549 cells engineered to overexpress either B cell maturation antigen (BCMA) or CD33, in or without rapamycin. Activation of the iSynPro promoter and IL-15 secretion were analyzed by enzyme-linked immunosorbent assay (ELISA). Transduced T cells using constitutively expressed sIL-15 LVV secreted IL-15 in the absence of the target or in the presence of nonspecific BCMA, and IL-15 secretion was not affected by the addition of rapamycin (Figures 18A and 18B). However, co-culture of DARIC33.iSynPro T cells with CD33+ tumor cells resulted in IL-15 secretion only in the presence of rapamycin (Figure 18C). Both forward iSynPro orientation and reverse iSynPro orientation induced IL-15 production in tumors and after rapamycin co-culture.

[0249] The functionality of manipulated T cells was analyzed by quantifying IFNγ secretion in the culture supernatant. Manipulated T cells were co-cultured with manipulated A549 cells, and IFNγ production was analyzed by MSD. Transduced T cells using constitutively expressed soluble WT IL-15 LVV secreted large amounts of IFNγ when cultured with the control A549-BCMA cell line (Figure 19A). In contrast, manipulated T cells exhibited robust IFNγ production when co-cultured with the A549-CD33 cell line in the presence of rapamycin (Figure 19B).

[0250] T cells engineered to express DARIC33 or DARIC33.iSynPro.IL15 / IL-15.D8S were also cultured with AML-derived tumor cell lines, and cytokine production was analyzed via MSD. Engineered T cells exhibited high IFNγ production when co-cultured with HL60 tumor cells in the presence of rapamycin. However, inclusion of either WT or the D8S IL-15 variant led to higher levels of secretion when T cells were co-cultured with the CD33low OCI-AML tumor line (Figure 20A). Inclusion of either IL-15 or IL-15(D8S) also led to higher IL-2 secretion when T cells were stimulated with either the OCI-AML or HL-60 tumor line (Figure 20B). As previously observed, T cells cultured without antigen did not produce any IL15. However, DARIC33.iSynPro T cells cultured with HL60 in the presence of rapamycin showed inducible IL15 production (Figure 20C).

[0251] Example 4. In vitro proliferation of manipulated cells. T cells were activated, transduced, and proliferated as described in Examples 1-3. After a 10-day proliferation protocol, transduced T cells were cultured with CD33+ MV4-11 in the presence of rapamycin in different media. T cells were counted, and the medium was changed on days 3, 7, 10, and 15 after activation. Addition of exogenous IL-2 and IL-15 to the culture resulted in sustained T cell proliferation (Figure 21A), while expression of IL-15 or IL-15(D8S) transgene resulted in higher levels of peak T cell proliferation compared to the control sample, but with similar dynamics of T cell contraction (Figures 21B and 21C).

[0252] Example 5. Characterization of manipulated cells in vivo. In vivo activity of IL15-secreting T cells was analyzed using a xenograft tumor model. T cells were activated, transduced, and proliferated as described in Example 1. Immunodeficient NSG mice were engrafted with CD33+MV4-11 tumor cells expressing firefly luciferase for in vivo follow-up. Similar tumor growth was observed in all animals treated with DARIC33 T cells in the absence of rapamycin (Figure 22). High 10 × 10 6 At T cell doses, the addition of rapamycin (0.1 mg / kg, mwf (Monday, Wednesday, Friday)) resulted in equivalent tumor control in all DARIC33 T cells (Figure 23). 6 At T cell doses, inclusion of the D8S IL15 transgene resulted in improved tumor control compared to DARIC33 T cells (Figure 24).

[0253] Example 6. Construction of a rapamycin-inducible engineered T cell receptor (eTCR). Lentiviral vectors comprising constructs encoding at least a multimerizing domain (e.g., rapamycin-inducible dimerizing domain), a CD3 subunit (e.g., CD3ε), and at least one extracellular antigen targeting domain were designed, cloned, and sequence-validated. The construct comprises or encodes various combinations of the following units: a signal sequence (e.g., a CD8α or IgK-derived signal sequence), one or more multimerization domains (e.g., FK506-binding protein (FKBP12 or FKBP) and FKBP-rapamycin-binding protein (FRB or FRB*)), a CD3ε subunit, one or more viral autocleavage peptides (e.g., P2A or T2A autocleavage peptides), one or more extracellular antigen targeting domains (e.g., antibody-derived targeting domain or native ligand-derived targeting domain), one hinge and transmembrane domain (e.g., derived from CD4), and one secreted cytokine molecule expressed behind either a conventional promoter (e.g., MND) or a T cell activation-inducible promoter (see Figures 25A and 25B).

[0254] Example 7. Evaluation of rapamycin-inducible eTCR T cells using IL-15 D8S in vivo. T cells expressing the rapamycin-inducible T cell receptor (TEA-T cells) were generated using a 7-day transduction and proliferation process, and then their expression and biological activity against specific target antigens were evaluated. Briefly, enriched CD4+ and CD8+ T cells were cultured in IL-2-containing medium and activated with human CD3 and human CD28 preparations. A lentiviral vector encoding a rapamycin-inducible eTCR, with a CD4 hinge and transmembrane domain, and immobilizing an FKBP12 multimerizing domain and two antigen-targeting domains, was used with or without IL-15, along with a D8S mutation driven by an inducible promoter (TEA+ / - IL-15 D8S). Enriched T cells were transduced one day after the start of culture, and the cells were then transferred to a 1L G-REX® (Wilson Wolf Corporation, St. Paul, Minnesota) culture system 24 hours later. After a total of 7 days in culture, TEA-T cells were evaluated for rapamycin-dependent, antigen-dependent activity, and rapamycin-independent, antigen-independent background activity.

[0255] Female NSG-MHCI / MHCII knockout mice were intravenously administered CD33+CLL1+ MV-411 xenograft tumor cells expressing firefly luciferase. After 8 days of tumor growth, 12 × 10⁶ cells were administered. 6 Untransduced T cells or 6 × 10 6 TEA+ / -IL-15 D8S T cells were administered intravenously either without rapamycin or with rapamycin administered three times per week (Figures 26A-26D). Survival was monitored for 70 days (Figure 27). TEA+IL-15 D8S was superior to TEA (without IL15-D8S) in both overall tumor growth and the percentage of mice that survived to the end of the study.

[0256] L. Conclusion The nucleic acid sequences and amino acid sequences provided herein are indicated using letter abbreviations for nucleotide bases and amino acid residues, as defined in 37 CFR §1.831-1.835 and as shown in WIPO Standard ST.26 (implemented July 1, 2022). Only single strands of each nucleic acid sequence are shown; however, complementary strands are understood to be included in embodiments where appropriate.

[0257] The sequence variants disclosed and referenced herein also include. Guidance for determining which amino acid residues can be substituted, inserted, or deleted without loss of biological activity can be found using computer programs well known in the art, such as DNASTAR® (Madison, Wisconsin) software. Preferably, the amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similar charged or uncharged amino acids. Conservative amino acid changes include substitutions of one of the families of amino acids associated in their side chains.

[0258] Appropriate conservative amino acid substitutions in peptides or proteins are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art generally recognize that single amino acid substitutions in the non-essential region of a polypeptide do not substantially alter its biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Natural amino acids are generally divided into the following conserved substitution families: Group 1: alanine (Ala), glycine (Gly), serine (Ser), and threonine (Thr); Group 2: (acidic): aspartic acid (Asp) and glutamic acid (Glu); Group 3: (acidic; also classified as polar, positively charged residues, and their amides): asparagine (Asn), glutamine (Gln), Asp, and Glu; Group 4: Gln and Asn; Group 5: (basic; also classified as polar, positively charged residues): arginine (Arg), lysine (Lys), and histidine (His); Group 6: (large aliphatic, nonpolar residues): isoleucine (Il e) Leucine (Leu), methionine (Met), valine (Val), and cysteine ​​(Cys); Group 7 (non-charged): tyrosine (Tyr), Gly, Asn, Gln, Cys, Ser, and Thr; Group 8 (large aromatic residues): phenylalanine (Phe), tryptophan (Trp), and Tyr; Group 9 (non-polar): proline (Pro), Ala, Val, Leu, Ile, Phe, Met, and Trp; Group 11 (aliphatic): Gly, Ala, Val, Leu, and Ile; Group 10 (small aliphatic, non-polar, or slightly polar residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing): Met and Cys. Additional information can be found in Creighton (1984) Proteins, WH Freeman and Company.

[0259] When creating such changes, the hydrophobicity and hydrophilicity indices of amino acids may be considered. The importance of the hydrophobicity and hydrophilicity amino acid index in conferring interactive biological functions on proteins is generally understood in this art (Kyte and Doolittle, 1982, J.Mol.Biol.157(1),105-32). Each amino acid has been assigned a hydrophobicity and hydrophilicity index based on its hydrophobicity and charge properties (Kyte and Doolittle, 1982). These values ​​are as follows: Ile(+4.5); Val(+4.2); Leu(+3.8); Phe(+2.8); Cys(+2.5); Met(+1.9); Ala(+1.8); Gly(-0.4); Thr(-0.7); Ser(-0.8); Trp(-0.9); Tyr(-1.3); Pro(-1.6); His(-3.2); Glutamate(-3.5); Gln(-3.5); Aspartic acid(-3.5); Asn(-3.5); Lys(-3.9); and Arg(-4.5).

[0260] It is known in the art that certain amino acids can be substituted with other amino acids having similar hydrophobicity or hydrophilicity indices or scores, resulting in proteins with similar biological activity, i.e., proteins that are still biologically and functionally equivalent. When making such changes, substitutions of amino acids with hydrophobicity or hydrophilicity indices within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that substitutions of similar amino acids can be effectively made based on hydrophilicity.

[0261] As detailed in US 4,554,101, the following hydrophilic values ​​have been assigned to amino acid residues: Arg (+3.0); Lys (+3.0); Aspartic acid (+3.0±1); Glutamic acid (+3.0±1); Ser (+0.3); Asn (+0.2); Gln (+0.2); Gly (0); Thr (-0.4); Pro (-0.5±1); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Val (-1.5); Leu (-1.8); Ile (-1.8); Tyr (-2.3); Phe (-2.5); Trp (-3.4). It is understood that amino acids can be substituted for other amino acids with similar hydrophilic values, and that biologically equivalent, and especially immunologically equivalent, proteins can still be obtained. In such changes, substitution of amino acids with a hydrophilicity value within ±2 is preferred, amino acids within ±1 are particularly preferred, and amino acids within ±0.5 are even more particularly preferred.

[0262] As outlined above, amino acid substitutions may be based on the relative similarity of amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, and similar characteristics. As shown elsewhere, gene sequence variants may include codon-optimizing variants, sequence polymorphisms, splice variants, and / or mutations that do not affect the function of the encoded product to a statistically significant degree.

[0263] Variants of proteins, nucleic acids, and gene sequences disclosed herein also include sequences having at least 70% sequence identity, 80% sequence identity, 85% sequence identity, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity with the proteins, nucleic acids, or gene sequences disclosed herein.

[0264] "% sequence identity" refers to the relationship between two or more sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relevance between proteins, nucleic acids, or gene sequences, as determined by the agreement between strings of such sequences. "Identity" (often referred to as "similarity") can be readily calculated by known methods, including those described below: Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Methods for determining identity are designed to give the best match among the sequences tested. Methods for determining identity and similarity are systematized in publicly available computer programs. Sequence alignment and percentage identity calculations can be performed using the Megalign program of the LASERGENE Bioinformatics Computing Suite (DNASTAR, Inc., Madison, Wisconsin). Multiple sequence alignments can also be performed using the Clustal method of alignment with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10) (Higgins and Sharp CABIOS, 5, 151-153 (1989)).Related programs also include the GCG suite program (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul, et al., J.Mol.Biol.215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput.Methods Genome Res., [Proc.Int.Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New Including York, NY. In the context of this disclosure, when sequence analysis software is used for analysis, it will be understood that the results of the analysis will be based on the “default values” of the referenced program. “Default values” means any set of values ​​or parameters that are originally loaded using the software when it is first initialized.

[0265] The variants also include nucleic acid molecules that hybridize to the sequences disclosed herein under stringent hybridization conditions and provide the same functionality as the reference sequence. Exemplary stringent hybridization conditions involve an overnight incubation at 42°C in a solution containing 50% formamide, 5XSSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5X Denhardt solution, 10% dextran sulfate, and 20 μg / ml denatured, sheared salmon sperm DNA, followed by washing the filter in 0.1XSSC at 50°C. Variations in the stringency of hybridization and signal detection are achieved primarily through manipulation of formamide concentration (lower percentages of formamide result in lower stringency); salt conditions; or temperature. For example, moderately high stringency conditions include an overnight incubation at 37°C in a solution containing 6×SSPE (20×SSPE = 3M NaCl; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, and 100 μg / ml salmon sperm blocking DNA; followed by washing at 50°C with 1×SSPE and 0.1% SDS. Furthermore, to achieve even lower stringency, washing performed after stringent hybridization may be carried out with higher salt concentrations (e.g., 5×SSC). Variations in the above conditions can be achieved through the inclusion and / or substitution of alternative blocking reagents used to suppress background noise in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of certain blocking reagents may require modifications to the hybridization conditions described above due to compatibility issues.

[0266] "To bind" refers to the association of a binding domain (e.g., a CAR-binding domain) with its homologous molecule. "To preferentially bind" refers to the association of a binding domain (e.g., a recombinant receptor-binding domain) with its homologous molecule. 5M -1 Affinity or K equal to or greater than that. a This refers to an association involving a specific binding interaction equilibrium binding constant (i.e., a unit of 1 / M) while not significantly associating with any other molecules or components in the relevant environmental sample. Binding domains can be classified as "high affinity" or "low affinity." In certain embodiments, "high affinity" binding domains are at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , or at least 10 13 M -1 K a This refers to those binding domains accompanied by 10 7 M -1 Until 10 6 M -1 Until 10 5 M -1 up to K a This refers to the binding domain that is associated with it. Alternatively, affinity is expressed in units of M (e.g., 10 -5 M~10 -13 The equilibrium dissociation constant (K) of a specific bond interaction with M) d ) may be defined as. In certain embodiments, the binding domain may have “enhanced affinity,” which refers to a selected or manipulated binding domain that binds more strongly to a congenerally bound molecule than the wild-type (or parent) binding domain. For example, enhanced affinity is stronger for congenerally bound molecules than the reference binding domain. a (Equilibrium association constant) for fewer congenerally bound molecules than those with reference binding domains d (Dissociation constant) may be the cause, or the off-rate (K) for congenerally linked molecules may be less than that of the reference binding domain. offThis can be due to [unspecified cause]. Various assays are known to detect binding domains that preferentially bind to specific homologous molecules and to determine binding affinity, such as Western blotting, ELISA, and BIACORE® analysis (see, e.g., Scatchard, et al., 1949, Ann. NYAcad. Sci. 51:660; and U.S. Patents 5,283,173, 5,468,614, or equivalents).

[0267] Unless otherwise indicated, the practices of this disclosure can utilize conventional techniques of immunology, molecular biology, microbiology, cell biology, and recombinant DNA. These methods are described in the following publications: see, for example, Sambrook, et al. Molecular Cloning: A Laboratory Manual, 2nd Edition (1989); FMAusubel, et al. eds. Current Protocols in Molecular Biology, (1987); the series Methods IN Enzymology (Academic Press, Inc.); M. MacPherson, et al. PCR: A Practical Approach, IRL Press at Oxford University Press (1991); MacPherson et al., eds. PCR 2: Practical Approach, (1995); Harlow and Lane, eds. Antibodies, A Laboratory Manual, (1988); and RIFreshney, ed. Animal Cell Culture (1987).

[0268] As will be understood by those skilled in the art, each embodiment disclosed herein includes, constitutes, or may constitute the specific element, process, component, or ingredient described herein. “Include” or “including” should be interpreted as enumerating “comprise,” “consist of,” or “essentially of.” The transitional terms “comprise” or “comprises” mean, have, or enable, the inclusion of, but not limited to, non-specific elements, processes, components, or ingredients, even in a major quantity. The transitional phrase “consising of” excludes any unspecified element, process, component, or ingredient. The transitional phrase “consisting essentially of” limits the scope of the embodiment to specific elements, processes, components, or ingredients, and does not substantially affect the embodiment. The material effects will result in a statistically significant reduction in immune cell function, as described herein.

[0269] Unless otherwise indicated, all numbers used in this specification and the claims to express quantities, properties, etc., of the components, such as molecular weight and reaction conditions, should be understood in all instances to be modified by "about". Therefore, unless otherwise indicated, the numerical parameters expressed in this specification and the appended claims are approximations that may vary depending on the desired properties to be obtained by the invention. At the very least, each numerical parameter should be interpreted by applying the usual rounding technique, at least in light of the reported number of significant figures, not as an attempt to limit the application of the doctrine of equivalents to the claims. Where further clarity is required, “approximately” when used in conjunction with a stated number or range has a meaning reasonably attributable to those skilled in the art, namely, indicating something somewhat more or somewhat less than the stated value or range, within the range of ±20%; ±19%; ±18%; ±17%; ±16%; ±15%; ±14%; ±13%; ±12%; ±11%; ±10%; ±9%; ±8%; ±7%; ±6%; ±5%; ±4%; ±3%; ±2%; or ±1% of the stated value.

[0270] Although the numerical ranges and parameters representing the broad scope of the present invention are approximations, the numerical values ​​shown in specific embodiments are reported as accurately as possible. Any numerical value, however, inherently includes a certain error that inevitably arises from the standard deviation found in each of its test measurements.

[0271] The grouping of alternative elements or embodiments of the Invention disclosed herein should not be construed as limiting. Each group member may be referenced and claimed individually or in any combination with other members or elements of the group found herein. One or more members of the group may be included in or removed from the group for convenience and / or patentability reasons. In the event of any such inclusion or removal, this specification shall be deemed to include the group as modified and thus satisfy the written description of all Markush groups used in the appended claims.

[0272] Certain embodiments of the present invention are described herein and include the best modes known to the inventors for carrying out the invention. Of course, variations relating to these described embodiments will be apparent to those skilled in the art when reading the preceding description. The inventors expect that those skilled in the art will use such variations where appropriate, and the inventors intend that the invention will be practiced in ways other than those specifically described herein. Accordingly, the invention includes all modifications and equivalents of the subject matter enumerated in the claims appended herein, as permitted by applicable law. Furthermore, any combination of the elements described above in all possible variations is encompassed by the invention unless otherwise shown herein or otherwise clearly contradicts the context.

[0273] Furthermore, numerous references have been made throughout this specification to patents, printed publications, scholarly articles, and other written texts (reference materials within this specification). Each of the referenced materials is individually incorporated into this specification by reference in whole for the purpose of the teachings they refer to.

[0274] In conclusion, the embodiments of the present invention disclosed herein should be understood as illustrative examples of the principles of the present invention. Other modifications that may be used are within the scope of the invention. Thus, as an example, but not limiting, alternative configurations of the present invention may be used in accordance with the teachings herein. Therefore, the present invention is not limited to those precisely shown and described herein.

[0275] The details provided herein are, for example, for illustrative purposes of an illustrative consideration of preferred embodiments of the invention and are presented in order to provide what is considered to be the most useful and readily understandable explanation of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt has been made to describe the structural details of the invention in more detail than is necessary for a basic understanding of the invention, and the explanations taken with reference to the drawings and / or examples will be made clear to those skilled in the art how some forms of the invention can be embodied in practice.

[0276] The definitions and descriptions used in this disclosure are intended to be controlled in any future structure unless explicitly and expressly modified in the examples, or in any case where the application of the meaning would render any structure meaningless or essentially meaningless. Where the construction of a term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, Third Edition, or a dictionary known to those skilled in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Eds. Attwood T et al., Oxford University Press, Oxford, 2006).

[0277] In general, the terms used in the following claims should not be construed as limiting the claims to specific embodiments disclosed herein and herein, but rather as including all possible embodiments along with the full scope of the equivalents for which rights are granted. Therefore, the claims are not limited by this disclosure.

Claims

1. A method for improving the function of adoptive cell therapy (ACT), comprising transducing immune effector cells or a population of immune effector cells using a polynucleotide encoding mutant interleukin 15 (IL-15) and a recombinant receptor, wherein the sequence encoding the mutant IL-15 is under the regulatory control of a promoter having the sequence shown in SEQ ID NO:

15.

2. A method for improving the persistence (or function) of adoptive cell therapy (ACT), comprising transducing immune effector cells or a population of immune effector cells using a polynucleotide encoding an exogenous mutant IL-15 polypeptide, wherein the exogenous mutant IL-15 polypeptide binds to an IL-15 receptor complex containing IL-15Rα compared to an IL-15 receptor complex without IL-15Rα.

3. The method according to claim 2, wherein the exogenous mutant IL-15 polypeptide has a lower affinity for a complex containing IL2Rβ and the common gamma receptor without IL-15Rα compared to IL2Rβ and the common gamma receptor with IL-15Rα.

4. The artificial expression construct according to claim 2, wherein the exogenous mutant IL-15 polypeptide has a lower affinity for the common gamma receptor and / or atypical binding to IL2Rβ compared to wild-type IL-15.

5. The method according to claim 2, wherein the mutant IL-15 is unable to bind to the common gamma receptor.

6. The method according to claim 2, wherein the mutant IL-15 polypeptide contains a D-to-S mutation at position 8 compared to wild-type IL-15.

7. The method according to claim 6, wherein the wild-type IL-15 includes the sequence shown in SEQ ID NO: 8 or SEQ ID NO:

41.

8. The method according to claim 6, wherein the wild-type IL-15 is coded by the sequence shown in sequence number 10 or sequence number 43.

9. The method according to claim 2, wherein the mutant IL-15 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 42; or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 9 or SEQ ID NO:

42.

10. The method according to claim 2, wherein the mutant IL-15 polypeptide is encoded by the sequence shown in SEQ ID NO: 11 or SEQ ID NO: 44; or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 11 or SEQ ID NO:

44.

11. The method according to claim 2, wherein the method results in increased durability and improved functionality.

12. The method according to claim 11, wherein the persistence is increased compared to immunoeffector cells or a population of immunoeffector cells transduced using exogenous wild-type IL-15 polypeptide.

13. The method according to claim 11, wherein the improved function includes reduced antigen-independent IFNγ release compared to an immune effector cell or population of immune effector cells containing a polynucleotide encoding an exogenous wild-type IL-15 polypeptide.

14. The method according to claim 11, wherein the improved function includes increased immune cell proliferation compared to immune effector cells containing a polynucleotide encoding an exogenous wild-type IL-15 polypeptide.

15. The method according to claim 11, wherein the immune effector cells or population of immune effector cells include recombinant receptors or exogenous lymphocyte receptors.

16. The method according to claim 15, wherein the recombinant receptor comprises a chimeric antigen receptor (CAR), an engineered T cell receptor (eTCR), a dimerizing agent-regulated immunoreceptor complex (DARIC), or a hybrid thereof.

17. The method according to claim 15, wherein the exogenous lymphocyte receptor includes a T cell receptor (TCR) or a B cell receptor (BCR).

18. The method according to claim 15, wherein the recombinant receptor binds to a target antigen.

19. The method according to claim 18, wherein the target antigen includes CD33, CLL1, CD19, CD20, CD22, EGFR, EphA2, Her2, IL13Ra2, ROR1, CD133, mesothelin, CD123, or L1-CAM.

20. The method according to claim 16, wherein the DARIC comprises a signal transduction component comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a sequence having at least 95%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2.

21. The method according to claim 16, wherein the DARIC comprises a targeted component comprising the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4; or a sequence having at least 95%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 3 or SEQ ID NO:

4.

22. The method according to claim 16, wherein DARIC comprises the amino acid sequence shown in SEQ ID NO: 5; or a sequence having at least 95%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:

5.

23. The recombinant receptor, (a) a signaling component comprising (i) a first multimerization domain, (ii) a first linker, and (iii) an intracellular component; and The method according to claim 16, comprising (b) a targeting component including (i) a binding domain, (ii) a second linker, (iii) a second polymerizing domain, and (iv) a transmembrane domain.

24. The recombinant receptor, (a) a signaling component comprising (i) a first polymerizing domain containing an FRB polypeptide or FKBP polypeptide, (ii) a first linker, and (iii) CD3ε; and The method according to claim 16, comprising (b) a targeting component including (i) an anti-CLL1 binding domain, (ii) an anti-CD33 binding domain, (iii) a second linker, (iv) a second polymerizing domain comprising an FRB polypeptide or FKBP, (v) a spacer, and (vi) a transmembrane domain.

25. The recombinant receptor, (a) a signaling component comprising (i) a first polymerizing domain containing an FRB polypeptide or FKBP polypeptide, (ii) a first linker, and (iii) CD3ε; and The method according to claim 16, comprising (b) a targeting component comprising (i) an anti-CLL1 binding domain, (ii) an anti-CD33 binding domain, (iii) a second linker, (iv) a second polymerization domain comprising an FRB polypeptide or an FKBP polypeptide, (v) a CD4 hinge region, (vi) a CD4 transmembrane domain, and (vii) a cleaved CD4 intracellular polypeptide.

26. The method according to claim 23, wherein the targeted component does not include a functional intracellular domain or a co-stimulatory domain having signal transduction capabilities.

27. The method according to claim 25, wherein the CD4 hinge region includes an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in Sequence ID No.

84.

28. The method according to claim 25, wherein the CD4 hinge region includes the amino acid sequence shown in SEQ ID NO:

84.

29. The method according to claim 24, wherein the CD3ε comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in Sequence ID No.

85.

30. The method according to claim 24, wherein the CD3ε comprises the amino acid sequence shown in SEQ ID NO:

85.

31. The method according to claim 24, wherein the CD3ε comprises both the extracellular and intracellular portions of the CD3ε.

32. The method according to claim 24, wherein the FRB polypeptide and FKBP polypeptide are localized extracellularly when the signaling component and targeting component are expressed.

33. The method according to claim 23, wherein the first polymerizing domain and the second polymerizing domain are different.

34. The method according to claim 23, wherein the first polymerizing domain comprises an FRB polypeptide, and the second polymerizing domain comprises an FKBP polypeptide.

35. The method according to claim 23, wherein the first polymerizing domain comprises an FKBP polypeptide, and the second polymerizing domain comprises an FRB polypeptide.

36. The method according to claim 24, wherein the FRB polypeptide comprises the FRB T2098L variant.

37. The method according to claim 24, wherein the FRB polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 86, or comprising the sequence shown in SEQ ID NO:

86.

38. The method according to claim 24, wherein the FRB polypeptide has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 87, or comprises an amino acid sequence that includes the sequence shown in SEQ ID NO:

87.

39. The method according to claim 24, wherein the FKBP polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 88, or comprising the sequence shown in SEQ ID NO:

88.

40. The method according to claim 24, wherein the FKBP polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 89, or including the sequence shown in SEQ ID NO:

89.

41. The method according to claim 23, wherein the first and second polymerizing domains associate with a drug.

42. The method according to claim 41, wherein the drug comprises rapamycin or its rapalog.

43. The method according to claim 41, wherein the drug is AP1903, AP20187, AP21967 (also known as C16-(S)-7-methylindolerapamycin), everolimus, novolimus, pimecrolimus, ridafololimus, sirolimus, tacrolimus, temsirolimus, umilolimus, zotarolimus, or BPC015.

44. The method according to claim 23, wherein the first linker is a linker having a length of 2 to 40 amino acids.

45. The method according to claim 44, wherein the first linker is selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, G4S, 2xG4S, 3xG4S, 4xG4S, 5xG4S, and any combination thereof.

46. The method according to claim 44, wherein the first linker is a 3xG4S linker.

47. The method according to claim 23, wherein the second linker is a linker having a length of 2 to 40 amino acids.

48. The present invention according to claim 47, wherein the second linker is selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, G4S, 2xG4S, 3xG4S, 4xG4S, and any combination thereof.

49. The method according to claim 47, wherein the second linker is a G4S linker.

50. The method according to claim 25, wherein the CD4 transmembrane domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in Sequence ID No.

90.

51. The method according to claim 25, wherein the CD4 transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:

90.

52. The method according to claim 25, wherein the cleaved intracellular CD4 polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 91 or SEQ ID NO:

92.

53. The method according to claim 25, wherein the cleaved intracellular CD4 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 91 or SEQ ID NO:

92.

54. The method according to claim 24, wherein the anti-CLL1 binding domain comprises a single-domain antibody (sdAb) or a single-chain variable fragment (scFv).

55. The method according to claim 54, wherein the sdAb is a VHH antibody or an antibody consisting only of the heavy chain (HcAb).

56. The method according to claim 54, wherein the sdAb is a camelid VHH.

57. The method according to claim 54, wherein scFv or sdAb is human or humanized.

58. The method according to claim 54, wherein the anti-CLL1 binding domain comprises a complementarity-determining region (CDR) 1 containing the sequence shown in SEQ ID NO: 97, a CDR 2 containing the sequence shown in SEQ ID NO: 98, and a CDR 3 containing the sequence shown in SEQ ID NO:

99.

59. The method according to claim 54, wherein the anti-CLL1 binding domain comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in Sequence ID No.

100.

60. The method according to claim 54, wherein the anti-CLL1 binding domain includes the sequence shown in Sequence ID No.

100.

61. The method according to claim 24, wherein the anti-CD33 binding domain includes sdAb or scFv.

62. The method according to claim 61, wherein the sdAb is a VHH antibody or an antibody consisting only of the heavy chain (HcAb).

63. The method according to claim 61, wherein the sdAb is a camelid VHH.

64. The method according to claim 61, wherein scFv or sdAb is human or humanized.

65. The method according to claim 61, wherein the anti-CD33 binding domain comprises CDR1 containing the sequence shown in SEQ ID NO: 93, CDR2 containing the sequence shown in SEQ ID NO: 94, and CDR3 containing the sequence shown in SEQ ID NO:

95.

66. The method according to claim 61, wherein the anti-CD33 binding domain comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in Sequence ID No.

96.

67. The method according to claim 61, wherein the anti-CD33 binding domain includes the sequence shown in SEQ ID NO:

96.

68. The method according to claim 23, wherein the signal transduction component further comprises a signal sequence.

69. The method according to claim 68, wherein the signal sequence is a CD8 signal sequence.

70. The method according to claim 69, wherein the CD8 signal sequence includes an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:

101.

71. The method according to claim 69, wherein the CD8 signal sequence includes the amino acid sequence shown in SEQ ID NO:

101.

72. The method according to claim 23, wherein the targeting component further comprises a signal sequence.

73. The method according to claim 72, wherein the signal sequence is an IgK signal sequence.

74. The method according to claim 73, wherein the IgK signal sequence includes an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:

102.

75. The method according to claim 73, wherein the IgK signal sequence includes the amino acid sequence shown in SEQ ID NO:

102.

76. The method according to claim 23, wherein the signal transduction component includes a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:

103.

77. The method according to claim 23, wherein the signal transduction component includes the sequence shown in SEQ ID NO:

103.

78. The method according to claim 23, wherein the targeted component includes a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:

104.

79. The method according to claim 23, wherein the targeted component includes the sequence shown in SEQ ID NO:

104.

80. The method according to claim 23, wherein the recombinant receptor comprises a fusion polypeptide containing the targeting component and the signal transduction component of the recombinant receptor.

81. The method according to claim 80, wherein the fusion polypeptide comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:

105.

82. The method according to claim 80, wherein the fusion polypeptide comprises the sequence shown in SEQ ID NO:

105.

83. The aforementioned immune effector cells or population of immune effector cells a) T cells, αβ T cells, or γδ T cells; b) CD3+, CD4+, and / or CD8+ cells; c) Cytotoxic T lymphocytes (CTLs), tumor-infiltrating lymphocytes (TILs), or helper T cells; or d) The method according to claim 2, comprising natural killer (NK) cells or natural killer T (NKT) cells.

84. The method according to claim 2, wherein the polynucleotide comprises an exogenous promoter operably linked to the polynucleotide encoding the exogenous mutant IL-15 polypeptide.

85. The method according to claim 84, wherein the exogenous promoter is a constitutive promoter.

86. The aforementioned constitutive promoters include: cytomegalovirus immediate-type gene promoter (CMV); elongation factor 1 alpha promoter (EF1-α); phosphoglycerate kinase-1 promoter (PGK); ubiquitin-C promoter (UBQ-C); cytomegalovirus enhancer / chicken beta-actin promoter (CAG); polyoma enhancer / herpes simplex thymidine kinase promoter (MC1); beta-actin promoter (β-ACT); monkey virus 40 promoter (SV40); myeloproliferative sarcoma virus enhancer The method according to claim 85, selected from the group consisting of a sir, negative regulatory region deletion, dl587rev primer binding site substitution (MND) U3 promoter; mouse mammary tumor virus (MMTV) promoter; human immunodeficiency virus (HIV) long-terminal repeat (LTR) promoter; MoMuLV promoter; avian leukemia virus promoter; Epstein-Barr virus immediate-type promoter; Rous sarcoma virus promoter; actin promoter; myosin promoter; hemoglobin promoter; and creatine kinase promoter.

87. The method according to claim 85, wherein the constitutive promoter includes an MNDU3 promoter or an EF1-α promoter.

88. The method according to claim 87, wherein the EF1-α promoter includes the first intron of the human EF1-α gene.

89. The method according to claim 87, wherein the EF1-α promoter lacks the first intron of the human EF1-α gene.

90. The method according to claim 85, wherein the constitutive promoter includes a nucleotide sequence shown in any of SEQ ID NOs: 12, 13, or 14.

91. The method according to claim 84, wherein the exogenous promoter is an inducible promoter.

92. The method according to claim 91, wherein the inducible promoter is an iSynPro promoter.

93. The method according to claim 92, wherein the iSynPro promoter includes the sequence shown in SEQ ID NO: 15, or a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO:

15.

94. The method according to claim 92, wherein the iSynPro promoter includes a minimal promoter operably linked to a sequence having at least 95% sequence identity with the sequence shown in any of SEQ ID NOs: 45 to 83.

95. The method according to claim 92, wherein the iSynPro promoter includes a minimal promoter operably coupled to the sequence shown in any of SEQ ID NOs: 45 to 83.

96. An artificial expression construct comprising a sequence encoding mutant interleukin 15 (IL-15), wherein the sequence encoding mutant IL-15 is under the regulatory control of a promoter comprising: i) a minimal promoter operably linked to a sequence having at least 95% sequence identity with the sequence shown in any of SEQ ID NOs: 45 to 83; or ii) a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO:

15.

97. The artificial expression construct according to claim 96, wherein the promoter has at least 98% sequence identity with the sequence shown in Sequence ID No.

15.

98. The artificial expression construct according to claim 96, wherein the promoter has at least 99% sequence identity with the sequence shown in Sequence ID No.

15.

99. The artificial expression construct according to claim 96, wherein the promoter has the sequence shown in Sequence ID No.

15.

100. The artificial expression construct according to claim 96, comprising the minimal promoter operably linked to a sequence having at least 98% or at least 99% sequence identity with the sequence shown in any of SEQ ID NOs: 45 to 83.

101. The artificial expression construct according to claim 96, wherein the promoter comprises the minimal promoter operably linked to the sequence shown in any of sequence numbers 45 to 83.

102. The artificial expression construct according to claim 96, wherein the minimum promoter is the IL2 minimum promoter.

103. The artificial expression construct according to claim 102, wherein the IL2 minimum promoter includes the sequence shown in SEQ ID NO: 107, or a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO:

107.

104. The artificial expression construct according to claim 96, wherein the mutant IL-15 binds to an IL-15 receptor complex containing IL-15Rα.

105. The artificial expression construct according to claim 96, wherein the mutant IL-15 has a lower affinity for a complex containing IL2Rβ and the common gamma receptor without IL-15Rα compared to IL2Rβ and the common gamma receptor with IL-15Rα.

106. The artificial expression construct according to claim 96, having lower affinity to the common gamma receptor and / or atypical binding to IL2Rβ compared to wild-type IL-15.

107. The artificial expression construct according to claim 96, wherein the mutant IL-15 is unable to bind to the common gamma receptor.

108. The artificial expression construct according to claim 96, wherein the mutant IL-15 contains a D to S mutation at position 8, compared to wild-type IL-15.

109. The artificial expression construct according to claim 108, wherein the wild-type IL-15 comprises the sequence shown in SEQ ID NO: 8 or SEQ ID NO:

41.

110. The artificial expression construct according to claim 108, wherein the wild-type IL-15 is encoded by the sequence shown in sequence number 10 or sequence number 43.

111. The artificial expression construct according to claim 96, wherein the mutant IL-15 comprises the sequence shown in SEQ ID NO: 9 or SEQ ID NO: 42; or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the sequence shown in SEQ ID NO: 9 or SEQ ID NO:

42.

112. The artificial expression construct according to claim 96, wherein the mutant IL-15 is encoded by the sequence shown in SEQ ID NO: 11 or SEQ ID NO: 44; or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the sequence shown in SEQ ID NO: 11 or SEQ ID NO:

44.

113. The artificial expression construct according to claim 96, wherein the artificial expression construct comprises a sequence having at least 95% sequence identity with the sequence shown in Sequence ID No. 19 (iSynPro.IL15.D8S.Fwd).

114. The artificial expression construct according to claim 96, wherein the artificial expression construct comprises a sequence having at least 98% sequence identity with the sequence shown in Sequence ID No. 19 (iSynPro.IL15.D8S.Fwd).

115. The artificial expression construct according to claim 96, wherein the artificial expression construct comprises a sequence having at least 99% sequence identity with the sequence shown in Sequence ID No. 19 (iSynPro.IL15.D8S.Fwd).

116. The artificial expression construct according to claim 96, wherein the artificial expression construct includes the sequence shown in Sequence ID No. 19 (iSynPro.IL15.D8S.Fwd).

117. The artificial expression construct according to claim 96, further comprising a sequence encoding a recombinant receptor or an exogenous lymphocyte receptor, wherein the recombinant receptor or exogenous lymphocyte receptor comprises a binding domain that binds to an antigen expressed on the surface of a target cell.

118. The artificial expression construct according to claim 117, wherein the recombinant receptor comprises a chimeric antigen receptor (CAR), an engineered TCR (eTCR), a dimerizing agent-regulated immunoreceptor complex (DARIC), or a hybrid thereof.

119. The artificial expression construct according to claim 117, wherein the exogenous lymphocyte receptor comprises a T cell receptor (TCR) or a B cell receptor (BCR).

120. The artificial expression construct according to claim 117, wherein the sequence encoding the recombinant receptor or exogenous lymphocyte receptor is operably linked to a second promoter.

121. The aforementioned second promoter is the cytomegalovirus immediate-type gene promoter (CMV); elongation factor 1 alpha promoter (EF1-α); phosphoglycerate kinase-1 promoter (PGK); ubiquitin-C promoter (UBQ-C); cytomegalovirus enhancer / chicken beta-actin promoter (CAG); polyoma enhancer / herpes simplex thymidine kinase promoter (MC1); beta-actin promoter (β-ACT); simian virus 40 promoter (SV40); myeloproliferative sarcoma virus enhancer, An artificial expression construct according to claim 120, selected from the group consisting of a negative regulatory region deletion, dl587rev primer binding site substitution (MND) U3 promoter; mouse mammary tumor virus (MMTV) promoter; human immunodeficiency virus (HIV) long-terminal repeat (LTR) promoter; MoMuLV promoter; avian leukemia virus promoter; Epstein-Barr virus immediate-type promoter; Rous sarcoma virus promoter; actin promoter; myosin promoter; hemoglobin promoter; and creatine kinase promoter.

122. The artificial expression construct according to claim 120, wherein the second promoter comprises an MNDU3 promoter or an EF1-α promoter.

123. The artificial expression construct according to claim 121, wherein the EF1-α promoter includes the first intron of the human EF1-α gene.

124. The artificial expression construct according to claim 121, wherein the EF1-α promoter lacks the first intron of the human EF1-α gene.

125. The artificial expression construct according to claim 117, wherein the binding domain is part of an extracellular component.

126. The artificial expression construct according to claim 117, wherein the binding domain comprises an anti-CD33 binding domain or an anti-CLL1 binding domain.

127. The artificial expression construct according to claim 117, wherein the target cells include cancer cells, or cells infected with bacteria, viruses, fungi, parasites, or arthropods.

128. The artificial expression construct according to claim 117, further comprising intracellular components of the recombinant receptor.

129. The artificial expression construct according to claim 128, wherein the intracellular component comprises a CD3ζ signaling domain and / or a 4-1BB signaling domain.

130. The artificial expression construct according to claim 128, wherein the intracellular component is linked to the extracellular component via a transmembrane domain.

131. The artificial expression construct according to claim 130, wherein the transmembrane domain includes a CD8α transmembrane domain, a CD4 transmembrane domain, or a CD28 transmembrane domain.

132. The artificial expression construct according to claim 117, wherein the recombinant receptor further comprises a multimerization domain.

133. The artificial expression construct according to claim 117, wherein the recombinant receptor is polymerized upon administration of a drug.

134. The artificial expression construct according to claim 133, wherein the drug comprises rapamycin or its rapalog.

135. The artificial expression construct according to claim 133, wherein the drug is AP1903, AP20187, AP21967 (also known as C16-(S)-7-methylindolerapamycin), everolimus, novolimus, pimecrolimus, ridafololimus, sirolimus, tacrolimus, temsirolimus, umilolimus, zotarolimus, or BPC015.

136. The artificial expression construct according to claim 132, wherein the polymerizing domain comprises an FK506-binding protein (FKBP) polymerizing domain or a variant thereof, and an FKBP-rapamycin-binding (FRB) polymerizing domain or a variant thereof.

137. The artificial expression construct according to claim 118, wherein the DARIC comprises a signaling component comprising the amino acid sequence shown in SEQ ID NO: 1 or 2, or a sequence having at least 95%, at least 98%, or at least 99% sequence identity with the sequence shown in SEQ ID NO: 1 or 2.

138. The artificial expression construct according to claim 118, comprising a targeted component wherein DARIC comprises the amino acid sequence shown in SEQ ID NO: 3 or 4; or a sequence having at least 95%, at least 98%, or at least 99% sequence identity with the sequence shown in SEQ ID NO: 3 or 4.

139. The artificial expression construct according to claim 118, wherein DARIC comprises the amino acid sequence shown in SEQ ID NO: 5; or a sequence having at least 95%, at least 98%, or at least 99% sequence identity with the sequence shown in SEQ ID NO:

5.

140. The recombinant receptor, (a) a signaling component comprising (i) a first multimerization domain, (ii) a first linker, and (iii) an intracellular component; and (b) The artificial expression construct according to claim 118, comprising a targeting component including (i) a binding domain, (ii) a second linker, (iii) a second polymerizing domain, and (iv) a transmembrane domain.

141. The recombinant receptor, (a) a signaling component comprising (i) a first polymerizing domain containing an FRB polypeptide or FKBP polypeptide, (ii) a first linker, and (iii) CD3ε; and (b) The artificial expression construct according to claim 118, comprising a targeted component including (i) an anti-CLL1 binding domain, (ii) an anti-CD33 binding domain, (iii) a second linker, (iv) a second polymerizing domain comprising an FRB polypeptide or an FKBP polypeptide, (v) a spacer, and (vi) a transmembrane domain.

142. The recombinant receptor, (a) a signaling component comprising (i) a first polymerizing domain containing an FRB polypeptide or FKBP polypeptide, (ii) a first linker, and (iii) CD3ε; and (b) an artificial expression construct according to claim 118, comprising a targeted component including (i) an anti-CLL1 binding domain, (ii) an anti-CD33 binding domain, (iii) a second linker, (iv) a second polymerization domain comprising an FRB polypeptide or an FKBP polypeptide, (v) a spacer comprising a CD4 hinge region, (vi) a CD4 transmembrane domain, and (vii) a cleaved CD4 intracellular polypeptide.

143. The artificial expression construct according to claim 140, wherein the targeted component does not include a functional intracellular domain or a co-stimulatory domain having signal transduction capability.

144. The artificial expression construct according to claim 142, wherein the CD4 hinge region comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in Sequence ID No.

84.

145. The artificial expression construct according to claim 142, wherein the CD4 hinge region comprises the amino acid sequence shown in Sequence ID No.

84.

146. The artificial expression construct according to claim 141, wherein the CD3ε comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in Sequence ID No.

85.

147. The artificial expression construct according to claim 141, wherein the CD3ε comprises the amino acid sequence shown in Sequence ID No.

85.

148. The artificial expression construct according to claim 141, wherein the CD3ε comprises both the extracellular and intracellular portions of CD3ε.

149. The artificial expression construct according to claim 141, wherein the FRB polypeptide and FKBP polypeptide are localized extracellularly when the signaling component and the targeting component are expressed.

150. The artificial expression construct according to claim 140, wherein the first and second polymerizing domains are different.

151. The artificial expression construct according to claim 140, wherein the first polymerizing domain comprises an FRB polypeptide, and the second polymerizing domain comprises an FKBP polypeptide.

152. The artificial expression construct according to claim 140, wherein the first polymerizing domain comprises an FKBP polypeptide, and the second polymerizing domain comprises an FRB polypeptide.

153. The artificial expression construct according to claim 141, wherein the FRB polypeptide comprises the FRB T2098L variant.

154. The artificial expression construct according to claim 141, wherein the FRB polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 86, or comprising the sequence shown in SEQ ID NO:

86.

155. The artificial expression construct according to claim 141, wherein the FRB polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 87, or including the sequence shown in SEQ ID NO:

87.

156. The artificial expression construct according to claim 141, wherein the FKBP polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 88, or comprising the sequence shown in SEQ ID NO:

88.

157. The artificial expression construct according to claim 141, wherein the FKBP polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 89, or including the sequence shown in SEQ ID NO:

89.

158. The artificial expression construct according to claim 140, wherein the first polymerizing domain and the second polymerizing domain associate with a drug.

159. The artificial expression construct according to claim 158, wherein the drug comprises rapamycin or its rapalog.

160. The artificial expression construct according to claim 158, wherein the drug is AP1903, AP20187, AP21967 (also known as C16-(S)-7-methylindolerapamycin), everolimus, novolimus, pimecrolimus, ridafololimus, sirolimus, tacrolimus, temsirolimus, umilolimus, zotarolimus, or BPC015.

161. The artificial expression construct according to claim 140, wherein the first linker is a linker having a length of 2 to 40 amino acids.

162. The artificial expression construct according to claim 161, wherein the first linker is selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, G4S, 2xG4S, 3xG4S, 4xG4S, 5xG4S, and any combination thereof.

163. The artificial expression construct according to claim 161, wherein the first linker is a 3xG4S linker.

164. The artificial expression construct according to claim 140, wherein the second linker is a linker having a length of 2 to 40 amino acids.

165. The artificial expression construct according to claim 164, wherein the second linker is selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, G4S, 2xG4S, 3xG4S, 4xG4S, and any combination thereof.

166. The artificial expression construct according to claim 164, wherein the second linker is a G4S linker.

167. The artificial expression construct according to claim 142, wherein the CD4 transmembrane domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in Sequence ID No.

90.

168. The artificial expression construct according to claim 142, wherein the CD4 transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:

90.

169. The artificial expression construct according to claim 142, wherein the cleaved intracellular CD4 polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 91 or SEQ ID NO:

92.

170. The artificial expression construct according to claim 142, wherein the cleaved intracellular CD4 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 91 or SEQ ID NO:

92.

171. The artificial expression construct according to claim 141, wherein the anti-CLL1 binding domain comprises a single-domain antibody (sdAb) or a single-chain variable fragment (scFv).

172. The artificial expression construct according to claim 171, wherein the sdAb is a VHH or heavy chain-only antibody (HcAb).

173. The artificial expression construct according to claim 171, wherein the sdAb is a camelid VHH.

174. The artificial expression construct according to claim 171, wherein the scFv or sdAb is human or humanized.

175. The artificial expression construct according to claim 141, wherein the anti-CLL1 binding domain comprises a complementarity-determining region (CDR) 1 containing the sequence shown in SEQ ID NO: 97, a CDR 2 containing the sequence shown in SEQ ID NO: 98, and a CDR 3 containing the sequence shown in SEQ ID NO:

99.

176. The artificial expression construct according to claim 141, wherein the anti-CLL1 binding domain comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:

100.

177. The artificial expression construct according to claim 141, wherein the anti-CLL1 binding domain comprises the sequence shown in Sequence ID No.

100.

178. The artificial expression construct according to claim 141, wherein the anti-CD33 binding domain comprises sdAb or scFv.

179. The artificial expression construct according to claim 178, wherein the sdAb is a VHH or a heavy chain-only antibody (HcAb).

180. The artificial expression construct according to claim 178, wherein the sdAb is a camelid VHH.

181. The artificial expression construct according to claim 178, wherein the scFv or sdAb is human or humanized.

182. The artificial expression construct according to claim 141, wherein the anti-CD33 binding domain comprises CDR1 containing the sequence shown in SEQ ID NO: 93, CDR2 containing the sequence shown in SEQ ID NO: 94, and CDR3 containing the sequence shown in SEQ ID NO:

95.

183. The artificial expression construct according to claim 141, wherein the anti-CD33 binding domain comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in Sequence ID No.

96.

184. The artificial expression construct according to claim 141, wherein the anti-CD33 binding domain comprises the sequence shown in Sequence ID No.

96.

185. The artificial expression construct according to claim 140, wherein the signal transduction component further comprises a signal sequence.

186. The artificial expression construct according to claim 185, wherein the signal sequence is a CD8 signal sequence.

187. The artificial expression construct according to claim 186, wherein the CD8 signal sequence comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in Sequence ID No.

101.

188. The artificial expression construct according to claim 186, wherein the CD8 signal sequence includes the amino acid sequence shown in SEQ ID NO:

101.

189. The artificial expression construct according to claim 140, wherein the targeted component further comprises a signal sequence.

190. The artificial expression construct according to claim 189, wherein the signal sequence is an IgK signal sequence.

191. The artificial expression construct according to claim 190, wherein the IgK signal sequence comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:

102.

192. The artificial expression construct according to claim 190, wherein the IgK signal sequence includes the amino acid sequence shown in SEQ ID NO:

102.

193. The artificial expression construct according to claim 140, wherein the signal transduction component comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:

103.

194. The artificial expression construct according to claim 140, wherein the signal transduction component includes the sequence shown in SEQ ID NO:

103.

195. The artificial expression construct according to claim 140, wherein the targeted component comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in Sequence ID No.

104.

196. The artificial expression construct according to claim 140, wherein the targeted component comprises the sequence shown in Sequence ID No.

104.

197. The artificial expression construct according to claim 117, wherein the recombinant receptor comprises a fusion polypeptide containing an eTCR targeting component and a signaling component.

198. The artificial expression construct according to claim 197, wherein the fusion polypeptide comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:

105.

199. The artificial expression construct according to claim 197, wherein the fusion polypeptide comprises the sequence shown in SEQ ID NO:

105.

200. The artificial expression construct according to claim 96, further comprising a first control element.

201. The artificial expression construct according to claim 200, wherein the first control element includes or encodes a transduction marker, a selection cassette, or a suicide gene.

202. The artificial expression construct according to claim 201, wherein the transduction marker comprises cleaved HER2 protein (Her2tG), epidermal growth factor receptor (EGFRt), or cleaved CD19 (tCD19).

203. The artificial expression construct according to claim 201, wherein the selection cassette comprises a dihydrofolate reductase double mutant (DHFRdm).

204. The artificial expression construct according to claim 96, further comprising a first skip sequence.

205. The artificial expression construct according to claim 204, wherein the first skip sequence is located between the sequence encoding the mutant IL-15 and the first control element.

206. The artificial expression construct according to claim 204, wherein the first skip sequence encodes a 2A self-cleaving polypeptide.

207. The artificial expression construct according to claim 206, wherein the 2A self-cleaving polypeptide comprises T2A, P2A, E2A, or F2A.

208. The artificial expression construct according to claim 200, further comprising a second control element.

209. The artificial expression construct according to claim 204, further comprising a second skip sequence.

210. The artificial expression construct according to claim 209, wherein the second skip sequence is located between the mutant IL-15 and the recombinant receptor.

211. The artificial expression construct according to claim 209, wherein the second skip sequence encodes a 2A self-cleaving polypeptide.

212. The artificial expression construct according to claim 211, wherein the 2A skip self-cleaving polypeptide comprises T2A, P2A, E2A, or F2A.

213. A method comprising transducing an immune effector cell or a population of immune effector cells using the artificial expression construct described in claim 96.

214. Nanoparticles for encapsulating the artificial expression construct described in claim 96.

215. Non-natural cells or population thereof, comprising the artificial expression construct described in claim 96.

216. The non-natural cells or group thereof according to claim 215, wherein the cells or group thereof include self-cells or allogeneic cells with respect to the target.

217. The non-natural cells or population thereof according to claim 215, comprising in vivo or ex vivo cells or population thereof.

218. The non-natural cells or population thereof according to claim 215, wherein the cells or population thereof include immune cells.

219. The non-natural cells or population thereof according to claim 218, wherein the immune cells are lymphocytes.

220. The non-natural cells or population thereof according to claim 219, wherein the lymphocytes include T cells, B cells, natural killer (NK) cells, or NK-T cells.

221. The non-native cells or population thereof according to claim 215, wherein the cells or population thereof include T cells selected from CD3+ T cells, CD4+ T cells, CD8+ T cells, central memory T cells, effector memory T cells, and / or naive T cells.

222. The non-native cells or population thereof according to claim 215, wherein the cells or population thereof include CD8+ T cells.

223. The non-native cells or population thereof according to claim 215, wherein the cells or population thereof include CD4+ T cells.

224. A composition comprising non-natural cells or a population thereof as described in claim 215, and a pharmaceutically acceptable carrier.

225. A method for treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the artificial expression construct according to claim 96, the nanoparticles according to claim 214, the non-natural cells or population thereof according to claim 215, or the composition according to claim 224, thereby treating the subject in need thereof.

226. The method according to claim 225, wherein the subject requiring it has cancer or an infectious disease.

227. The method according to claim 226, wherein the cancer includes hematological malignancies.

228. The method according to claim 227, wherein the hematological malignancy includes leukemia, lymphoma, or multiple myeloma.

229. The method according to claim 228, wherein the leukemia includes acute myeloid leukemia (AML).

230. The method according to claim 226, wherein the cancer includes solid tumors.

231. The method according to claim 230, wherein the solid tumor includes lung cancer, squamous cell carcinoma, colorectal cancer, pancreatic cancer, breast cancer, thyroid cancer, bladder cancer, cervical cancer, esophageal cancer, ovarian cancer, gastric cancer, endometrial cancer, or brain cancer.

232. The method according to claim 231, wherein the lung cancer is non-small cell lung cancer.

233. The method according to claim 217, wherein the brain cancer includes glioma, glioblastoma, or oligodendroglioma.

234. The method according to claim 211, wherein the administration of a therapeutically effective amount includes administration into the bladder, vein, skin, artery, parenchyma, node, lymphatic vessel, abdominal cavity, lesion, prostate, vagina, rectum, local, intrathecal, tumor, muscle, or subcutaneously.

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