Co-stimulatory domain for use in genetically modified cells

Abstract

This invention provides a novel co-stimulatory domain useful in genetically modified cells for promoting cell proliferation and / or promoting cytokine secretion after antigen recognition. [Solution] For example, this specification discloses genetically modified cells comprising a chimeric antigen receptor or an inducible regulatory construct incorporating the costimulatory domain disclosed herein. This specification also discloses plasmids and viral vectors comprising nucleic acid sequences encoding the costimulatory domain, as well as methods for administering compositions comprising the novel costimulatory domain to a target for mitigating the symptoms, progression, or development of diseases such as cancer.

Description

[Technical Field]

[0001] This disclosure relates to the fields of molecular biology and recombinant nucleic acid technology. In particular, this disclosure relates to T cell proliferation. This disclosure relates to a novel co-stimulatory domain engineered to stimulate cell proliferation and avoid T cell depletion. Furthermore, it includes genetically modified cells containing novel costimulatory domains, and the treatment of diseases such as cancer. Regarding the use of cells like this.

[0002] References to sequence listings submitted as text files via EFS-Web This application includes a sequence listing submitted in ASCII format via EFS-Web. The entire text is incorporated herein by reference. Created on October 4, 2017. The above ASCII copy is named P109070017WO00SEQ.txt The size is 124,207 bytes. [Background technology]

[0003] T-cell adoptive immunotherapy is a promising approach for cancer treatment. This strategy is specific Isolated human cells that have been genetically modified to enhance their specificity for tumor-associated antigens. T cells are used. Genetic modification is performed to transfer antigen specificity to T cells, resulting in chimeric antigen receptors. This may include the expression of somatic (CAR) or exogenous T cell receptors. In contrast to exogenous T cell receptors... Therefore, CARs extract their specificity from the variable domains of monoclonal antibodies. Therefore, T cells that express CAR have a non-restrictive mode of major histocompatibility complex (MHC). This induces tumor immune response. To date, T-cell adoptive immunotherapy has been used for B-cell malignancies (for example) For example, acute lymphoblastic leukemia (ALL), B-cell non-Hodgkin lymphoma (NHL), and chronic chronic lymphocytic leukemia), multiple myeloma, neuroblastoma, glioblastoma, progressive glioma, ovarian cancer It has been used in the clinical treatment of many cancers, including mesothelioma, melanoma, and pancreatic cancer.

[0004] T cell activation is initiated by the endogenous T cell receptor, thereby providing the cell with antigen specificity as well. Signal transduction by T cells is amplified by cell surface co-stimulatory receptors such as CD28. Without co-stimulation, stimulation via the T cell receptor is insufficient to promote T cell proliferation and can lead to cell anergy. Co-stimulation also helps to avoid T cell exhaustion and some forms of activation-induced cell death. The initial chimeric T cell activation receptor was generated by fusing the ζ chain of CD3 to the extracellular domains of T cell co-receptors including CD4, CD8, and CD25.

[0005] Next, by fusing the CD3ζ chain to a single-chain variable fragment (scFv), the so-called "first-generation" chimeric antigen receptor was generated, thereby obtaining antigen specificity and antigen-induced T cell activation. The first-generation CAR was able to mediate cytotoxicity but could not direct the expansion of antigen-induced responses in modified primary T cells. In fact, studies in early transgenic mouse models revealed that T cells expressing first-generation CARs had only moderate effects on tumor progression in vivo due to anergy and the production of small amounts of interferon gamma ( IFN-γ). The "second-generation" chimeric antigen receptor was generated by further fusing a single co-stimulatory domain in cis to the cytoplasmic CD3ζ chain. Studies have shown that the addition of co-stimulatory domains allows for the expansion of antigen-induced responses in primary It has been demonstrated to enable the expansion of CAR-T cells and the increased secretion of cytokines such as IFN-γ. In fact, early clinical trials using second-generation CAR-T cells have shown significantly enhanced persistence and expansion in patients suffering from B-cell lymphoma, CLL, and B-cell ALL. A number of costimulatory domains, such as the CD28, 4-1BB, or OX-40 elements, have been introduced into the CAR and have been utilized in CAR-T cells administered to patients with B-cell malignancies. The so-called "third-generation" chimeric antigen receptor has introduced two costimulatory domains together with the cytoplasmic CD3ζ chain, enabling further enhancement of cell expansion and / or cytokine secretion after repeated antigen exposure. In addition to the use of costimulatory domains within the CAR construct, the group has also incorporated costimulatory domains into various "safety switches" that can be separated from the CAR. One such safety switch contains MyD88 and CD40 signaling domains fused to a binding domain that dimerizes when bound by a small molecule such as rimiducid. This safety switch is used together with a CAR construct that contains only the cytoplasmic CD3ζ chain to promote cell activation. By administering the small molecule, the safety switch dimerizes, enabling MyD88 / CD40 costimulatory signaling to promote CAR-T cell expansion and cytokine secretion after antigen recognition by a separate CAR construct. A number of costimulatory domains have been previously disclosed in both patents and the literature. For example, Patent Document 1 discloses both the 4-1BB signaling domain and the CD3ζ signaling domain. have been introduced into the CAR and have been utilized in CAR-T cells administered to patients with B-cell malignancies. The so-called "third-generation" chimeric antigen receptor has introduced two costimulatory domains together with the cytoplasmic CD3ζ chain, enabling further enhancement of cell expansion and / or cytokine secretion after repeated antigen exposure.

[0006] In addition to the use of costimulatory domains within the CAR construct, the group has also incorporated costimulatory domains into various "safety switches" that can be separated from the CAR. One such safety switch contains MyD88 and CD40 signaling domains fused to a binding domain that dimerizes when bound by a small molecule such as rimiducid. This safety switch is used together with a CAR construct that contains only the cytoplasmic CD3ζ chain to promote cell activation. By administering the small molecule, the safety switch dimerizes, enabling MyD88 / CD40 costimulatory signaling to promote CAR-T cell expansion and cytokine secretion after antigen recognition by a separate CAR construct.

[0007] A number of costimulatory domains have been previously disclosed in both patents and the literature. For example, Patent Document 1 discloses both the 4-1BB signaling domain and the CD3ζ signaling domain. ​​​​​​​They are claiming a patent for a polynucleotide encoding a chimeric antigen receptor. However, , the domain of this disclosure, or any domain having 80% sequence identity with respect to the domain of this disclosure No domain names have been disclosed. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] U.S. Patent No. 8,399,645 [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] This disclosure relates to genetics for promoting cell proliferation and / or cytokine secretion. This disclosure provides a novel co-stimulatory domain useful for providing modified offspring cells. Novel co-stimulation that promotes different degrees of cell proliferation and / or cytokine secretion after cell activation We advance technology by providing a highly sensitive domain. For example, the co-stimulation disclosed herein. The domain exhibits superior activity compared to co-stimulatory domains such as 4-1BB and CD28. Provided. Furthermore, this specification incorporates one or more of the co-stimulatory domains disclosed herein. Genetically modified cells containing chimeric antigen receptors (CARs) have also been disclosed. The genetically modified cells disclosed herein are composed of one or more co-stimulatory domains disclosed herein. It includes an inductive modulating construct that is incorporated. Furthermore, in this specification, the co-stimulatory domain is called nucleic acid molecules containing nucleic acid sequences, recombinant DNA constructs (e.g., plasmids), and viruses Lus vectors, and novel co-stimulatory devices for reducing the symptoms, progression, or onset of disease. Methods for administering a composition containing the main component are also provided. In some embodiments, as specified herein Genetically modified cells containing the novel costimulatory domains disclosed may be formulated as pharmaceutical compositions, for example. It is used as an immunotherapy in the treatment of cancer. [Means for solving the problem]

[0010] Therefore, in one embodiment, the present disclosure relates to the amino acid sequence described in any one of SEQ ID NOs: 5 to 8. A costimulatory domain or its active variant having at least 80% sequence identity with respect to The present disclosure provides nucleic acid molecules containing nucleotide sequences encoding fragments. Various aspects of this disclosure Therefore, the active variants or fragments of the co-stimulatory domains disclosed herein are of sequence numbers 5-8. At least 80%, at least 85%, and at least one of the amino acid sequences described in any one of them. 90%, at least 95%, at least 96%, at least 97%, at least 98% or have at least 99% sequence identity.

[0011] In some embodiments, an active mutant or fragment amino acid encoding a costimulatory domain is used. The column contains at least one TRAF binding motif. In certain embodiments, TRAF binding The motif is selected from the group consisting of sequence numbers 9-11. In a particular embodiment, co-stimulation The active mutant or fragment amino acid sequence encoding the domain is separated by a spacer sequence. It contains two TRAF-binding motifs. In some such embodiments, The α sequence is selected from the group consisting of sequence numbers 12 to 15. In certain embodiments, co-stinging The mutant or fragment amino acid sequence encoding the stimuli domain was separated by a spacer sequence. It contains two TRAF binding motifs selected from the group consisting of row numbers 9-11, and a spacer. The sequence is selected from the group consisting of sequence numbers 12 to 15.

[0012] In certain embodiments, the variant or fragment amino acid sequence encoding the co-stimulatory domain is distributed The spacer arrays, such as the spacer array described in column number 12, separated by the spacer arrays, and It contains 11 TRAF-binding motifs. In some embodiments, it encodes a co-stimulatory domain. The resulting mutant or fragment amino acid sequence is the spacer sequence described in any of SEQ ID NOs: 13-15. TRAF-bound motifs of sequence numbers 10 and 11, separated by spacer sequences such as columns. Includes.

[0013] In certain embodiments, the nucleic acid molecule is an amino acid compound as described in any one of SEQ ID NOs. 5 to 8. It contains a nucleotide sequence that codes for a co-stimulatory domain that includes a column.

[0014] In some embodiments, the nucleic acid molecule is the nucleotide described in any one of SEQ ID NOs: 1 to 4. A small number of nucleotide sequences are found in the ocidal sequence or one of the sequence numbers 1-4. It contains variants or fragments with at least 80% identity, and the nucleotide sequence is co-synchronized. It encodes the domain. In some embodiments, the nucleic acid molecule is one of sequence numbers 1 to 4. For any one of the nucleic acid sequences described, at least 80%, at least 85%, and at least 9% 0%, at least 95%, at least 96%, at least 97%, at least 98%, and This is a variant of any one of sequence numbers 1-4 that has at least 99% sequence identity or It contains a nucleotide sequence containing a fragment, and the nucleotide sequence encodes an active costimulatory domain. ru.

[0015] In some embodiments, the nucleic acid molecule encodes a chimeric antigen receptor (CAR). The CAR contains a rheotide sequence and has the amino acid sequence described in any one of SEQ ID NOs. 5-8. In contrast, at least one co-stimulator described herein having at least 80% sequence identity The domain or its active variant or fragment. In some such embodiments, Nucleic acid molecules contain at least one amino acid sequence described in one of SEQ ID NOs. 5-8. It contains a nucleotide sequence encoding a CAR that includes a co-stimulatory domain.

[0016] In other such embodiments, the CAR includes an extracellular antigen-binding domain. Specific implementation In this configuration, the extracellular antigen-binding domain is a single-stranded variable fragment (scFv). Various embodiments In this context, the antigen-binding domain has specificity for cancer or tumor antigens. Specific implementation In this state, the encoded CAR contains a CD19-specific antigen-binding domain.

[0017] In further such embodiments, the coded CAR is at least two co-stimulatory domains Includes n. In such embodiments, at least two co-stimulatory domains are described herein. The co-stimulatory domain described herein, or at least one co-stimulatory domain described herein. and at least one additional co-stimulatory domain known in the field (e.g., 4-1) (BB, CD28, OX40, ICOS). In some embodiments, coded CAR It further comprises at least one intracellular signaling domain. In certain embodiments, it comprises at least one At least one intracellular signaling domain is the CD3ζ domain.

[0018] In other embodiments, the nucleic acid molecule is an amino acid sequence described in any one of SEQ ID NOs: 5-8. For at least one co-stimulatory domain having at least 80% sequence identity or Includes a nucleotide sequence encoding an inducible regulatory construct containing an active mutant or fragment of In some embodiments, the nucleic acid molecule is an amino acid as described in any one of SEQ ID NOs. 5-8. Nuclei encoding an inducible regulatory construct containing at least one co-stimulatory domain including an acid sequence. It contains a rheotide sequence. In certain embodiments, the encoded inductive modulatory construct contains two induces The conductive regulatory construct further includes a binding domain that enables dimerization, and the dimerization is fine It initiates a co-stimulatory signal to the cell. In some embodiments, the binding domain is a small molecule ( For example, it binds to a rimiducid, an antibody, or another molecule that enables dimerization. In a specific embodiment in which the binding domain binds to a small molecule, the binding domain is of the FKBP12 type. It contains analogues (e.g., F36V substitution), and the small molecule is rimiducid (i.e., AP) (1903)

[0019] In certain embodiments, nucleic acid molecules include mRNA, recombinant DNA constructs (e.g., plasmin It is either (a type of virus) or contained within the viral genome of the viral vector.

[0020] In another aspect, the disclosure provides recombinant DNA constructs, which are sequence number At least 80% sequence identity to the amino acid sequence described in any one of numbers 5-8 Nu encoding at least one co-stimulatory domain or an active variant or fragment thereof This specification includes nucleic acid molecules containing a cleotide sequence. In certain embodiments, recombinant D The NA construct contains the amino acid sequence described in any one of SEQ ID NOs: 5-8. It contains a nucleotide sequence that codes for DNA. In some embodiments, recombinant DNA constructs This includes a nucleotide sequence encoding a CAR as described herein, where the CAR is the sequence number. It has at least 80% sequence identity with the amino acid sequence described in any one of 5-8. It comprises at least one co-stimulatory domain or an active variant or fragment thereof. Morphologically, recombinant DNA constructs are based on the amino acid sequence described in one of sequence numbers 5-8. For at least one co-stimulatory domain having at least 80% sequence identity or Nuclei encoding the inducible regulatory construct described herein, which includes an active mutant or fragment of Includes the Otid sequence.

[0021] In some such embodiments, recombinant DNA constructs are represented by SEQ ID NOs. 5-8. A less than 80% sequence identity is found in the amino acid sequence described in one of the following documents. Each nucleotide pair encodes one co-stimulatory domain or its active variant or fragment. It encodes a viral vector containing a column. In some embodiments, the viral vector is retroviral vectors, lentiviral vectors, adenovirus vectors, or A It is a deno-associated virus (AAV) vector. In certain embodiments, the viral vector is This is a recombinant AAV vector.

[0022] In some embodiments, this disclosure refers to the amino acid sequence described in any one of SEQ ID NOs: 5-8. For at least one co-stimulatory domain having at least 80% sequence identity or Nucleic acid components described herein, comprising nucleotide sequences encoding active variants or fragments thereof. A viral vector containing offspring is provided. In a particular embodiment, the viral vector is sequence Nuclei encoding a co-stimulatory domain containing the amino acid sequence described in one of numbers 5-8 It contains a rheotide sequence. In some embodiments, the viral vector is C as described herein. The CAR contains a nucleotide sequence encoding AR, and one of the sequence numbers 5-8 At least one co-amino acid having at least 80% sequence identity with the described amino acid sequence The stimulating domain or its active variant or fragment is included. In other embodiments, the viral domain The ter is at least 80% of the amino acid sequence described in any one of SEQ ID NOs. 5-8. At least one co-stimulatory domain having sequence identity, or its active variant or fragment. The nucleotide sequence comprises a nucleotide sequence encoding an inducible modulo construct described herein, which includes the following:

[0023] In certain embodiments, the viral vector is a retroviral vector, lentiviral vector It is a vector, adenovirus vector, or adeno-associated virus (AAV) vector. In certain embodiments, the viral vector is a recombinant AAV vector.

[0024] In another aspect, the present disclosure provides genetically modified cells, which are sequence numbers 5-8. A small number of amino acid sequences that have at least 80% sequence identity with any one of the amino acid sequences described in Nucleotides encoding at least one co-stimulatory domain or its active variant or fragment. This specification includes nucleic acid molecules containing a sequence. In some embodiments, genetically modified molecules The cell comprises an expression cassette containing a nucleotide sequence encoding the CAR described herein. CAR has at least 8 amino acids relative to the amino acid sequence described in any one of SEQ ID NOs. 5-8. At least one co-stimulatory domain or its active variant having 0% sequence identity Includes fragments. In certain embodiments, genetically modified cells are used in the inducible regulatory constructs described herein. The inducible regulatory construct includes an expression cassette containing a nucleotide sequence encoding the sequence number At least 80% sequence identity to the amino acid sequence described in any one of numbers 5-8 It comprises at least one co-stimulatory domain or an active variant or fragment thereof. In such an embodiment, the nucleic acid molecule described herein, or the generated The current cassette is either present within the genome of genetically modified cells or incorporated into the cell genome. Not found. In some such embodiments, the nucleic acid molecules described herein, The expression cassettes described herein are in recombinant DNA constructs, mRNA, or viruses. It exists in genetically modified cells within the genome, but is not integrated into the cell's genome.

[0025] In further embodiments, the genetically modified cell comprises (i) the costimulatory domain described herein. A CAR expression cassette containing a nucleotide sequence encoding a non-CAR, and (ii) sequence At least 80% sequence identity to the amino acid sequence described in any one of numbers 5-8. The present invention comprises at least one co-stimulatory domain or an active variant or fragment thereof having the above It includes a regulatory expression cassette encoding an inducible regulatory construct as described in the details. In the embodiments, the genetically modified cells (i) incorporate the costimulatory domain described herein. (ii) a CAR that is not included and (ii) comprising at least one co-stimulatory domain as described herein It includes an inducible regulatory domain. In some embodiments, the genetically modified cells are (i) Honmei Nucleotide sequences encoding CARs that do not include the costimulatory domain described in the detailed document, and (ii ) Encoding an inductive regulatory domain comprising at least one co-stimulatory domain as described herein It includes an expression cassette containing a nucleotide sequence. In each embodiment, the CAR expression cassette The and / or regulatory expression cassettes are located within the genome of genetically modified eukaryotic cells, or are located within the genome of genetically modified eukaryotic cells. It is not integrated into the cell genome. In some such embodiments, CAR expression Cassettes and / or regulatory expression cassettes are present in recombinant DNA constructs, mRNA, or via It exists in genetically modified eukaryotic cells within the Rus genome, but it is not integrated into the cell's genome.

[0026] In some embodiments, the genetically modified cells described herein are genetically modified eukaryotic cells. Yes. In certain embodiments, the cells are T cells or natural killer (NK) cells. Other In this embodiment, the genetically modified cells are primary human T cells or primary human NK cells. In this embodiment, the genetically modified cells are human CAR-T cells or human CAR-NK cells.

[0027] In certain embodiments, the genetically modified cells described herein are co-stimulated as described herein. Compared to control cells that do not contain the main component, these cells show increased proliferation and / or cytokine secretion. In some embodiments, increased proliferation and / or cytokine secretion are observed in vitro. and / or shown in vivo. In certain embodiments, increased cytokine secretion is shown as intended. Compared to control cells that do not contain the co-stimulatory domains described in the document, cell activation and / or proliferation were observed. This includes increases in IFN-γ, IL-2, TNF-alpha, or other cytokines related to [the condition]. nothing.

[0028] In another aspect, this disclosure may refer to at least one co-stimulatory domain or its This invention provides a method for producing genetically modified cells containing active mutants or fragments, and this method is sequence number At least 80% sequence identity to the amino acid sequence described in any one of numbers 5-8 This code encodes at least one co-stimulatory domain or an active variant or fragment thereof. This includes introducing at least one nucleic acid molecule described in the specification into a cell.

[0029] In some embodiments of this method, the introduced nucleic acid molecule is one of SEQ ID NOs. 5-8. At least one amino acid sequence having at least 80% sequence identity The CAR described herein, comprising one costimulatory domain or an active variant or fragment thereof Do it.

[0030] In some embodiments of this method, the introduced nucleic acid molecule is one of SEQ ID NOs. 5-8. At least one amino acid sequence having at least 80% sequence identity Inducible regulation according to this specification, comprising one costimulatory domain or an active variant or fragment thereof. Code structures.

[0031] In some embodiments, this method further involves (i) encoding the manipulated nuclease A second nucleic acid molecule in which the manipulated nuclease is expressed in the cell. (ii) introducing the child or a modified nuclease protein into a cell, The manipulated nuclease then recognizes the recognition sequence, cleaves it, and leaves the cleavage site in the cell's genome. It generates and encodes at least one co-stimulatory domain or its active variant or fragment. Nucleic acids are inserted into the genome at the cleavage site. In some embodiments, manipulated nucleic acids Rease is a modified meganuclease, recombinant zinc finger nuclease (ZF N), recombinant transcription activator-like effector nuclease (TALEN), CRISPR / This refers to a specific embodiment of the present method. Therefore, the manipulated nuclease is a manipulated meganuclease. In certain embodiments, Therefore, the manipulated meganuclease is a single-stranded meganuclease.

[0032] In one such embodiment of this method, any one of SEQ ID NOs. 5-8 is introduced into the cells. At least one amino acid sequence having at least 80% sequence identity with the amino acid sequence described above Nucleic acid molecules encoding the co-stimulatory domain or its active variant or fragment are nucleic acid molecules Adjacent to the nuclease cleavage site, so that it can be inserted into the genome at the cleavage site by homologous recombination. The sequence further includes sequences homologous to the sequence. In another such embodiment of the present method, the nucleic acid molecule Nuclease cleavage sites so that nucleic acid molecules can be inserted into the genome by non-homologous end joining. It lacks substantial homology to [the other].

[0033] In some embodiments of this method, the cells are eukaryotic cells. In certain embodiments, the cells This refers to primary human T cells, or T cells such as primary human NK cells, or natural killer (NK) cells. ) These are cells. In certain embodiments, the genetically modified cells produced by this method are HI These are CAR-T cells or human CAR-NK cells.

[0034] In a specific embodiment of this method, the amino acid sequence described in any one of SEQ ID NOs. 5 to 8 is used. In contrast, at least one co-stimulatory domain or its sequence identity having at least 80% sequence identity Nucleic acid molecules encoding active mutants or fragments are mRNAs as described herein. Using the recombinant DNA construct described herein, or the viral vector described herein, intracellularly It will be introduced to [location / platform].

[0035] In some embodiments, the amino acid sequence described in any one of SEQ ID NOs: 5-8 and at least one co-stimulatory domain or its activity having at least 80% sequence identity The introduction of at least one nucleic acid molecule described herein that encodes a variant or fragment is, To increase the activation, proliferation, and / or cytokine secretion of genetically modified cells. Specific implementation Morphologically, increased cytokine secretion is a control that does not contain the co-stimulatory domains described herein. Compared to cells, IFN-γ, IL-2, and T are associated with cell activation and / or proliferation. This includes increased secretion of NF-alpha or any other cytokine.

[0036] In another aspect, the present disclosure relates to a pharmaceutically acceptable carrier and one of Sequence IDs 5-8 At least one amino acid sequence having at least 80% sequence identity with the amino acid sequence described above Genetically modified cells according to this specification, comprising a co-stimulatory domain or its active variant or fragment. The present invention provides a pharmaceutical composition comprising the following: In a particular embodiment, the genetically modified cells are sequence numbers 5- It has at least 80% sequence identity with respect to the amino acid sequence described in any one of the eight. The foregoing includes at least one co-stimulatory domain or an active variant or fragment thereof. Includes CAR. In other embodiments, the genetically modified cell is one of sequence numbers 5-8. At least one amino acid sequence having at least 80% sequence identity with the amino acid sequence described above Inducible regulatory constructs according to this specification, comprising a co-stimulatory domain or its active variant or fragment. Includes objects.

[0037] In certain embodiments, the genetically modified cells are CAR-T cells. In this embodiment, CAR-T cells have an amino acid sequence described in any one of SEQ ID NOs. 5-8. The co-stimulatory domain described herein having at least 80% sequence identity with respect to or The CAR comprises an active mutant or fragment of the CAR- T cells that do not contain the costimulatory domain or its active variant or fragment as described herein. It contains CAR, and furthermore, it contains less than the amino acid sequence described in any one of SEQ ID NOs. 5-8. At least one co-stimulatory domain described herein having at least 80% sequence identity or This includes inducible regulatory constructs containing the active mutant or fragment thereof. CAR-T cells have specificity for cancer or tumor-specific antigens, and the pharmaceutical composition is cancer immunoassay. It is useful in the methods of epidemic treatment.

[0038] In another aspect, the present disclosure provides a method for administering genetically modified cells, and genetically modified The cells have at least 80% of the amino acid sequence described in any one of SEQ ID NOs. 5-8. At least one co-stimulatory domain having sequence identity, or its active variant or fragment. This includes the methods described herein. In some embodiments of this method, genetically modified cells This is a combination of at least 80% of the amino acid sequence described in any one of SEQ ID NOs. 5-8. It contains at least one co-stimulatory domain having column identity, or an active variant or fragment thereof. This includes CARs as described herein. In other embodiments of this method, genetically modified cells are sequenced At least 80% sequence identity to the amino acid sequence described in any one of numbers 5-8. This specification includes at least one costimulatory domain having or an active variant or fragment thereof. This includes the inductive regulatory structures described in the document.

[0039] In a specific embodiment of this method, the subjects to whom genetically modified cells are administered have diseases such as cancer. In some embodiments of this method, the genetically modified cells are CAR-T cells. In such embodiments, CAR-T cells have specificity for cancer or tumor-specific antigens. It has and is useful in cancer immunotherapy methods. In some embodiments, cancer is B-cell malignancies (e.g., acute lymphoblastic leukemia (ALL), B-cell non-Hodgkin's disease) Parker's disease (NHL, or chronic lymphocytic leukemia), multiple myeloma, neuroblastoma, glioblastoma , advanced glioma, ovarian cancer, mesothelioma, melanoma, or pancreatic cancer. In certain embodiments, It is a B-cell lymphoma.

[0040] In another aspect, this disclosure relates to the genetically modified cells described herein for use as pharmaceuticals. The disclosure provides cells. The disclosure further provides pharmaceuticals for treating diseases in subjects that require them. This specification provides for the use of genetically modified cells in the manufacture of such products. Therefore, pharmaceuticals are useful for cancer immunotherapy in those who need them.

[0041] In another aspect, the Disclosure relates to immunotherapy for treating cancer in a population that requires it. The method provides for the production of genetically modified cells and pharmaceuticals by the method disclosed herein. This includes administering a pharmaceutical composition containing a suitably acceptable carrier to a target.

[0042] In some embodiments, cancer is carcinoma, lymphoma, sarcoma, blastoma, and leukemia. It is selected from the group consisting of n.

[0043] In some embodiments, cancers include B-cell origin cancers, breast cancer, gastric cancer, and neuroblastoma. Osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, The group is selected from the group consisting of B-cell origin and Hodgkin lymphoma. In some embodiments, the origin is B-cell origin. These cancers include B-line acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell non-Hypoblastic leukemia. The patient is selected from a group consisting of dikin lymphomas.

[0044] The foregoing and other aspects and embodiments of the present invention are described below in the detailed description and claims. By examining them, a more complete understanding can be achieved. Separate embodiments for clarity. The specific features of the present invention described in the context of this invention can be combined in a single embodiment. It can also be provided. All combinations of embodiments are specifically included by the present invention. , as if every single combination were disclosed individually and explicitly. Disclosed herein. Conversely, described in the context of a single embodiment for the sake of brevity. The various features of the present invention can also be provided separately or in any suitable combination of sub-features. All subcombinations of the features listed in the embodiments also specifically encompass the present invention. Included, as if each of all such subcombinations were individually and explicitly stated herein. The present invention is disclosed herein as if it were explicitly disclosed. Each embodiment of the present invention can be applied to other embodiments of the present invention with necessary modifications. [Brief explanation of the drawing]

[0045] [Figure 1]Alignments of novel co-stimulatory domains novel 1 (SEQ ID NO: 5), novel 3 (SEQ ID NO: 6), novel 5 (SEQ ID NO: 7), and novel 6 (SEQ ID NO: 8) are shown. Individual TRAF-binding motifs are identified, and spacer regions can be found between TRAF-binding motifs within each listed co-stimulatory domain. [Figure 2] The CAR constructs include anti-CD19scFv, CD8 hinge and transmembrane domains, a co-stimulatory domain, and a CD3ζ intracellular signaling domain. The co-stimulatory domains shown include CD28, 4-1BB, novel 1(N1), novel 3(N3), novel 5(N5), and novel 6(N6). CAR constructs lacking co-stimulatory domains are also shown. [Figure 3A-H] We report the results of GFP analysis demonstrating CAR expression after lentiviral transduction of donor human T cells. Results are reported for each of the novel co-stimulatory domains: novel 1 (N1), novel 3 (N3), novel 5 (N5), and novel 6 (N6). Figure 3A) shows pseudotransduction. Figure 3B) shows transduction by a CAR containing the 4-1BB co-stimulatory domain. Figure 3C) shows transduction by a CAR containing the CD28 co-stimulatory domain. Figure 3D) shows transduction by a (BB-)CAR without a co-stimulatory domain. Figure 3E) shows transduction by a CAR containing the novel 1 (N1) co-stimulatory domain. Figure 3F) shows transduction by a CAR containing the novel 3 (N3) co-stimulatory domain. Figure 3G) shows transduction by a CAR containing the novel 5 (N5) co-stimulatory domain. Figure 3H) shows transduction by a CAR containing the novel 6 (N6) co-stimulatory domain. [Figure 4A-C] Figure 4A shows the number of CAR-T cells expanded over time after repeated antigen-induced activation in Raji cultures. Results are reported for each of the novel costimulatory domains: novel 1 (N1), novel 3 (N3), novel 5 (N5), and novel 6 (N6). Figure 4A shows the results obtained for a mixed population of CD4+ and CD8+ CAR-T cells. Figure 4B shows the results obtained for the CD4+ CAR-T cell population. Figure 4C shows the results obtained for the CD8+ CAR-T cell population. [Figure 5A-C]The following figures show cytokine secretion in transduced CAR-T cell populations on days 3, 7, 10, and 14 after transduction. Figure 5A shows interferon-gamma (IFN-γ) secretion. Figure 5B shows TNF-alpha (TNF-α) secretion. Figure 5C shows IL-2 secretion. [Figure 6A-C] The number of CAR-T cells expanded over time after repeated antigen-induced activation in Raji cultures is shown, using more frequent antigen encounters and higher target:effector ratios. Results are reported for each of the novel costimulatory domains: novel 1 (N1), novel 3 (N3), novel 5 (N5), and novel 6 (N6). Figure 5A shows the results obtained for a mixed population of CD4+ and CD8+ CAR-T cells. Figure 5B shows the results obtained for the CD4+ CAR-T cell population. Figure 5C shows the results obtained for the CD8+ CAR-T cell population. [Figure 7] The donor template construct is shown from 5' to 3', containing a 5' reverse terminal repeat (ITR), a 5' homology arm, a promoter, an anti-CD19scFv coding sequence, a CD8 hinge and transmembrane domain, a co-stimulatory domain, and a CD3ζ intracellular signaling domain, an SV40 poly(A) signaling domain, a 3' homology arm, and a 3' ITR. The co-stimulatory domains shown include 4-1BB, novel 1(N1), and novel 6(N6). [Figure 8A-B] This figure shows the time-course CAR-T cell proliferation in response to antigens using CAR-T cells prepared from two different donors. Proliferation was measured for CAR-T cells containing 4-1BB, N1, or N6 costimulatory domains. Novel costimulatory signaling domains were found to support proliferation levels equivalent to or higher than those supported by 4-1BB signaling. Figure 8A shows the results using CAR-T cells prepared from donor K799. Figure 8B shows the results using CAR-T cells prepared from donor z4100. [Figure 9A-B]This shows the killing of K19 cells at various effector:target (E:T) ratios in 24-hour and 72-hour co-cultures with CAR-T cells containing 4-1BB, N1, or N6 costimulatory domains. Figure 9A shows CAR-T cells prepared from donor K799. Figure 9B shows CAR-T cells prepared from donor z4100. [Figure 10A-C] Histograms of the results of cell proliferation assays used to determine the relative proliferation of CAR-T cells containing 4-1BB, N1, or N6 costimulatory domains in response to CD19+ target cells are shown. Figure 10A shows the proliferation of CAR-4-1BB CAR-T cells compared to negative control TRC KO T cells at two different E:T ratios. Figure 10B shows the proliferation of CAR-4-1BB T cells compared to CAR-N1 T cells. Figure 10C shows the proliferation of CAR-4-1BB T cells compared to CAR-N6 T cells. [Figure 11] The 7241, 7205, and 7206 donor template constructs are shown, containing a 5' reverse terminal repeat (ITR), a 5' homology arm, a promoter, an anti-CD19scFv coding sequence, a CD8 hinge and transmembrane domain, a co-stimulatory domain, and a CD3ζ intracellular signaling domain, an SV40 poly(A) signal or an SV40 bipoly(A) signal, a 3' homology arm, and a 3' ITR from 5' to 3'. The co-stimulatory domains shown include 4-1BB (construct 7241) and novel 6 (N6; constructs 7205 and 7206). [Figure 12A-G]Figure 12A shows the dorsal and ventral total flux values ​​observed in vivo in mice after engraftment and growth of Raji-ffluc cells and subsequent treatment with TCR KO cells or CAR T cells containing CAR constructs 7205, 7206, or 4-1BB. Figure 12A shows the dorsal flux after treatment with TCR KO cells. Figure 12B shows the dorsal flux after treatment with 7205 CAR T cells. Figure 12C shows the dorsal flux after treatment with 7206 CAR T cells. Figure 12D shows the dorsal flux after treatment with 4-1BB CAR T cells. Figure 12E shows the ventral flux after treatment with TCR KO cells. Figure 12F shows the ventral flux after treatment with 7205 CAR T cells. Figure 12G shows the ventral flux after treatment with 7206 CAR T cells. Figure 12H shows the ventral flux after treatment with 4-1BB CAR T cells. [Figure 13A-D] This shows images of dorsal and ventral total flux observed in vivo in mice after engraftment and growth of Raji-ffluc cells and subsequent treatment with TCR KO cells or CAR T cells containing CAR constructs 7205, 7206, or 4-1BB. Figure 13A shows images of dorsal flux at days 7, 10, and 16 in groups treated with TCR KO cells, 7205 CAR T cells, 7206 CAR T cells, and 4-1BB CAR T cells. Figure 13B shows images of dorsal flux at days 24, 31, and 38 in groups treated with 7205 CAR T cells, 7206 CAR T cells, and 4-1BB CAR T cells. Figure 13C shows images of ventral flux at days 7, 10, and 16 in groups treated with TCR KO cells, 7205 CAR T cells, 7206 CAR T cells, and 4-1BB CAR T cells. Figure 13D shows imaging of ventral flux at days 24, 31, and 38 in groups treated with 7205CAR T cells, 7206CAR T cells, and 4-1BB CAR T cells. [Figure 14] The survival curves of mice after engraftment and growth of Raji-ffluc cells, and subsequent treatment with TCR KO cells or CAR T cells containing CAR constructs 7205, 7206, or 4-1BB are shown. [Figure 15] The 7240 donor template construct is shown from 5' to 3', containing a 5' reverse terminal repeat (ITR), a 5' homology arm, a promoter, an anti-CD19scFv coding sequence, a CD8 hinge and transmembrane domain, a costimulatory domain, a MyD88 costimulatory domain, a novel 6(N6) costimulatory domain, as well as a CD3ζ intracellular signaling domain, an SV40 bipoly A signaling domain, a 3' homology arm, and a 3' ITR. [Figure 16A-F] This report presents the results of a cell-trace violet proliferation assay using human T cells transfected to express an anti-CD19 CAR containing a novel 6(N6) costimulatory domain, or a CAR containing both MyD88 and the novel 6(N6) costimulatory domain. Transfected cells were labeled with cell-trace violet and co-cultured with CD19-negative K562 cells or engineered CD19-positive K562 cells (K19 cells). Proliferation was evaluated by flow cytometry for CD4+ and CD8+ subsets of T cells. Figure 16A shows CD4+ cells transfected with TRC1-2x.87EE meganuclease alone. Figure 16B shows CD8+ cells transfected with TRC1-2x.87EE meganuclease alone. Figure 16C shows CD4+ cells transfected with TRC1-2x.87EE meganuclease and an N6 CAR donor template. Figure 16D shows CD8+ cells transfected with TRC1-2x.87EE meganuclease and N6 CAR donor template. Figure 16E shows CD4+ cells transfected with TRC1-2x.87EE meganuclease and MyD88 / N6 CAR donor template. Figure 16F shows CD8+ cells transfected with TRC1-2x.87EE meganuclease and MyD88 / N6 CAR donor template. [Figure 17]From 5' to 3', the 7235 donor template construct is shown, containing a 5' reverse terminal repeat (ITR), a 5' homology arm, a promoter, a coding sequence for the MyD88 costimulatory domain, a novel 6(N6) costimulatory domain, and sequences encoding the tandem ligand-binding FKBP12v36 domain (Fv), a T2A element, an anti-CD19scFv, a CD8 hinge, and a transmembrane domain, as well as a CD3ζ intracellular signaling domain, an SV40 bipoly A signal, a 3' homology arm, and a 3' ITR. [Figure 18A-B] This report presents the results of a cell-trace violet proliferation assay using human T cells transfected to express only TRC1-2x.87EE meganuclease without a donor template. Transfected cells were labeled with cell-trace violet and co-cultured with CD19-negative K562 cells or engineered CD19-positive K562 cells (K19 cells). Proliferation was evaluated by flow cytometry for CD4+ and CD8+ subsets of T cells. Figure 18A shows the CD4+ subset of cells. Figure 18B shows the CD8+ subset of cells. [Figure 19A-D] This report presents the results of a cell-trace violet proliferation assay using human T cells transfected to express an anti-CD19 CAR containing a novel 6(N6) domain. Transfected cells were labeled with cell-trace violet and co-cultured with CD19-negative K562 cells or engineered CD19-positive K562 cells (K19 cells). Proliferation was evaluated by flow cytometry for CD4+ and CD8+ subsets of T cells. Furthermore, cells were evaluated in K19 co-culture in and without rimiducid. Figure 19A shows the CD4+ subset of cells cultured on K19 or K562 cells. Figure 19B shows the CD4+ subset of cells cultured on K19 cells in and without rimiducid. Figure 19C shows the CD8+ subset of cells cultured on K19 or K562 cells. Figure 19D shows the CD8+ subset of cells cultured on K19 cells in and without rimiducid. [Figure 20A-D]This report presents the results of a cell-trace violet proliferation assay using human T cells transfected to express an anti-CD19 CAR lacking the costimulatory domain, combined with an inducible construct containing both MyD88 and a novel 6(N6) costimulatory domain (iMyD88 / N6 CAR). Transfected cells were labeled with cell-trace violet and co-cultured with CD19-negative K562 cells or engineered CD19-positive K562 cells (K19 cells). Furthermore, cells were evaluated in K19 co-culture in and without rimiducid. Proliferation was assessed by flow cytometry for CD4+ and CD8+ subsets of T cells. Figure 20A shows the CD4+ subset of cells cultured on K19 or K562 cells. Figure 20B shows the CD4+ subset of cells cultured on K19 cells in and without rimiducid. Figure 20C shows the CD8+ subset of cells cultured on K19 or K562 cells. Figure 20D shows the CD8+ subset of cells cultured on K19 cells in the presence or absence of rimiducid.

[0046] A brief explanation of arrays Sequence ID 1 shows a nucleic acid sequence encoding a novel single costimulatory domain.

[0047] Sequence ID 2 shows the nucleic acid sequence encoding a novel tricostimulatory domain.

[0048] Sequence ID 3 shows a nucleic acid sequence encoding a novel 5-costimulatory domain.

[0049] Sequence ID 4 shows a nucleic acid sequence encoding a novel 6-costimulatory domain.

[0050] Sequence ID 5 shows the amino acid sequence of a novel co-stimulatory domain.

[0051] Sequence ID 6 shows the amino acid sequence of the novel three costimulatory domains.

[0052] Sequence ID 7 shows the amino acid sequence of the novel 5-co-stimulatory domain.

[0053] Sequence ID 8 shows the amino acid sequence of the novel 6-co-stimulatory domain.

[0054] Sequence ID 9 is the amino acid sequence of the TRAF-binding motif found in a novel costimulatory domain. This indicates.

[0055] Sequence ID 10 describes the amino acid configuration of the TRAF-binding motif found in a novel costimulatory domain. Show the columns.

[0056] Sequence ID 11 shows the amino acid configuration of the TRAF-binding motif found in a novel costimulatory domain. Show the columns.

[0057] Sequence ID 12 shows the amino acid sequence of the spacer sequence found in the novel co-stimulatory domain. .

[0058] Sequence ID 13 shows the amino acid sequence of the spacer sequence found in the novel co-stimulatory domain. .

[0059] Sequence ID 14 shows the amino acid sequence of the spacer sequence found in the novel co-stimulatory domain. .

[0060] Sequence ID 15 shows the amino acid sequence of the spacer sequence found in the novel co-stimulatory domain. .

[0061] Sequence ID 16 shows the amino acid sequence of the chimeric antigen receptor signal peptide.

[0062] Sequence ID 17 shows the amino acid sequence of the anti-CD19 chimeric antigen receptor scFv.

[0063] Sequence ID 18 shows the amino acid sequences of the chimeric antigen receptor CD8 hinge and transmembrane region. .

[0064] Sequence ID 19 shows the amino acid sequence of the CD3-ζ intracellular signaling domain.

[0065] Sequence ID 20 shows the amino acid sequence of the CD28 costimulatory domain.

[0066] Sequence ID 21 shows the amino acid sequence of the 4-1BB costimulatory domain.

[0067] Sequence ID 22 shows the amino acid sequence of an anti-CD19 chimeric antigen receptor lacking a co-stimulatory domain. show.

[0068] Sequence ID 23 is an amino acid of an anti-CD19 chimeric antigen receptor containing a CD28 costimulatory domain. The acid sequence is shown.

[0069] Sequence ID No. 24 is an anti-CD19 chimeric antigen receptor containing a 4-1BB costimulatory domain. The anoacid sequence is shown.

[0070] Sequence ID 25 is an amino acid of an anti-CD19 chimeric antigen receptor containing a novel co-stimulatory domain. Show the array.

[0071] Sequence ID 26 is an amino acid of an anti-CD19 chimeric antigen receptor containing a novel 3-costimulatory domain. Show the array.

[0072] Sequence ID 27 is an amino acid of an anti-CD19 chimeric antigen receptor containing a novel 5-costimulatory domain. Show the array.

[0073] Sequence ID 28 is an amino acid of an anti-CD19 chimeric antigen receptor containing a novel 6-costimulatory domain. Show the array.

[0074] Sequence ID 29 encodes an anti-CD19 CAR containing a 4-1BB costimulatory domain. The nucleic acid sequence of the actor is shown.

[0075] Sequence ID 30 is a vector encoding an anti-CD19 CAR containing a novel co-stimulatory domain. This shows the nucleic acid sequence.

[0076] Sequence ID 31 is a vector encoding an anti-CD19 CAR containing a novel 6-costimulatory domain. This shows the nucleic acid sequence.

[0077] Sequence ID 32 shows the nucleic acid sequence of the JeT promoter.

[0078] Sequence ID 33 shows the nucleic acid sequence of the SV40 poly(A) signal sequence.

[0079] Sequence ID 34 shows the nucleic acid sequence of the first SV40 bipoly A signal sequence.

[0080] Sequence ID 35 shows the nucleic acid sequence of the second SV40 bipoly A signal sequence.

[0081] Sequence ID 36 contains 724 co-stimulatory domains and SV40 polyA signaling pathways. The nucleic acid sequence of the vector encoding the anti-CD19 CAR construct is shown.

[0082] Sequence ID 37 contains a novel 6-costimulatory domain and SV40 polyA signaling pathway, along with 7205 anti-inflammatory drugs. The nucleic acid sequence of the vector encoding the CD19 CAR construct is shown.

[0083] Sequence ID 38 contains 7206 novel costimulatory domains and SV40 bipoly A signaling pathway. The nucleic acid sequence of the vector encoding the anti-CD19 CAR construct is shown.

[0084] Sequence ID 39 shows the amino acid sequence of the MyD88 costimulatory domain.

[0085] Sequence ID 40 contains MyD88 and a novel 6-costimulatory domain, along with 7240 anti-CD19 C The nucleic acid sequence of the vector encoding the AR construct is shown.

[0086] Sequence ID 41 shows the amino acid sequence of the tandem ligand-binding FKBP12v36 domain. show.

[0087] Sequence ID 42 is the 7235 anti-CD19 CAR construct which codes for the first generation CAR, MyD88 and novel 6 costimulatory domains and tandem ligand-binding FKBP12v36 The nucleic acid sequences of the vector encoding the inducible regulatory construct, including the main component, are shown. [Modes for carrying out the invention]

[0088] 1.1 References and Definitions The patents and scientific literature referenced herein establish knowledge available to those skilled in the art. The issued U.S. patents, granted applications, published foreign applications, and Gen patents cited in the details are as follows: The references, including the sequences in the Bank database, appear as if each one is specifically and individually referenced. Incorporated herein by reference to the same extent as indicated by which it is to be incorporated. .

[0089] This disclosure can be embodied in different forms and is not limited to the embodiments described herein. It should not be interpreted that way. Rather, these embodiments demonstrate that this disclosure is thorough and It is provided to be complete and to fully convey the scope of this disclosure to those skilled in the art. For example, The features exemplified in terms of implementation can be incorporated into other embodiments, and in a specific embodiment... Features illustrated in this specification can be removed from the embodiment. Furthermore, the features suggested herein Numerous modifications and additions to the embodiments described herein that do not depart from this disclosure may be made in light of this disclosure. It should be obvious to the contractors.

[0090] Unless otherwise defined, all technical and scientific terms used herein are defined in this disclosure. This has the same meaning as it is generally understood by those skilled in the art to which it belongs. The terms used in this disclosure are intended solely to describe specific embodiments. This disclosure is not intended to limit the scope of this disclosure.

[0091] All publications, patent applications, patents, and other references mentioned herein are considered in their entirety. This is incorporated herein by reference.

[0092] As used herein, "a," "an," or "the" means one or more. It is possible. For example, "a" cells can refer to a single cell or a group of cells.

[0093] As used herein, unless otherwise specifically indicated, the word "or" means "and / It is used in the comprehensive sense of "or," and not in the exclusive sense of "either / or." .

[0094] As used herein, "co-stimulatory domain" refers to a domain that, upon activation, transmits intracellular proliferation signals. Refers to polypeptide domains that transmit signals and / or cell survival signals. Co-stimulatory domain Activation may occur following homodimerization of the two co-stimulatory domain polypeptides. Sexualization also involves constructs containing, for example, a co-stimulatory domain (e.g., a chimeric antigen receptor or induction). This can also occur after activation of sex regulatory constructs. Generally, the co-stimulatory domain is a transmembrane co-stimulatory receptor. It can be induced from the body, particularly from the intracellular portion of the costimulatory receptor. Limited examples include the co-stimulatory domains described herein, 4-1BB, CD28, ICOS, This includes, but is not limited to, OX-40 and CD27.

[0095] As used herein, “chimeric antigen receptor” or “CAR” means an antigen or other The specificity for the ligand or molecule is determined by the immune effector cells (e.g., T cells or NK cells). This refers to a modified receptor that is transplanted into cells. Chimeric antigen receptors are typically at least This also includes an extracellular ligand-binding domain or portion, and one or more signaling domains and / or includes intracellular domains containing co-stimulatory domains.

[0096] In some embodiments, the extracellular ligand-binding domain or portion is a specific epithet A pepper or antigen (for example, on the surface of cells such as cancer cells or other disease-causing cells or particles) A monoclonal antibody that provides specificity for an epitope or antigen that preferentially exists on top of it. This is the form of a single-chain variable fragment (scFv) derived from a body. In some embodiments, scF v is bound via a linker sequence. In some embodiments, extracellular ligand binding occurs. The domain is specific to any target antigen or epitope. In some embodiments, scFv is humanized. In some embodiments, the extracellular dormancy of the chimeric antigen receptor The drug contains autoantigens (Payne et al. (2016) Science, Vol.353(6) (See 295):179-184) This is because it is linked to autoantigen-specific B cell receptors on B lymphocytes. Therefore, it is recognized, and thus autoreactive B lymphocytes are specifically identified in antibody-mediated autoimmune diseases. They instruct T cells to target and kill them in an unusual way. Such CARs are chimeric autoantibody receptors. It may be called a CAAR, and one or more of the components to such a CAAR as described herein. The incorporation of the stimulation domain is included in this disclosure.

[0097] The intracellular signaling domain, following the binding of the extracellular domain, transmits an activation signal to the cell. It is a cytoplasmic domain that transmits signals. Intracellular signal transduction domains are used in this technology. It may be any intracellular signaling domain for any known purpose. Such cytoplasmic signaling The transmission domain is not limited, but may include CD3ζ.

[0098] In some embodiments, the intracellular domain also promotes cell proliferation after binding of the extracellular domain. This specification transmits co-stimulatory signals that promote cell survival and / or cytokine secretion. It contains one or more intracellular co-stimulatory domains, such as those described in the book. The insulators are, but are not limited to, any co-stimulatory domains or in the art disclosed herein. In the case of publicly known domains, for example, CD28 domains, 4-1BB domains, OX-40 domains This may include the main, ICOS domain, or CD27 domain, etc. In some embodiments, The chimeric antigen receptor binds to the extracellular ligand-binding domain via a hinge or junction sequence. It further includes additional structural elements, including a combined transmembrane domain.

[0099] As used herein, “inducible regulatory construct” means a construct that promotes cell proliferation, cell survival, and / Alternatively, it provides inducible costimulatory signals to promote cytokine secretion, which are expressed within cells. This refers to transmembrane or intracellular constructs that are activated by co-stimulatory signals. One of the following provides the information described herein and / or other information known in the art: Includes the above co-stimulatory domain. In some embodiments, the co-stimulatory signals are, for example, two It is induced by homodimerization of the inducible regulatory construct polypeptide. This generally enables homodimerization of small molecules, antibodies, or two construct polypeptides. It contains a binding domain that enables homodimerization after binding to other molecules.

[0100] As used herein, “co-stimulatory signal” means in vitro and / or in vivo This promotes cell proliferation and expansion of cell populations, enhances cell survival, and regulates cytokine secretion. For example, it controls (upward or downward) and / or the production and / or secretion of other immunomodulatory molecules. This refers to intracellular signals induced by regulatory, co-stimulatory domains. Several implementation forms In this state, the co-stimulatory signal follows the homodimerization of two co-stimulatory domain polypeptides. It is induced. In some embodiments, the co-stimulatory signal is a construct containing a co-stimulatory domain. It is induced after activation of (for example, a chimeric antigen receptor or an inducible regulatory construct).

[0101] As used herein, the term “activation” means inducing a detectable effector function. This refers to the state of cells (e.g., T cells) that have been sufficiently stimulated to perform certain actions. Therefore, activation leads to induced cytokine production and / or induced cell proliferation and expansion. It is related to this.

[0102] As used herein, the terms “antitumor activity” or “antitumor effect” refer to the effect of a tumor volume. Decrease, decrease in tumor cell count, decrease in metastasis, extension of life expectancy, or other characteristics associated with cancer. This refers to the biological effects that can be demonstrated by the improvement of various physiological symptoms. "Antitumor effect" is a more accurate term. Firstly, the ability of the genetically modified cells of this disclosure to prevent tumor development is evident. It is possible.

[0103] As used herein, the terms “manipulated” or “recombinant” are used with respect to proteins. This involves genetic engineering of nucleic acids that encode proteins, and cells or organisms that express proteins. This means that the amino acid sequence has been altered as a result of the application of scientific technology. Regarding nucleic acids... The terms "manipulated" or "recombinant" have changed as a result of the application of genetic engineering techniques. This means that it has a nucleic acid sequence. Genetic engineering techniques include PCR and DNA cloning. Technology, transfection, transformation and other gene transfer techniques, homologous recombination, site-specific This includes, but is not limited to, targeted mutagenesis and gene fusion. According to this definition, Although it has the same amino acid sequence as naturally occurring proteins, clonin in heterologous hosts Proteins produced by pharmacokinetics and expression are not considered recombinants.

[0104] As used herein, the term “wild type” refers to the most common type involved in a given phenotype. This refers to naturally occurring polynucleotide or polypeptide sequences. The wild-type allele or Polypeptides can confer a normal phenotype to an organism, but they can also confer a variant or mutant contrast. Genes or polypeptides can, in some cases, confer altered phenotypes.

[0105] When used herein in relation to recombinant proteins, the term “modified” means the reference sequence (e.g., For example, any insertion or deletion of amino acid residues in a recombinant sequence (from the wild-type or natural sequence) It means, or substitution.

[0106] As used herein, the terms “homologous recombination” or “HR” mean “homologous recombination” using a donor template. This refers to a natural cellular process in which double-strand DNA breaks are repaired using the same DNA sequence (for example) Cahill et al. (2006), Front. Biosci. 11:1958-197 (See 6). Homologous DNA sequences are endogenous chromosome or episome sequences or extrasomal sequences delivered to cells. It could be a causative nucleic acid.

[0107] As used herein, the terms “non-homologous end joining” or “NHEJ” refer to double-stranded DN A cleavage is repaired by the direct joining of two non-homologous DNA segments in a natural cellular process. This refers to (for example, Cahill et al. (2006), Front. Biosci 11:1 (See 958-1976).

[0108] As used herein, the term “reduced” means any reduction in the symptoms or severity of a disease. This refers to a decrease in the proliferation or number of cancer cells. In any case, such a decrease The maximum percentages are 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 9%. It can be 0%, 95%, or up to 100%. Therefore, the term "decreased" refers to a portion of the condition. This includes both partial reduction and complete reduction.

[0109] As used herein, the term “increase” refers to the co-stimulatory domain disclosed herein. , or activation, proliferation, of cells genetically modified to contain the active fragment or variant thereof Or it refers to any increase in cytokine signaling. Such an increase is up to 5%, 10%. %, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or It can be up to 100% or more. Cell activation, proliferation, or increased cytokine signaling. Any method can be used to measure the addition. For example, activation and / or cy Increased tokine expression is associated with IFN-γ, IL-2, TNF-α, or cell activation and / or This is any other cytokine that may be used to determine changes in proliferation. It can include an increase in one expression. Alternatively, it may include an increase in cell division and an expansion of the cell population.

[0110] When used herein in relation to both amino acid sequences and nucleic acid sequences, the term "identity "Percent," "Sequence Identity," "Similarity Percent," "Sequence Similarity," etc., refer to aligned sequences. To maximize the similarity between amino acid residues or nucleotides, and to select identical or similar residues or nucleotides. The number of ocides, the total number of residues or nucleotides, and gaps in sequence alignment. The degree of similarity between two arrays, based on array alignment, which is a function of existence and length. Refers to a measure. Various algorithms for determining sequence similarity using standard parameters. Sequences and computer programs are available. When used herein, sequences The similarity lies in the BLASTp program for amino acid sequences and BLA for nucleic acid sequences. These were measured using the STn program, and both are from the National Biotechnology Information Center. It is available through the center (www.ncbi.nlm.nih.gov), for example , Altschul et al. (1990), J.Mol.Biol.215:403~410; Gish and States (1993), Nature Genet.3:266 -272; Madden et al. (1996), Meth. Enzymol. 266:131~ 141;Altschul et al. (1997), Nucleic Acids Res.25 :33 89-3402); Zhang et al. (2000), J.Comput. Biol7 (1-2):203-14 is described herein. When used herein, two amino acids The similarity percentage of acid sequences is calculated using the parameters of the BLASTp algorithm, i.e., word length = 3. Gap opening penalty = -11, Gap extension penalty = -1 The score is based on the scoring matrix = BLOSUM62. If so, the similarity percentage of the two nucleic acid sequences is a parameter of the BLASTn algorithm. , i.e., word length = 11, gap opening penalty = -5, gap extension Scores based on: penalty = -2, match reward = 1, mismatch penalty = -3 It is A.

[0111] When used herein in relation to the modification of two proteins or amino acid sequences, the term " "Corresponding" means that a specific modification in the first protein corresponds to a modification in the second protein. This demonstrates that the substitution is the same amino acid residue as in (for example) When standard sequence alignment is performed (using the BLASTp program), the The amino acid position of the modification in protein 1 corresponds to the amino acid position of the modification in protein 2. Used to indicate that they correspond or are aligned. Therefore, for the alignment of the array In this case, if residues X and Y correspond to each other, then X and Y may be different numbers. Despite this fact, the modification of amino acid "A" by residue "X" in the first protein is This corresponds to the modification of amino acid "A" by residue "Y" in the second protein.

[0112] Terms: "recombinant DNA construct," "recombinant construct," "expression cassette," "expression construct" "Chimera constructs," "constructs," and "recombinant DNA fragments" are interchangeable in this specification. Used, they are linear or cyclic nucleic acid molecules. Recombinant constructs may be regulated and coded without restriction. The recombinant construct includes artificial or naturally occurring combinations of nucleic acid molecules, including columns. While not found in nature as a whole, parts of the construct can be found in nature. For example, recombinant DN Structure A may have regulatory and coding sequences originating from different sources, or originating from the same source. This may include regulating and chord arrangements that are arranged in a manner different from those found in nature. Such structures may be used on their own or in conjunction with vectors. .

[0113] As used herein, “vector” or “recombinant DNA vector” means a given host Includes a replication system and sequence capable of transcription and translation of polypeptide coding sequences in chief cells. It may be a structure. If vectors are used, the selection of vectors should be as well known to those skilled in the art. It depends on the method that will be used to transform the host cells. The vector is p Rasmid vectors and recombinant lentiviruses or recombinant AAV vectors, or the present disclosure Suitable for delivering genes encoding the co-stimulatory domain to target cells, known in the field This may include, but is not limited to, any other vectors. A person skilled in the art can understand any of the terms of this disclosure. Successfully transform, select, and grow host cells containing separated nucleotides or nucleic acid sequences. I am well aware of the genetic elements that must be present on the vector in order to achieve this.

[0114] As used herein, “vector” may also refer to a viral vector. Viral vectors include retroviral vectors, lentiviral vectors, and adenowicin vectors. may include, but is not limited to, retroviral vectors and adeno-associated viral vectors (AAVs). not limited thereto

[0115] As used herein, the term "operably linked" is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a nucleic acid sequence encoding a nuclease and a regulatory sequence (e.g., a promoter) as disclosed herein is a functional linkage that enables the expression of the nucleic acid sequence encoding the nuclease. The operably linked elements may be continuous or discontinuous. When used to refer to the joining of two protein coding regions, operably linked means that the coding regions are intended to be within the same reading frame.

[0116] As used herein, "transfected" or "transformed" or "transduced" or "nucleofected" refers to the process by which exogenous nucleic acid is introduced or transferred into a host cell. A "transfected" or "transformed" or "transduced" cell has been transfected, transformed, or transduced with exogenous nucleic acid. Cells include primary subject cells and their progeny.

[0117] As used herein, "human T cell" or "T cell" refers to a T cell isolated from a human donor. Human T cells, and cells derived therefrom, include isolated T cells that have not been passaged in culture, T cells that have been passaged and maintained under cell culture conditions without immortalization, and T cells that have been immortalized and can be maintained indefinitely under cell culture.

[0118] ​​​​​​​​​​​​​As used herein, "human natural killer cells" or "human NK cells" or "Natural killer cells" or "NK cells" are cells that cause important cell damage to the innate immune system. It refers to a type of NK cell. The role of NK cells is crucial in the adaptive immune response of vertebrates. This is similar to the role of cytotoxic T cells. NK cells rapidly react against virus-infected cells. It shows a response, reacts to tumor formation, and acts approximately 3 days after infection. Human NK cells and related cells. The cells used are isolated NK cells that have not been passaged in culture, and cells cultured under conditions without immortalization. NK cells that have been passaged and maintained under the following conditions, and immortalized and maintained indefinitely in cell culture. It contains NK cells that can be produced.

[0119] As used herein, “control” or “control cell” means the genes of a genetically modified cell. This refers to cells that provide a reference point for measuring changes in type or phenotype. A control cell is, for example, (a) wild-type cells, i.e., starting material for the gene mutation that resulted in the genetically modified cells (b) Cells with the same genotype, (b) Genetically modified cells with the same genotype, but null constructs (i.e.) Cells transformed with a construct that does not have a known effect on the target trait, or (c) Genetically identical to genetically modified cells, but expressing altered genotypes or phenotypes. This may include cells that are not exposed to conditions, stimuli, or further genetic modification that would induce such changes.

[0120] The term “effective dose” or “therapeutic effective dose” refers to a beneficial or desirable biological and / or clinical dose. This refers to a quantity sufficient to produce results. The quantity is for therapeutic purposes (e.g., genetically modified cells, CAR- T cell, CAR-NK cell) preparation or composition, disease and its severity, and the treatment It will vary depending on the age, weight, physical condition, and responsiveness of the elephant. In certain embodiments, the effective amount of cells containing the costimulatory domain disclosed in the present specification or the pharmaceutical composition disclosed in the present specification reduces the progression of at least one symptom or disease.

[0121] As used herein, the term "treat" or "treatment" means reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0122] As used herein, the term "cancer" should be understood to encompass any neoplastic disease characterized by abnormal cell growth (whether invasive or metastatic or not). Invasive or metastatic cancers can spread to other parts of the body. Cancers involving uncontrolled cell division can cause malignant tumors or cancers, while cancers with slowly dividing cells can cause benign tumors or cancers.

[0123] As used herein, the term "carcinoma" refers to a malignant tumor composed of epithelial cells.

[0124] As used herein, the term "leukemia" refers to a malignant tumor of the hematopoietic organs / hematopoietic system, generally characterized by abnormal proliferation and development of white blood cells and their precursors in the blood and bone marrow.

[0125] As used herein, the term "sarcoma" refers to a tumor composed of substances such as embryonic connective tissue, generally composed of dense cells embedded in fibrillar, heterogeneous, or homogeneous substances.

[0126] As used herein, the term "melanoma" refers to the melanocyte system of the skin and other organs It refers to a tumor that arises from [unclear / unclear].

[0127] As used herein, the term "lymphoma" refers to a group of blood cells that originate from lymphocytes. It refers to a tumor.

[0128] As used herein, the term "blastoma" means progenitor cells or blast cells (immature tissue or This refers to a type of cancer caused by malignant tumors of embryonic tissue.

[0129] As used herein, the term “meganucleases” means those with more than 12 base pairs. This refers to an endonuclease that binds to double-stranded DNA at its recognition sequence. In some embodiments, The recognition sequence of the meganuclease of this disclosure is 22 base pairs. The meganuclease is I- It may be an endonuclease derived from CreI, and for example, it exhibits DNA binding specificity and DNA cleavage characteristics. Modifications compared to natural I-CreI in terms of activity, DNA binding affinity, or dimerization properties. This can refer to an engineered variant of I-CreI. Methods for producing I-CreI mutants are known in the art (e.g., international (Published No. 2007 / 047859). The meganucleases used herein are heterozygous. It binds to double-stranded DNA as a dimer. Meganucleases also bind to a pair of DNA-binding dormant molecules. "Single-chain meganucleus" is a molecule in which the peptide linker is used to bind to a single polypeptide. It may be "rease". The term "homing endonuclease" is the same as the term "meganuclea". This is synonymous with "-ase". The meganucleases disclosed herein are expressed in cells, particularly human T cells. If measured using the method described herein, it is substantially nontoxic. In combination, adverse effects on cell viability or a significant decrease in meganuclease cleavage activity were observed. It can be transfected into cells without any need to be transfected and maintained at 37°C.

[0130] As used herein, the term “single-chain meganuclease” means linked by a linker. Refers to a polypeptide containing a pair of nuclease subunits. Single-stranded meganuclea The -se has an N-terminal subunit-linker-C-terminal subunit configuration. Two mega Nuclease subunits are generally not identical in amino acid sequence, and non-identical DNs It will recognize the A sequence. Therefore, single-stranded meganucleases are typically pseudoparies. Cleaves the palindrom recognition sequence or the non-palindrom recognition sequence. Single-stranded meganuclease It is not actually a dimer, but is a "single-stranded heterodimer" or "single-stranded heterodimer megadimer". It is sometimes called a "nuclease". For clarity, unless otherwise specified, the term " The term "meganuclease" can refer to a dimer or a single-chain meganuclease.

[0131] As used herein, the term “linker” means two meganuclease subunits This refers to an exogenous peptide sequence used to bind a peptide to a single polypeptide. The inker may have a sequence found in natural proteins, or any natural protein It may be an artificial arrangement not found in nature. The linker is flexible and lacks secondary structure. Phosphorus may also have a tendency to form specific three-dimensional structures under physiological conditions. Carr is included in U.S. Patent Nos. 8,445,251 and 9,434,931. This includes, but is not limited to, any of the following:

[0132] As used herein, the terms "zinc finger nuclease" or "ZFN" are used interchangeably. These include restrictive endonucleases, homing endonucleases, S1 nucleases, and man. Gulpin nuclease, pancreatic DNase I, Micrococcus nuclease, and yeast HO Endonucleases, including but not limited to endonucleases, or exonucleases. The zinc finger DNA-binding domain fused to the nuclease domain from rease Refers to chimeric proteins containing zinc finger nucleases. Zedmain includes, but is not limited to, FokI, FoM, and StsI restriction enzymes. Contains substances derived from type IIs restriction endonucleases. Additional type IIs restriction endonucleases Rease is described in International Publication No. 2007 / 014275, which is the whole story. It is incorporated by reference. The structure of the zinc finger domain is determined by the coordination of the zinc ion. This stabilizes it. DNA-binding proteins containing one or more zinc finger domains It binds to DNA in a sequence-specific manner. Is the zinc finger domain a naturally occurring sequence? Or it is reasonable to produce a protein that binds to a given DNA sequence of about 18 base pairs in length. It may be redesigned through conventional or experimental means. For example, U.S. Patent No. 5,789,538. No. 5,925,523, No. 6,007,988, No. 6,013,453, No. 6,2 Publication No. 00,759, and International Publication No. 95 / 19431, International Publication No. 96 / 06166, International Publication No. 98 / 53057, International Publication No. 98 / 54311, International Publication No. 00 / 27 Publication No. 878, International Publication No. 01 / 60970, International Publication No. 01 / 88197, and International Publication See Publication No. 02 / 099084 (each of which is included in its entirety for reference). This is what I want. This manipulated protein domain is then processed by nucleases such as FokI nuclease. By fusing to a domain, DNA cleavage can be targeted with genome-level specificity. This is possible. Selection of target sites, zinc finger proteins, and zinc finger proteins. Methods for designing and constructing Clease are known to those skilled in the art, as published in U.S. Patent Application No. 20. No. 030232410, No. 20050208489, No. 2005064474, No. 2 Publication No. 0050026157, No. 20060188987, and International Publication No. 07 / 0142 These are described in detail in issue 75, and each of them is incorporated by reference in its entirety.

[0133] As used herein, the term "TALEN" means a restrictive endonuclease, holopropyl alcohol. Mingendonuclease, S1 nuclease, mangobean nuclease, pancreatic DNA It contains -ase I, Micrococcus nuclease, and yeast HO endonuclease, but these Nuclease domains from endonucleases or exonucleases, not limited to those mentioned above. It contains a DNA-binding domain that includes multiple TAL domain repeats fused to the active portion of or the TAL domain. It refers to an endonuclease. For example, the whole thing is included as reference, Chri See Stian et al. (2010) Genetics 186:757-761. Nuclease domains useful for ALEN design include FokI, FoM, StsI, and Hh Contains aI, HindIII, Nod, BbvCI, EcoRI, BglI, and AlwI. However, this does not include those derived from type IIs restriction endonucleases. These are not the only ones. Further type IIs restricting endonucleases are described in International Publication No. 2007 / 0 It is described in Patent No. 14275. In some embodiments, nucleotides of TALEN The main component is the FokI nuclease domain or its active portion. TAL domain repeats. TALE is a protein used in the infection process by plant pathogens of the genus Xanthomonas. It may originate from the family of transcription activators (effectors). The TAL domain repeats are different It is a 33-34 amino acid sequence with amino acids at positions 12 and 13. Repeat variable dipeptide These two positions, called RVD, are highly variable and are specific nucleotides. It shows a strong correlation with recognition. Each base pair in the DNA target sequence is in contact by a single TAL repeat. This specificity arises from RVD. In some embodiments, TALEN is 16 to 22 Includes TAL domain repeats. DNA cleavage by TALENs is adjacent to a nonspecific central region. It requires two DNA recognition regions (i.e., "spacers"). Regarding TALEN, the term A "spacer" is a set of two nucleic acids that are recognized and bound by each monomer that makes up a TALEN. This refers to nucleic acid sequences that separate sequences. TAL domain repeats are naturally occurring TALE proteins. It may be a naturally occurring sequence derived from a substance, or it may produce a protein that binds to a specific DNA sequence. It can be redesigned through rational or experimental means (for example, Boch et al. (2009) )Science 326(5959):1509-1512 and Moscow and Bog danove (2009) Science 326 (5959):1501 (each in its entirety) (See references) RVD and their corresponding target nucleotides. For methods of manipulating TALENs to recognize specific sequences and examples, see the United States. See also Patent Application Publication No. 20110145940 and International Publication No. 2010 / 079430. Please be illuminated. In some embodiments, each nuclease (e.g., FokI) monomer is It can fuse with TAL effector sequences that recognize different DNA sequences, thus enabling two recognition Only when the recognition sites are in very close proximity do inactive monomers combine to form a functional enzyme. put out.

[0134] As used herein, the term "compact TALEN" refers to I-TevI ​​homing Gendonuclease or U.S. Patent Application No. 20130117869 (the whole is for reference only) Any part of any of the endonucleases listed in Table 2 (which can be incorporated) A DNA-binding domain having one or more TAL domain repeats fused in any direction. This refers to endonucleases including MmeI, EndA, End1, I-BasI, and IT. evII, I-TevIII, I-TwoI, MspI, MvaI, NucA, and Nu Including but not limited to cM, compact TALENs have DNA processing activity. Therefore, the need for a dual target site that does not require dimerization and has an intervening DNA spacer. To reduce the impact. In some embodiments, the compact TALEN has 16 to 22 TALD Includes the main iteration.

[0135] As used herein, the term "CRISPR" refers to caspases such as Cas9, and By hybridizing to the recognition site in genomic DNA, caspase DNA cleavage is performed. This refers to a caspase-based endonuclease containing a guide RNA that directs the process. The caspase component of R is an RNA-induced DNA endonuclease. In certain embodiments, Caspases are class II Cas enzymes. In some of these embodiments, The caspase is a class II, type II enzyme, such as Cas9. Caspases are class II, type V enzymes, such as Cpf1. Guide RNA is a target. Direct repeats and guide sequences complementary to the target recognition site (in the context of the endogenous CRISPR system) This includes (often called a spacer). In certain embodiments, CRISPR is Direct repetitive sequences present on guide RNA (sometimes called tracr-mate sequences) (Completely or partially) complementary tracrRNA (trans-activated CRISP) It further includes R RNA. In certain embodiments, the caspase functions as a nickasase. The ability of enzymes to cleave only one strand of target DNA or to cleave only one strand of target polynucleotides. It is possible to mutate the corresponding wild-type enzyme so that it is lacking, as nickase. A non-limiting example of a functional caspase enzyme is the D10A augmentation within the RuvCl catalytic domain. C has a mutation, or has the H840A, N854A, or N863A mutation. It contains the AS9 enzyme.

[0136] As used herein, the term "megaTAL" refers to a manipulated sequence-specific homocyte Along with ngendonucleases, transcription activator-like effectors (TALEs) DNA binding dormant This refers to a single-chain nuclease containing yn.

[0137] As used herein, the term “recognition sequence” means a sequence bound by an endonuclease. This refers to the DNA sequence that is cleaved. In the case of meganucleases, the recognition sequence is 4 base pairs. It contains a pair of inverted 9-base pairs of "half-parts" that are separated. Single-stranded meganuclear In the case of ze, the N-terminal domain of the protein is in contact with the first semi-region, and the C-terminal domain of the protein The main part makes contact with the second half-part. The cleavage by meganuclease involves four base pairs at 3'. This creates an "overhang." An "overhang" or "sticky end" is a double-stranded DNA sequence. Short single-stranded DNA segments that can be produced by endonuclease cleavage. Yes. In the case of meganucleases and single-chain meganucleases derived from I-CreI, over The hang contains 10-13 bases of the 22-base pair recognition sequence. In the case of a compact TALEN, recognition The recognition sequence is the first CNNNGN sequence recognized by the I-TevI ​​domain, followed by... A nonspecific spacer with a length of 4 to 16 base pairs, followed by a TAL-effector. The main sequence (which typically has a 5'T base) is 16-2 It can contain a second sequence of 2 bp. Cleavage by compact TALEN results in two salts. This results in a 3' overhang between the base pairs. In the case of CRISPR, the recognition sequence is directly connected to the guide RNA. The sequence that binds to tectonic Cas9 cleavage, typically consisting of 16-24 base pairs. It is recognized as a guide sequence. Complete complementarity between sequences is not necessarily required to cause a cleavage. R-mediated cleavage occurs depending on the caspase, resulting in blunt ends (class II, type II caspases). (e.g., by class II, type V caspases) or protruding ends may be produced. Cpf In embodiments utilizing I-caspase, a CRISPR complex containing CpfI caspase is used. Cutting by the body results in a 5' overhang, and in certain embodiments, 5 nucleotides This results in a 5' overhang. Each caspase enzyme also performs complementary recognition of the guide RNA. Recognition of PAM (protospacer adjacent motif) sequences that are close to the sequence is required. The length required for PAM and the distance from the target sequence differ depending on the caspase enzyme, AM is typically a sequence of 2-5 base pairs adjacent to the target / recognition sequence. The PAM sequences of the enzymes are known in the art (for example, their entirety can be found by referencing each of them). This is incorporated in U.S. Patent No. 8,697,359 and U.S. Patent Application Publication No. 20160. (See No. 208243), the PAM sequences of novel or manipulated caspase enzymes are PAM depleted. Dryness assay (for example, the entire assay is incorporated herein by Karvelis et al. (20 17) Methods 121-122:3-8) and other methods known in the art. It can be identified using the law.

[0138] As used herein, the terms “target site” or “target sequence” refer to the nuclease This refers to the region of chromosomal DNA in a cell that contains the recognition sequence.

[0139] As used herein, the term “T cell receptor alpha gene” or “TCR alpha” The "α gene" is interchangeable and is inherited in T cells that encode the T cell receptor α subunit. It refers to the zodiac sign. The T cell receptor alpha is NCBI gene ID number 6 before or after rearrangement. It can refer to 955. After rearrangement, the T cell receptor alpha gene is endogenously promo T, reconstructed V and J segments, endogenous splice donor sites, introns, endogenous T cell receptor alpha, including the splice receptor site and subunit coding exons. It includes the constant region gene locus.

[0140] As used herein, the term “T cell receptor alpha constant region gene” means T cell This refers to the coding sequence of the cell receptor alpha gene. The constant region of the T cell receptor alpha gene contains , the wild-type sequence and its functional variant identified by NCBI gene ID number 28755 It contains foreign substances.

[0141] When used herein, the enumeration of numerical ranges for a variable is within the scope of this disclosure. The intention is to convey that it can be done with a variable equal to any of the values. Therefore For essentially discrete variables, the variable is within a numerical range that includes the endpoints of its range. It can be equal to any integer value of . Similarly, for a variable that is essentially continuous, that variable It can be equal to any real number within that numerical range, including the endpoints of that range. For example, a variable described as having values ​​between 0 and 2 is essentially discrete. If it is true, it can take the value 0, 1, or 2, and if the variable is essentially continuous. It can take values ​​of 0.0, 0.1, 0.01, 0.001, or any other real number between 0 and 2 (inclusive). It is possible.

[0142] 2.1 Principle of the Invention This disclosure, in part, shows that the manipulated co-stimulatory domain is superior to the conventional co-stimulatory domain. It is based on the discovery that it can exhibit equivalent or superior activity. In certain cases, The co-stimulatory domains disclosed herein are used for cell proliferation and / or cytokinesiology after antigen-induced activation. It has equivalent or superior co-stimulatory activity with respect to secretion. In some embodiments, this specification A nucleic acid molecule is provided that contains a nucleic acid sequence encoding one of the co-stimulatory domains disclosed. In some embodiments, the co-stimulatory domain is, for example, a chimeric antigen receptor or an inducible It is expressed in genetically modified cells as part of a construct such as a regulatory construct. Therefore, in this specification Cells containing the novel costimulatory domain and methods for creating cells containing the novel costimulatory domain. This is provided. Furthermore, this specification is provided for the purpose of reducing the symptoms or severity of the disease. A method for administering genetically modified cells containing the indicated co-stimulatory domain is disclosed. Several implementations In terms of form, administration of genetically modified cells containing the costimulatory domain disclosed herein is used for cancer, autologous Symptoms of diseases such as immune disorders and other conditions that may be targeted by the genetically modified cells of this disclosure. Or reduce the severity. This specification refers to genetically modified cells and pharmaceutically acceptable cells as disclosed herein. This includes administering a pharmaceutical composition containing a carrier to a target that requires it. Methods of immunotherapy for treating cancer will also be disclosed.

[0143] 2.2 Nucleic acid molecules encoding the co-stimulatory domain This specification describes nucleic acid molecules encoding novel costimulatory domains, and molecules having costimulatory activity. A variant (i.e., an active variant) is provided. The co-stimulatory activity of each domain is determined by the immune cells. This technology is known for measuring the activation, proliferation, and cytokine secretion of cells such as [list of cells]. It can be determined using a method of choice. One example of such a method is by Linsley et al. , Journal of Experimental Medicine176(199 2) Disclosed in 1595-604. Further examples include cell proliferation and cy This specification includes methods for measuring tokine secretion.

[0144] Therefore, nucleic acid molecules containing nucleic acid sequences encoding the co-stimulatory domain described in Sequence IDs 5-8 and its active variants are provided. Specifically, the nucleic acid sequence described in Sequence ID No. 1 is provided in this specification. This code encodes the co-stimulatory domain of sequence number 5, which is referred to as a novel domain in the book. (Described in sequence number 2) The nucleic acid sequence encodes the co-stimulatory domain of sequence number 6, which we refer to as the three novel domains in this specification. The nucleic acid sequence described in Sequence ID No. 3 is a co-incision of Sequence ID No. 7, which is referred to herein as the five novel domains. Encodes the domain. The nucleic acid sequence described in Sequence ID No. 4 is a novel domain in this specification and This codes for the co-stimulatory domain of sequence number 8.

[0145] This specification also includes active variants of nucleic acid sequences encoding the costimulatory domains disclosed herein. The provided mutant nucleic acid sequence encodes a domain that possesses co-stimulatory activity. Further variants of the co-stimulatory domain disclosed herein are provided. In this context, "mutant" is intended to mean a substantially similar sequence. Polypeptides are formed when one or more amino acids are deleted from one or more internal sites of a natural protein. By / or addition, and / or at one or more sites of a natural polypeptide, one or more A The substitution of amino acids signifies a polypeptide derived from a "natural" polypeptide. This is intended to be the case. Similarly, "mutant" polynucleotides have one or more in the natural nucleotide sequence. The deletion and / or addition of one or more nucleic acids in the above region constitutes a “natural” polynucleotide. It is a polynucleotide derived from. As used herein, "natural" polynucleotide The rheotide or polypeptide contains the parent sequence from which the variant is derived. In certain embodiments, co The parent nucleic acid sequences of the mutant polynucleotides encoding the stimulating domain include SEQ ID NOs: 1-4. Similarly, in some embodiments, mutant polypeptides encoding novel costimulatory domains The parent polypeptide sequence of the do includes sequence numbers 5-8.

[0146] The mutant polypeptides included in the embodiments are biologically active. That is, they are naturally Natural proteins continue to possess the desired biological activity, namely, co-stimulatory activity. Mutants can arise, for example, from human manipulation. The organisms of the natural co-stimulatory domain in the embodiment. Scientifically active variants (e.g., SEQ ID NOs. 5-8), or the co-stimulatory domains disclosed herein. Variants of the natural nucleic acid sequences encoding (e.g., SEQ ID NOs. 1-4) are described elsewhere in this specification. As determined by the sequence alignment program and parameters described herein. less than the amino acid sequence of natural polypeptides or the nucleic acid sequence of natural polynucleotides. Approximately 40%, 45%, 50%, 55%, 60%, 65%, 70%, and 75% respectively. %, approximately 80%, approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95% It will have approximately 96%, 97%, 98%, or 99% sequence identity. Biologically active mutants of the morphological co-stimulatory domain are similar to such co-stimulatory domains, and only Approximately 1-20 amino acid residues, just about 1-10, just about 1-5, just about 4 These can differ by as few as three, two, or even one amino acid residue.

[0147] The polypeptides of the embodiments are modified by various methods including amino acid substitution, deletion, cleavage, and insertion. It can be changed in this way. Such methods of manipulation are generally known in the field. For example, amino acid sequence variants can be prepared by mutations in DNA. Methods for inducing natural mutagenesis and modifying polynucleotides are well known in this field. For example, Kunkel(1985)Proc.Natl.Acad.Sci.USA82:488 -492; Kunkel et al. (1987) Methods in Enzymol. 154 :367-382; U.S. Patent No. 4,873,192; Walker and Gaastra (1983) Techniques in Molecular Biology (M acMillan Publishing Company (New York State) and there Please refer to the references cited. This affects the biological activity of the target protein. Guidelines for appropriate amino acid substitutions that do not cause problems can be found in Dayhoff et al. (1978) Atlas. of Protein Sequence and Structure(Natl. The model was found in Biomed.Res.Found (Washington, DC). This can be done, and is incorporated herein by reference. Amino acids have similar properties. Conservative substitution, such as replacing an element with another element, may be the optimal approach.

[0148] Depending on the context, "fragment" refers to a part of the amino acid sequence of a polypeptide or protein. , or polynucleotides encoding a portion of the amino acid sequence of a polypeptide or protein It refers to a cydo. The fragment can retain the activity of the original protein, and therefore such a " The "active" fragment is, for example, one of the fragments of SEQ ID NOs. 5-8 that retains co-stimulatory activity. Contains a fragment of the co-stimulus domain. A co-stimulus domain such as one of the fragments of sequence numbers 1-4. A fragment of a nucleotide sequence encoding a bioactive protein fragment can encode a bioactive protein fragment. A biologically active nucleotide fragment is a part of a nucleic acid sequence that encodes a co-stimulatory domain. The costimulatory domain-encoding portion was isolated and expressed, and the costimulatory domain-encoding portion It can be prepared by evaluating the activity of the affected region. The co-stimulatory domain fragment is represented by SEQ ID NO: 5 It contains ~8 fragments. The co-stimulatory domain fragments number at least 15, 20, 30, 35, and 3. Contains amino acids 6, 37, 38, 39, 40, 41, or 42.

[0149] In certain embodiments, variants or fragments of the co-stimulatory domains disclosed herein are In this specification, a TRAF motif refers to at least one TNFR-related factor (TRAF ) Includes a bonding motif. Examples of TRAF motifs provided herein include QMED (bonding motif). It includes column number 9), QEED (sequence number 10), and EEEG (sequence number 11), This is not limited to these. For example, in some embodiments, the co-stimulatory dormancy disclosed herein Active variants or fragments of Yin include SEQ ID NOs. 9 and 11 or SEQ ID NOs. 10 and 11.

[0150] In some embodiments, the co-stimulatory domain or its variant or fragment is a spacer domain. Includes two TRAF motifs separated by region. Where used herein, the term The "spacer region" refers to the area between two predictive TRAF motifs in the co-stimulatory domain. In certain embodiments, the spacer region is the amino acid sequence ASSCRCPQ (SEQ ID NO: 12 ), ASSCRFPE (SEQ ID NO: 13), ASSCRFPQ (SEQ ID NO: 14), and AS Contains one of the SCRAPS (SEQ ID NO: 15). In a specific active mutant costimulatory domain In this context, the spacer region of sequence number 12 is the TRAF bond between sequence number 9 and sequence number 11. It is located between the -f. In other active mutant co-stimulatory domains, the spacer of SEQ ID NO: 13 The region is located between the TRAF binding motifs of SEQ ID NO: 10 and SEQ ID NO: 11. In the active mutant co-stimulatory domain, the spacer region of SEQ ID NO: 14 is related to SEQ ID NO: 10 It is located between the TRAF-binding motifs of sequence number 11. In this context, the spacer region of sequence number 15 is the TRAF coupling region of sequence number 10 and sequence number 11. It is located between the chiefs. Alternatively, the spacer region maintains the co-stimulatory activity of the mutant domain. It can be any array.

[0151] In certain embodiments, the expression cassette or expression construct is used within cells, with less disclosure herein. It is provided to express at least one co-stimulatory domain or its active variant. In several embodiments, the cassette encodes a novel co-stimulatory domain or an active variant thereof. This specification includes 5' and 3' regulatory sequences operably linked to nucleic acid molecules provided herein. "Operationally linked" is intended to mean a functional link between two or more elements. For example, nucleic acid sequences and regulatory sequences encoding the co-stimulatory domains disclosed herein (e.g.) For example, the operable link between the promoter and the nucleic acid sequence encoding the co-stimulatory domain It is a functional link that enables expression. The operably linked elements are continuous or discontinuous. It may be. It is operable when used to refer to the binding of two protein-coding regions. Being linked means that the code areas are within the same reading frame.

[0152] In some embodiments, the cassette further comprises at least one cell that is simultaneously transformed Further genes are included. In further embodiments, further genes are placed on multiple expression cassettes. Provided. In some embodiments, such expression cassettes are used for transcriptional regulation of regulatory regions. Multiple restriction sites and / or recombinant polynucleotides for insertion, as shown below. It comprises a site. In some embodiments, the expression cassette further includes a selection marker gene. nothing.

[0153] In some embodiments, the expression cassette is transcribed and flipped in the 5'-3' direction of transcription. Translation initiation region (i.e., promoter), coding a co-stimulatory domain as disclosed herein. Nucleic acid sequences or their active variants, and functional transcription in the genetically modified cells of this disclosure and includes a translation termination region (i.e., termination region). The modulo region provided herein (i.e., p The romodea, transcriptional regulatory region, and translation termination region) and / or nucleic acid molecules are either natural or inherited. The principal cell may be similar to or relative to the principal cell. Alternatively, the regulatory region provided herein. The regions and / or nucleic acid molecules may be heterogeneous to or from the host cell. When used, "different species" in relation to the sequence means either derived from an alien species or derived from the same species. In such cases, intentional human intervention may cause the composition and / or the genomic locus to change its natural form. It is a sequence that has been substantially modified from the original. For example, a sequence that has been operably linked to a different nucleic acid molecule. Promoters originate from a different species than the one from which the nucleic acid molecule originated, or from the same / similar species. If derived from a species, one or both are substantially derived from its original form and / or genomic locus. Modified, or its promoter is operatively linked to nucleic acid molecules. It is not a natural promoter. Or, the regulatory region and / or nucleic acid molecule provided herein. It may be completely synthetic.

[0154] The termination region is a nucleic acid molecule that is either naturally occurring or operably linked to the transcription initiation region. Whether it may be naturally occurring to the cell host, or whether it may be naturally occurring to the promoter, nucleic acid A molecule, a cell host, or any combination thereof, from another source (i.e., foreign or It may originate from a different species. When preparing the expression cassette, the DNA sequence is oriented appropriately. Various DNA fragments can be manipulated. For this purpose, an adapter or phosphorus You can use a Kerr to join DNA fragments, or use other methods as needed. This can provide a restriction site, removal of excess DNA, removal of restriction sites, etc. For in vitro mutagenesis, primer repair, restriction, annealing, and resubstitution. For example, transitions and transversions may be involved.

[0155] In some embodiments, several processes are used in the expression cassette provided herein. A motor is used. An example of a suitable motor is pre- and early-stage cytomegalovirus (CMV). This is a promoter array. This promoter array has any poly connected to it in an operable manner. A potent constitutive promoter sequence capable of driving high levels of nucleotide sequence expression. That is. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However Simian virus 40 (SV40) early promoter, mouse mammary gland tumor virus (MMTV), Human Immunodeficiency Virus (HIV) Long-Term Repeat (LTR) Promoter, Mo MuLV promoter, avian leukemia virus promoter, Epstein-Barr virus pre-early stage Promoters, Roussarcoma virus promoters, and, for example, not limited to, This includes the actin promoter, myosin promoter, hemoglobin promoter, and creatine. Other components, including but not limited to human gene promoters such as kinase promoters. Constitutive promoter arrays may also be used. Furthermore, this disclosure is limited to the use of constitutive promoters. It should not. Inductive promoters are also intended as part of this disclosure. Use involves the expression of a operably linked polynucleotide sequence, where such expression is desired. It can be turned on at times, or its expression can be turned off when expression is not desired. It provides a molecular switch that can be activated. Examples of inductive promoters include the metallothione promoter. Glucocorticoid promoter, progesterone promoter, and tetracycline pro Motors are included but not limited to them. Synthetic motors, e.g., JeT motors. This is also intended as part of this disclosure (see International Publication No. 2002 / 012514).

[0156] In some embodiments, the promoter is selected based on the desired results. To modulate the timing, location and / or level of expression of the disclosed polynucleotides By using different promoters in the expression cassette, different applications can be enhanced. It is recognized that such expression constructs may also, if desired, have a promoter regulatory region. (For example, regulated inductively, constitutively, environmentally, or developmentally, or cellularly or (Tissue-specific / selective expression confers), transcription start site, ribosome binding site, RNA This may include a processing signal, a transcription termination site, and / or a polyadenylation signal.

[0157] To evaluate the expression of a co-stimulatory domain or a CAR polypeptide containing a co-stimulatory domain The expression cassette also contains a select marker gene, a reporter gene, or both. Includes cells intended to be transfected or infected via a viral vector. This makes it easier to identify and select expressing cells from a population. In other embodiments, The selection marker is loaded onto a separate DNA fragment and used in the simultaneous transfection procedure. This is possible. Both the selection marker and reporter genes enable expression in host cells. It may be adjacent to an appropriate regulatory sequence for this purpose. Useful selection markers include, for example, antibiotic resistance. This includes sex genes and fluorescent marker genes.

[0158] In certain embodiments, at least one co-stimulatory domain or its activity as disclosed herein An expression cassette is provided that contains nucleic acid molecules encoding CARs, including mutants. When used, a "CAR expression cassette" refers to at least one nucleus that encodes a CAR. This refers to an expression cassette containing an acid molecule. In some embodiments, the CAR expression cassette is co It encodes a CAR that does not contain a stimulating domain. The CAR expression cassette is also disclosed herein. CARs that may encode the indicated co-stimulus domain, or co-stimuli not disclosed herein. A CAR containing a domain may be encoded. For example, in some embodiments, an expression cassette As disclosed herein, it includes an extracellular ligand-binding domain and a costimulatory domain. It contains one or more sequences encoding an intracellular stimulating domain or an active variant thereof. In this embodiment, the extracellular ligand-binding domain is, for example, an antigen specific to B-cell lymphoma. It is specific to the antigens of cancer cells.

[0159] In certain embodiments, the expression cassette contains anti-CD19scFv, a novel costimulatory domain ( Sequence ID No. 5) or its active variant, and a CAR containing the CD3ζ signaling domain In other embodiments, the expression cassette contains anti-CD19scFv, novel 3 costimulatory dormant In (SEQ ID NO: 6) or its active variant, and CA containing the CD3ζ signaling domain In other embodiments, the expression cassette is anti-CD19scFv, novel 5-co-stimulatory. The active domain (SEQ ID NO: 7) or its active variant, and the CD3ζ signaling domain are included. It codes for CAR. In other embodiments, the expression cassette is anti-CD19scFv, novel 6. Co-stimulatory domain (SEQ ID NO: 8) or its active variant, and CD3ζ signaling domain These expression cassettes encode CARs containing any appropriate disease-specific antigen or It is thought that this can be manipulated to have specificity for molecules.

[0160] In other specific embodiments, at least one costimulatory domain or the one disclosed herein An expression cassette is provided that contains nucleic acid molecules encoding inducible regulatory constructs, including active mutants. As used herein, “regulatory expression cassette” means encoding an inducible regulatory construct. This refers to an expression cassette containing nucleic acid molecules. Expression cassettes include CAR expression cassettes and regulatory It can be either an expression cassette or an expression cassette. In some embodiments, a single expression cassette is sequence One of the co-stimulatory domains numbered 5-8, or its active fragment or variant, and Honmei CARs that do not contain nucleotide sequences encoding the inducible regulatory constructs described in the details. It may contain the following nucleotide sequence.

[0161] For example, in some embodiments, the expression cassette is as disclosed herein. A sequence encoding a syntactic domain and at least one co-stimulatory domain, or an active variant thereof. It contains small molecules, antibodies, or other molecules that bind to the binding domain to form two inducible regulatory constructs. This induces dimerization. In some embodiments, such dimerization is a co-stimulation of the cell. It initiates a signal to promote proliferation, survival, and / or cytokine secretion. In some embodiments in which the nucleotide can bind to small molecules, the binding domain is FKBP1 It includes analogs of 2 (e.g., including F36V substitution), and the small molecule is rimiducid (i.e.) , AP1903) CAR-T cell safety switches and other such inducible regulatory constructs Any binding domain that is known in the art to be useful in this disclosure is applicable to this disclosure. It is intended to be done in this way.

[0162] In certain embodiments, the expression cassette comprises a binding domain and a novel costimulatory domain (sequence). This code includes inductive regulatory constructs containing (5) or its active variant. In other embodiments, The expression cassette contains the binding domain and the novel 13 costimulatory domains (SEQ ID NO: 6) or their activity It encodes an inducible regulatory construct containing a mutant. In other embodiments, the expression cassette binds Inducible regulation including the domain and novel 5-costimulatory domain (SEQ ID NO: 7) or its active variant. It encodes a construct. In other embodiments, the expression cassette contains a binding domain and a novel 6-co-spine. This encodes an inducible regulatory construct containing the stimulating domain (SEQ ID NO: 8) or its active variant.

[0163] This specification also provides vectors containing nucleic acid molecules encoding the novel costimulatory domains of this disclosure. Provided. In some embodiments, the vector is novel as disclosed herein. It includes a nucleic acid molecule encoding a co-stimulatory domain or expression cassette. In some embodiments, The nucleic acids encoding the co-stimulatory domains disclosed herein can be converted into several types of vectors. It is roned. For example, in some embodiments, nucleic acids are plasmids, phagem Vectors including, but not limited to, phage derivatives, animal viruses, and cosmids. -It is cloned. The vectors that are particularly relevant are expression vectors, replication vectors, and Includes lobe generation vectors and sequence determination vectors.

[0164] In certain embodiments, the nucleic acid molecule encoding the co-stimulatory domain is a retroviral vector - Lentiviral vectors, adenovirus vectors, and adeno-associated viruses (AA) V) Provided on viral vectors such as vectors. Viral vector technology is in this field. This is well known, for example, Sambrook et al. (2001, Molecular Clonin g:A Laboratory Manual, Cold Spring Harbor Laboratory (New York), as well as other virology and molecular biology maniacs. It is described in the manual. Useful viruses as vectors include retroviruses and adenoviruses. This includes viruses, adeno-associated viruses, herpesviruses, and lentiviruses. These are not the only options. Generally, a suitable vector is functional in at least one organism. A replication origin, promoter sequence, convenient restriction endonuclease site, and one or more Includes selection markers (e.g., International Publication No. 01 / 96584, International Publication No. 01 / 29 (Patent No. 058, and U.S. Patent No. 6,326,193).

[0165] 2.3 Chimeric Antigen Receptors (CARs) and Inducible Regulatory Constructs This specification provides genetically modified cells that express cell surface chimeric antigen receptors (CARs). Generally, the CARs of this disclosure include at least an extracellular domain and an intracellular domain. In some embodiments, the extracellular domain is a ligand-binding domain or a partial domain. It includes a target-specific binding element, also known as an intracellular domain, or The cytoplasmic domain is, as disclosed herein, at least one co-stimulatory domain It includes or an active variant thereof, and one or more signal transduction domains such as CD3ζ. For example, in some embodiments, the CARs disclosed herein are provided in SEQ ID NOs. 5-8. Intracellular domains containing at least one co-stimulatory domain, or their activating mutations, such as those mentioned above. Including the body. In certain embodiments, the CAR disclosed herein includes at least two co-stimulatory units. Main (at least one co-stimulatory domain is described in SEQ ID NOs. 5-8) or main This includes the active fragments or variants disclosed in the details.

[0166] In some embodiments, the CARs of this disclosure are also referred to as ligand-binding domains or portions. It contains extracellular target-specific binding elements. The selection of the ligand-binding domain is determined by the surface of the target cell. It depends on the type and number of ligands that define the plane. For example, the ligand-binding domain is specific To recognize ligands that act as cell surface markers on target cells related to the disease state. It can be selected for the ligand-binding domain in the CAR of this disclosure. Examples of cell surface markers that can act as markers include viral infections, bacterial infections, and parasitic infections. This may include parasitic infections, autoimmune diseases, and those related to cancer cells. Several implementations Morphologically, the CAR of this disclosure is a desired ligand binding mechanism that specifically binds to antigens on tumor cells. By manipulating a portion, it is engineered to target the desired tumor antigen. In this context, "tumor antigen" refers to an antigen common to certain hyperproliferative disorders such as cancer.

[0167] In some embodiments, the extracellular ligand-binding domain of the CAR is any target It is specific to tumor antigens or epitopes. As a non-limiting example, several implementations... In this state, the target antigen is a tumor-associated surface antigen, such as ErbB2 (HER2 / neu), cancer Embryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGF) R), EGFR variant III (EGFRvIII), CD19, CD20, CD30, C D40, disialoganglioside GD2, mammary ductal epithelial mucin, gp36, TAG-72, S Sphingoglycolipids, glioma-associated antigens, human chorionic gonadotropin B, alpha-fetopro Thein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2(AS), intestinal carboxylase Terase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PS) A) PAP, NY-ESO-1, LAGA-la, p53, Prostein, PSMA, Survival and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M Neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGFl)-I It presents IGF-II, IGF receptor, mesoserine, and tumor-specific peptide epitopes. Major histocompatibility complex (MHC) molecules, 5T4, ROR1, Nkp30, NKG2 D, tumor stromal antigen, extra domain A (EDA) of fibronectin and extra Domain B (EDB), as well as the Al domain of tenascin C (TnC Al) and fibroblasts. Cell-associated proteins (FAPs); lineage-specific or tissue-specific antigens, e.g., CD3, CD4 , CD8, CD24, CD25, CD33, CD34, CD133, CD138, CTL A-4, B7-1 (CD80), B7-2 (CD86), Endoglin, Major tissue compatibility Gene complex (MHC) molecules, BCMA (CD269, TNFRSF17), CS1, or Virus-specific surface antigens such as HIV-specific antigens (e.g., HIV gpl20); EBV-specific Heterogeneous antigens, CMV-specific antigens, HPV-specific antigens such as E6 or E7 oncoproteins, Las SE virus-specific antigen, influenza virus-specific antigen, and their surface marks It is any derivative or variant of Kerr. In certain embodiments of this disclosure, ligand-bound domain The drug is specific to CD19.

[0168] In some embodiments, the extracellular domain of the chimeric antigen receptor is located on an autologous B lymphocyte. It further includes self-antigens that can be recognized by antigen-specific B cells (Payne et al. (2016)). See Science, Vol.353(6295):179-184), via antibodies. In autoimmune diseases, T cells are instructed to specifically target and kill autoreactive B lymphocytes. Such CARs can be called chimeric autoantibody receptors (CAARs), and they The incorporation of one or more co-stimulatory domains into the CAAR as described herein is not included in this disclosure. It can be done.

[0169] In some embodiments, the CAR disclosed herein has an extracellular ligand-binding domain or It is a transmembrane domain that links the autoantigen with the intracellular signaling domain and the co-stimulatory domain. It further includes. In some embodiments, the transmembrane domain is CD8α transmembrane polypeptide. That is the case.

[0170] The intracellular signaling domain of the CAR disclosed herein is normal in cells where the CAR is located. Activation of at least one effector function and / or proliferation pathway and cell survival pathway It is involved in activation. The term "effector function" refers to a specialized function of a cell. The effector function of T cells includes, for example, cell-lytic activity or cytokine secretion. It may be peractive. Intracellular signaling domains such as CD3ζ are connected to the extracellular domain. In response to this, it can provide an activation signal to the cell. As discussed, activation signals Nal induces cellular effector functions, such as cell lysis activity or cytokine secretion. It is possible.

[0171] In some embodiments, the intracellular domain, after binding to the extracellular domain, promotes cell proliferation and denaturation. This specification transmits co-stimulatory signals that promote cell survival and / or cytokine secretion. It contains one or more intracellular co-stimulatory domains, such as those described. In some embodiments, The intracellular co-stimulatory domains are, but are not limited to, any co-stimulatory domains disclosed herein. Or domains known in the art, such as the CD28 domain, the 4-1BB domain, or OX. - Includes 40 domains, ICOS domains, or CD27 domains, etc.

[0172] This specification also provides genetically modified cells that express inducible regulatory constructs. In that embodiment, the inducible regulatory construct is used to promote cell proliferation, cell survival, and / or cytokines. Transmembrane or intracellularly expressed transmembrane or that provide inductive costimulatory signals to promote secretion These are intracellular constructs. In some embodiments, the inducible regulatory constructs co-sting upon activation. Provides a strong signal, such as those described herein and / or others known in the art. It contains one or more co-stimulatory domains. Generally, a co-stimulatory signal is, for example, two inductive tunic This can be induced by homodimerization of the construct polypeptide. Inducible regulatory construct Typically, this enables homodimerization of small molecules, antibodies, or two construct polypeptides. It contains a binding domain that allows homodimerization after binding to other molecules. Dimerization occurs in cells. Initiating co-stimulatory signals to promote proliferation, survival, and / or cytokine secretion. This is possible. In some embodiments in which the binding domain binds to a small molecule, the binding domain is It includes analogues of FKBP12 (e.g., including F36V substitution), and the small molecule is rimiduc The ID is (i.e., AP1903). Such inducible CAR-T cell safety switches, etc. Any binding domain known in the art to be useful in regulatory structures This disclosure intends to convey the following.

[0173] In certain embodiments, the intracellular signaling domain of the CAR of this disclosure is derived from CD3ζ. The signaling domain and at least one novel co-stimulatory domain, e.g., SEQ ID NO: 5~ 8, or their active variants.

[0174] In other specific embodiments, the inductive modulating construct disclosed herein is a two-component construct. A binding domain that enables embolization, and at least one novel co-stimulatory domain (e.g., Includes (series numbers 5-8) or their active variants.

[0175] 2.4 Method for preparing recombinant viral vectors In some embodiments, the present disclosure provides recombinant AAV vectors for use in the methods of the present disclosure. The vector is provided. Recombinant AAV vectors are typically mammalian vectors such as HEK-293. It is produced in the cell line. The virus's cap gene and rep gene self-replicate. Prevents the delivery of therapeutic genes (e.g., endonuclease genes) and creates space for their delivery. Recombinant AAV vectors are removed from the vector for packaging purposes within the cell line. It needs to be provided in transformer. Furthermore, a "helper" is needed to support the replication (for example) Adenovirus) components need to be provided (Cots D, Bosch A, Chil lon M(2013)Curr.Gene Ther.13(5):370-81). In many cases, recombinant AAV vectors contain a first plasmid that encodes a "helper" component. , a second plasmid containing the cap gene and rep gene, and the virus packaged A third plasmid containing a viral ITR with an intervening DNA sequence is used to control the cell line. It is fabricated using triple transfection, which then... The virus particle containing the genome (ITR and target intervening gene) enclosed in a psid is frozen and thawed. Cells can be removed by decyclization, sonication, surfactants, or other means known in the art. Isolate the particles. Then, separate them using cesium chloride density gradient centrifugation or affinity chromatography. Purification using Raffy, followed by the application of the desired gene to organisms such as cells, tissues, or human patients. Delivered to the gene. Therefore, at least one encoding the co-stimulatory domain described herein Recombinant AAV vectors containing nucleic acid sequences, e.g., SEQ ID NOs. 5-8, or their active variants. A method for producing - is provided herein. Similarly, CAR, or described herein At least one of the listed co-stimulatory domains, e.g., SEQ ID NOs: 5-8, or their active variants. A method for preparing recombinant AAV vectors encoding inducible regulatory constructs is provided.

[0176] In some embodiments, gene transfer is achieved via a lentiviral vector. In contrast to other retroviruses, in some situations lentiviruses are specific It can be used to transduce non-dividing cells. A non-limiting example of a lentiviral vector. Examples include human immunodeficiency virus 1 (HIV-1), HIV-2, and simian immunodeficiency virus. (SIV), human T lymphotropic virus 1 (HTLV-1), HTLV-2, or equine This includes those derived from lentiviruses such as the infectious anemia virus (E1AV). Lentiviral vectors are produced by multiple attenuation of HIV toxic genes. For example, the genes env, vif, vpr, vpu, and nef are deleted for therapeutic purposes. This makes the vector safer. Lentiviral vectors are used in this technology. This is publicly known, as Naldini et al. (1996 and 1998); Zufferey et al. (199 7); Dull et al., 1998, U.S. Patent No. 6,013,516; and No. 5,994,1 See issue 36. In some embodiments, these viral vectors are plasmids. It is either virus-based or virus-based, and is used for incorporating foreign nucleic acids, for selection, and nucleic acids. It is configured to possess essential sequences for transfer into host cells. Known lentivir The American Type Culture Collection (ATCC); 10801 University Depository institutions such as Tibulbard, Manassas, Virginia (20110-2209) or It can be readily obtained from collections or known materials using generally available techniques. It can be isolated from the source.

[0177] In certain embodiments, the lentiviral vector is human immunodeficiency virus (HIV) or Plasmi, which encodes the cloned gag, pol, tat, and rev genes, D, and E from vesicular stomatitis virus (VSV-G) used for pseudotyped viral particles. It is prepared using a second plasmid encoding a pleurope protein. pCDH-E Transfer vectors such as F1-MCS vectors use JeT Promotor or EF1 Promotor. It can be used with appropriate motors such as electric motors. Next, CAR signaling Domains, such as the co-stimulatory domain disclosed herein and its active variants, under the promoter It can be inserted into the flow, followed by the insertion of IRES and GFP. Then all three p Rasmid is transfected into lentiviral cells such as Lenti-X-293T cells. This allows for the lentivirus to be recovered after an appropriate incubation period and concentrated. It can be reduced and screened. Therefore, in this specification, at least one nucleic acid Sequences, co-stimulatory domains described herein, e.g., SEQ ID NOs. 5-8, or their activating mutations. A method for producing recombinant lentiviral vectors containing the body is provided. Similarly, this specification provides , at least one co-stimulatory domain described herein (e.g., SEQ ID NOs. 5-8) or the Recombinant lentiviral vectors encoding CARs or inducible regulatory constructs containing active mutants. A method for producing the following is provided.

[0178] 2.5 Genetically modified cells and their populations including novel costimulatory domains This specification describes at least one novel costimulatory domain (e.g., as disclosed herein). For example, cells genetically modified to contain sequence numbers 5-8) or their active variants are provided. In certain embodiments, the genetically modified cells are at least one novel as described herein. C containing a co-stimulatory domain (e.g., SEQ ID NOs. 5-8) or its active variant. Includes nucleic acid molecules encoding AR or inducible regulatory constructs. In different variations of this disclosure, The nucleic acid molecules or expression cassettes encoding the novel costimulatory domains described herein are gene It is either present in the genome of the modified cell or not incorporated into the cell's genome. Nucleic acid components In some embodiments where the offspring or expression cassette is not incorporated into the genome, the nucleic acid molecule or Expression cassettes are expressed in recombinant DNA constructs, mRNA, viral genomes, or cells. It is present in genetically modified cells containing other nucleic acids that are not integrated into the genome. In certain embodiments, Genetically modified cells contain nucleic acid molecules encoding the costimulatory domain described herein. Furthermore, it is possible to encode a CAR that does not contain the co-stimulatory domain disclosed herein. This specification includes nucleotide sequences encoding creotide sequences and / or inducible regulatory constructs. It may include at least one expression cassette disclosed in the document.

[0179] In some of the genetically modified cells embodied herein, at least as described herein A CAR or inductive modulatory construct incorporating one novel co-stimulatory domain is used. The nucleic acid molecules involved are located together with the cell's endogenous T cell receptor alpha gene. In some embodiments, the nucleic acid molecule is an exogenous gene of the T cell receptor alpha constant region. It is located within the endogenous T cell receptor alpha constant region gene, such as within Son 1.

[0180] In certain embodiments, cells containing a novel costimulatory domain or its active variant are eukaryotic cells. Yes. In certain embodiments, cells containing a novel costimulatory domain or its active variant are T cells. Cells or NK cells, particularly human T cells or NK cells. In some embodiments, the cells are These are primary T cells or primary NK cells.

[0181] T cells and NK cells are found in peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, and intestinal It can be obtained from numerous sources, including tissue from the stained area, ascites, pleural fluid, splenic tissue, and tumors. Yes, it is possible. In certain embodiments of this disclosure, any number of T cell lines and N cells available in the art are used. K cell lines may be used. In some embodiments of this disclosure, T cells and NK cells are , obtained from units of blood collected from a subject using any number of techniques known to those skilled in the art In one embodiment, cells derived from the circulating blood of an individual are obtained by apheresis.

[0182] Genetically modified cells containing the novel costimulatory domains or their active variants disclosed herein are novel When compared to appropriate control cells that do not contain the co-stimulatory domain or its active variant, proliferation An increase in can be shown. In some embodiments, the novel co-stimulatory dormancy disclosed herein Cells containing 1 or its active variants are stimulated with an appropriate antigen in vitro or in vitro. Further increases in activation and proliferation are observed. For example, CAR-T cells and CAR-NK cells. The cells are control cells that do not contain the novel costimulatory domains or their active variants disclosed herein. Compared to [another factor], it can show increased activation, proliferation, and / or cytokine secretion. Increased itokine secretion is particularly associated with increased secretion of IFN-γ, IL-2, and TNF-α. It may include. Methods for measuring cell activation and cytokine production are well known in the art. Several suitable methods are provided in the examples herein.

[0183] This disclosure further includes at least one novel costimulatory domain described herein (e.g., sequence CARs or inducible regulatory constructs incorporating numbers 5-8) or their active mutants are used This specification includes a plurality of genetically modified cells containing nucleic acid molecules in their genome. A population of genetically modified cells is provided. Therefore, in various embodiments of the present invention, genetically modified cells A population is provided, and at least 10%, at least 15%, and at least 20% of the population's cells are provided. %, at least 25%, at least 30%, at least 35%, at least 40%, less At least 45%, at least 50%, at least 55%, at least 60%, at least 6 5%, at least 70%, at least 75%, at least 80%, at least 85%, small At least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% are novel co-stimulatory domains disclosed herein. These are genetically modified cells that include [specific characteristics]. In certain embodiments, a population of genetically modified cells is provided. at least 10%, at least 15%, at least 20%, at least 25% of the cells in the population %, at least 30%, at least 35%, at least 40%, at least 45%, less At least 50%, at least 55%, at least 60%, at least 65%, at least 7 0%, at least 75%, at least 80%, at least 85%, at least 90%, small At least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100%, express CARs containing the novel co-stimulatory domain described herein. In other embodiments, a population of genetically modified cells is provided, and at least one of the cells of the population 0%, at least 15%, at least 20%, at least 25%, at least 30%, small At least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, At least 80%, at least 85%, at least 90%, at least 95%, and 96%, at least 97%, at least 98%, at least 99%, or up to 100% However, the inducible modulatory constructs containing the novel co-stimulatory domains disclosed herein and the co-stimulatory constructs described herein It expresses a CAR that does not contain a stimulating domain.

[0184] 2.6 Method for producing genetically modified cells This disclosure includes genetic modifications comprising novel costimulatory domains or their active variants disclosed herein. A method for producing cells is provided. In a particular embodiment, at least one novel co-stimulatory domain (For example, SEQ ID NOs. 5-8) or the CARs that incorporate their active variants. A method is provided for modifying cells to contain nucleic acid sequence molecules. In other embodiments, fewer If at least one novel costimulatory domain (e.g., SEQ ID NOs. 5-8) or its active variant is incorporated A method for modifying cells to contain nucleic acid molecules encoding the inducible regulatory constructs that are being introduced. A novel co-stimulatory domain or its Nucleic acid molecules or expression cassettes encoding active mutants are integrated into the cell's genome. Or, it is not integrated into the cell's genome.

[0185] In some embodiments, a novel co-stimulatory domain (or CAR) disclosed herein may be used. DNA or RNA encoding an inducible regulatory construct is known in the art. It is introduced into cells using the technology described herein. In certain embodiments, novel co-stimuli disclosed herein A vector containing nucleic acid encoding a domain (or CAR or inducible regulatory construct) or Expression cassettes are introduced into cells using viral vectors. Such vectors are Known in the technical field, lentiviral vectors, adenovirus vectors, and Includes adeno-associated virus (AAV) vector (Vannucci et al., (2013) (Overviewed in New Microbiol. 36:1-22). Recombinations useful in this disclosure AAV vectors facilitate the transduction of viruses into cells and the introduction of nuclease genes into cells. Insertion of any serum, and in certain embodiments, insertion into the cell genome. It may have a type. In certain embodiments, the recombinant AAV vector is AAV2 or They have the AAV6 serotype. Recombinant AAV vectors also have a second chain in host cells. It may be self-complementary, thus not requiring DNA synthesis (McCarty et al. (2001)). Gene Ther. 8:1248-54).

[0186] In some embodiments, the nucleic acid molecule or expression cassette disclosed herein is DNA In the form of (e.g., circular or linear plasmid DNA or PCR product) or RNA within cells The manipulated nuclease gene is delivered in DNA form (e.g., plasmid) and / or delivered via a viral vector (e.g., AAV or lentiviral vector) In some embodiments, they are operably connected to a promoter or as specified herein The expression cassette is found in the disclosure. In some embodiments, the promoter is a virus Endogenous promoters derived from vectors (e.g., LTRs of lentiviral vectors) or well known In the cytomegalovirus or SV40 virus initial promoter, etc. Yes. In other embodiments, the promoter is a composite promoter such as a JeT promoter. In certain embodiments, genes encoding novel costimulatory domains or CARs disclosed herein The offspring preferentially drives gene expression in target cells (e.g., human T cells) It is operably connected to the terminal.

[0187] In some embodiments, the co-stimulatory domain (or CAR or) disclosed herein Nucleic acid molecules or expression cassettes encoding inductive regulatory constructs are used by methods known in the art. Using this method, covalent or non-covalent bonding is performed on the nanoparticles, or encapsulation is performed within such nanoparticles. (Sharma et al., (2014) Biomed Res Int. 2014). Nano The particles are nanoscale delivered with a length scale of <1 μm, preferably <100 nm. It is a system. Such nanoparticles are composed of metals, lipids, polymers, or biomacromolecules. It can be designed using a core that allows multiple copies of nucleic acid molecules or expression cassettes to be used. It can be attached to or encapsulated in a particle core. This delivers DN to each cell. By increasing the copy number of A, and therefore increasing the intracellular expression of each manipulated nuclease... , maximizing the possibility of co-stimulatory domains (or CARs or inductive regulatory constructs) being expressed The surface of such nanoparticles is coated with a polymer or lipid (e.g., chitosan, cationic polymer). Further modification with a rimer or cationic lipid, so that its surface is suitable for cell delivery and payload acquisition. It may be possible to form core-shell nanoparticles that provide further functionality to enhance penetration (Jian et al. (2012) Biomaterials, 33(30):7621-30). Nanoparticles To direct them towards the appropriate cell type and / or to increase the likelihood of cell uptake, Nanoparticles can be further advantageously bound to targeting molecules. Examples of ligating molecules include antibodies specific to cell surface receptors and natural ligatures that target cell surface receptors. It contains ligands (or parts of natural ligands).

[0188] In some embodiments, the co-stimulatory domain (or CAR or) disclosed herein Nucleic acid molecules or expression cassettes encoding inducible regulatory constructs are encapsulated within liposomes. It is converted or complexed using cationic lipids (e.g., LIPOFECTAMI). NE, Life Technologies Corp. Carlsbad, CA; Zu ris et al. (2015) Nat Biotechnol. 33:73-80; Mishra (See et al. (2011) J Drug Deliv. 2011:863734). Liposo The mol and lipoplex formulations protect the payload from degradation and facilitate fusion with the cell membrane. This can also enhance cell uptake and delivery efficiency by disrupting the cell membrane.

[0189] In some embodiments, the co-stimulatory domain (or CAR or) disclosed herein A nucleic acid molecule or expression cassette encoding an inducible regulatory construct is supported by a polymer scaffold (e.g., P Encapsulated within LGA or using cationic polymers (e.g., PEI, PLL) They are then complexed (Tamboli et al. (2011) Ther Deliv. 2(4):5 23-536). In some embodiments, the co-stimulatory domain (or CA) disclosed herein is used. Nucleic acid molecules or expression cassettes encoding R or inducible regulatory constructs are self-inducible to micelles. It combines with amphiphilic molecules that assemble (Tong et al. (2007) J Gene Med .9(11):956-66). Polymer micelles prevent aggregation and reduce charge interactions. Hydrophilic polymers that can reduce extracellular nonspecific interactions (for example) It may include micelle shells formed of polyethylene glycol.

[0190] In some embodiments, the co-stimulatory domain (or CAR or induction) disclosed herein is used. Nucleic acid molecules or expression cassettes encoding sex regulatory constructs are delivered to cells in an emulsion. It is prescribed as an emulsion. The term "emulsion" is not limited to but includes a lipid structure. This refers to any oil-in-water, water-in-oil, water-in-oil-in-water, or oil-in-water-in-oil-in-oil-in-oil-in-a-water dispersion or droplet. When the water-immiscible phase is mixed with the aqueous phase, nonpolar residues (e.g., long hydrocarbon chains) are absorbed by the water. This can be formed as a result of hydrophobic forces that move the polar head groups away from water and direct them toward it. Other lipid structures include monolayers, sparse layers, and multilayer lipid vesicles, micelles, and lamellar phases. However, it is not limited to these. Emulsions consist of an aqueous phase and a lipophilic phase (typically oil and organic solvent). It consists of (including a medium). Emulsions also often contain one or more surfactants. Nanoemulsion formulations are well known, for example, U.S. Patent Application No. 2002 / 00 Patent No. 45667 and No. 2004 / 0043041, and U.S. Patent No. 6,015,832 Nos. 6,506,803, 6,635,676, and 6,559,189 These are described, and their entirety is incorporated herein by reference.

[0191] In some embodiments, the co-stimulatory domain (or CAR or induction) disclosed herein is used. Nucleic acid molecules or expression cassettes encoding sex regulatory constructs are multifunctional polymer conjugates. (M astorakos et al. (2015) Nanoscale.7(9):3845-56;C heng et al. (2008) J Pharm Sci. 97(1):123-43). Dend The generation of the rimer allows control over payload capacity and size, and high payload capacity Furthermore, it is possible to improve stability by utilizing the display of multiple surface groups, and non-specific This can reduce unwanted interactions.

[0192] Methods for introducing and expressing genes into cells are well known in this field. Expression vectors In this context, the vector is transmitted to a host cell, e.g., a mammal, by any method in the art. It can be easily introduced into bacterial, yeast, or insect cells. For example, the expression vector is a substance Polynucleotides can be introduced into host cells by scientific, chemical, or biological means. Physical methods for introducing othiocyanate into host cells include calcium phosphate precipitation and lipofect. This includes injection, particle impact, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. For example, Sambrook et al. (2001, Molecular Cloning: AL) aboratory manual, Cold Spring Harbor Labo See (ratory, New York). Introducing polynucleotides into host cells. A preferred method is calcium phosphate transfection. Biological methods for introducing a drug into host cells include the use of DNA and RNA vectors. Viral vectors, especially retroviral vectors, are used to deliver genetically modified cells to mammals, such as human cells. It has become the most widely used method for inserting offspring. Other viral vectors lentivirus, poxvirus, herpes simplex virus I, adenovirus, and It may originate from adeno-associated viruses, etc. For example, U.S. Patent No. 5,350,674 and No. 5 See issues 585 and 362. Chemistry for introducing polynucleotides into host cells. The means include colloidal dispersion of polymer composites, nanocapsules, microspheres, and beads. Lipid systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes Includes. Exemplary colloidal systems for use as delivery media in vitro and in vivo. These are liposomes (for example, artificial membrane vesicles).

[0193] In some embodiments, the present invention further includes nucleic acid molecules or expressions disclosed herein. The cassette provides introduction into the T cell receptor alpha gene. In a specific embodiment, nucleic acid The molecule or expression cassette contains the coding sequence for the T cell receptor α subunit. It is introduced into the recognition sequence present in the alpha constant region of a gene. In this way, nucleic acid molecules or The introduction of the current cassette disrupts the expression of the endogenous T cell receptor α subunit, resulting in internal It disrupts the expression of the intrinsic T cell receptor. In certain embodiments, such a recognition sequence is used to destroy the T cell receptor. It may be located within exon 1 of the cellular receptor alpha constant region gene.

[0194] In certain embodiments, nucleic acid molecules encoding the costimulatory domain disclosed herein are used with T cells. Alternatively, by introducing it into cells such as NK cells, the co-stimulatory domain disclosed herein is not included. Compared to control cells, it increases cell activation, proliferation, and / or cytokine secretion. It can be made possible. In some embodiments, it can activate, proliferate, and / or site cells. Cain secretion involves introducing a nucleic acid molecule encoding a costimulatory domain disclosed herein. Therefore, it can be increased in vitro or in vivo.

[0195] In some embodiments, at least one novel costimulatory domain or its active fragment is Alternatively, introducing the mutant into cells such as T cells or NK cells may lead to the novel co-infection disclosed herein. Compared to control cells that do not contain the aggressive domain, the cell proliferation period and / or cell population expansion Prolongs cell growth and / or slows cell depletion. Cell expansion and depletion (T cells or NK cells) Methods for measuring cell enlargement and depletion, etc., are known in the art and are not described herein. It is disclosed in that section.

[0196] 2.7 Pharmaceutical Compositions In some embodiments, the present disclosure may refer to genetically modified cells or a collection of genetically modified cells. The present invention provides a pharmaceutical composition comprising a compound and a pharmaceutically acceptable carrier. It can be prepared according to the technology of knowledge. For example, Remington, The Sci ence and Practice of Pharmacy(21sted.200 See 5). In the manufacture of pharmaceutical formulations according to this disclosure, cells are typically pharmaceutically The composition is mixed with an acceptable carrier and administered to the subject. The carrier is, of course, manufactured It must be acceptable in the sense that it is compatible with any other component in the agent, and for the subject It must not be harmful. In some embodiments, the pharmaceutical compositions of this disclosure are used in relation to the target. It further includes one or more additional drugs useful for treating the disease. Genetically modified cells are genetically modified In a further embodiment, which is T cells or NK cells (or cells derived therefrom), The disclosed pharmaceutical composition contains cytokines (e.g.,) that promote cell proliferation and engraftment in vivo. This further contains biomolecules such as IL-2, IL-7, IL-15, and / or IL-21. The disclosed pharmaceutical composition containing genetically modified cells contains additional drugs or biological molecules in the same composition. It may be administered as a single unit, or it may be administered simultaneously in separate compositions.

[0197] This disclosure also relates to genetically modified cells described herein for use as pharmaceuticals or Provides a population. This disclosure further provides medical treatment for diseases in those who need it. This specification provides for the use of genetically modified cells or populations thereof in the manufacture of pharmaceuticals. In one such aspect, the drug is useful for cancer immunotherapy in those who need it. ru.

[0198] In some embodiments, the pharmaceutical compositions and medicinal products of this disclosure are used in T-cell adoptive immunotherapy. This is useful for treating any disease that could be targeted. In certain embodiments, this The disclosed pharmaceutical compositions and drugs are useful as immunotherapies in cancer treatment. Non-limiting examples of cancers that can be treated with the composition and pharmaceuticals include carcinomas, lymphomas, and sarcomas. This includes, but is not limited to, melanoma, blastoma, leukemia, and germ cell tumors, B-cell tumors. Carcinomas of cystic origin, neuroblastoma, osteosarcoma, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, liver cancer, gastric cancer Bone cancer, pancreatic cancer, skin cancer, head and neck cancer, breast cancer, lung cancer, malignant melanoma of the skin or eye, kidney cancer, child Uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer Endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endocrine cancer Phosphate cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumors, phosphorus Phasmatic lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, neoplasms of the central nervous system (CNS), CNS lymphoma, tumor angiogenesis, spinal axial tumor, brainstem glioma, pituitary adenoma, Kaposi's sympathetic gland Tumors, epidermoid carcinoma, squamous cell carcinoma, and environmentally induced cancers including those caused by asbestos, Multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid lymphoma, chronic myelopathy Lymphoblastic leukemia, chronic lymphocytic leukemia, immunoblastic large cell lymphoma, acute lymphoblastic leukemia mycosis fungoides, anaplastic large cell lymphoma, and T-cell lymphoma, as well as any of these cancers This includes combinations of the following: In certain embodiments, cancers of B cell origin are, non-limitingly, B-cell lineage cancers. Lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-cell lymphoma, diffuse large cell type B-cell lymphoma, pre-B ALL (indicated for pediatric use), mantle cell lymphoma, follicular lymphoma This includes lymphoma, marginal zone lymphoma, Burkitt lymphoma, and B-cell non-Hodgkin lymphoma.

[0199] 2.8 Method of administering genetically modified cells Other embodiments disclosed herein may be used for subjects requiring the genetically modified cells of this disclosure. This is the administration of the drug. In certain embodiments, the pharmaceutical compositions described herein require the treatment of the drug. It is administered to elephants. For example, it includes the novel costimulatory domain or its active variant described herein. An effective amount of cell population can be administered to a subject with the disease. In certain embodiments, The disease may be cancer, for example, cancer of B cell origin. Therefore, this disclosure also applies to mammals. A process comprising administering CAR-T cells to a target cell population or tissue in a mammal. This provides a method for providing a cell-mediated immune response, in which the CAR specifically interacts with a given target. Interacting extracellular ligand-binding domains (e.g., tumor antigens), and at least one A signaling domain (e.g., CD3ζ) and at least one novel co- Includes an intracellular domain containing a stimulus signaling domain or an active variant thereof. Other implementations Morphologically, CAR does not contain the novel costimulatory domain described herein, but the cells are as described herein. The invention further includes an inducible regulatory construct containing at least one novel co-stimulatory domain described in the document, Dimerization of the conductive regulatory construct initiates co-stimulatory signals to the cell. In such embodiments... In this context, the method aims to promote cell proliferation and expansion of CAR-T cell populations in vivo. Inducible regulation to induce proliferative and / or survival signals in CAR-T cells. The procedure further includes the administration of a small molecule, antibody, or other molecule that induces dimerization of the construct. CAR-T cells either decrease in proliferation or decrease in number in the recipient, or It can kill target cells. Unlike antibody therapy, the genetically modified cells of this disclosure can kill target cells. It can replicate and proliferate in the biomass, which may lead to sustained control of the disease over the long term. It provides sustainability.

[0200] Possible routes of administration include parenteral administration (e.g., intravenous (IV), intramuscular (IM), intradermal, This includes subcutaneous (SC) or infusion administration. Furthermore, administration may be by continuous infusion, single or multiple infusions. This may be achieved by several bolus doses. In certain embodiments, one or both drugs Injected over a period of approximately 12 hours, 6 hours, 4 hours, 3 hours, 2 hours, or less than 1 hour. In yet another embodiment, the injection occurs slowly at first and then increases over time.

[0201] In some embodiments, the genetically modified cells of this disclosure are used to treat tumor antigens for the purpose of treating cancer. It targets the following cancers, though not limited to them: carcinoma, adenocarcinoma, lymphoma, sarcoma, black This may include, but is not limited to, chromomas, blastomas, leukemias, and germ cell tumors. B-cell origin cancer, neuroblastoma, osteosarcoma, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, liver cancer Stomach cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, breast cancer, lung cancer, skin, or intraocular malignant melanoma, kidney Thrusting cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer , fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer Endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors Cancer, lymphoid lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, new central nervous system (CNS) cancers Biology, primary CNS lymphoma, tumor angiogenesis, spinal axial tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, and environmentally induced cancers including those caused by asbestos. Myeloma, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid lymphoma, chronic Myeloid leukemia, chronic lymphocytic leukemia, immunoblastic large cell lymphoma, acute lymphoblastic Leukemia, mycosis fungoides, anaplastic large cell lymphoma, and T-cell lymphoma, and these This includes any combination of the above. In certain embodiments, cancers of B cell origin are, without limitation, B-cell acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-cell lymphoma, diffuse Large B-cell lymphoma, pre-B ALL (indicated for pediatric use), mantle cell lymphoma, follicular This includes B-cell lymphoma, marginal zone lymphoma, Burkitt lymphoma, and B-cell non-Hodgkin lymphoma. nothing.

[0202] In these embodiments in which cancer is treated with the genetically modified cells disclosed herein, In some cases, subjects who have been administered genetically modified cells are subjected to radiation, surgery, or chemotherapy. Additional therapeutic drugs, such as anticoagulants, may be administered.

[0203] Where an "effective dose" or "therapeutic dose" is indicated, the exact amount of the composition of this disclosure administered is: Age, weight, tumor size (if present), degree of infection or metastasis, and the patient's (subject's) condition. This may be determined by a physician, taking into account individual differences in physical condition. In some embodiments, this specification The pharmaceutical composition containing the genetically modified cells described is 10 4 ~10 9 Administer at a dose of cells / kg body weight. The dose is given and includes all integer values ​​within this range. In a further embodiment, the dose is 10 5 ~1 0 6 This represents cells per kg of body weight and includes all integer values ​​within this range. In some embodiments, The cell composition is administered multiple times at these dosages. Cells are generally known in immunotherapy. It can be administered using injection techniques (for example, Rosenberg et al., N See ew Eng.J.of Med319:1676, 1988. For specific patients The optimal dosage and treatment plan should be determined by monitoring the patient for signs of the disease and treating accordingly. By adjusting this, it can be easily determined by those skilled in the medical field.

[0204] In some embodiments, administration of the genetically modified cells of this disclosure may reduce the severity of a target disease or condition. At the very least, it alleviates one symptom. For example, administration of the genetically modified cells of this disclosure is of B cell origin. It can alleviate at least one symptom of cancer, such as B-cell origin cancer. The symptoms of cancer are well known in this field and can be determined by known techniques. [Examples]

[0205] experiment The present disclosure will be further illustrated by the following embodiments, which should be construed as limiting. No. A person skilled in the art can use simple, routine experiments to determine the specifics described herein. It will be possible to recognize or verify numerous equivalents of substances and procedures. The eel equivalent is intended to be included in the claims following the embodiments below.

[0206] Example 1 Construction of lentiviral vectors for expressing CARs with novel co-stimulatory domains The purpose of this study is to promote CAR-T cell expansion and cytokine secretion after antigen stimulation. The objective was to evaluate and characterize novel co-stimulatory domains developed for this purpose.

[0207] As shown in Figure 1, four novel costimulatory domains containing two TRAF-binding motifs were created. These domains were analyzed. New 1 (N1; SEQ ID NO: 5), New 3 (N3; SEQ ID NO: 6) These are called new 5 (N5; Sequence ID 7) and new 6 (N6; Sequence ID 8). To evaluate the aggressive domain, we used a lentiviral vector to detect anti-CD19 CAR-T Cells were prepared. Each CAR had a signal peptide (SEQ ID NO: 16) from 5' to 3', The FMC63 antibody has heavy and light chain variable regions linked to the G4S)3 polypeptide linker. The anti-CD19scFv (SEQ ID NO: 17), CD8 hinge region and transmembrane domain (SEQ ID NO: 17) It included, number 18), and an intracellular region containing two intracellular signaling domains. The SV40 polyadenylated (polyA) sequence (sequence number 22) is positioned 3' downstream of the CAR sequence. Some lentiviral vectors have an intracellular domain that (i) is a novel costimulatory domain. (ii) an anti-CD19 containing the CD3-ζ signaling domain (SEQ ID NO: 19) A CAR was encoded. A negative control vector encoding a CAR lacking a co-stimulatory domain (null) was used. Prepare the CD28 (SEQ ID NO: 20) or 4-1BB (SEQ ID NO: 21) costimulatory domain. and prepare further vectors encoding CARs having a CD3-ζ signaling domain. Table 1 summarizes the lentiviral vectors prepared for this study. Each CAR This is shown in Figure 2, and the respective sequences are described in sequence numbers 22-28.

[0208] [Table 1]

[0209] gag, pol, tat, and Using plasmids encoding birev, a second-generation approach was used to extract lentiviral vectors A ter was prepared. Envelope protein (VSV-G) derived from vesicular stomatitis virus was used. The virus particles were pseudotyped using a second plasmid that encodes the virus. I purchased the pCDH-EF1-MCS (from SystemBiosciences) from E Modified to include JeT promoters (Sequence ID 32) instead of F1 promoters, CAR The signal transduction mutant was cloned downstream of the promoter, followed by IRES and GFP. I cloned all three plasmids into Lenti-X-293T cells (ClonT). Transfect (purchased from ech / Takara), and after 3 days, extract lentil virus from the supernatant. The virus particles were recovered. The virus particles were collected using the Lenti-X enrichment device (ClonTech / Takar a) Concentrate using the Lenti-XqRT-PCR titration kit (ClonTech / T The viral genome count / ml was determined by quantification using Akeda, and 293T cells (A The transduction units / ml were determined by titration using TCC.

[0210] Example 2 Expression of a novel chimeric antigen receptor containing a costimulatory domain in human T cells and antigen induction Characterization in the Ress test

[0211] 1. Preparation of CAR-T cells and antigen-induced stress testing The purpose of this study was to evaluate novel co-stimulatory domains in antigen-induced stress. In short, the lentiviral vector was prepared as described in Example 1. To prepare donor human T cells for thivirus transduction, T cells are subjected to ImmunoCu lt anti-CD2 / CD3 / CD28 multimer (StemCell Technologies) and stimulated for 4 days in 20 ng / ml of IL-2. Subsequently, the cells were collected and placed in separate wells for transduction with individual lentiviral vector. 5 transduction units per T cell were added to the culture. Transduction was performed in X-VIVO15 medium (Lonza ) supplemented with only IL-2 (20 ng / ml) and 8 μg / ml of polybrene (Sigma). Prior to medium exchange, co-incubation of the vector and T cells was carried out overnight (X-Vivo15 + 20 ng / ml IL-2 + 5% normal human serum).

[0212] CAR expression began 4 days after lentiviral transduction and was confirmed by GFP analysis (Figure 3). Samples of each lentiviral-transduced T cell culture were obtained and the GFP signal was measured with a Becton-Dickinson LSR:Fortessa flow cytometer

[0213] + The GFP T cell population in each culture was identified by the region labeled CAR-GFP+ in Figure 3, and the frequency of GFP+ events is listed on each dot plot. Subsequently, 5 × 10 4 CAR-T cells were cultured with an equal number of Raji cells. At the indicated time points (d3, 6, 10, 14, 17, and 20) shown in Figure 4, cell number and viability were measured by automated cell counting and trypan blue exclusion. CAR-T cells were identified as CD4 or CD8 + using antibodies against human CD4 and CD8, and the GFP signal was identified using flow cytometry. The number of CAR-T cells was calculated and 1 × 10 + 5 4 + + 5 5 5 5 CAR-T cells were plated in 5 ×10 4 The cells were recultured with an additional number of Raji cells (2:1 effector:target ratio). D4 + CD8 + The total number of CAR-T cells was tracked and plotted over time. Then, 50 μl of the culture supernatant was collected and stored at -20°C for the triple cytokine secretion assay. It was found. The cytokine levels in the supernatant were Ultrasensitive human IL-2. Manufacturing of TNFα and IFNγ magnetic bead kits (LifeTechnologies) Measurements were taken using the original recommended method. Data was collected using a LuminexMagPix instrument. Acquired.

[0214] In the second study, CAR-T cells containing the inventors' novel costimulatory domain were used as described above. It is constructed as follows: 1 x 10 5 1 × 10¹⁶ CAR-T cells 5 Cultured together with individual Raji tumor cells At the time shown on the X-axis in Figure 6, the cell number and viability were measured using automated cell counting and tripandometry. - Measured by elimination. CAR-T cells were measured using antibodies against human CD4 and CD8. The cells were identified, and the GFP signal was identified using flow cytometry. The CAR-T count was calculated. , 1 x 10 5 1 × 10¹⁶ CAR-T cells 5 Recultured with an additional number of Raji cells Furthermore, in Figure 6, the target:effector ratio was adjusted to 1:1, and Raji cells were more frequently d Cultured at 3, 5, 7, 10, 12, 14, 17, 19, 21, 24, 26, 28, and 31. Added to the substance. CD4 + CD8 + , and track the total number of CAR-T cells over time. I did.

[0215] Four days after transduction, but before co-culturing with Raji cells, the T cell culture was prepared as described above. CAR-GFP expression was evaluated using flow cytometry. 5 T cells per cell. Transduction was performed using MOIs of individual transduction units, and the results were almost identical for all lentivirus samples. The following efficiency was observed.

[0216] 2. Experimental Results Part 1 The number of CAR-T cells was measured over time and plotted in Figure 4. The difference in the number of CAR-T cells was 1 This became apparent from day 0 onward. CAR T cells possessing the 41BBz signaling domain. It showed more sustained proliferation than those possessing the CD28z domain. In this experiment, The BB null control showed the lowest level of CAR-T expansion. New domains N5 and N6 showed high levels. This demonstrated the sustained proliferation of Bell's cells. Since each subset proliferated at a similar rate, CD4 (Figure 4B) No preference for ) or CD8 (Figure 4C) T cell expansion was observed in this proliferation assay. In descending order of performance, the results are N6>N5>41BBz>N1>>BBnull=CD2 8z > N3.

[0217] IFNγ, TNFα, and IL-2 secretion were analyzed using the Luminex multiplex assay. The measurements were taken and are shown in Figures 5A, 5B, and 5C, respectively. In general, all site capacities The secretion levels of IFNγ decreased over time. Ranking of domains related to IFNγ secretion at d3 The order of ng is 41BBz=CD28z>N1>N5>BBnull=N3>N6. IFNγ The level decreased by more than 50% in all experimental groups at d7, and continued to decrease in the rest of the experiment. I continued for a bit. The ranking of domains related to TNFα secretion is CD28z > N5 = 41B Bz.BB null > N1 = N6 > N3. Between day 3 and day 7 of culture, TNFα production Bell is reduced by approximately 50% in CD28z, BB null, N3, and N5 cultures, but 41BB z, N1, and N6 cultures maintain TNFα production levels until day 7. On the other hand, IL-2 Compared to 41BBz and CD28z, the production of all novel domain co-cultures was higher. The levels were low. IL-2 levels are measured by rapidly proliferating T cells present in some cultures. This high rate of IL-2 consumption could cause disruption (see Figure 4).

[0218] 3. Experimental Results Part 2 In the second experiment (Figure 6), more frequent antigen encounters and a higher target:effector ratio were used. Using this method, the results are in descending order: N3 > N5 > CD28z > N1 > N6 > 41BBz > BB Null. In this experiment, CAR-T cells expanded continuously throughout the culture period. CD4CA RT cells stopped expanding after about 12 days in Raji co-culture (Figure 6B), but CD8CA The RT cells continued to expand (Figure 6C).

[0219] 4. Conclusion Some novel signaling domains exhibit relatively low (Figure 4) or high (Figure 6) antigen-negative reactions. Under load, it delivers performance equivalent to or better than 41BBz and / or CD28z. N5 Signature CARs possessing a primary transmission domain are more efficient than 41BBz and CD28z under both conditions. It was excellent. N5 was similar to 41 BBs in the CD4 compartment in both experiments. Although it appears to perform well, the N5 in both experiments used the CD8 compartment. It's superior to the 41BBZ inside.

[0220] Example 3 Stress using AAV for targeted insertion of CARs in the 41BB, N1, or N6 costimulatory domains S Test

[0221] 1. Preparation of CAR-T cells and antigen-induced stress testing To evaluate novel intracellular signaling domains, CAR-T cells were generated and antigen-affected. We measured their responses to the exposure. To generate CAR-T cells, we used StemCel Using the lTechnologies CD3 Positive Selection Kit, harvested from a healthy human donor. T cells were isolated from the collected apheresis samples. In this assay, K799 and z4 Two different donors, referred to as 100, were used. T cells were treated with Immunocultan Activation and expansion were performed for 3 days using ti-CD2 / 3 / 28 (StemCellTech). After that, nucleofection using TRC1-2x87EE (Lonza4Dnuc A leofector was performed. Immediately after nucleofection, different intracellular signaling pathways were observed. AAV6 vector encoding anti-CD19 CAR, characterized by its domain, is used to phenotype cells. The CAR mutants included in this experiment are 4-1BB, N1, or N6 costimulatory dormancy. It contained . CAR expression was promoted in all vectors using the JeT promoter. Each CAR donor template is applied to the upstream and downstream regions of the TRC1-2x.87EE recognition sequence. The homologous 5' and 3' homologous arms were then placed adjacent to each other. The CAR donor mold was further 5 The ' and 3' reverse terminal repeat sequences were placed adjacent to each CAR. The donor template for each CAR is shown in Figure 7, and the CD 19-4-1BBCAR, CD19-N1 CAR, and CD19-N6 CAR The vector sequences used to generate the AAV are assigned to sequence numbers 29-31 respectively. show.

[0222] The infection multiplicity was 50,000. Five days after nucleofection / transduction, non-edited Clustered CD3+ cells were removed by magnetic removal (StemCellTechCD3+). Sex selection kit). Next, the purity of the CD3 fraction of the cells was evaluated, and anti-CD3-Bri llianViolet-711 (Biolegend), and CD19-Fc-Bioch First, using (Acro), followed by streptavidin-PE (BioLegend), the flow - CAR expression was evaluated by cytometry. The following day, T cells and CD19 expression were evaluated. A co-culture containing K562 ("K19" cells) that had been manipulated in this manner was assembled. Using the CD19-Fc+ frequency determined by the flow cytometry assay described above, CAR- The number of T cell inputs was calculated, and an effector:target (E:T) ratio of 2:1 was established. On days 3, 6, 8, and 10, the flow rate of tumor cells and CAR-T cells in the co-culture was measured. Samples were collected for tubulation evaluation. The number of CAR-T cells at each time point was measured after antigen encounter. Figure 8 plots the calculated CAR-T cells as proof of their expansion. To re-establish the 2:1 E:T ratio using the number and remaining tumor cells detected at each time point. Then, the required number of fresh K19 cells were returned to the co-culture. Furthermore, the parallel co-culture plates were changed to variable E: The T ratio was set to (2:1, 1:1, and 1:2). Samples of these co-cultures were left for 24 hours. Cells were collected after 72 hours, and the number of CD19+ cells was determined by flow cytometry. Results Figure 9 shows the number of CD19+ cells that survived co-culture with CAR-T cells, compared to the number of target cells. It serves as an indicator of lethality.

[0223] 2. Results of antigen-induced stress tests The input T cell population was normalized to the frequency of CAR+ cells, and the same number of CAR-T cells were added to E: T and T2 cells were attacked with K19 target. CAR-T cell proliferation was controlled using donor K799 (Figure 8A) and T cells generated from donor z4100 (Figure 8B) were evaluated. The T cells included TRAC. Cells edit but do not contain CAR insertions (TRCKO) and proliferate in response to antigen encounter. In contrast, CAR-T cells constructed using the 4-1BB signaling domain were not found. The cells proliferated strongly during the first week of co-culture and then contracted on day 12. N1 or N6 mutants The proliferation rate of CAR-T cells produced using this method is the rate supported by 4-1BB. No substantial differences were found. Killing of CD19+ target cells also occurred during 24-hour co-culture. The culture samples were evaluated at various E:T ratios after 1 hour and 72 hours, and the remaining CD19+ We analyzed the number of cells and compared them to a control that contained the same number of K19 cells but no CAR-T cells. The results were plotted against the wells. The number of K19 cells was lower than that of the non-T cell control, indicating cell killing and Interpret. CAR-T cells created using material derived from donor K799 are... At that point, it showed almost no cell lysis and only significant killing at the least severe E:T ratio of 2:1. As shown (Figure 9, Panel A). However, by 72 hours, a wide range of E:T ratios Lethality was observed. N1 and N6 were comparable to or worse than 4-1BB. N1 was 4- It appeared to have superior cell lysis activity compared to 1BB. It was prepared from donor z4100. In co-cultures containing CAR-T cells, a wide range of activity was observed at both 24 and 72 hours. Killing was observed (Figure 9B). As described above, N1 and N6 were compared with 4-1BB. In this case, the cell lysis activity was equivalent or superior. Generally, it was prepared from donor z4100. More widespread killing was observed in CAR-T cells. CD4 in donor K799: Note that the CD8 ratio is approximately 3:1, while the ratio in the z4100 is 1:1. Therefore, a fixed number of total CAR-T cells (as in these experiments) are important. Samples containing [the specified substance] had different numbers of cytotoxic CD8+ T cells, and z4100 was [the specified substance]. Containing approximately twice the number of CD8+ T cells, this donor-derived cell showed enhanced lethality. Explain.

[0224] 3. Conclusion The novel co-stimulatory signaling domain is supported by 4-1BB signaling at a level It was found to support proliferation and target cell killing levels equivalent to or higher than those of [another drug]. Important Furthermore, the inventors have discovered that CAR can be transmitted by randomly inserted lentiviral vectors. In addition to other data delivered, CARs produced by the inventors' targeted insertion strategy -These characteristics of N1 and N6 in T cells are shown here. This method is CA against the antigen. The difference in RT response may be due to the difference in the cumulative copy number between different CAR-T preparations. Reduce. Importantly, both random insertion and targeted insertion methods, especially N6, are natural 4-1 This demonstrated a viable alternative to the co-stimulatory support provided by BB.

[0225] Example 4 Proliferation in CAR-T cells having 41BB, N1, or N6 as a co-stimulatory domain Issei

[0226] 1. Preparation and proliferation assay of CAR-T cells containing a costimulatory domain. CAR elements featuring novel co-stimulatory domains are being extracted from lentiviral transfer vectors. The CAR element was processed and bound to a pDI vector. CAR element expression was induced by the JeT promoter. To control and enable target gene insertion, this element is positioned adjacent to the TRAC homology arm. To enable packaging into AAV6 particles, this donor template is reversed at the end. Place them adjacent to the repetitive sequence. These plasmids are first digested with restriction endonucleases and then... The cells were linearized by tanol precipitation. Subsequently, the T cells that received the initial antigen stimulation were subjected to TRC1-2× Nucleofect with 87EE, linearized CAR plasmid, and STINGsiRNA It reduced toxicity mediated by intracellular nucleic acid sensors. Lonza4D Nucleof Nucleic acid delivery was performed using a chromosome. Edited T cells were delivered to pooled 5% human serum and 30 In XVIVO-15 medium (Lonza) supplemented with ng / ml IL-2 (Gibco) They were grown using a human CD3-positive selection kit (StemCellT). After culturing for 7 days, they were selected using a human CD3-positive selection kit (StemCellT). Using echnologies, unedited CD3+ cells are magnetically removed. The cells were left to stand overnight in 2 ng / ml IL-2, and then 2 μM was administered according to the manufacturer's recommendations. Labeled with CellTrace Violet (LifeTechnologies). Violet (CTV) is a substrate for intracellular esterases, and carboxyfluoroses It functions very similarly to in-succinimidyl ester (CFSE). CTV crosses the membrane. It then diffuses into the cytoplasm, where it is cleaved by esterase enzymes, which are abundant in the cytoplasm of living cells. It is cleaved. The cleaved products do not diffuse across the cell membrane, unlike the free-moving proteins found in cytoplasmic proteins. It reacts very strongly with the amino group and is fluorescent. When labeled cells divide, the fluorescent cells... The protein divides evenly between daughter cells, with each having two cells that are half as bright as the parent generation. It becomes a cell. CTV fluorescence is measured temporally (e.g., control on day 0) or biologically (e.g., unstimulated). By comparing with cells, flow cytometry can be used to determine the frequency of dim events in CTV. By comparing the degree, the proliferation rate of various cell populations can be measured. Next, CAR, C T cells that do not express AR-4-1BB, CAR-N1, or CAR-N6 The CTV-labeled CD3 fraction was attacked with antigen-carrying tumor cells. For this assay, K562 cells that stably express CD19 were subjected to two different effector-versus-target (E:T) ratio tests. They were used in 2:1 and 1:1 ratios. Importantly, the CAR+ frequency was compared to biotinylated CD19- The results for each co-culture were determined using Fc and streptavidin-PE. T cell input The numbers were normalized based on their CAR+ frequencies. Co-culture of T cells and CD19+K562. After performing the procedure for 5 days, flow cytometry analysis was performed on CD4-PE and CD8-APC. T cells were clearly identified using antibodies (BioLegend). FlowJo software Data was acquired and analyzed using TreeStar, and proliferation was evaluated by dye dilution. did.

[0227] 2. Results of the proliferation assay The results from the proliferation assay are shown in Figure 10. The superimposed histogram in Figure 10A is 2 Proliferation of CAR-4-1BBCAR-T cells and negative control TRCK at different E:T ratios This shows a lack of proliferation of OT cells. Slightly more than half of the CAR-4-1BBT cells are CD1 9+ cells proliferated in response to target cells, while 13% of the control sample divided. (See Figure 10B superimposition) The combined histogram shows the proliferation rate of CAR-4-1BB cells relative to CAR-N1 cells. As shown, dividing cells are observed in each culture at approximately equal frequencies. Figure 10C shows superimposed histograms. The togram shows the proliferation rate of CAR-4-1BBT cells compared to CAR-N6T cells. N6 supported proliferation in 77% of cells, while 4-1BB supported proliferation in 56% of cells. This supports cell proliferation. The table below shows the frequency of dividing cells in each culture.

[0228] Table 2. Division frequency of CAR-T cells co-cultured with antigen-carrying tumor cells. [Table 2]

[0229] 3. Conclusion Lentivirus screening is an alternative and / or alternative for incorporation into CAR constructs. In an attempt to identify superior signaling domains, N6 was identified as a primary candidate. This experiment was conducted under a random insertion / variable copy number scenario typical of lentiviral delivery. Furthermore, it demonstrates the ability of a novel signaling domain to function in a single-copy target insertion scenario. These data support the possibility of designing and delivering functional signaling variants. This demonstrates that N6 is a co-stimulatory signal associated with CD3z after encounter with the antigen. It was found to be particularly superior to 4-1BB as a transducer.

[0230] Example 5 Co-stimulatory domains of 4-1BB and N6 in a mouse xenograft model of disseminated B-cell lymphoma The effectiveness of CART cells that possess

[0231] 1. Preparation of CAR-T cells and injection into tumor-bearing mice The purpose of this study is to express an anti-CD19 CAR construct containing the N6 costimulatory domain. The efficacy of manipulated CART cells was evaluated, and 4- cells incorporated into the CAR were evaluated. The objective was to compare these cells with CART cells that possess a 1BB costimulatory domain.

[0232] Anti-CD19 CAR sequences characterized by 4-1BB or N6 costimulatory domains are pDI-plated It was cloned into Sumid and used to create an AAV6 virus vector. CAR Element expression is controlled by a JeT promoter, and this donor template is TRC1-2x.87 When delivered with EE site-specific endonucleases, the TRAC inheritance of the target gene To enable insertion into the stroma, the transgene was positioned adjacent to the TRAC homology arm. The CAR-transformed gene donor template enables packaging into AAV6 particles. It was placed adjacent to the reverse terminal repeat sequence.

[0233] For the N6 co-stimulatory domain, two different CAR transgenes were created for testing. These were packaged into different AAV vectors. These two N6-containing CAR elements were To evaluate whether these polyA sequences affect the function of CAR T cells, It includes different polyadenylation (polyA) sequences that are utilized at the 3' end of the CAR-transformed gene. The poly(A) sequence used in the 7241(4-1BB) and 7205(N6) structures was present. This was the SV40 poly(A) sequence containing sequence number 33 in the 7206(N6) construct. The polyA sequences used were the first sequence containing sequence number 34 and the second sequence containing sequence number 35. This is an SV40 bipolyA sequence having the following characteristics. Table 3 shows the characteristics of the CAR constructs used in this study. These are outlined and shown in Figure 11. 7241(4-1BB) Structure, 72 To generate AAVs that encode the 05(N6) construct and the 7206(N6) construct The sequences of the vectors used are provided in sequence numbers 36-38, respectively.

[0234] AAV Vector [Table 3]

[0235] T cells that have received initial antigen stimulation are electroporated with TRC1-2×87EEmRNA. Nucleic acid delivery was performed using Lonza4DNucleofector. After tropization, the cells are pseudotransduced, or 4-1BB or N6 costimulated. AA with a donor template containing an anti-CD19 CAR transgene including one of the main ones Transduction was performed using the V6 vector.

[0236] Edited T cells were mixed with pooled 5% human serum and 30 ng / ml IL-2 (Gibc). Cells were grown in XVIVO-15 medium (Lonza) supplemented with (o). Cells were cultured for 5 days. After that, a human CD3-positive selection kit (StemCell Technologies) was used. Then, unedited CD3+ cells were removed magnetically. The cells were cultured for a further 3 days.

[0237] NSG mice (n=5 per group) were given 2e5 Ra mice expressing firefly luciferase. ji cells (Raji-ffluc) were injected. Three days later, each mouse was given a 1e6 control TCR. KO cells, or 7205(N6), 7206(N6), or 7241(4-1BB) vectors 1e6CAR T cells prepared using a ter were injected. On the day shown, living MAU Luciferin substrate (150 mg / kg in saline) was injected intraperitoneally into the patient, anesthetized, and 7 minutes later... Lucifer using IVIS spectra (PerkinElmer, Waltham, MA) Gelase activity was measured. LivingImagesoftware4.5.2(Per The data was analyzed and exported using kinElmer, Waltham, and MA. The luminescence signal intensity in the image is expressed as radiance in p / sec / cm² / sr. Using the entire object as the target area, LivingImagesoftware4.5.2( The total flux was also calculated using PerkinElmer, Waltham, MA. Mice are monitored for signs of disease progression and euthanized according to predetermined criteria if necessary. Set.

[0238] 2. Results of the mouse xenograft model Mouse xenotransplantation for disseminated B-cell lymphoma to evaluate the effectiveness of CAR constructs listed in Table 3. The results of the model are shown in Figures 12 to 14.

[0239] Ventral and dorsal imaging of mice 7 days after Raji-ffluc injection revealed that TC Engraftment and growth of Raji-ffluc cells were possible in control mice administered RKO cells. These are visualized and indicated by the increase in luminescence signals on day 10 and day 16. Rapid growth of Raji cells was observed in these mice (Figures 12, 13A, and 13C). In contrast, structures 7205(N6), 7206(N6), and 7241(4-1BB) Treatment of mice with CAR T cells containing the specified gene resulted in delayed tumor growth (Figure 12 and (Figure 13). As evidenced by dorsal and ventral imaging of the animals, Raji growth is A subset of mice from groups 7205(N6) and 7241(4-1BB)CAR T was found to have odor It was detected that it would start around the 20th day. However, 7206(N6)C No significant tumor growth was observed in the AR T treatment group during the 40-day study.

[0240] As shown in Figure 14, all five mice in the TRCKO control group developed diseases including complete hindlimb paralysis. Due to the rapid onset of disease-related symptoms, the animal was euthanized on the 19th day. However, 7205(N6 Treatment with CAR T cells produced using ) and 7206(N6) vectors was performed on mice. This enhanced survival, and all mice in these groups remained alive on day 40 (720 One mouse in group 6 died unrelated to tumor growth or CAR T injection, and was removed from the study. (Excluded). Mice treated with the 4-1BB-containing CAR construct of 7241 were also included in this treatment group. The lifespan of the mice was extended, with one mouse requiring euthanasia on day 38, and the other four... The mice survived throughout the 40-day study.

[0241] 3. Conclusion Raji-fflucCD19 + Mice transplanted with cells possessing the N6 costimulatory domain When treated with anti-CD19 CAR T cells expressing second-generation CARs (7205 and 7 In both configurations, the survival time of mice was compared to mice treated with TCRKO cells. This resulted in prolongation and a dramatic reduction in tumor burden. Importantly, throughout the 40-day study... The 7205(N6) structure exhibits performance equivalent to that of 72414-1BB-containing CAR. It appears that the 7206(N6) construct is effective in sustained suppression of Raji cell growth. It appeared to be superior to both the 05(N6) configuration and the 7241(4-1BB) configuration. Overall, these data suggest that the N6 costimulatory domain functions as a costimulatory domain. Inconsistent with experiments evaluating the in vitro activity of the construct, CD19 was detected in vivo. + Kill the target This supports in vitro findings that support the capabilities of CAR T cells. Furthermore, N6 coscinate Constructs with a stimulation domain are active CAR T constructs with a 4-1BB co-stimulation domain It matches or exceeds the sex.

[0242] Example 6 Characterization of third-generation CARs containing multiple co-stimulatory domains

[0243] 1. Creation of CAR T cells expressing third-generation CARs To evaluate novel costimulatory domains included in the present invention as part of third-generation CAR Further constructs were prepared, in which the intracellular signaling domain has two co-stimulatory domains and It contains one CD3-ζ signaling domain.

[0244] In a specific embodiment, the signal sequence (sequence number 16) and FMC63 are located at 5' to 3'. The base CD19-specific scFv (SEQ ID NO: 17), the above CD8 hinge and transmembrane suppository In (SEQ ID NO: 18), followed by the MyD88 co-stimulatory domain (SEQ ID NO: 39; internationally publicly available). (Sequence obtained from No. 2016 / 036746), N6 costimulatory domain (SEQ ID NO: 8), and a third-generation anti-CD19 C containing the CD3-ζ signaling domain (SEQ ID NO: 19). An AR was prepared. The SV40 bipoly A signal sequence, including SEQ ID NOs. 34 and 35, is It was located 3' downstream of the CAR array. As described in the previous example, this structure was The sequence to be expressed was cloned into a pDI plasmid, and CAR expression was directed to the JeT promoter. Therefore, it was controlled. Furthermore, this donor template was TRC1-2x.87EE site-specific endodontic When delivered together with crease, it enables the insertion of the target gene into the TRAC locus. To achieve this, the transgene was placed adjacent to the TRAC homology arm. - The template was further positioned adjacent to the reverse terminal repeat sequence. MyD88 / N6 CAR donor template As shown in Figure 15, the sequence of the vector containing the donor template is provided as sequence number 40.

[0245] In some experiments, the CAR donor template linearized DN after linearization of the pDI plasmid. It will be delivered as A. In other cases, the donor template will be AAV for virus delivery. It will likely be packaged into 6 particles.

[0246] 2. Evaluation of MyD88 / N6CAR T cells regarding cytotoxicity, proliferation, and cytokine secretion. price In several experiments, MyD88 / N6CAR T cells received initial antigen stimulation. T cells are linearized using a CAR template plasmid, TRC1-2x.87EE meganuclease, and Nucleofection is performed with STINGsiRNA, and the process is mediated by intracellular nucleic acid sensors. By reducing the toxicity, it is manufactured as described above in Example 4. MyD88 / N Further growth and expansion of 6CAR T cells as described.

[0247] Regarding the cytotoxicity, proliferation, and cytokine secretion of MyD88 / N6CAR T cells Characterize the cells. To evaluate their cytotoxicity and proliferation, MyD88 / N6CAR T cells are selected. As described above in Example 3, the antigen-induced stress test was performed, and here MyD88 / N6CAR T cells express CD19 ("K19" cells) in various effector:target ratios. Co-culture with manipulated K562 cells. For proliferation, use cell trace violet. Cells are labeled and co-cultured with antigen-carrying K19 cells in various effector:target ratios. The evaluation is performed as described in Example 4. Cytokine secretion (e.g., human IL) -2, TNFα, and IFNγ) are co-cultured with K19 cells in various effector:target ratios. After that, the decision is made as described in Example 2 above.

[0248] Similar experiments investigating cell killing, proliferation, and cytokine secretion have shown that recombinant AAV particles possess certain characteristics. Using induction, the CAR donor template is delivered to T cells that have received initial antigen stimulation, and TRC1-2 This is further nucleofected with mRNA encoding ×.87EE meganuclease. conduct.

[0249] 3. Proliferation of MyD88 / N6 CAR T cells To compare the functions of N6 and MyD88 / N6 costimulatory domains in CAR T cells Therefore, a novel third-generation CA containing the N6 costimulatory domain or MyD88 / N6 costimulatory domain Linear plasmid DNA expressing R is used in TRC1-2x.87EE site-specific endoplasma Human T cells were nucleofected with crease and STINGsiRNA. MyD The 88 / N6CAR code construct is shown in Figure 15. The construct that codes for N6CAR is 720 It is called 6 and provided as sequence number 38. The construct that codes MyD88 / N6CAR It is called 7240 and provided as sequence number 40.

[0250] After nucleofection, cells were treated with 5% FBS and 30 ng / ml IL-2 (Gibc). The cells were grown for 5 days in X-Vivo medium (Lonza) supplemented with (o). On the 5th day, the remaining CD3 + T cell selection kit II for human CD3-positive cells (StemCellTechnolo Labeled using gies) and magnetically removed according to the manufacturer's recommendations. Remaining CD3 depletion. The fractions were supplemented with 10 ng / ml of IL-15 and 3 ng / ml of IL-21 (Gibco). The samples were resuspended in the added X-Vivo medium and grown for a further 2 days. Samples for the assay were then prepared. To do so, 2e from N6 and MyD88 / N6 conditions 6 T cells, and TRC1-2x. Control T cells treated only with 87EE were lysed in vitro with cell trace violet (CTV). Labeled with a 2 μM solution of the liquid. After incubation, CD19-biotin Fc(Acro After staining with Biosystems and streptavidin PE (BD), CTV labeling was performed. Consistency and CAR T cell frequency in Becton-Dickinson LSR: Fort Evaluation was performed using an ESSA flow cytometer. For the proliferation assay, cytokines were added. In X-Vivo medium, 2e in 2 wells on a 96-well round-bottom plate. 5 All T-shirts are thin. Cell (2e 3 Adding CAR T cells, and adding the CAR T cell frequency, 1% of the total T cell population Normalized to %. To evaluate antigen-specific CAR T cell proliferation, non-specific background data was used. 4e 3 Add K19 cells to one well, 4e 3 K562 thin The cells were added to the second well. The cells were mixed and incubated for a total of 6 days.

[0251] Six days after co-culture, the cells were centrifuged and washed twice with PBS. Individual T cell subsets To analyze proliferation, samples were treated with CD4BV711 and CD8BV7 to eliminate dead cells. 85 antibody (Biolegend), and ghost dye BV510 (TONBObios The sample was stained with (sciences). After staining, the sample was flushed and the data was collected using Becton-Dickey. nsonLSR:Collected using a Fortessa flow cytometer.

[0252] 4. Results of proliferation studies Figure 16 shows the results of a growth assay comparing the N6 and MyD88 / N6 costimulatory domains. To measure background proliferation in the CAR-negative population, TRC-only nucleooff was used. CTV dilutions in T cells derived from the test control sample were used in K19 cells or K562 cells. They were compared in wells co-cultured with K1 Similar levels of nonspecific proliferation were observed in the presence of cells 9 (faint shadow) and K562 cells (dark shadow). This suggests that all proliferation processes are CD19-independent (Figures 16A and 16B). In comparison, linear plasmid DNA expressing the N6 costimulatory domain is used for nucleofects. CD4 + and CD8 + Both T cells show the presence of K19 cells compared to the K562 control. The lower values ​​showed greater CTV dilution and substantial antigen-specific proliferation (Figures 16C and 16C and 16C). 6D). In particular, the same evaluation performed in T cells expressing the MyD88 / N6 costimulatory domain However, K19 cells showed greater proliferation of CAR T cells in response (Figure 16E and (Figure 16F). Overall dilution of CTVs is observed in CAR T cells expressing the N6 costimulatory domain alone. Compared to cytoplasm, MyD88 / N6-expressing T cells had fewer of the same co-stimulatory domains. Expression of TRC resulted in greater dilution of CTVs compared to control T cells with TRC alone.

[0253] 5. Conclusion T cells that have received initial antigen stimulation are stimulated by the N6 or third-generation MyD88 / N6 costimulatory domain. Nucleofection with linearized plasmid DNA expressing either of these results in rare CTVs. As shown by the analysis, CART cells capable of antigen-specific proliferation were obtained. Furthermore, proliferation CD4 + and CD8 + This occurs in both T cell subsets and in non-CAR-expressing control cells. It occurred above the background level where it was observed. In summary, this study found that N6 and Both MyD88 and N6 can function as costimulatory domains in CAR T cells. This was demonstrated.

[0254] Example 7 Characterization of novel co-stimulatory domains in inducible constructs

[0255] 1. Inducible construction of CAR T cells expressing first-generation CARs and novel costimulatory domains. Manufacturing of objects Co-expressed with first-generation anti-CD19 CARs containing the CD3-ζ signaling domain. To evaluate novel co-stimulatory domains included in the present invention as part of an inducible co-stimulatory construct Further structures were prepared.

[0256] In a specific embodiment, the expression cassette for the inducible co-stimulatory construct is located at 5' to 3'. A structure including a T2A element and a CAR expression cassette encoding a first-generation anti-CD19 CAR. The structure was prepared.

[0257] The first-generation anti-CD19 CAR encoded by the CAR expression cassette is 5' to 3'. ', signal sequence (sequence number 16), FMC63-based CD19-specific scFv (distribution Column number 17), and the intracellular region contains the CD3-ζ signaling domain (SEQ ID NO: 19). It contained the above-mentioned CD8 hinge and transmembrane domain (SEQ ID NO: 18). SEQ ID NO: 34 The SV40 bipoly A signal sequence, including sequence number 34, is located 3' downstream of the CAR sequence. They were doing it.

[0258] The inductive co-stimulus construct has the N6 co-stimulus domain alone (SEQ ID NO: 8) from 5' to 3', This includes a tandem of the MyD88 domain (sequence number 39) and the N6 domain (sequence number 8). Two tandem ligand-binding FKBP12v36 domains (SEQ ID NO: 41; International Publication) The Fv domain, which contains the sequence obtained from No. 2015 / 123527, is then subdivided It binds to the rimiducid and induces construct dimerization and activation of co-stimulatory signaling. To guide.

[0259] As described in the previous example, these constructs were cloned into a pDI plasmid, The expression of both the inducible co-stimulatory construct and the anti-CD19 CAR can be suppressed by the JeT promoter. It was controlled. Furthermore, these structures are TRC1-2x.87EE site-specific endonuclei. To enable the insertion of the target gene into the TRAC locus when delivered with the enzyme. These structures were placed adjacent to the TRAC homology arms. These structures were further adjacent to the reverse terminal repeat sequence. I made them do it.

[0260] In some experiments, the donor template was linearized DNA after linearization of the pDI plasmid. It will be delivered as follows. In other cases, the donor template will be used for AAV6 virus delivery. It will likely be packaged into particles.

[0261] Anti-CD19 CAR expression along with an inducible costimulatory construct containing only the N6 costimulatory domain. These cells are called iN6 CAR T cells. Both the MyD88 and N6 costimulatory domains are involved. Cells expressing anti-CD19 CAR together with an inducible costimulatory construct containing iMyD88 These are called N6CAR T cells.

[0262] 2. CART cells using inducible constructs for cell killing, proliferation, and cytokine secretion. evaluation In some experiments, iN6CAR T cells or iMyD88 / N6CAR T cells The cells are linearized using a template plasmid, TRC1-2x.87EE, to form T cells that have received initial antigen stimulation. Nucleofection with meganuclease and STINGsiRNA to obtain intracellular nucleic acids. By reducing the toxicity mediated by the sensor, the fabrication described above in Example 4 was achieved. To do so, further describe iN6CAR T cells or iMyD88 / N6CAR T cells as described. To grow and expand.

[0263] Small molecule ri induces dimerization of inducible constructs and initiates co-stimulatory signaling within cells. Cytotoxicity, proliferation, and cytokine secretion in the presence and absence of miducid Regarding this, we will characterize iN6CAR T cells and iMyD88 / N6 CAR T cells. To evaluate cytotoxicity and proliferation, CAR T cells were subjected to the antigen induction described in Example 3. CAR T cells were subjected to stress tests and targeted at various effectors:CD19("K") Co-culture with K562 cells manipulated to express "19" cells. Regarding proliferation, Cells are labeled with cell trace violet, and antigen-carrying K19 cells and various effects are observed. The ter is evaluated by co-culturing at the target ratio, as described in Example 4. Infusion secretion (e.g., human IL-2, TNFα, and IFNγ) acts as a various effector: After co-culturing with K19 cells at the target ratio, the determination is made as described in Example 2 above.

[0264] Similar experiments investigating cell killing, proliferation, and cytokine secretion have shown that recombinant AAV particles possess certain characteristics. Using induction, the donor template is delivered to T cells that have received initial antigen stimulation, and TRC1-2×.8 This is further nucleofected with mRNA encoding 7EE meganuclease.

[0265] 3. Proliferation of iMyD88 / N6CAR T cells To characterize the functionality of novel co-stimulatory domains in inducible constructs, initial antigen stimulation is performed. Degenerate T cells expressing the iMyD88 / N6-inducible costimulatory construct using a linear plasmid DN A, or as a control, the N6 costimulatory domain expressed as part of CAR, The cells were treated with TRC1-2x.87EE site-specific endonuclease as described above. Furthermore, nucleofectation was performed with STINGsiRNA. As a negative control, TRC1-2 Using only x.87EE site-specific endonuclease and STINGsiRNA, The sample was nucleofected. The CAR donor mold structure is shown in Figure 17. As described above. The construct that codes for N6 CAR is called 7206 and is provided as sequence number 38. The construct encoding the MyD88 / N6 costimulatory domain is called 7235, and sequence number 42 and To provide.

[0266] After nucleofection, T cell samples were treated with 5% FBS and 30 ng / ml IL-2 ( The mixture was left to stand for 6 hours in X-Vivo(Lonza) medium supplemented with Gibco. Then, The sample was divided in half into two separate wells, and one well was filled with rimid at a final concentration of 5 nanomoles. I added ucid to one well and left the other well untreated. Then I incubated the cells for 5 days. I did it. On the 5th day, I used the remaining CD3 + T cells selected using the Human CD3-Positive Selection Kit II (StemC Labeled using ellTechnologies, and magnetically removed according to the manufacturer's recommendations. The remaining CD3-depleted fraction was treated with 10 ng / ml IL-15 and 3 ng / ml IL- The nucleotides were resuspended in X-Vivo medium supplemented with 21 (Gibco). After nucleofection... Samples to which rimiducid was added on day 0 were given fresh rimiducid at a final concentration of 5 nanomoles. Ducid was spiked, but the untreated samples were re-treated only with cytokine-added X-Vivo. The solution was then clouded. Next, the cells were incubated for another two days.

[0267] Whether or not rimiducid was added to the sample for the assay. iMyD88 / N6 conditions and N6 conditions 2e 6 T cells and TRC1-2x.87EE only Control T cells were treated with a 2 μM solution of cell trace violet (CTV) in vitro. Labeled with CD19-biotinFc(AcroBiosyst). After incubation, CD19-biotinFc(AcroBiosyst) After staining with ems and streptavidin PE (BD), the consistency of the CTV label and C AR T cell frequency in Becton-Dickinson LSR:Fortessa flow CAR T cell frequency was evaluated using a cytometer. CAR T cell frequency was measured using XV cells without cytokine supplementation. 2e in two separate wells on a 96-well round-bottom plate in IVO medium 5 Total T cells (2e 3 CAR T cells were added and normalized to 1% of the total T cell population to which the cells were added. , 4e in one well 3 The target K19 cells were placed in one well, and 4e cells were placed in the other well as a control. 3 of K562 cells were introduced. Next, rim was used on day 0 and day 5 after nucleofection. Rimiducid was added to the sample containing iducid. The cells were mixed and left for a total of 6 days. It was incubated.

[0268] On day 6 after co-culture, the samples were stained for flow cytometry analysis. To quantify the proliferation of Busset, the sample was treated with CD4BV711 and CD8BV785 antibodies ( Stained with Biolegend, and ghost dye BV510 (TONBObioscien Dead cells were eliminated during analysis by adding CES to the staining cocktail. After staining, the test The data is sent via the Becton-DickinsonLSR:Fortessa flow. The data was collected using a cytometer.

[0269] 4. Results of proliferation studies Results of a growth assay comparing the novel inducible costimulator iMyD88 / N6 with N6CAR The results are shown in Figures 18-20. CD from nucleofect cells containing only non-CAR-expressing TRCs. 4 + and CD8 + T cells are either K19 cells (light shadow) or K562 cells (dark shadow). When co-cultured with either of them, they showed similar levels of nonspecific growth (Figures 18A and 18A). B).

[0270] In contrast, proliferation of CAR T cells expressing N6CAR is observed in K562 cells (dark shadow). Conversely, when co-cultured with K19 cells (faint shadow), the dilution of CTV was greater, It was antigen-dependent (Figures 19A and 19C). Furthermore, C expressed non-inducible N6CAR. AR T cells are observed in the presence or absence of rimiducid and K19 cells (dark shadow). (Faint shadows) indicate substantial proliferation of both T cell subsets (Figures 19B and 19D). In the absence of the switch-dependent costimulatory domain, rimiducid has no function whatsoever. They showed that they would not do so.

[0271] Regarding the inducible co-stimulatory construct, CD4 expressing iMyD88 / N6 + and CD8 + T Both types of cell proliferation were observed in K19 cells (lighter shadow) compared to K562 control cells (dark shadow). This was more frequent when co-cultured with (Figures 20A and 20C). Importantly, CTV dilution The increase was observed when both T cell subsets analyzed were cultured with K19 cells. Compared to the sample without miducid treatment (faint shadow), the rimiducid sample (dark shadow) (shadow) relied on supporting the induction function of the iMyD88 / N6 switch (Figures 20B and 20) D).

[0272] 5. Conclusion Expression of the novel inducible costimulatory construct iMyD88 / N6 on CAR T cells is antigen-dependent. CD4 + and CD8 + This resulted in the proliferation of both T cells. Surprisingly, the cultured T The dilution of CTVs on cells is greatest in the presence of rimiducid, and in CAR T cells. This shows the inducible properties of the co-stimulatory domain construct when expressed. Co-stimulatory domain When expressed as part of CAR, rimiducid has an effect on CAR T cell proliferation. Because it had no effect, these data represent a novel switch on CAR T cell function. This supports the functionality of the co-stimulatory domain.

Claims

1. For the amino acid sequence described in any one of SEQ ID NOs: 8, 5, 6, or 7, at least Nucleic acids containing nucleotide sequences encoding a costimulatory domain with 80% sequence identity. molecule.

2. The claim 1 states that the costimulatory domain comprises at least one TRAF-binding motif. nucleic acid molecules.

3. The TRAF binding motif is selected from the group consisting of SEQ ID NOs: 10, 9, and 11. The nucleic acid molecule according to claim 2.

4. The aforementioned co-stimulatory domain consists of two TRAF-binding motifs separated by a spacer sequence. A nucleic acid molecule according to any one of claims 1 to 3, comprising:

5. Claim 4, the spacer is selected from the group consisting of sequence numbers 15 and 12-14. The nucleic acid molecules described.

6. The aforementioned co-stimulatory domains are separated by spacer sequences, sequence numbers 10, 9, and 11 The spacer array includes two TRAF-binding motifs selected from the group consisting of the following, and the spacer array is an array The group consisting of numbers 15 and 12 to 14, as described in any one of claims 1 to 5. nucleic acid molecules.

7. The aforementioned co-stimulatory domains are separated by spacer sequences, TRs 9 and 11 A nucleic acid molecule according to any one of claims 1 to 6, comprising an AF-binding motif.

8. The nucleic acid molecule according to claim 7, wherein the spacer sequence includes sequence number 12.

9. The nucleic acid molecule has at least 80% sequence identity with respect to Sequence ID No.

1. Claim 7 or the claim that includes a nucleotide sequence, wherein the nucleotide sequence encodes a co-stimulatory domain. The nucleic acid molecule described in item 8.

10. The nucleotide sequence comprises a costimulatory domain containing the amino acid sequence described in Sequence ID No.

5. A nucleic acid molecule according to any one of claims 7 to 9.

11. The aforementioned co-stimulatory domains are separated by spacer sequences, as shown in sequence numbers 10 and 11. A nucleic acid molecule according to any one of claims 1 to 6, comprising an RAF-binding motif.

12. The spacer array is selected from the group consisting of sequence numbers 15, 13, and 14. The nucleic acid molecule described in item 11.

13. The nucleic acid molecule contains at least 80% of any one of SEQ ID NOs: 4, 2, or 3. It contains a nucleotide sequence having sequence identity, and the nucleotide sequence has a co-stimulatory domain A nucleic acid molecule according to claim 11 or 12, which codes for.

14. The nucleotide sequence is the amino acid sequence described in any one of SEQ ID NOs: 8, 6, or 7. Nucleic acid component according to any one of claims 11 to 13, which encodes a co-stimulatory domain including a column child.

15. The nucleic acid molecule comprises a nucleotide sequence encoding a chimeric antigen receptor (CAR), The CAR comprises at least one co-stimulatory domain as described in any one of claims 1 to 14. The nucleic acid molecule according to any one of claims 1 to 14.

16. The CAR contains the amino acid sequence described in any one of SEQ ID NOs: 8, 5, 6, or 7. The nucleic acid molecule according to claim 15, comprising at least one co-stimulatory domain.

17. The CAR comprises at least two co-stimulatory domains, and at least one of the co-stimulatory domains The other is a co-stimulatory domain as described in any one of claims 1 to 14, claim 15 or The nucleic acid molecule according to claim 16.

18. The CAR is at least two co-stimulatory domains as described in any one of claims 1 to 14. A nucleic acid molecule according to claim 17, comprising:

19. Claims 15- A nucleic acid molecule as described in any one of item 18.

20. The nucleic acid according to claim 19, wherein the intracellular signaling domain is a CD3ζ domain. molecule.

21. The nucleic acid molecule comprises a nucleotide sequence encoding an inducible regulatory construct, and the inducible regulatory construct The segmental construct comprises at least one co-stimulatory domain as described in any one of claims 1 to 14. The nucleic acid molecule according to any one of claims 1 to 14.

22. The inductive regulatory construct is a bond that dimerizes two of the inductive regulatory constructs. The main further comprises the main, and dimerization initiates a co-stimulatory signal to the cell, as described in claim 21. Nucleic acid molecule.

23. The nucleic acid molecule according to claim 22, wherein the binding domain binds to a small molecule or an antibody.

24. The nucleic acid component according to claim 22 or 23, wherein the binding domain includes an analogue of FKBP12. child.

25. The nucleic acid molecule is mRNA, recombinant DNA construct, or viral vector. A nucleic acid molecule according to any one of claims 1 to 24, which is a nom.

26. A recombinant DNA construct comprising the nucleic acid molecule according to any one of claims 1 to 24.

27. The recombinant DNA construct encodes a viral vector, and the viral vector is required Recombinant DN according to claim 26, comprising the nucleic acid molecule described in any one of claims 1 to 24. A structure.

28. The viral vector is a recombinant adeno-associated virus (AAV) vector, according to the claim. Recombinant DNA constructs as described in 27.

29. A viral vector comprising the nucleic acid molecule according to any one of claims 1 to 24.

30. The viral vector according to claim 29, wherein the viral vector is a recombinant AAV vector. Kuta.

31. Genetically modified cells containing the nucleic acid molecule described in any one of claims 1 to 24.

32. The gene-modified cells contain the nucleic acid molecule described in any one of claims 1 to 24. Genetically modified cells according to claim 31, including the current cassette.

33. The nucleic acid molecule or the expression cassette is present in the genome of the genetically modified cell, claim Genetically modified cells as described in item 32.

34. The nucleic acid molecule or the expression cassette is not incorporated into the genome of the genetically modified cell. i. Genetically modified cells according to claim 32.

35. The nucleic acid molecule or the expression cassette constructs the recombinant DNA in the genetically modified cells. In a substance, it is present in mRNA or in the viral genome, and in the genome of the genetically modified cell. Genetically modified cells according to claim 34, which are not incorporated into the above.

36. The gene-modified cell has the costimulatory domain described in any one of claims 1 to 24 Claims 31-35 include an expression cassette containing a nucleotide sequence encoding a CAR. Genetically modified cells as described in any one of the items.

37. The aforementioned genetically modified cells (i) A CAR that does not contain the co-stimulatory domain described in any one of claims 1 to 24 A CAR expression cassette containing a nucleotide sequence, (ii) Regulation encoding an inductive regulatory structure according to any one of claims 21 to 24 Expression cassette and A genetically modified cell according to any one of claims 31 to 35, including the above.

38. The aforementioned genetically modified cells (i) A CAR that does not contain the co-stimulatory domain described in any one of claims 1 to 24 The nucleotide sequence that is used, (ii) Nucleus encoding the inductive modulating structure according to any one of claims 21 to 24 The leotide sequence and Genetically modified cells according to any one of claims 31 to 35, comprising an expression cassette containing 。

39. The aforementioned genetically modified cells (i) A CAR that does not include the costimulatory domain as described in any one of claims 1 to 24 、 (ii) an inductive regulatory domain according to any one of claims 21 to 24, A genetically modified cell according to any one of claims 31 to 35, including the above.

40. The gene-modified cell is a gene-modified eukaryotic cell, according to any one of claims 31 to 39. Genetically modified cells as described.

41. Any one of claims 31 to 40, wherein the genetically modified cells are genetically modified human T cells. Genetically modified cells as described above.

42. The aforementioned genetically modified cells contain the amino acids described in any one of Sequence ID No. 8, 5, 6, or 7. Control cells lacking a co-stimulatory domain that exhibits at least 80% sequence identity with respect to the sequence. Compared to, any of claims 31 to 41 shows increased proliferation and / or cytokine secretion. Genetically modified cells as described in item 1.

43. A gene comprising at least one co-stimulatory domain as described in any one of claims 1 to 24. A method for producing modified cells, wherein the method is described in at least one of claims 1 to 24. A method that involves introducing a single nucleic acid molecule into a cell.

44. The nucleic acid molecule codes for CAR, as described in any one of claims 15 to 20. The method according to claim 43, wherein the molecule is a nucleic acid molecule.

45. The nucleic acid molecule is described in any one of claims 21 to 24 as encoding an inducible regulatory construct. The method according to claim 43, wherein the nucleic acid molecule is as described above.

46. A method according to any one of claims 43 to 45, wherein the method is (i) A second nucleic acid molecule encoding an engineered nuclease, wherein the engineered A nuclease is expressed in the aforementioned cell as a second nucleic acid molecule, or (ii) Manipulated nuclease proteins The further method includes introducing the following into the cells: The manipulated nuclease recognizes the cell's genome to generate cleavage sites. Recognizes and cuts the sequence, The nucleic acid molecule encoding the at least one co-stimulatory domain is preceded by the cleavage site. A method for inserting into the genome of a cell.

47. The manipulated nuclease is a manipulated meganuclease, recombinant zinc finger - Nuclease (ZFN), recombinant transcription activator-like effector nuclease (TALE) N), CRISPR / Cas nuclease, or megaTAL nuclease, The method according to claim 46.

48. Claim 46 or 4, wherein the manipulated nuclease is a manipulated meganuclease. The method described in 7.

49. The nucleic acid molecule according to any one of claims 1 to 24 is characterized in that the nucleic acid molecule undergoes homologous recombination. Therefore, the adjacent parts of the cleavage site are arranged so that they are inserted into the genome of the cell at the cleavage site. The method according to any one of claims 46 to 48, further comprising homologous sequences in the columns.

50. The nucleic acid molecule according to any one of claims 1 to 24, wherein the nucleic acid molecule has non-homologous terminals Substantial homology with respect to the cleavage site so that it is inserted into the genome of the cell by binding. The method according to any one of claims 46 to 48, which lacks the property.

51. The gene-modified cell is a gene-modified eukaryotic cell, according to any one of claims 43 to 50. Method of description.

52. The genetically modified cell is a genetically modified human T cell, any one of claims 43 to 51. Methods used.

53. The nucleic acid molecule according to any one of claims 1 to 24 is the mR according to claim 25. The method according to any one of claims 43 to 52, wherein the cells are introduced using NA.

54. The nucleic acid molecule according to any one of claims 1 to 24, which is any one of claims 26 to 28. Claims 43 to 52, which are introduced into the cells using the recombinant DNA construct described in paragraph 1. The method described in any one of the items.

55. The nucleic acid molecule according to any one of claims 1 to 24, according to claim 29 or claim 30 Any of claims 43 to 52, which are introduced into the cells using the viral vector described above. The method described in item 1.

56. The genetically modified cells, compared to control cells that do not contain a costimulatory domain, After introducing any one of the nucleic acid molecules described in items 1 to 24, activation, proliferation, and / or cytochemistry occur. The method according to any one of claims 43 to 55, which shows an increase in insulin secretion.

57. A pharmaceutically acceptable carrier and the gene modification described in any one of claims 31 to 42. A pharmaceutical composition containing cells.

58. A method of immunotherapy for treating cancer in a person who requires it, the aforementioned The law administers the genetically modified cells described in any one of claims 36 to 42 to the subject. A method that includes doing so.

59. The method includes administering the pharmaceutical composition described in claim 57, and the pharmaceutical composition The gene-modified cell described in any one of claims 36 to 42, as described in claim 58. Method of loading.

60. Claim 58 or Claims, wherein the cancer is carcinoma, lymphoma, sarcoma, blastoma, or leukemia. The method described in item 59.

61. The aforementioned cancers include B-cell origin cancers, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, From prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, and Hodgkin lymphoma The method according to any one of claims 58 to 60, selected from the group.

62. The aforementioned B-cell origin cancers include B-lineage acute lymphoblastic leukemia and B-cell chronic lymphocytic leukemia. The method according to claim 61, selected from the group consisting of , and B-cell non-Hodgkin lymphoma.

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