Anti-EGFR chimeric antigen receptor

Abstract

Disclosed herein is a polynucleotide comprising a human-codon-optimized sequence encoding a polypeptide comprising EGFR806CAR. To optimize its expression, the human-codon-optimized sequence may be incorporated into a construct comprising an optimally functioning promoter, spacer, intracellular signaling domain, transmembrane domain, selectable marker, at least one self-cleaving peptide, and EFGRt. This sequence may then be expressed in cells, such as T cells, for the treatment or suppression of cancers, such as glioblastoma, liquid tumors, and solid tumors.

Description

Technical Field

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 234,090, entitled "Anti-EGFR Chimeric Antigen Receptor," filed on August 17, 2021, and U.S. Provisional Patent Application No. 63 / 122,839, entitled "Therapeutic Sequences of Codon-Optimized EGFR806CAR," filed on December 8, 2020, the entire disclosures of which are hereby expressly incorporated by reference herein.

[0002] Sequence listing reference This application is filed with an electronic sequence listing. This sequence listing is provided as an approximately 11 kilobyte file created on December 1, 2021, with the file name SCRI348WOSEQLIST. The information set forth in this electronic sequence listing is hereby expressly incorporated by reference herein in its entirety.

[0003] Aspects of the disclosure generally relate to anti-EGFR chimeric antigen receptors (CARs) and T cells comprising such CARs. Some embodiments relate to enhancing the expression of anti-EGFR CARs, methods of expressing anti-EGFR CARs, and methods of enhancing the expression of anti-EGFR CARs to target cancers such as glioblastoma, liquid tumors, and solid tumors.

Background Art

[0004] Numerous treatment methods are used to treat cancer. Common treatments currently employed include surgery, chemotherapy, radiation therapy, hormone therapy, targeted drug therapy, and cell therapy. Cell therapy for treating patients with cancer or other diseases involves injecting cellular materials, such as living cells, into the patient in need of treatment. Cell therapy may include the injection of polyclonal T cells, antigen-specific T cells, activated killer cells, natural killer cells, dendritic cells, or macrophages. Recently, the development of T cells carrying chimeric antigen receptors (CARs) has progressed and is considered a promising treatment pathway for cancer immunotherapy and viral therapy.

[0005] CAR T-cell therapy is a type of immunotherapy in which T cells are isolated in a laboratory, genetically modified to express synthetic receptors that recognize specific antigens or proteins presented on cells such as cancer cells, and then reinjected into the patient. Although promising antitumor activity has been demonstrated in clinical trials, CAR T-cell therapy still suffers from drawbacks such as insufficient cell activation, a short half-life, and inefficient targeting of cancer tissue. Therefore, further approaches to CAR T-cell therapy are strongly needed. [Overview of the Initiative] [Means for solving the problem]

[0006] The various embodiments provided herein relate to various polynucleotides comprising human-codon-optimized sequences encoding anti-EGFR chimeric antigen receptors (CARs). In some embodiments, the human-codon-optimized sequence comprises the sequence shown in Sequence ID No. 1. In some embodiments, the polynucleotide further comprises an operablely linked promoter. In some embodiments, the promoter comprises an EF1a sequence or an EF1a / HTLV sequence, preferably a human EF1a sequence or a human EF1a / HTLV sequence. In some embodiments, the promoter comprises the sequence shown in Sequence ID No. 2.

[0007] In some embodiments, the polynucleotide further comprises at least one sequence encoding a self-cleaving peptide or IRES, preferably the sequence encoding the self-cleaving peptide or IRES being codon-optimized for human expression. In some embodiments, the self-cleaving peptide is a 2A self-cleaving peptide, e.g., P2A or T2A or both. In some embodiments, the sequence encoding the self-cleaving peptide comprises the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0008] In some embodiments, the polynucleotide further comprises a sequence encoding one or more selection markers, preferably the sequence encoding the one or more selection markers is codon-optimized for human expression. In some embodiments, the one or more selection markers include DHFRdm. In some embodiments, the one or more selection markers include the sequence shown in SEQ ID NO: 5.

[0009] In some embodiments, the polynucleotide further comprises a sequence encoding EGFRt, preferably the sequence encoding EGFRt is codon-optimized for human expression. In some embodiments, the sequence encoding EGFRt comprises the sequence shown in SEQ ID NO: 6.

[0010] In some embodiments, the polynucleotide further comprises a sequence encoding one or more intracellular signaling domains, preferably the sequence encoding the one or more intracellular signaling domains is codon-optimized for human expression. In some embodiments, the intracellular signaling domain comprises 41BB or CD3ζ or both. In some embodiments, the sequence encoding the one or more intracellular signaling domains comprises the sequence shown in Sequence ID No. 7.

[0011] In some embodiments, the polynucleotide further comprises a sequence encoding a transmembrane domain, preferably the sequence encoding the transmembrane domain being codon-optimized for human expression. In some embodiments, the transmembrane domain comprises CD28tm. In some embodiments, the sequence encoding the transmembrane domain comprises the sequence shown in Sequence ID No. 8.

[0012] In some embodiments, the polynucleotide further comprises a sequence encoding a spacer, preferably the spacer-encoding sequence is codon-optimized for human expression. In some embodiments, the spacer comprises a portion of IgG4, such as the hinge region of IgG4. In some embodiments, the spacer-encoding sequence is the sequence shown in SEQ ID NO: 9. In some embodiments, the polynucleotide-encoding sequence is the sequence shown in SEQ ID NO: 10.

[0013] Furthermore, as disclosed herein, various embodiments provide isolated cells containing the polynucleotides described in the embodiments. In some embodiments, the cells are immune cells. In some embodiments, the cells are progenitor T cells or hematopoietic stem cells. In some embodiments, the cells are T cells, B cells, natural killer cells, antigen-presenting cells, dendritic cells, macrophages, or granulocytes (e.g., basophils, eosinophils, neutrophils, or mast cells). In some embodiments, the cells are CD4+ T cells or CD8+ T cells. In some embodiments, the cells are CD8+ cytotoxic T cells selected from the group consisting of CD8+ naive T cells, CD8+ memory T cells, CD8+ central memory T cells, CD8+ regulatory T cells, iPS cell-derived CD8+ T cells, CD8+ effector memory T cells, and CD8+ bulk T cells. In some embodiments, the cells are CD4+ helper T cells selected from the group consisting of CD4+ naive T cells, CD4+ memory T cells, CD4+ central memory T cells, CD4+ regulatory T cells, iPS cell-derived CD4+ T cells, CD4+ effector memory T cells, and CD4+ bulk T cells. In some embodiments, the cells are cells of the same species as the subject or autologous cells obtained from the subject. In some embodiments, the cells are ex vivo cells. In some embodiments, the cells are in vivo cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells.

[0014] Furthermore, as disclosed herein, in various embodiments, isolated cells comprising polynucleotides disclosed herein further express antibodies or their conjugate fragments or scFv specific to B cell-specific cell surface molecules, where the B cell-specific cell surface molecules are, for example, CD19, CD20, CD1d, CD5, CD19, CD20, CD21, CD22, CD23 / FcεRII, CD24, CD25 / IL-2Rα, CD27 / TNFRSF7, CD32, CD34, CD35, CD38, CD40(TNFRSF5), CD44, CD45, CD45.1, CD45.2, CD54(ICAM-1), CD69, CD72, CD79, CD80, CD84 / SLAMF5, LFA-1, CALLA, BCMA, B cell receptor (BCR), IgM, IgD, B220 / CD45R, C1q The present invention provides isolated cells characterized by being R1 / CD93, CD84 / SLAMF5, BAFF R / TNFRSF13C, B220 / CD45R, B7-1 / CD80, B7-2 / CD86, TNFSF7, TNFRSF5, ENPP-1, HVEM / TNFRSF14, BLIMP1 / PRDM1, CXCR4, DEP-1 / CD148, or EMMPRIN / CD147. In some embodiments, the cells are immune cells. In some embodiments, the cells are progenitor T cells or hematopoietic stem cells. In some embodiments, the cells are T cells, B cells, natural killer cells, antigen-presenting cells, dendritic cells, macrophages, or granulocytes (e.g., basophils, eosinophils, neutrophils, or mast cells). In some embodiments, the cells are CD4+ T cells or CD8+ T cells. In some embodiments, the cells are CD4+ helper T cells selected from the group consisting of CD4+ naive T cells, CD4+ memory T cells, CD4+ central memory T cells, CD4+ regulatory T cells, iPS cell-derived CD4+ T cells, CD4+ effector memory T cells, and CD4+ bulk T cells. In some embodiments, the cells are cells of the same species as the subject or autologous cells obtained from the subject. In some embodiments, the cells are cells of the same species as the subject or autologous cells obtained from the subject.In some embodiments, the cells are ex vivo cells. In some embodiments, the cells are in vivo cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells.

[0015] Furthermore, as disclosed herein, in various embodiments, methods are provided for suppressing or treating cancer in subjects, preferably humans, who require suppression or treatment of cancer, comprising administering polynucleotides or cells disclosed herein to said subjects. In some embodiments, said administration is performed by intracranial injection. In some embodiments, said cancer is glioblastoma. In some embodiments, said cancer is a solid tumor. In some embodiments, said solid tumor is selected from the group consisting of breast cancer, brain tumor, lung cancer, liver cancer, stomach cancer, spleen cancer, colorectal cancer, kidney cancer, pancreatic cancer, prostate cancer, uterine cancer, skin cancer, head cancer, cervical cancer, sarcoma, neuroblastoma, and ovarian cancer.

[0016] Furthermore, as disclosed herein, various embodiments provide the use of the polynucleotides or cells disclosed herein as pharmaceuticals. In some embodiments, the polynucleotides or cells according to any of the embodiments are intended for use in the treatment or suppression of cancers such as glioblastoma, leukemia, lymphoma, hematological malignancies, humoral malignancies, and solid tumors.

[0017] Furthermore, as disclosed herein, in various embodiments, a method is provided for suppressing, mitigating, or treating cancer in a subject, preferably a human, requiring suppression, mitigation, or treatment of cancer, comprising administering a polynucleotide or cells described herein to the subject in combination with an effective amount of at least one additional anticancer agent, thereby providing a combination therapy having an enhanced therapeutic effect. In some embodiments, the cancer is glioblastoma. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a lymphoma, hematological malignancy, or humoral malignancy. In some embodiments, the anticancer agent is delivered with a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof. [Brief explanation of the drawing]

[0018] [Figure 1A] A schematic diagram of the EGFR806CAR construct sequence controlled by a short promoter is shown (hereafter referred to as "short promoter" or "SP" in the diagram).

[0019] [Figure 1B] A schematic diagram of the EGFR806CAR construct sequence controlled by a long promoter is shown (hereafter referred to as "long promoter" or "LP" in the diagram).

[0020] [Figure 1C] A schematic diagram of the human codon-optimized EGFR806CAR construct sequence controlled by a long promoter is shown (hereafter referred to as "codon-optimized", "Co", or Sequence ID No. 10 in the diagram).

[0021] [Figure 2] This specification shows a timeline of the methodologies used in the initial human T cell research disclosed herein.

[0022] [Figure 3]The results of flow cytometry analysis are shown. Six days after transfection and four days after selection with MTX, T cells were stained with biotinylated anti-EGFR antibody and APC-labeled streptavidin. Mock T cells were used as a negative control.

[0023] [Figure 4A] The results of flow cytometry analysis are shown. Eleven days after transfection, T cells were stained with biotinylated anti-EGFR antibody and APC-labeled streptavidin. Mock T cells were used as a negative control.

[0024] [Figure 4B] The results of flow cytometry analysis are shown. Eleven days after transfection, T cells were stained with anti-EGFRvIII-his antibody and APC-labeled anti-his antibody. Mock T cells were used as a negative control.

[0025] [Figure 4C] The results of flow cytometry analysis are shown. Eleven days after transfection, T cells were stained with biotinylated protein L and BV405-labeled streptavidin. Mock T cells were used as a negative control.

[0026] [Figure 5A] The results of flow cytometry analysis are shown. Seven days after rapid expansion culture, T cells were stained with biotinylated anti-EGFR antibody and APC-labeled streptavidin. Mock T cells were used as a negative control.

[0027] [Figure 5B] The results of flow cytometry analysis are shown. Seven days after rapid expansion culture, T cells were stained with biotinylated protein L and BV4*05-labeled streptavidin.

[0028] [Figure 5C] FACS analysis of the staining intensity of cells stained with EGFRVIII antigen is shown.

[0029] [Figure 5D] Figure 5C shows a bar graph illustrating the median fluorescence intensity (MFI) of PE dye quantified in CD8+ cells.

[0030] [Figure 5E] The FACS analysis of staining intensity using Erbitux, which binds to EGFRt markers co-expressed with each CAR, is shown.

[0031] [Figure 5F] Figure 5E shows a bar graph illustrating the median MFI values ​​obtained by quantifying PE dye in CD8+ cells.

[0032] [Figure 6A] Western blot analysis using anti-CD3ζ antibody is shown. The molecular weight of CAR is approximately 50 kD, while the molecular weight of endogenous CD3ζ is approximately 15 kD. Mock T cells were used as a negative control, and H9 cells expressing 806CAR were used as a positive control.

[0033] [Figure 6B] Western blot analysis using anti-CD3ζ antibody is shown.

[0034] [Figure 6C] Figure 6B shows a bar graph illustrating the quantitative determination of the band density of CAR CD3ζ.

[0035] [Figure 6D] This bar graph shows the gene copy number of each construct in cells, measured using droplet digital (dd) PCR.

[0036] [Figure 6E] Figure 6C shows a bar graph illustrating the corrected CD3ζ intensity, re-corrected by the average copy number per cell. It shows cells containing constructs with long promoters (HIV7.3).

[0037] [Figure 7A] The results of the IL2 cytokine release assay (BioPlex) are shown. The K562 parental cell line was used as the negative control, and K562 / OKT3 was used as the positive control. The K562 / EGFRvIII cell line is a target cell line for 806CAR T cells.

[0038] [Figure 7B] The TNF cytokine release assay (BioPlex) is shown. The K562 parental strain was used as the negative control, and K562 / OKT3 was used as the positive control.

[0039] [Figure 7C] The results of the IFN-γ cytokine release assay (BioPlex) are shown. The K562 parental strain was used as the negative control, and K562 / OKT3 was used as the positive control.

[0040] [Figure 7D] This shows cytokine release assays (MSD assays for IL2, TNF-α, and IFN-γ) in another human donor cell line. The K562 parental cell line was used as the negative control, and K562 / OKT3 was used as the positive control.

[0041] [Figure 8A] The chromium release assay (cytotoxicity assay) is shown. The K562 parental cell line was used as the negative control, and K562 / OKT3 was used as the positive control. The K562 / EGFRvIII cell line is a recombinant target cell line of 806CAR T cells and expresses exogenous EGFRvIII. The ratio of effector cells to target cells was set to 30:1 to 1:1.

[0042] [Figure 8B] This shows a chromium release assay (cytotoxic assay) using another human donor cell as an effector cell.

[0043] [Figure 8C]This paper presents incucyte analysis of various target tumor cell lines treated with human donor-derived effector cells.

[0044] [Figure 9] This section describes gene copy number analysis using droplet digital PCR. In this analysis, WPRE primers were used to target the lentiviral scaffold region integrated with the target gene (806CAR-DHFRdm-EGFRt) within the genome.

[0045] [Figure 10A] This shows an in vivo intracranial glioblastoma model.

[0046] [Figure 10B] This study quantifies the bioluminescence signal of tumors in mice. Codon-optimized sequences, sequences with short promoters, or sequences with long promoters were transduced into T cells and tested in an intracranial NSG mouse model. T cells were used at low doses (concentrations not therapeutically effective) to allow for the detection of differences between test groups. Each test group contained five mice. U87 glioma cells (target cells of 806CAR) expressing GFP:ffluc (a fusion protein of GFP and firefly luciferase) were injected intracranially (ic). One week later, T cells were injected intracranially. Bioluminescence images were taken at least once a week, and the quantified signals are shown graphed.

[0047] [Figure 10C] This graph shows Kaplan-Meier survival analysis in mice. T cells were transduced with codon-optimized sequences, sequences with short promoters, or sequences with long promoters, and tested in an intracranial NSG mouse model. T cells were used at low doses (concentrations not therapeutically effective) to allow for the detection of differences between test groups. Each test group contained 5 mice. The data in this graph shows the percentage of surviving mice in each group per unit of time.

[0048] [Figure 10D]This shows the quantitative analysis of tumor bioluminescence.

[0049] [Figure 10E] This shows the Kaplan-Meier survival analysis in mice. (*) These mice were euthanized due to developing tumor-related symptoms such as hunched posture, weight loss, piloerection, lethargy, and labored breathing. [Modes for carrying out the invention]

[0050] This specification discloses a polynucleotide comprising a human-codon-optimized sequence encoding a polypeptide containing an anti-EGFR chimeric antigen receptor (CAR). This human-codon-optimized sequence may be incorporated into a construct comprising an operablely linked promoter, preferably an optimized promoter, a spacer, an intracellular signaling domain, a transmembrane domain, a selection marker, at least one self-cleaving peptide, and EFGRt to optimize its expression. This sequence may then be expressed in cells, such as T cells, for the purpose of treating or suppressing cancers such as glioblastoma, humoral neoplasms, and solid tumors.

[0051] Definition of Terms Unless otherwise stated, the technical and scientific terms used herein have the meanings generally understood by those skilled in the art. All patents, applications, published applications, and other publications referenced herein are incorporated herein by reference in their entirety unless otherwise stated. If there are multiple definitions for a term used herein, the definitions given in this section shall prevail unless otherwise stated.

[0052] In this specification, "a" or "an" may mean one or more.

[0053] In this specification, the term "about" has the ordinary meaning that would be understood by those skilled in the art, and therefore indicates that a particular numerical value includes variations in error or variations between multiple measurements that are inherently associated with the method used to determine that value.

[0054] In this specification, the terms “modify” or “alter,” or any form thereof, mean modification, alteration, replacement, deletion, substitution, removal, change, or transformation.

[0055] In this specification, the terms “function” and “functional” have the general meanings understood in light of this specification and refer to biological function, enzymatic function, or therapeutic function.

[0056] In this specification, the terms “transduction” and “transfection” are used interchangeably and mean the introduction of nucleic acids into cells by artificial means, including methods using viruses and methods not using viruses.

[0057] In this specification, the term “isolated” has the general meaning understood in light of this specification and refers to (1) substances and / or objects separated from at least some of the components present at the time of their initial production (in their natural state and / or under experimental conditions), and / or (2) substances and / or objects manufactured, prepared and / or produced by human hands. “Isolated” nucleic acid molecules and proteins include nucleic acid molecules and proteins purified by standard purification methods. Furthermore, isolated nucleic acid molecules and proteins include nucleic acid molecules and proteins produced by recombinant expression in host cells, as well as chemically synthesized proteins and nucleic acids. The isolated substance and / or object may be separated from the other components initially present in proportions of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100%, approximately these proportions, at least these proportions, at least approximately these proportions, less than or equal to these proportions, or approximately less than or equal to these proportions (or a range including these numbers and / or a range with these numbers as upper and lower limits). In some embodiments, the purity of the isolated substance is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% purity, approximately these purities, at least these purities, at least approximately these purities, less than or equal to these purities, or approximately less than or equal to these purities (or a range of purities including these values ​​and / or a range of purities with these values ​​as upper and lower limits). In this specification, “isolated” substance may be “pure” (e.g., substantially free of other components). In this specification, “isolated cells” may refer to cells isolated from an organism or tissue consisting of many cells.

[0058] In this specification, “in vivo” has the general meaning as understood herein and refers to performing a particular method within the body of an organism, typically an animal, a mammal such as a human, or a plant, or within the living cells that make up these organisms, rather than in tissue extracts or dead organisms.

[0059] In this specification, “exvivo” has the general meaning as understood herein and refers to performing a specific method outside of a living organism with minor modifications to its natural state.

[0060] In this specification, “in vitro” has the general meaning as understood herein and refers to carrying out a particular method outside of biological conditions, for example, in a petri dish or test tube.

[0061] The term “gene” has the general meaning as understood herein and usually refers to a portion of nucleic acid that codes for a protein or functional RNA, but the term may also include regulatory sequences. Those skilled in the art will understand that the term “gene” may include gene regulatory sequences (e.g., promoters and enhancers) and / or intron sequences. Furthermore, it will be understood that the definition of “gene” also includes nucleic acids that code for functional RNA molecules, such as tRNA and miRNA, but do not code for proteins. In some cases, a gene includes regulatory sequences involved in transcription, message generation, or composition. In another embodiment, a gene includes a transcribed sequence that codes for a protein, polypeptide, or peptide. In accordance with the definitions of terms set forth herein, “isolated gene” may include transcribed nucleic acids, regulatory sequences, coding sequences, etc., substantially isolated from other sequences, such as other natural genes, other natural regulatory sequences, other natural sequences that code for polypeptides or peptides. In this regard, the term “gene” is used simply to refer to nucleic acids containing transcribed nucleotide sequences and their complementary strands. As understood in the art, the functional term "gene" encompasses not only genome sequences but also RNA sequences, cDNA sequences, or smaller recombinant nucleic acid segments, which include, but are not limited to, non-transcribed gene regions, such as promoter or enhancer regions in a gene. These smaller recombinant gene nucleic acid segments may also be used to express proteins, polypeptides, domains, peptides, fusion proteins, mutants, and / or similar entities using nucleic acid manipulation techniques, or may be configured to express such entities.

[0062] In this specification, “nucleic acid” or “nucleic acid molecule” has the general meaning understood in light of this specification and refers to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, DNA or RNA or oligonucleotides found in natural cells, fragments obtained by polymerase chain reaction (PCR), and fragments obtained by ligation, cleavage, endonuclease activity, and exonuclease activity. Nucleic acid molecules may consist of natural nucleotide monomers (such as DNA or RNA), analogs of natural nucleotides (e.g., enantiomers of natural nucleotides), or combinations thereof. Modified nucleotides may have modifications to the sugar moiety and / or the pyrimidine base moiety or purine base moiety. Modifications to the sugar moiety include, for example, the substitution of one or more hydroxyl groups with halogens, alkyl groups, amines, or azide groups, and the sugar moiety may be etherified or esterified. Furthermore, the entire sugar moiety may be substituted with a structure that is stereochemically or electronically similar, such as aza sugars and carbocyclic sugar analogs. Modified base moieties include alkylated purines, alkylated pyrimidines, acylated purines, acylated pyrimidines, and other known heterocyclic substituents. Nucleic acid monomers can be linked by phosphodiester bonds or similar bonds. Similar bonds to phosphodiester bonds include phosphorothioate bonds, phosphorodithioate bonds, phosphoroselenoate bonds, phosphorodiselenoate bonds, phosphoranilothioate bonds, phosphoranilidate bonds, and phosphoramidate bonds. The term "nucleic acid molecule" also includes so-called "peptide nucleic acids," which include native or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids may be single-stranded or double-stranded. The term "oligonucleotide" can be used interchangeably with "nucleic acid" and may refer to double-stranded DNA or double-stranded RNA, or single-stranded DNA or single-stranded RNA.Nucleic acids may be contained in nucleic acid vectors or nucleic acid constructs (e.g., plasmids, viruses, retroviruses, lentiviruses, bacteriophages, cosmids, fosmids, phagemids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or human artificial chromosomes (HACs)) that can amplify and / or express nucleic acids in various biological systems. Typically, nucleic acid vectors or nucleic acid constructs also include, but are not limited to, elements such as promoters, enhancers, terminators, inducers, ribosome binding sites, translation start sites, start codons, stop codons, polyadenylation signals, replication start sites, cloning sites, multicloning sites, restriction enzyme sites, epitopes, reporter genes, selection markers, antibiotic selection markers, target sequences, peptide purification tags or accessory genes, or any combination thereof.

[0063] The nucleic acids described herein include nucleic acid bases. Basic bases, i.e., standard bases, natural bases, or unmodified bases, are adenine, cytosine, guanine, thymine, and uracil. Other nucleic acid bases include, but are not limited to, purines, pyrimidines, modified nucleic acid bases, 5-methylcytosine, pseudouridine, dihydrouridine, inosine, 7-methylguanosine, hypoxanthine, xanthine, 5,6-dihydrouracil, 5-hydroxymethylcytosine, 5-bromouracil, isoguanine, isocytosine, aminoallyl bases, dye-labeled bases, fluorescently labeled bases, and biotin-labeled bases.

[0064] A nucleic acid or nucleic acid molecule may contain one or more sequences encoding multiple types of peptides, polypeptides, or proteins. These one or more sequences may be linked adjacently within a single nucleic acid or nucleic acid molecule, or they may be linked with another nucleic acid in between. This other nucleic acid may be, for example, a linker, a repeat sequence, or a restriction enzyme site, or sequences of 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 25 nucleotides, 30 nucleotides, 35 nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, 70 nucleotides, 75 nucleotides, 80 nucleotides, 81 nucleotides, or 81 nucleotides. Examples include sequences of base length, 82 nucleotides, 83 nucleotides, 84 nucleotides, 85 nucleotides, 80 nucleotides, 81 nucleotides, 82 nucleotides, 83 nucleotides, 84 nucleotides, 85 nucleotides, 90 nucleotides, 95 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides, or 300 nucleotides in length, sequences of approximately these lengths, sequences of at least these lengths, sequences of at least approximately these lengths, sequences of less than or equal to these lengths, or sequences of approximately less than or equal to these lengths, or sequences of a length within the range of any two of the aforementioned lengths as upper and lower limits. In this specification, the term “downstream” with respect to nucleic acids has the general meaning understood in light of this specification, and in the case of a double-stranded nucleic acid, refers to a sequence located at the 3' end of a given sequence on the strand containing the coding sequence (sense strand). In this specification, the term “upstream” with respect to nucleic acids has the general meaning understood in light of this specification, and in the case of a double-stranded nucleic acid, refers to a sequence located at the 5' end of a given sequence on the strand containing the coding sequence (sense strand). In this specification, the term “grouped” with respect to nucleic acids has the general meaning understood in light of this specification and refers to two or more adjacent sequences that are directly linked or two or more adjacent sequences linked with another nucleic acid in between.These other nucleic acids include, for example, linkers, repeat sequences, or restriction enzyme sites, or sequences of 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 25 nucleotides, 30 nucleotides, 35 nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, 70 nucleotides, 75 nucleotides, 80 nucleotides, 81 nucleotides, 82 nucleotides, 83 nucleotides, 84 nucleotides, 85 nucleotides, 86 nucleotides, 87 nucleotides, 88 nucleotides, 89 nucleotides, 90 nucleotides, 95 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides, or Examples include sequences of 300 base pairs in length, sequences of approximately that length, sequences of at least that length, sequences of at least approximately that length, sequences of less than or equal to that length, or sequences of approximately less than or equal to that length, or sequences of a length within the range of any two of the aforementioned lengths as upper and lower limits. Sequences sandwiched between two or more of the aforementioned sequences are not typically sequences that encode functional or catalytic polypeptides, proteins, or protein domains.

[0065] In this specification, the term “codon” has the ordinary meaning that would be understood by those skilled in the art, and refers to an RNA or DNA sequence consisting of three nucleotides corresponding to a specific amino acid or stop signal. Examples of such codons include, but are not limited to, the 61 natural codons, the three stop codons, the start codon, and synthetic codons corresponding to non-standard amino acids.

[0066] In this specification, the term “polynucleotide” has the ordinary meaning that can be understood by those skilled in the art, and refers to a class of compounds including polydeoxynucleotides, polydeoxyribonucleotides, and polyribonucleotides. Thus, “polynucleotide” refers to oligomers or polymers of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), or their mimetic forms, and includes polynucleotides composed of natural nucleic acid bases and sugar-phosphodiester (PO) nucleoside (skeletal) bonds, and further includes “recombinant” polynucleotides or substituted polynucleotides that contain non-natural parts having similar functions to those of the natural ones.

[0067] In this specification, “peptide,” “polypeptide,” and “protein” have the general meanings understood in light of this specification and refer to macromolecules composed of amino acids linked by peptide bonds. “Peptide” refers to both short-chain (i.e., peptides, oligopeptides, and oligomers) and long-chain peptides. Numerous functions of peptides, polypeptides, and proteins are known in the art, including, but are not limited to, enzymatic, structural, transport, defense, hormone, or signal transduction functions. Examples of peptides resulting from codon translation include, but are not limited to, combinations of amino acids selected from the 20 common amino acids, norleucine, ornithine, norvaline, homoserine, selenocysteine, pyrrolicin, non-coding amino acids, more than 140 amino acids known to be found in proteins, and synthetic amino acids constructed in the laboratory. Peptides, polypeptides, and proteins are often biologically generated by ribosome complexes using nucleic acid templates, but are not always generated in this way; they can also be produced by chemical synthesis. By genetically modifying nucleic acid templates, mutations in peptides, polypeptides, or proteins can be induced. Such mutations include substitutions, deletions, cleavages, additions, duplications, or fusions of two or more peptides, polypeptides, or proteins.The fusion of two or more peptides, polypeptides, or proteins can be carried out so that they are linked adjacently within a single molecule, or they can be linked with another amino acid in between, such as a linker, repeat sequence, epitope or tag, or 1 nucleotide, 2 nucleotide, 3 nucleotide, 4 nucleotide, 5 nucleotide, 6 nucleotide, 7 nucleotide, 8 nucleotide, 9 nucleotide, 10 nucleotide, 11 nucleotide, 12 nucleotide, 13 nucleotide, 14 nucleotide, 15 nucleotide, 16 nucleotide, 17 nucleotide, 18 nucleotide, 19 nucleotide, 20 nucleotide, or 25 salts. Examples of sequences include those of a base length, 30 nucleotides, 35 nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, 70 nucleotides, 75 nucleotides, 80 nucleotides, 85 nucleotides, 90 nucleotides, 95 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides, or 300 nucleotides in length, sequences of approximately these lengths, sequences of at least these lengths, sequences of at least approximately these lengths, sequences of less than or equal to these lengths, or sequences of approximately less than or equal to these lengths, or sequences of a length within the range of any two of the aforementioned lengths as upper and lower limits. In this specification, the term “downstream” on a polypeptide has the general meaning understood in light of this specification and refers to the sequence following the C-terminus of the preceding sequence. In this specification, the term “upstream” on a polypeptide has the general meaning understood in light of this specification and refers to the sequence preceding the N-terminus of the following sequence. Proteins may contain amino acids other than the 20 amino acids encoded by the gene. Proteins include proteins modified by processes found in nature (e.g., processing and other post-translational modifications) and proteins modified by chemical modification techniques. The same type of modification may be present in similar or varying degrees at several sites on a single protein, and a single protein may contain numerous modifications. Modifications may be located on the peptide backbone, amino acid side chains, or at the amino or carboxyl terminus.Examples of modifications include acetylation; acylation; ADP-ribosylation; amidation; covalent bonding of flavin, heme moiety, nucleotide or nucleotide derivative, lipid or lipid derivative, sugar chain or phosphotidylinositol; crosslinking; cyclization; formation of disulfide bonds; demethylation; formation of covalent crosslinks; glycosylation; hydroxylation; iodization; methylation; myristoylation; oxidation; proteolysis; phosphorylation; S-nitrosation; racemization; lipid bonding; sulfation; γ-carboxylation of glutamic acid residues; and hydroxylation.

[0068] In this specification, the term “codon optimized” has the usual meaning as understood by those skilled in the art and refers to the optimization of a sequence for gene expression or protein production using molecular biological methods. For example, the half-life of mRNA may be affected by substituting less common codons with more common codons, or the structure of mRNA may be modified by introducing secondary structures that interfere with mRNA translation. In some embodiments, codons can be optimized using computer software and algorithms that can predict sequences that will result in optimal expression efficiency. Nucleic acids can be optimized for expression in cells depending on the type of cell into which they are incorporated. Examples of suitable host cells include, but are not limited to, prokaryotic cells such as Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and V. natriegens, eukaryotic cells such as Saccharomyces cerevisiae, plant cells, insect cells, nematode cells, amphibian cells, fish cells, and mammalian cells such as human cells, such as T cells. An AU or a portion of a gene can be optimized. In some embodiments, desired expression regulation can be achieved by optimizing substantially the entire gene. In another embodiment, the desired regulation is achieved by optimizing a portion of the gene, rather than the entire gene.

[0069] In this specification, the term “promoter” has the ordinary meaning that can be understood by those skilled in the art and refers to a DNA sequence to which a protein binds, thereby regulating the transcription of a polynucleotide. Examples of promoters include, but are not limited to, EF1a, HTLV1, EF1a / HTLV (SEQ ID NO: 2), CMV, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, and Ptac. Typically, promoters are located in the 5' region of the polynucleotide being transcribed. More typically, a promoter is defined as the upstream region of the first exon. Promoters can be of any length. For example, short promoters such as the EF1a core promoter are 100–300 base pairs long, while long promoters such as EF1a / HTLV are 400–1000 base pairs long. In some embodiments, the promoter is a native promoter. In other embodiments, the promoter is a chemically synthesized promoter using commonly available techniques. See, for example, Beaucage et al. (1981) Tet. Lett. 22:1859 and U.S. Patent No. 4,668,777. Promoters can regulate constitutive transcription, in which a gene product is expressed sequentially, or inductive transcription, in which the expression of a gene product is affected by specific conditions, such as light, temperature, chemical concentration, protein concentration, or conditions in an organism, cell, or organelle. Promoters may be eukaryotic or prokaryotic promoters and may be recombinant to increase the expression rate or total copy number of a gene product. Promoters may further include at least one regulatory element, such as an upstream element. Such elements may further include other DNA sequences that affect the transcription of polynucleotides, such as a UAR and a synthesized upstream element. In this specification, “promoter regulatory element” means an element that affects the activity of the promoter.Promoter control elements include, but are not limited to, sequencers of transcription factors and regulatory factors, such as enhancers, scaffold / matrix attachment regions, TATA boxes, transcription start regions, locus regulatory regions (LCRs), UARs, URRs, other transcription factor binding sites, and reverse repeat sequences.

[0070] In this specification, the term “regulatory sequence” has the ordinary meaning that can be understood by those skilled in the art and refers to a nucleotide sequence that affects the initiation of transcription or translation, the rate of transcription or translation, or the stability and / or transmissibility of a transcript or polypeptide product. Regulatory sequences include, but are not limited to, promoters, promoter regulatory elements, protein-binding sequences, 5'UTR and 3'UTR, transcription start sites, stop sequences, polyadenylation sequences, introns, and certain sequences in amino acid coding sequences (such as secretory signals and protease cleavage sites).

[0071] In this specification, the term “operably linked” has the ordinary meaning that would be understood by those skilled in the art, and refers to two or more elements, such as polypeptide sequences or polynucleotide sequences, being physically or functionally linked so that these elements can operate as intended. For example, the operable linkage of a polynucleotide of interest and a regulatory sequence (e.g., a promoter) is a functional linkage that enables the expression of the polynucleotide of interest. In this regard, the term “operably linked” refers to a positional relationship between a regulatory region and a coding sequence such that the transcription or translation of the coding sequence of interest to be transcribed is effectively controlled by the regulatory region. In some embodiments disclosed herein, the term “operably linked” means a configuration in which a regulatory sequence is appropriately positioned relative to a sequence encoding a polypeptide or functional RNA such that the expression or intracellular localization of mRNA encoding a polypeptide, polypeptide, and / or functional RNA is controlled or regulated by the regulatory sequence. Thus, when the transcription of a nucleic acid sequence is induced by a promoter, the promoter is operably linked to this nucleic acid sequence. The operably linked elements may or may not be contiguous. Furthermore, with respect to polypeptides, "operably linked" means that the polypeptide exerts a predetermined activity through the physical linkage (e.g., different segments, different regions, or different domains) of amino acid sequences (e.g., via direct or indirect linkage). In this disclosure, various segments, regions, or domains of the chimeric polypeptides of this disclosure may be operably linked so that the chimeric polypeptides of this disclosure maintain appropriate folding, processing, targeting, expression, binding, and other functional properties in cells. Unless otherwise stated, the various regions, domains, and segments of the chimeric polypeptides of this disclosure are operably linked to one another. In the chimeric polypeptides of this disclosure, the operably linked regions, domains, and segments may be contiguous or non-contiguous (e.g., linked to one another via linkers).DNA operably ligated to a promoter either has its transcription initiation regulated by the promoter or exerts its function in combination with the promoter.

[0072] In this specification, the term “self-cleaving peptide” has the usual meaning as understood by those skilled in the art, and refers to a peptide sequence that cleaves the peptide bond between two constituent amino acids, thereby separating itself from two surrounding proteins. This cleavage is thought to occur by ribosome “skipping” of the peptide bond between proline and glycine at the C-terminus of the 2A peptide sequence. Polycistronic mRNA regulatory elements were discovered in the course of virology research. Internal ribosome introduction site (IRES) elements were reported in 1988 by two independent laboratories studying poliovirus and encephalomyocarditis virus.

[0073] In this specification, the term “internal ribosome entry site (IRES)” has the general meaning understood in light of this specification and refers to an element that recruits the 40S subunit to facilitate translation of downstream mRNA. Similarly, the 2A family of self-cleaving peptides, P2A (SEQ ID NO: 3), E2A, F2A, and T2A (SEQ ID NO: 4), were discovered in porcine tesiovirus-1, equine rhinitis A virus, foot-and-mouth disease virus, and thosea asigna virus, respectively. Both 2A elements and IRES elements can produce multiple peptides from a single mRNA strand.

[0074] In this specification, the terms “selection marker” or “selectable marker” have the ordinary meanings that can be understood by those skilled in the art, and refer to genes encoding polypeptides that confer a phenotype to cells, enabling positive selection, negative selection, or screening of cells containing the selection marker gene. The selection marker gene may be used to distinguish between transformed and untransformed cells, or to identify recombinant cells or other types of genetically modified cells. In some embodiments, the selection marker is co-expressed with a codon-optimized sequence corresponding to the EGFR806CAR construct (SEQ ID NO: 1). Under this condition, the presence of the marker enables the selection of cells containing the codon-optimized sequence. Examples of selection markers include, but are not limited to, DHFRdm (SEQ ID NO: 5), DHFR, HER2T, MDR1, MRP1, O6-MGMT, cytidine deaminase, glutathione transferase Yc, and aldehyde dehydrogenase.

[0075] In this specification, the term “intracellular signaling domain” has the ordinary meaning that will be understood by those skilled in the art, and refers to a portion of a protein facing the inside of a cell that, when activated, positively or negatively regulates one or more signaling pathways in a host cell. Examples of signaling pathways include, but are not limited to, proliferation, cytokine release, survival, cytotoxicity, phagocytosis, and metabolism. In some embodiments, the signaling domain comprises a first signaling domain. In some embodiments, the signaling domain comprises a first signaling domain and a second signaling domain. In some embodiments, the protein having the intracellular signaling domain is a transmembrane receptor, such as, but is not limited to, EFGR, the EGFR variant EGFR806CAR construct, or the codon-optimized EGFR variant EGFR806CAR construct. Examples of intracellular signaling domains include, but are not limited to, 41BB, CD3ζ, OX40, CD27, and CD28. In some embodiments, the sequence of the present invention comprises two intracellular signaling domains, such as, but is not limited to, the combination of 41BB and CD3ζ (SEQ ID NO: 7).

[0076] In this specification, the term “transmembrane domain” has the ordinary meaning that can be understood by those skilled in the art, and refers to a portion of a protein that, if present, is incorporated into the cell membrane region of a cell or the membrane region of an organelle. In some embodiments, the membrane into which the transmembrane domain is incorporated is the cell membrane of a cell. Thus, a “transmembrane” protein is a protein that contains a transmembrane domain. The majority of transmembrane domains consist of α-helices. The transmembrane domain of a protein spans the entire thickness of the membrane. A protein may contain multiple transmembrane domains, in which case each independently spans the membrane. Other domains of a protein are called “extracellular domains” or “intracellular domains,” depending on their relative position to the cytoplasm or the outside of the cell. Examples of transmembrane domains include, but are not limited to, CD28tm (SEQ ID NO: 8), CD8αtm, CD4tm, CD3ζtm, and T cell receptor (TCR)-associated ζ chains.

[0077] In this specification, the terms “hinge” and “spacer” are used interchangeably and have the usual meanings that would be understood by those skilled in the art, referring to domains that, by their length, position, and structure, confer flexibility to a protein. In certain cases, altering the spacing between an antigen-recognition domain and another domain can reduce activation-induced cell death. Hinge regions are found in the IgG, IgA, and IgD immunoglobulin classes, for example, in IgG4 (SEQ ID NO: 9), but are not limited to these.

[0078] Cells, T cells, and CAR-T cells In this specification, the terms “individual,” “subject,” or “patient” have their ordinary meanings as understood by those skilled in the art, and therefore include humans and non-human mammals. The term “mammal” is used in the biological sense that the term normally implies. Thus, “mammal” specifically includes, but is not limited to, primates such as monkeys (chimpanzees, apes, monkeys) and humans; as well as cattle, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice, and guinea pigs.

[0079] In this specification, the term “cell” has the general meaning understood in light of this specification and may refer to any type of cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0080] In this specification, the term “immune cells” has the general meaning understood in light of this specification and refers to any cell involved in adaptive or innate immune responses. Immune cells originate from stem cells in the bone marrow and develop into various types of white blood cells. Examples of immune cells include, but are not limited to, memory cells, progenitor T cells, progenitor B cells, hematopoietic stem cells, myeloid progenitor cells, lymphoid progenitor cells, T cells, helper T cells, thymocytes, regulatory T cells, effector T cells, cytotoxic T lymphocytes, γ / δ T cells, B cells, plasma cells, natural killer cells, antigen-presenting cells, dendritic cells, macrophages, histiocytes, astrocytes, myeloid cells, monocytes, and granulocytes (e.g., basophils, eosinophils, neutrophils, or mast cells).

[0081] The term "T cell" is used in the biological sense that the term usually implies. Therefore, a T cell is a lymphocyte involved in the adaptive immune system that has a T cell receptor on its cell surface. Examples of T cells include, but are not limited to, CD4+ T cells, CD8+ T cells, CD8+ cytotoxic T cells, CD8+ naive T cells, CD8+ memory T cells, CD8+ central memory T cells, CD8+ regulatory T cells, iPS cell-derived CD8+ T cells, CD8+ effector memory T cells, CD8+ bulk T cells, CD4+ helper T cells, CD4+ naive T cells, CD4+ memory T cells, CD4+ central memory T cells, CD4+ regulatory T cells, iPS cell-derived CD4+ T cells, CD4+ effector memory T cells, and CD4+ bulk T cells.

[0082] The term "B cell" is used in its usual biological sense. Therefore, B cells are lymphocytes that are involved in the adaptive immune system and secrete antibodies. B cells can produce various types of antibodies and cell surface markers, including, for example, CD19, CD20, CD1d, CD5, CD21, CD22, CD23 / FcεRII, CD24, CD25 / IL-2Rα, CD27 / TNFRSF7, CD32, CD34, CD35, CD38, CD40(TNFRSF5), CD44, CD45, CD45.1, CD45.2, CD54(ICAM-1), CD69, CD72, CD79, CD80, CD84 / SLAMF5, LFA-1, CALLA, BCMA, B cell receptor (BCR), IgM, IgD, B220 / CD45R, C1q R1 / CD93, CD84 / SLAMF5, BAFF Examples include, but are not limited to, R / TNFRSF13C, B220 / CD45R, B7-1 / CD80, B7-2 / CD86, TNFSF7, TNFRSF5, ENPP-1, HVEM / TNFRSF14, BLIMP1 / PRDM1, CXCR4, DEP-1 / CD148, and EMMPRIN / CD147.

[0083] In this specification, “antibody” refers to an immunoglobulin molecule that can specifically bind to a specific epitope on an antigen. The antibody may be a naturally occurring intact immunoglobulin, a recombinant immunoglobulin, or the immunoreactive portion of an intact immunoglobulin. The antibodies useful in the present invention may take various forms, for example, polyclonal antibodies, monoclonal antibodies, humanized monoclonal antibodies, intracellular antibodies ("intrabody"), Fv, Fab or F(ab)2, or single-chain antibodies (scFv), camel antibodies or humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0084] In this specification, the term “antigen” has the general meaning understood in light of this specification and refers to a compound, composition, or substance that can stimulate antibody production or a T-cell response in the body of an animal, including compositions injected or absorbed into the body of an animal. Antigens react to specific humoral or cellular immune products, such as products induced by heterologous immunogens. “Antigen” includes all relevant antigenic epitopes. Examples of antigens include, but are not limited to, EGFR, EGFRvIII, HER2, MSLN, PSMA, CEA, GD2, IL13Rα2, GPC3, CAIX, L1-CAM, CA125, CD133, FAP, CTAG1B, MUC1, and FR-α.

[0085] In this specification, the term “T cell receptor” (or “TCR”) has the general meaning understood in light of this specification and refers to a transmembrane protein found on the surface of a T cell that is capable of recognizing an antigen. TCRs are described according to the TCR nomenclature of International Immunogenetics (IMGT) and are related to the publicly available database of TCR sequences from IMGT. Natural T cell receptors consist of two polypeptide chains, most commonly a combination of α and β chains, or a combination of γ and δ chains. Generally, each chain contains a variable region, a linking region, and a constant region. Each variable region contains three CDRs (complementarity-determining regions) embedded in a framework sequence, one of which is a hypervariable region called CDR3. Similarly, the linking region of a TCR is defined according to the IMGT-specific TRAJ and TRBJ nomenclature, and the constant region of a TCR is defined according to the IMGT-specific TRAC and TRBC nomenclature. The unique sequences defined by IMGT nomenclature are widely known and are available to those skilled in the art of the TCR field. For example, the IMGT nomenclature is available from the publicly available IMGT database. Sequences defined by the IMGT nomenclature are also disclosed in "T cell Receptor Factsbook", (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8, but due to the time lag between its publication date and the current information, the information contained in this publication needs to be checked periodically by referring to the IMGT database.

[0086] In this specification, the term “chimeric antigen receptor” (or “CAR”) T cell has the general meaning understood in light of this specification and refers to T cells genetically modified to produce artificial T cell receptors used in immunotherapy. Examples of artificial T cell receptors include, but are not limited to, the EGFR806CAR construct and the codon-optimized EGFR806CAR construct. Chimeric antigen receptors recognize tumor-associated antigens on the cell surface independently of human leukocyte antigens and activate recombinant T cells via one or more signaling molecules to kill, proliferate, and produce cytokines (Jena et al., 2010). In this specification, the term “chimeric antigen receptor (CAR)” may also refer to, for example, artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors, and encompass recombinant receptors that can be given artificial specificity to specific immune effector cells. Adoptive transfer of CAR-expressing T cells has shown promise in several clinical trials. Currently, it is possible to produce clinical-grade recombinant T cells using modular methods. In certain embodiments, the CAR directs cell specificity to tumor-associated antigens, for example. Examples of tumor-associated antigens include, but are not limited to, EGFR, EGFRvIII, HER2, MSLN, PSMA, CEA, GD2, IL13Rα2, GPC3, CAIX, L1-CAM, CA125, CD133, FAP, CTAG1B, MUC1, and FR-α. In some embodiments, the CAR comprises an intracellular activation domain, a transmembrane domain, and an extracellular domain containing a tumor-associated antigen binding region. In certain cases, the CAR further comprises a co-stimulatory signaling domain such as CD3ζ, FcR, CD27, CD28, CD137, DAP10, and / or OX40. In some cases, other molecules can be co-expressed with CAR, and such molecules include costimulatory molecules, imaging reporter genes (e.g., positron emission tomography reporter genes), gene products that conditionally eliminate T cells by the addition of prodrugs, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.

[0087] CAR T cells can also express cleaved epidermal growth factor receptor (EGFRt) on the surface of T cells (SEQ ID NO: 6). Targeting EGFRt with the IgG1 monoclonal antibody cetuximab has been shown to eliminate CD19 CAR T cells in adoptive mice in the early or late stages, thereby permanently and completely restoring normal functional B cells without tumor recurrence. EGFRt can be used in a variety of clinical applications to regulate the survival of recombinant cells. See, for example, Paszkiewicz et al. (2016) J Clin Invest 126(11): 4262-4272.

[0088] Cancer and related CAR-T therapy targeting Furthermore, this specification discloses a method of administering the novel polynucleotides of the present invention and / or cells expressing said polynucleotides as a treatment for cancer. The term “cancer” is used in the biological sense that it normally implies. Therefore, "cancer" includes, but is not limited to, cancers of any type of cell, such as glioblastoma, astrocytoma, meningioma, craniopharyngioma, medulloblastoma and other brain tumors, leukemia, skin cancer, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal cancer, Hodgkin lymphoma, hematological malignancies, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, lymphoma, mesothelioma, malignant melanoma, multiple myeloma, neuroblastoma, nasopharyngeal cancer, ovarian cancer, osteosarcoma, pancreatic cancer, pituitary cancer, retinoblastoma, salivary gland cancer, stomach cancer, small intestine cancer, testicular cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, Wilms' tumor, solid tumors and humoral tumors. In this specification, the term “solid tumor” has the general meaning understood in light of this specification and refers to an abnormal mass of tissue that does not contain a fluid portion or cyst. Examples of solid tumors include, but are not limited to, sarcomas, carcinomas, and lymphomas. Solid tumors can form in many types of cancerous tissue, such as, but are not limited to, breast cancer, brain tumors, lung cancer, liver cancer, stomach cancer, spleen cancer, colorectal cancer, kidney cancer, pancreatic cancer, prostate cancer, uterine cancer, skin cancer, head cancer, cervical cancer, sarcomas, neuroblastomas, and ovarian cancers.

[0089] In this specification, the term “anticancer agent” has the general meaning understood in light of this specification and refers to small molecules, compounds, proteins or other pharmaceuticals used to treat, suppress or prevent cancer. Common examples of anticancer agents that can be used in one or more embodiments described herein include alkylating agents, anti-EGFR antibodies, anti-Her-2 antibodies, antimetabolites, vinca alkaloids, platinum-based drugs, anthracyclines, topoisomerase inhibitors, taxanes, antibiotics, immunomodulators, antibodies against immune cells, interferons, interleukins, HSP90 inhibitors, antiandrogens, antiestrogens, hypercalcemia agents, apoptosis inducers, aurora kinase inhibitors, Bruton's tyrosine kinase inhibitors, calcineurin inhibitors, CaM kinase II inhibitors, CD45 tyrosine phosphatase CDC25 phosphatase inhibitors, CHK kinase inhibitors, cyclooxygenase inhibitors, bRAF kinase inhibitors, cRAF kinase inhibitors, Ras inhibitors, cyclin-dependent kinase inhibitors, cysteine ​​protease inhibitors, DNA intercalators, DNA cleavage agents, E3 ligase inhibitors, EGF pathway inhibitors, farnesyltransferase inhibitors, Flk-1 kinase inhibitors, glycogen synthase kinase 3 (GSK3) inhibitors, histone deacetylase (HDAC) inhibitors, IκBα kinase inhibitors, imidazotetrazinone, insulin tyrosine kinase inhibitors, c-Jun N-terminal kinase (JNK) inhibitors, mitogen-activated protein kinase (MAPK) inhibitors, MDM2 inhibitors, MEK inhibitors, ERK inhibitors, MMP inhibitors, mTor inhibitors, NGFR tyrosine kinase inhibitors, p38MAP kinase inhibitors, p56 tyrosine kinase inhibitors, PDGF pathway inhibitors, phosphatidylinositol 3-kinase inhibitors, phosphatase inhibitors, protein phosphatase inhibitors, PKC inhibitors, PKCδ kinase inhibitors, polyamine synthesis inhibitors, PTP1B inhibitors, protein tyrosine kinase inhibitors, SRC family tyrosine kinase inhibitors, Syk tyrosine kinase inhibitors, Janus (JAK-2 and / or JAK-3) tyrosine kinase inhibitors, retinoids, RNA polymerase II elongation inhibitors, serine / threonine kinase inhibitors, sterol biosynthesis inhibitors, VEGF pathway inhibitors, chemotherapeutic agents Examples include, but are not limited to, alitretinoin, altretamine, aminopterin, aminolevulinic acid, amsacrin, asparaginase, atrasentan, bexarotene, carbocon, demecoltin, efapoxial, erusamitrusine, etogluside, hydroxycarbamide, leucovorin, ronidamin, rucanson, masopropyl, methyl aminolevulinate, mitogluzone, mitotane, oblimersen, omasetaxin, pegaspargase, porfimer sodium, prednimustine, citimazine seradenovec, talaporfin, temoporfin, trabectedin, and verteporfin.

[0090] In this specification, the terms “administer” or “to administer” have the general meaning as understood in light of this specification and mean to provide or supply a drug, such as a composition disclosed herein, to a target via an effective route. Typical routes of administration include, but are not limited to, oral, injectable (e.g., intracranial, subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, percutaneous, intranasal, vaginal, intraocular, and inhalation routes.

[0091] In this specification, the terms “effective dose” or “effective amount” have the ordinary meaning that can be understood by those skilled in the art, and refer to the amount of the composition or compound described herein that produces an observable biological effect. The actual dose of the active ingredient contained in the active composition of the subject disclosed herein may be modified as appropriate so that an amount of the active composition or compound effective in obtaining the desired therapeutic effect in a particular subject and / or use is administered. The selected dose will depend on various factors such as the activity of the composition, formulation, route of administration, concomitant use of other drugs or treatments, the severity of the condition being treated, the health status and medical history of the subject being treated. In some embodiments, a minimum dose is administered, and it is preferable that this minimum dose be increased to the minimum effective dose in which no dose-limiting toxicity is observed. Determination or adjustment of the effective dose, and evaluation of when and how such adjustments should be made, are also included herein.

[0092] In this specification, the term “pharmaceutically acceptable” has the ordinary meaning that a person skilled in the art would understand, and refers to carriers, diluents, additives and / or stabilizers that are not toxic to cells or mammals at doses and concentrations used for use in cells or mammals, or carriers, diluents, additives and / or stabilizers that have acceptable toxicity. In this specification, the terms “diluent,” “additive,” and / or “carrier” have the ordinary meaning that a person skilled in the art would understand, and include any solvent, dispersion medium, coating agent, antibacterial agent, antifungal agent, isotonic agent, absorption retarder, etc., suitable for administration to human, mouse, rat, cat, dog or other vertebrate host.

[0093] Various pharmaceutically acceptable carriers, diluents, additives, or combinations thereof can be incorporated into pharmaceutical compositions. In one embodiment, the pharmaceutically acceptable diluents, additives, and / or carriers are those approved by government regulatory authorities for use in animals, including non-human mammals (such as cats, dogs, non-human primates, and mice) and humans, or are listed in the United States Pharmacopeia or other known pharmacopoeias. The pharmaceutically acceptable diluents, additives, and / or carriers may be sterile liquids, such as sterile water or oil, and may be mineral oil, animal or plant-derived, or synthetic. Water, saline solution, dextrose aqueous solution, and glycerol aqueous solution can be used as liquid diluents, additives, and / or carriers, particularly for injection solutions. Suitable pharmaceutical diluents and / or additives include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, and ethanol. Examples of pharmaceutically acceptable carriers include, but are not limited to, aqueous pH buffers. Furthermore, pharmaceutically acceptable carriers may also include antioxidants such as ascorbic acid; low molecular weight (less than approximately 10 residues) polypeptides; proteins such as serum albumin; gelatin; immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids; sugars such as glucose, mannose, and dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol and sorbitol; salt-forming counterions such as sodium; nonionic surfactants such as Tween®, polyethylene glycol (PEG), HEG, and PLURONICS®; and one or more of nanoparticles, nanosomes, micelles, lipid nanoparticles, and liposomes. Additionally, suitable pharmaceutical carriers include molecules or solvents that affect the delivery, uptake, and metabolism of drugs, proteins, or molecules. The composition may further contain, optionally, trace amounts of wetting agents, fillers, emulsifiers, or pH buffers. Such compositions may be in the form of solutions, suspensions, emulsions, sustained-release formulations, etc.The formulation should be appropriate for the method of administration.

[0094] specific sequence Table 1 below lists specific sequences useful for the particular embodiments provided herein. [Table 1] TIFF2023552810000003.tif207153TIFF2023552810000004.tif247153TIFF2023552810000005.tif118154

[0095] specific polynucleotides Some embodiments of the methods and compositions provided herein include polynucleotides comprising a sequence encoding a chimeric antigen receptor (CAR) that can specifically bind to EGFR. In some embodiments, the sequence encoding the CAR is codon-optimized for expression in human cells. In some embodiments, the codon-optimized sequence encoding the CAR for expression in human cells is operably ligated to a promoter comprising an E1a promoter, such as the E1a minimal promoter. The codon-optimized sequence encoding the CAR for expression in human cells is operably ligated to a promoter comprising an E1a promoter, such as the E1a minimal promoter, and an HTLV element.

[0096] Some embodiments include a polynucleotide comprising a human-codon-optimized sequence encoding a polypeptide containing EGFR806CAR scFv. In some embodiments, the human-codon-optimized sequence comprises the sequence shown in SEQ ID NO: 1. Some embodiments further include a promoter operably ligated to the human-codon-optimized sequence. In some embodiments, the promoter comprises an EF1a sequence or an EF1a / HTLV sequence, preferably a human EF1a sequence or a human EF1a / HTLV sequence. In some embodiments, the promoter comprises the sequence shown in SEQ ID NO: 2.

[0097] Some embodiments further include at least one sequence encoding a self-cleaving peptide or IRES, preferably the sequence encoding the self-cleaving peptide or IRES is codon-optimized for human expression. In some embodiments, the self-cleaving peptide is a 2A self-cleaving peptide, for example, P2A or T2A or both. In some embodiments, the sequence encoding the self-cleaving peptide includes the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0098] Some embodiments further include sequences encoding one or more selection markers, preferably the sequences encoding the one or more selection markers are codon-optimized for human expression. In some embodiments, the one or more selection markers include DHFRdm. In some embodiments, the sequences encoding the one or more selection markers include the sequence shown in Sequence ID No. 5.

[0099] Some embodiments further include a sequence encoding a cleaved EGFR polypeptide (EGFRt), preferably the sequence encoding the EGFRt being codon-optimized for human expression. In some embodiments, the sequence encoding the EGFRt includes the sequence shown in Sequence ID No. 6.

[0100] Some embodiments further include sequences encoding one or more intracellular signaling domains, preferably the sequences encoding the one or more intracellular signaling domains are codon-optimized for human expression. In some embodiments, the intracellular signaling domains include 41BB or CD3ζ or both. In some embodiments, the sequences encoding the one or more intracellular signaling domains include the sequence shown in Sequence ID No. 7.

[0101] Some embodiments further include a sequence encoding a transmembrane domain, preferably the sequence encoding the transmembrane domain is codon-optimized for human expression. In some embodiments, the transmembrane domain includes CD28tm. In some embodiments, the sequence encoding the transmembrane domain includes the sequence shown in Sequence ID No. 8.

[0102] Some embodiments further include a sequence encoding a spacer, preferably the spacer encoding sequence is codon-optimized for human expression. In some embodiments, the spacer includes a portion of IgG4, such as the hinge region of IgG4. In some embodiments, the spacer encoding sequence is the sequence shown in Sequence ID No. 9.

[0103] In some embodiments, the sequence encoding the polynucleotide is the sequence shown in Sequence ID No. 10.

[0104] specific cells Some embodiments of the methods and compositions provided herein include cells comprising polynucleotides provided herein. In some embodiments, the cells are immune cells. In some embodiments, the cells are progenitor T cells or hematopoietic stem cells. In some embodiments, the cells are T cells, B cells, natural killer cells, antigen-presenting cells, dendritic cells, macrophages, or granulocytes (e.g., basophils, eosinophils, neutrophils, or mast cells). In some embodiments, the cells are CD4+ T cells or CD8+ T cells. In some embodiments, the cells are CD8+ cytotoxic T cells selected from the group consisting of CD8+ naive T cells, CD8+ memory T cells, CD8+ central memory T cells, CD8+ regulatory T cells, iPS cell-derived CD8+ T cells, CD8+ effector memory T cells, and CD8+ bulk T cells. In some embodiments, the cells are CD4+ helper T cells selected from the group consisting of CD4+ naive T cells, CD4+ memory T cells, CD4+ central memory T cells, CD4+ regulatory T cells, iPS cell-derived CD4+ T cells, CD4+ effector memory T cells, and CD4+ bulk T cells. In some embodiments, the cells are cells of the same species as the subject or autologous cells obtained from the subject. In some embodiments, the cells are ex vivo cells. In some embodiments, the cells are in vivo cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells.

[0105] In some embodiments, the cells further express antibodies or their conjugate fragments or scFv specific to B cell-specific cell surface molecules, such as CD19, CD20, CD1d, CD5, CD19, CD20, CD21, CD22, CD23 / FcεRII, CD24, CD25 / IL-2Rα, CD27 / TNFRSF7, CD32, CD34, CD35, CD38, CD40(TNFRSF5), CD44, CD45, CD45.1, CD45.2, CD54(ICAM-1), CD69, CD72, CD79, CD80, CD84 / SLAMF5, LFA-1, CALLA, BCMA, B cell receptor (BCR), IgM, IgD, B220 / CD45R, C1q R1 / CD93, CD84 / SLAMF5, BAFF These are R / TNFRSF13C, B220 / CD45R, B7-1 / CD80, B7-2 / CD86, TNFSF7, TNFRSF5, ENPP-1, HVEM / TNFRSF14, BLIMP1 / PRDM1, CXCR4, DEP-1 / CD148, or EMMPRIN / CD147.

[0106] Specific treatment methods Some embodiments of the methods and compositions provided herein include methods for suppressing, mitigating, or treating cancer in subjects requiring suppression, mitigation, or treatment of cancer. Some embodiments include methods for suppressing, mitigating, or treating cancer in subjects, preferably humans, requiring suppression, mitigation, or treatment of cancer, comprising administering to the subjects polynucleotides provided herein or cells provided herein containing said polynucleotides.

[0107] Some embodiments involve the use of polynucleotides provided herein or cells provided herein containing said polynucleotides as pharmaceuticals or in the preparation of pharmaceuticals.

[0108] In some embodiments, the cancer is leukemia, lymphoma, hematological malignancy, humoral malignancy, or solid tumor. In some embodiments, the solid tumor is selected from the group consisting of breast cancer, brain tumor, lung cancer, liver cancer, stomach cancer, spleen cancer, colorectal cancer, kidney cancer, pancreatic cancer, prostate cancer, uterine cancer, skin cancer, head cancer, cervical cancer, sarcoma, neuroblastoma, and ovarian cancer. In some embodiments, the cancer is glioblastoma. In some embodiments, the administration is performed by intracranial injection.

[0109] As disclosed herein, the inventors have found that incorporating a codon-optimized EGFR806CAR construct sequence for human use into T cells can dramatically enhance its expression, thereby providing therapeutic advantages for cancer therapy. Specific examples of these findings are shown in the following examples. [Examples]

[0110] Example 1 - Design and insertion of a codon-optimized EGFR806CAR construct into T cells for human use. As disclosed herein, three novel sequences of the EGFR806CAR construct were designed and tested for expression in T cells and efficacy in cancer therapy. These constructs included an EGFR806CAR construct controlled by a short EF1a promoter (253 nucleotides long) (Figure 1A) (also referred to herein as the “HIV7.2 construct”); and an EGFR806CAR construct controlled by a long promoter consisting of EF1a and HTLV (544 nucleotides long) (Figure 1B) (also referred to herein as the “HIV7.3 construct”). The third construct was controlled by a long promoter consisting of EF1a and HTLV, and the entire length of the open reading frame was codon-optimized for human expression (Figure 1C) (SEQ ID NO: 10) (also referred to herein as the “coHIV7.2 construct”). Codon optimization was performed using the GeneArt algorithm, which is available online.

[0111] The open reading frames of these constructs included the leader sequence, the EGFR806CAR construct, the hinge region of IgG4, the transmembrane domain (CD28tm), the signaling domains (41BB and CD3ζ), the self-cleaving peptides (P2A and T2A), the drug selection marker (DHFRdm), and the cell surface marker (EGFRt). In SEQ ID NO: 10, the full length of this open reading frame was codon-optimized for human expression.

[0112] As disclosed herein, each of the three constructs was inserted into primary human T cells (Figure 2). Polyclonal proliferation of primary human T cells was stimulated using CD3:CD28 beads. After 24 hours, lentiviral particles containing one of the three sequences were mixed with protamine sulfate and added to primary T cells containing CD4 and CD8 cells in a 1:1 ratio. Protamine sulfate alone was added to "mock cells" as a negative control. Selection with methotrexate (MTX) was initiated a total of 3 days after stimulation. Fluorescence-activated cell sorting (FACS) flow cytometry analysis was initiated a total of 7 days after stimulation. FACS analysis was repeated a total of 12 days after stimulation, rapid expansion culture was performed, and cells were cryopreserved until the next in vivo study. Post-expansion culture assays, including FACS analysis, Western blotting, cytokine release, chromium release, and ddPCR, were performed a total of 26 days after stimulation.

[0113] Example 2 - Enhancement of T cell expression by a codon-optimized EGFR806CAR construct for humans. As disclosed herein, we were able to express all three sequence variants and the EGFR806CAR construct from mock controls in primary human T cells. Six days after transduction and four days after methotrexate (MTX) selection, primary human T cells were stained with biotin-labeled anti-EGFR antibody and APC-labeled streptavidin (Figure 3). Surprisingly, we found that expression was enhanced by the long promoter, and expression in T cells was even more dramatically enhanced by the human codon-optimized variant. Even 11 days after transduction, the trend of significantly enhanced expression of truncated EGFR (EGFRt) continued (Figure 4A). Furthermore, CAR expression was tested by staining with a combination of anti-EGFRvIII-his antibody and APC-labeled anti-his antibody (Figure 4B), or a combination of biotin-labeled protein L and BV405-labeled streptavidin (Figure 4C). Both EGFRvIII (the antigen for CAR) and protein L bind to 806CAR. Similar to EGFRt expression, CAR expression was shown to be significantly higher in T cells containing the human codon-optimized sequence. After 7 days of rapid expansion culture, the human codon-optimized sequence showed significantly higher expression of both EGFRt and CAR (Figure 5A, Figure 5B).

[0114] Furthermore, as disclosed herein, CAR expression and CAR protein processing were tested using Western blotting in parallel with flow cytometry (Figure 6A). Protein lysates were collected and Western blotting was performed according to standard procedures. Western blots were visualized using an anti-CD3ζ antibody. The molecular weight of this CAR is approximately 50 kD, while the molecular weight of endogenous CD3ζ is approximately 15 kD. H9 cells expressing 806CAR were used as a positive control (confirmed to have a purity of over 99% by flow cytometry). The results of Western blotting were similar to those observed by flow cytometry, with a slight enhancement of expression upon introduction of a long promoter and a significant enhancement of expression upon introduction of a long promoter and a codon-optimized open reading frame.

[0115] As a control experiment, we evaluated whether the high expression from the human codon-optimized EGFR806CAR construct was due to a higher gene copy number in cells. This was tested using droplet digital (dd) PCR according to a standard method (Figure 9). ddPCR was performed using WPRE primers that target the lentiviral scaffold region integrated with the target gene (806CAR-DHFRdm-EGFRt) within the genome. Surprisingly, as shown in the graph, the average copy number per cell of the human codon-optimized EGFR806CAR construct was lower than that of constructs with long or short promoter sequences. This result indicates that the aforementioned expression enhancement effect is due to high gene expression levels, not a higher gene copy number.

[0116] In additional experiments, human T cells obtained from multiple donors were transduced using the constructs shown in Figures 1A, 1B, and 1C, and selected with methotrexate. On day 14, the cells were stained with EGFRvIII-His, the antigen of 806CAR, and then secondary staining was performed using PE-labeled anti-His antibody. Staining intensity was measured using FACS analysis (Figure 5C). Figure 5D is a bar graph showing the median fluorescence intensity (MFI) of PE dye quantified in CD8+ cells. Furthermore, the EGFRt marker of the selected cells was stained using biotin-labeled Erbitux and PE-labeled streptavidin as a secondary reagent. Staining intensity was measured using FACS analysis (Figure 5E). Figure 5F is a bar graph showing the quantification of MFI of PE dye in CD8+ cells. Cell surface expression of 806CAR was significantly increased in cells containing a codon-optimized construct with a long promoter (coHIV7.3) compared to cells containing a long promoter construct (HIV7.3) or a short promoter construct (HIV7.2).

[0117] Furthermore, in parallel with flow cytometry analysis, CAR expression and CAR protein processing were examined using Western blot analysis (Figure 6B). The density of the bands for each CAR molecule on the Western blot was quantified, corrected for endogenous CD3ζ signaling, and the results were plotted as a bar graph (Figure 6C). The gene copy number of each construct in cells was examined using droplet digital (dd) PCR according to a standard method (Figures 6D and 6E). Figure 6E shows the results after correcting the CD3ζ intensity shown in Figure 6C again for the average copy number per cell. The copy number in cells containing the construct with a long promoter (HIV7.3) was similar to that in cells containing the codon-optimized construct for humans (coHIV7.3), but the copy number in cells containing the construct with a short promoter (HIV7.2) was about half that of these cells. Taking into account the results of FACS and Western blot analysis, the codon-optimized construct for humans (coHIV7.3) showed the highest expression efficiency among the three constructs. The construct with the long promoter (EF1a(L)) had higher transcriptional activity than the construct with the short promoter (EF1a(S)). Furthermore, codon optimization improved gene expression.

[0118] Example 3 - Increased cytokine release by a codon-optimized EGFR806CAR construct for humans As disclosed herein, we analyzed whether enhanced expression from a codon-optimized EGFR806CAR construct for human use enhances cytokine release. This was tested using a cytokine release assay (BioPlex) (Figures 7A, 7B, and 7C). The K562 parental strain was used as the negative control, and K562 / OKT3 was used as the positive control. K563 cells are antigen-presenting cells, and OKT3 is an anti-TCR antibody against the TCR expressed on K562 cells as the positive control. The K562 / EGFRvIII strain is a target cell line for 806CAR T cells. As shown in the graph, the codon-optimized EGFR806CAR construct for human use enhanced the release of IL2, TNF-α, and IFN-γ. A consistent correlation was observed between higher EGFR806CAR expression levels and greater cytokine release.

[0119] In additional experiments, human T cells obtained from multiple donors were transduced using the constructs shown in Figures 1A, 1B, and 1C, and selected with methotrexate. In cytokine release assays for IL2, TNF-α, and IFN-γ, transduced effector cells were challenged with target cells expressing CAR targets. The K562 parental strain was used as a negative control, and K562 / OKT3 was used as a positive control. The K562 / EGFRvIII strain was a target cell line for 806CAR T cells. As shown in Figure 7D, when stimulated with antigen-positive tumors, the cells containing the coHIV7.3 construct released the most cytokines.

[0120] Example 4 - Optimal functionality of a human-codon-optimized EGFR806CAR construct in actual tumor cells As disclosed herein, we analyzed whether enhanced expression from a codon-optimized EGFR806CAR construct for human use correlated with cytotoxicity (Figure 8A). Cytotoxicity was assessed using a chromium-releasing assay under standard conditions. Similar to the cytokine assay, the K562 parental cell line was used as a negative control and K562 / OKT3 as a positive control. The K562 / EGFRvIII cell line is a recombinant target cell line for 806CAR T cells and expresses exogenous EGFRvIII. The effector-to-target cell ratio was set to 30:1 to 1:1. All three CAR T cell lines showed high cytotoxicity against EGFRvIII-expressing K562 cells, as well as different cytotoxicity against Be2 and U87 tumor cells. The data disclosed in this embodiment demonstrate that there are advantages to using T cells containing a codon-optimized EGFR806CAR construct for treating cells that low-express antigens typically found in actual tumor cells, rather than artificial tumor cell lines such as K562 / EGFRvIII.

[0121] In additional experiments, human T cells obtained from multiple donors were transduced using the constructs shown in Figures 1A, 1B, and 1C, and selected with methotrexate. In a cytotoxicity assay (chromium release assay), transduced effector cells were challenged with target cells expressing the CAR target. As shown in Figure 8B, all 806CAR T cells demonstrated effective killing of target antigen-positive tumor cells. Cells containing coHIV7.2 showed the highest cytolytic activity against the native tumor lines Be2 and U87 compared to cells containing HIV7.3 or HIV7.2.

[0122] In additional experiments, human T cells obtained from multiple donors were transduced using the constructs shown in Figures 1A, 1B, and 1C, and selected with methotrexate. Transduced effector cells were challenged twice at 0 and 96 hours with various target tumor cells expressing the mCherry marker in an E:T ratio of 2:1. The mCherry signal was collected using the Incucyte analysis system. The K562 cell line was a negative control, K562 / OKT was a positive control, and K562 / EGFRVIII and Be2 were CAR-specific target cell lines. As shown in Figure 8C, after each tumor challenge, cells containing the coHIV7.3 construct significantly suppressed the proliferation of K562 / EGFRVIII and Be2 cells. Cells containing the HIV7.3 construct and cells containing the HIV7.2 construct were unable to suppress the proliferation of K562 / EGFRVIII tumor cells after the second challenge. Furthermore, cells containing the HIV7.3 construct and cells containing the HIV7.3 construct were unable to suppress the proliferation of Be2 tumor cells (cell lines that lowly express tumor antigens).

[0123] Example 5 - Conferring significant in vivo therapeutic benefits with a codon-optimized EGFR806CAR construct for humans. As disclosed herein, the performance of T cells containing a human-codon-optimized EGFR806CAR construct was tested in vivo. Sequences containing a construct with a short promoter, a construct with a long promoter, or a human-codon-optimized construct were transduced into T cells and tested in an intracranial NSG mouse model (Figure 10A). T cells were used at low doses (concentrations not therapeutically effective) to allow for the detection of differences between test groups. Each test group contained 5 mice. U87 glioma cells (target cells of 806CAR) expressing GFP:ffluc (a fusion protein of GFP and firefly luciferase) were injected intracranially (ic). One week later, T cells were injected intracranially. Bioluminescence images were taken at least once a week, and the quantified signals are shown graphed. As shown in the graph, mice injected with the human-codon-optimized EGFR806CAR construct showed a significant decrease in bioluminescence after T cell injection, indicating reduced tumor formation (Figure 10B). Similarly, these mice showed increased survival rates over time against tumor-related deaths compared to mice administered with a negative control or mice administered with the EGFR806CAR construct having a short promoter (Figure 10C). In summary, the human-codon-optimized EGFR806CAR construct with a long promoter (SEQ ID NO: 10) exhibits high expression levels, enhanced cytokine release, and superior performance against actual tumor cells, including in vivo.

[0124] In additional experiments, human T cells obtained from multiple donors were transduced using the constructs shown in Figures 1A, 1B, and 1C, and selected with methotrexate. Transduced cells were tested in an intracranial NSG mouse model. T cells were used at low doses (concentrations not effective for treatment) to allow for the detection of differences between test groups. Each test group consisted of 5 mice. U87 glioma cells (target cells of 806CAR) expressing GFP:ffluc (a fusion protein of GFP and firefly luciferase) were injected intracranially (ic). One week later, T cells were injected intracranially. Bioluminescence images were taken at least once a week, and the signal was quantified (Figure 10D). Kaplan-Meier analysis was performed (Figure 10E). All mice injected with cells containing the coHIV7.3 construct survived for 90 days without developing symptoms. The other mice were euthanized because they developed tumor-related symptoms.

Claims

1. A polynucleotide comprising a human codon-optimized sequence encoding an anti-epidermal growth factor receptor (anti-EGFR) chimeric antigen receptor (CAR), The CAR is (a) an scFv encoded by a nucleotide sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO:1 and capable of specifically binding to EGFR; (b) a polypeptide spacer; (c) a transmembrane domain; and (d) Intracellular signaling domain A polynucleotide comprising:

2. The polynucleotide of claim 1 , wherein the scFv is encoded by the nucleotide sequence shown in SEQ ID NO:

1.

3. The polynucleotide of claim 1, characterized in that it is at least one selected from the following (i) to (iii): (i) the polypeptide spacer comprises an IgG4 hinge domain; (ii) the transmembrane domain comprises a CD28tm domain; (iii) the intracellular signaling domain comprises a 41BB domain and / or a CD3ζ domain.

4. The polynucleotide of claim 3 , wherein the polypeptide spacer has a length of 12 amino acids or less.

5. The polynucleotide of claim 3, characterized in that it is at least one selected from the following (i) to (iii): (i) the polypeptide spacer is encoded by a nucleotide sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO:9; (ii) the transmembrane domain is encoded by a nucleotide sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO:8; (iii) the intracellular signaling domain is encoded by a nucleotide sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO:

7.

6. The polynucleotide of claim 1, further comprising at least one selected from the following (i) to (iv): (i) a promoter operably linked to the human codon-optimized sequence, the promoter comprising an EF1a promoter or an EF1a / HTLV promoter; (ii) a nucleic acid encoding an auto-cleaving peptide selected from the P2A and T2A auto-cleaving peptides; (iii) a nucleic acid encoding a selectable marker comprising a dihydrofolate reductase receptor (DHFR); (iv) a nucleic acid encoding a selectable marker comprising a truncated EGFR (EGFRt) polypeptide.

7. The polynucleotide of claim 1, comprising a nucleotide sequence having at least 95% sequence identity to the sequence shown in SEQ ID NO:

10.

8. A vector comprising the polynucleotide according to any one of claims 1 to 7.

9. An isolated cell comprising the polynucleotide according to any one of claims 1 to 7.

10. The isolated cell of claim 9 which is an immune cell.

11. The isolated cell of claim 10, which is a precursor T cell, a hematopoietic stem cell, a T cell, a B cell, a natural killer cell, an antigen presenting cell, a dendritic cell, a macrophage, a granulocyte, a basophil, an eosinophil, a neutrophil or a mast cell.

12. a CD4+ T cell or a CD8+ T cell, (i) CD8+ cytotoxic T cells selected from the group consisting of CD8+ naive T cells, CD8+ memory T cells, CD8+ central memory T cells, CD8+ regulatory T cells, iPS cell-derived CD8+ T cells, CD8+ effector memory T cells, and CD8+ bulk T cells; or (ii) The isolated cell of claim 11, which is a CD4+ helper T cell selected from the group consisting of CD4+ naive T cells, CD4+ memory T cells, CD4+ central memory T cells, CD4+ regulatory T cells, iPS cell-derived CD4+ T cells, CD4+ effector memory T cells and CD4+ bulk T cells.

13. The isolated cell of claim 9 , which expresses an antibody or an antigen-binding fragment thereof, or an scFv capable of specifically binding to a B cell-specific cell surface molecule.

14. The B cell specific cell surface molecule is CD19, CD20, CD1d, CD5, CD19, CD20, CD21, CD22, CD23 / FcεRII, CD24, CD25 / IL-2Rα, CD27 / TNFRSF7, CD32, CD34, CD35, CD38, CD40 (TNFRSF5), CD44, CD45, CD45.1, CD45.2, CD54 (ICAM-1), CD69, CD72, CD79, CD80, CD84 / SLAMF5, LFA-1, CALLA, BCMA, B cell receptor (BCR), IgM, IgD, B220 / CD45R, C1q R1 / CD93, CD84 / SLAMF5, BAFF The isolated cell of claim 13, selected from R / TNFRSF13C, B220 / CD45R, B7-1 / CD80, B7-2 / CD86, TNFSF7, TNFRSF5, ENPP-1, HVEM / TNFRSF14, BLIMP1 / PRDM1, CXCR4, DEP-1 / CD148 and EMMPRIN / CD147.

15. A pharmaceutical composition comprising the isolated cell according to any one of claims 9 to 14 and a pharma- ceutically acceptable excipient.

16. 15. The isolated cell of any one of claims 9 to 14 for use in a method for suppressing, alleviating or treating cancer in a subject.

17. 17. The isolated cell for use according to claim 16, which is an autologous cell obtained from the subject.

18. 17. The isolated cell for use according to claim 16, wherein the cancer is a solid tumor or a lymphoma.

19. 19. The isolated cell for use according to claim 18, wherein the solid tumor is selected from the group consisting of breast cancer, brain cancer, lung cancer, liver cancer, gastric cancer, spleen cancer, colon cancer, kidney cancer, pancreatic cancer, prostate cancer, uterine cancer, skin cancer, head cancer, neck cancer, sarcoma, neuroblastoma and ovarian cancer.

20. 20. The isolated cell for use according to claim 19, wherein the cancer is glioblastoma.

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