CN117136195A

CN117136195A - anti-EGFR chimeric antigen receptor

Info

Publication number: CN117136195A
Application number: CN202180089182.2A
Authority: CN
Inventors: 迈克尔·C·詹森; 嘉魏
Original assignee: Seattle Childrens Hospital
Current assignee: Seattle Childrens Hospital
Priority date: 2020-12-08
Filing date: 2021-12-03
Publication date: 2023-11-28

Abstract

Disclosed herein is a polynucleotide comprising a human codon optimized sequence encoding a polypeptide comprising an EGFR806 CAR. The codon optimized sequence may be integrated into a construct comprising an optimally functional promoter, spacer, intracellular signaling domain, transmembrane domain, selectable marker, at least one self-cleaving peptide, and EFGRt to optimize expression. The sequence may then be expressed in cells (e.g., T cells) for the treatment or inhibition of cancer, such as glioblastoma, liquid or solid tumors.

Description

anti-EGFR chimeric antigen receptor

Cross Reference to Related Applications

U.S. provisional patent application No.63/234090 entitled "anti-EGFR chip antigen receptor" filed on 8/17 of 2021; and U.S. provisional patent application No.63/122839 entitled "codon optimized EGFR806CAR treatment sequence," filed on 8 of month 12 in 2020, which is expressly incorporated herein by reference, respectively.

Sequence listing reference

The application is presented with a list of sequences in electronic format. The sequence listing is provided as a file named SCRI348WOSEQLIST, created at 12 months 1 of 2021, and about 11KB in size. The information in electronic format of the sequence listing is expressly incorporated herein by reference in its entirety.

Technical Field

Aspects of the invention generally relate to anti-EGFR Chimeric Antigen Receptors (CARs) and T cells containing such CARs. Some embodiments relate to methods of enhancing expression of an anti-EGFR-CAR, expressing an anti-EGFR CAR, and using enhanced expression to target cancers such as glioblastoma, liquid tumor, or solid tumor.

Background

Many therapies are used to treat cancer. Currently, popular therapeutic approaches include surgery, chemotherapy, radiation therapy, hormonal therapy, targeted drug therapy and cell therapy. Cell therapy for patients suffering from cancer or disease is the injection of cellular material, such as living cells, into a patient in need thereof. This may include infusion of polyclonal or antigen specific T cells, activated killer cells, natural killer cells, dendritic cells or macrophages. T cells containing Chimeric Antigen Receptors (CARs) are a promising therapeutic approach for cancer immunotherapy and viral therapy, and progress has been made in this regard in recent years.

CAR T cell therapy is an immunotherapy in which T cells are isolated in the laboratory and express synthetic receptors by genetic manipulation, which recognize specific antigens or proteins displayed on cells (e.g., cancer cells), and then the cells are reinjected into the patient. Clinical trials have shown promising evidence of antitumor activity; however, CAR T cell therapy still suffers from insufficient cell activation, short half-life, low targeting to cancer tissues, and the like. Thus, other CAR T cell therapy methods are urgently needed.

Disclosure of Invention

Various embodiments provided herein relate to polynucleotides comprising human codon optimized sequences encoding anti-EGFR Chimeric Antigen Receptors (CARs). In some embodiments, the human codon optimized sequence includes the sequence set forth in SEQ ID NO. 1. In some embodiments, the polynucleotide further comprises an operably 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 set forth in SEQ ID NO. 2.

In some embodiments, the polynucleotide further comprises at least one sequence encoding a self-cleaving peptide or IRES, preferably wherein the sequence encoding the self-cleaving peptide or IRES is codon optimized for expression in humans. In some embodiments, the self-cleaving peptide is a 2A self-cleaving peptide, such as P2A or T2A or both. In some embodiments, the sequence encoding the self-cleaving peptide comprises the sequences shown in SEQ ID NO. 3 and SEQ ID NO. 4.

In some embodiments, the polynucleotide further comprises a sequence encoding one or more selectable markers, wherein the sequence encoding one or more selectable markers is preferably codon optimized for expression in humans. In some embodiments, the one or more selection markers comprise DHFRdm. In some embodiments, the one or more selectable markers include the sequence set forth in SEQ ID NO. 5.

In some embodiments, the polynucleotide further comprises a sequence encoding EGFRt, preferably wherein the sequence encoding EGFRt is codon optimized for expression in humans. In some embodiments, the sequence encoding the EGFRt comprises the sequence set forth in SEQ ID NO. 6.

In some embodiments, the polynucleotide further comprises a sequence encoding one or more intracellular signaling domains, preferably wherein the sequence encoding the one or more intracellular signaling domains is codon optimized for expression in humans. In some embodiments, the intracellular signaling domain comprises 41BB or cd3ζ, or both. In some embodiments, the sequence encoding one or more intracellular signaling domains comprises SEQ ID NO 7.

In some embodiments, the polynucleotide further comprises a sequence encoding a transmembrane domain, preferably wherein the sequence encoding the transmembrane domain is codon optimized for expression in humans. In some embodiments, the transmembrane domain comprises CD28tm. In some embodiments, the sequence encoding the transmembrane domain comprises the sequence set forth in SEQ ID NO. 8.

In some embodiments, the polynucleotide further comprises a sequence encoding a spacer, preferably wherein the sequence encoding the spacer is codon optimized for expression in humans. In some embodiments, the spacer comprises a portion of IgG4, such as a hinge region of IgG 4. In some embodiments, the sequence of the coding spacer is set forth in SEQ ID NO. 9. In some embodiments, the polynucleotide has a sequence as set forth in SEQ ID NO. 10.

As further disclosed herein, various embodiments provide an isolated cell comprising any one of the polynucleotides described in the embodiments above. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a precursor T cell or a hematopoietic stem cell. In some embodiments, the cell is a T cell, B cell, natural killer cell, antigen presenting cell, dendritic cell, macrophage or granulocyte, e.g., a basophil, eosinophil, neutrophil or mast cell. In some embodiments, the cell is a cd4+ T cell or a cd8+ T cell. In some embodiments, the cell is a cd8+ cytotoxic T cell selected from the group consisting of naive cd8+ T cells, cd8+ memory T cells, central memory cd8+ T cells, regulatory cd8+ T cells, IPS-derived cd8+ T cells, effector memory cd8+ T cells, and mixed (bulk) cd8+ T cells. In some embodiments, the cell is a cd4+ T helper cell selected from the group consisting of naive cd4+ T cells, cd4+ memory T cells, central memory cd4+ T cells, regulatory cd4+ T cells, IPS-derived cd4+ T cells, effector memory cd4+ T cells, and mixed cd4+ T cells. In some embodiments, the cells are allogeneic to the subject, or autologous to the subject. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vivo. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

As further disclosed herein, various embodiments provide an isolated cell comprising any one of the polynucleotides disclosed herein, and wherein the isolated cell further expresses an antibody or binding fragment thereof or an scFv specific for a B cell-specific cell surface molecule, such as CD19, CD20, CDld, CD5, CD19, CD20, CD21, CD22, CD23/FcεRII, CD24, CD25/IL-2RαCD27/TNFRSF7, CD32, CD34, CD35, CD38, CD40 (TNFRSF 5), 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, B/CD 45R, clq R1/CD93, CD84/SLAMF5, BAFF NFRSF13C, B/CD 45R, B7-1/CD80, B7-2/CD86, TNFSF7, TNFRSF5, ENPP-1, HVEM/TNSF 14, BLIMP 1/DM 1, CXCR4, DEP-1/CD148 or EMM 148/PRIN 147. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a precursor T cell or a hematopoietic stem cell. In some embodiments, the cell is a T cell, B cell, natural killer cell, antigen presenting cell, dendritic cell, macrophage or granulocyte, e.g., a basophil, eosinophil, neutrophil or mast cell. In some embodiments, the cell is a cd4+ T cell or a cd8+ T cell. In some embodiments, the cell is a cd4+ T helper cell selected from the group consisting of naive cd4+ T cells, cd4+ memory T cells, central memory cd4+ T cells, regulatory cd4+ T cells, IPS-derived cd4+ T cells, effector memory cd4+ T cells, and mixed cd4+ T cells. In some embodiments, the cells are allogeneic to the subject, or autologous to the subject. In some embodiments, the cells are allogeneic to the subject, or autologous to the subject. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vivo. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

As further disclosed herein, various embodiments provide a method of inhibiting or treating cancer in a subject (preferably a human) in need thereof, comprising administering to the subject any one of the polynucleotides disclosed herein or the cells disclosed herein. In some embodiments, the administration is by intracranial injection. In some embodiments, the cancer is glioblastoma. In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is selected from the group consisting of breast cancer, brain cancer, lung cancer, liver cancer, stomach cancer, spleen cancer, colon cancer, kidney cancer, pancreatic cancer, prostate cancer, uterine cancer, skin cancer, head cancer, neck cancer, sarcoma, neuroblastoma, and ovarian cancer.

As further disclosed herein, various embodiments provide for the use of any of the polynucleotides disclosed herein or cells disclosed herein as a medicament. In some embodiments, the polynucleotide of any of the embodiments above or the cell of any of the embodiments above is used to treat cancer, such as glioblastoma, leukemia, lymphoma, hematological tumor, liquid tumor, or solid tumor.

As further disclosed herein, various embodiments provide a method of inhibiting, ameliorating, or treating cancer in a subject (preferably a human) in need thereof, comprising administering to the subject a polynucleotide as described herein or any of the cells as described herein in combination with an effective amount of at least one additional anti-cancer agent to provide a combination therapy with 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 tumor, or liquid tumor. In some embodiments, the anticancer agent is delivered with a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof.

Drawings

FIG. 1A depicts a schematic of EGFR806CAR construct sequences controlled by a short promoter (hereinafter referred to in the figures as a "short promoter" or "SP").

FIG. 1B depicts a schematic of EGFR806CAR construct sequences controlled by a long promoter (hereinafter referred to in the figures as a "long promoter" or "LP").

FIG. 1C depicts a schematic of the sequence of a human codon optimized EGFR806CAR construct controlled by a long promoter (hereinafter referred to in the figures as "codon optimized", "CO" or SEQ ID NO: 10).

FIG. 2 depicts a method timeline used in the primary human T cell studies disclosed herein.

Fig. 3 depicts a flow cytometry analysis. T cells were stained with anti-EGFR-biotin and streptavidin-APC 6 days after transduction and 4 days after MTX selection. As negative control, mock T cells were used.

Fig. 4A depicts a flow cytometry analysis. T cells were stained with anti-EGFR-biotin and streptavidin-APC 11 days after transduction. As negative control, mock T cells were used.

Fig. 4B depicts a flow cytometry analysis. 11 days after transduction, T cells were stained with EGFRvIII-his and anti-his-APC. As negative control, mock T cells were used.

Fig. 4C depicts a flow cytometry analysis. 11 days after transduction, T cells were stained with the proteins L-biotin and streptavidin-BV 405. As negative control, mock T cells were used.

Fig. 5A depicts a flow cytometry analysis. 7 days after rapid expansion, T cells were stained with anti-EGFR-biotin and streptavidin-APC. As negative control, mock T cells were used.

Fig. 5B depicts a flow cytometry analysis. 7 days after rapid expansion, T cells were stained with the proteins L-biotin and streptavidin-BV 405.

Fig. 5C depicts FACS analysis of cell staining levels using egfrvlll antigen.

Fig. 5D depicts a bar graph of Median Fluorescence Intensity (MFI) quantification of PE dyes in cd8+ cells shown in fig. 5C.

Fig. 5E depicts FACS analysis of staining levels using erbitux binding to EGFRt markers co-expressed with each CAR.

Figure 5F depicts a bar graph MFI quantification of PE dye in cd8+ cells shown in figure 5E.

FIG. 6A depicts Western blot analysis with anti-CD 3 zeta antibodies. The molecular weight of the CAR is about 50kD and the endogenous zeta is about 15kD. Mock T cells were used as negative control and H9 cells expressing 806CAR were used as positive control.

FIG. 6B depicts Western blot analysis with anti-CD 3 zeta antibodies.

Fig. 6C depicts a bar graph for quantifying CAR CD3 ζ band levels shown in fig. 6B.

FIG. 6D depicts a bar graph of gene copy number of constructs in cells using drop number (dd) PCR assays.

Fig. 6E depicts a bar graph of normalized CD3 ζ intensity from fig. 6C, and again normalized by the average copy number per cell. Cells containing the long promoter construct (HIV 7.3).

FIG. 7A depicts cytokine release assay for IL2 (BioPlax). The K562 parent and K562/OKT3 served as negative and positive controls, respectively. The K562/EGFRvIII line is the target line for 806CAR T cells.

FIG. 7B depicts cytokine release assay for TNF (BioPlax). The K562 parent and K562/OKT3 served as negative and positive controls, respectively.

FIG. 7C depicts the cytokine release assay of IFNg (BioPlex). The K562 parent and K562/OKT3 served as negative and positive controls, respectively.

FIG. 7D depicts cytokine release assays (MSDs) for IL2, TNFa, and IFNg in different human donor cells. The K562 parental line and K562/OKT3 were used as negative and positive controls, respectively.

Fig. 8A depicts a chromium release assay (cytotoxicity assay). The K562 parent and K562/OKT3 served as negative and positive controls, respectively. The K562/EGFRvIII line is an engineered target line for 806CAR T cells, expressing exogenous EGFRvIII. The ratio of effector cells to target cells varies from 30:1 to 1:1.

Fig. 8B depicts a chromium release assay (cytotoxicity assay) using different human donor cells as effector cells.

Fig. 8C depicts an incucyte assay using human donor cells as effector cells against various target tumor cell lines.

FIG. 9 depicts gene copy number analysis using droplet digital PCR. In this analysis, WPRE primers were used to target lentiviral backbone regions integrated with the target gene (806 CAR DHFRdm EGFRt) in the genome.

Fig. 10A depicts an intracranial in vivo glioblastoma model.

FIG. 10B depicts quantification of tumor bioluminescence signals in mice. Codon optimized short and long promoter sequences were transduced into T cells and studied in an intracranial NSG mouse model. T cells were used at low doses (non-therapeutic) to enable detection of differences between test groups. Each test group contained 5 mice. U87 glioma cells (806 CAR target) expressing GFP fluc (GFP and firefly luciferase fusion protein) were injected intracranially (i.c.). After one week, T cells were injected intraperitoneally. Bioluminescence images were taken at least once a week and signal quantification was displayed.

FIG. 10C depicts a Kaplan-Meier survival analysis in mice. Codon optimized short and long promoter sequences were transduced into T cells and studied in an intracranial NSG mouse model. T cells were used at low doses (non-therapeutic) to enable detection of differences between test groups. Each test group contained 5 mice. The data disclosed herein describe the percentage of mice surviving per unit time per group.

FIG. 10D depicts tumor bioluminescence quantification.

FIG. 10E a depicts a Kaplan-Meier survival analysis in mice. Mice were euthanized for tumor-related symptoms, including humpback, weight loss, hair erectile, somnolence, and dyspnea.

Detailed Description

Disclosed herein is a polynucleotide comprising a human codon optimized sequence encoding a polypeptide anti-EGFR Chimeric Antigen Receptor (CAR). The codon optimized sequence may be integrated into a construct comprising an operably linked promoter (preferably an optimized promoter), a spacer, an intracellular signaling domain, a transmembrane domain, a selectable marker, at least one self-cleaving peptide, and EFGRt to optimize expression. The sequence may then be expressed in cells (e.g., T cells) for the treatment or inhibition of cancer, such as glioblastoma, liquid or solid tumors.

Definition of the definition

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, published applications, and other publications cited herein are incorporated by reference in their entirety unless otherwise indicated. If a term in this document has multiple definitions, the definitions in this section control unless otherwise indicated.

As used herein, "a" or "an" may refer to one or more.

As used herein, the term "about" has its ordinary meaning as understood by those skilled in the art, and thus means that a value includes variations in the inherent error of the method used to determine the value, or variations that exist between determinations.

As used herein, the term "modify" or "transform," or any form thereof, refers to a modification, transformation, substitution, deletion, replacement, removal, variation, or transformation.

As used herein, the terms "function" and "functional" have a simple and ordinary meaning as understood from the specification and refer to biological, enzymatic or therapeutic functions.

As used herein, the terms "transduction" and "transfection" are used equivalently to refer to the introduction of nucleic acids into cells by artificial methods (including viral and non-viral methods).

As used herein, the term "isolated" has a simple and ordinary meaning as understood in the specification, and refers to (1) substances and/or entities that are separated from at least some of the components with which they are associated at the time of initial production (whether in nature and/or in an experimental environment), and/or (2) production, preparation, and/or artificial manufacture. "isolated" nucleic acid molecules and proteins include nucleic acid molecules or proteins purified by standard purification methods. The term also includes nucleic acid molecules and proteins prepared by recombinant expression in host cells, as well as chemically synthesized proteins and nucleic acids. The isolated substances and/or entities may be isolated from other components equal to, about, at least about, no more than, or no more than 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% (or ranges including and/or spanning the above values) with which they were originally associated. In some embodiments, the isolated agent is, about, at least about, no more than, or no more than 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% (or ranges including and/or spanning) pure. As used herein, an "isolated" substance may be "pure" (e.g., substantially free of other components). As used herein, the term "isolated cell" may refer to a cell that is not comprised in a multicellular organism or tissue.

As used herein, "in vivo (in vivo)" is given a simple and ordinary meaning as understood in the specification, and refers to a method performed in living bodies (typically animals, mammals, including humans and plants) or living cells constituting such living bodies, not tissue extracts or dead organisms.

As used herein, "ex vivo" is a simple and ordinary meaning as understood in the specification, and refers to a process that is performed ex vivo with little change to natural conditions.

As used herein, "in vitro" is given a simple and ordinary meaning as understood in the specification, referring to the performance of a method outside of biological conditions (e.g., in a culture dish or tube).

The term "gene" as used herein has a simple and ordinary meaning as understood in the specification and generally refers to a portion of a nucleic acid encoding a protein or functional RNA; however, the term may optionally include regulatory sequences. One of ordinary skill in the art will appreciate that the term "gene" may include gene regulatory sequences (e.g., promoters, enhancers, etc.) and/or intron sequences. It will be further understood that the definition of a gene includes references to nucleic acids that do not encode proteins, but rather encode functional RNA molecules such as trnas and mirnas. In some cases, the gene includes regulatory sequences involved in transcription or information generation or composition. In other embodiments, the gene comprises a transcribed sequence encoding a protein, polypeptide or peptide. According to the terms described herein, an "isolated gene" may include transcribed nucleic acids, regulatory sequences, coding sequences, etc., which are substantially isolated from other such sequences (e.g., other naturally occurring genes, regulatory sequences, polypeptides or peptide coding sequences), etc. In this regard, for simplicity, the term "gene" refers to a nucleic acid comprising a transcribed nucleotide sequence and its complement. Those skilled in the art will appreciate that the functional term "gene" includes genomic sequences, RNA or cDNA sequences, or smaller engineered nucleic acid fragments, including nucleic acid fragments of non-transcribed portions of a gene, including but not limited to non-reverse transcribed promoters or enhancer regions of a gene. Smaller engineered gene nucleic acid fragments may be expressed using nucleic acid manipulation techniques or suitable for expression of proteins, polypeptides, domains, peptides, fusion proteins, mutants, and/or the like.

The term "nucleic acid" or "nucleic acid molecule" as used herein has a general meaning as understood in the specification and refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, those naturally occurring in cells, fragments produced by the Polymerase Chain Reaction (PCR), and fragments produced by any one of ligation, cleavage, endonuclease action, and exonuclease action. The nucleic acid molecule can be composed of monomers that are naturally occurring nucleotides (e.g., DNA and RNA), or analogs of naturally occurring nucleosides (e.g., enantiomeric forms of naturally occurring nucleotides), or a combination of both. Modified nucleotides may alter the sugar moiety and/or the pyrimidine or purine base moiety. Sugar modifications include, for example, substitution of one or more hydroxyl groups with halogen, alkyl, amine, and azide groups, or the sugar may be functionalized as an ether or ester. In addition, the entire sugar moiety may be replaced with a sterically and electronically similar structure, such as aza-sugar and carbocyclic sugar analogs. Examples of modifications of the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well known heterocyclic substituents. The nucleic acid monomers may be linked by phosphodiester bonds or analogues of such bonds. Analogs of phosphodiester linkages include phosphorothioates, phosphorodithioates, phosphoroselenates, phosphorodiselenates, phosphoroaniliothioates (phosphonatoates), phosphoroanilinoates (phosphonatoates) or phosphoramidates (phosphonamides). The term "nucleic acid molecule" also includes so-called "peptide nucleic acids" which include naturally occurring or modified nucleobases attached to a polyamide backbone. The nucleic acid may be single-stranded or double-stranded. "oligonucleotide" may be used interchangeably with nucleic acid and may refer to double-stranded or single-stranded DNA or RNA. The one or more nucleic acids may be contained in a nucleic acid vector or nucleic acid construct (e.g., plasmid, virus, retrovirus, lentivirus, phage, cosmid, F-cosmid, phage, bacterial Artificial Chromosome (BAC), yeast Artificial Chromosome (YAC), or Human Artificial Chromosome (HAC)), which may be used to amplify and/or express the one or more nucleic acids in various biological systems. Typically, the vector or construct will also comprise elements including, but not limited to, promoters, enhancers, terminators, inducers, ribosome binding sites, translation initiation sites, start codons, stop codons, polyadenylation signals, replication origins, cloning sites, multiple cloning sites, restriction enzyme sites, epitopes, reporter genes, selectable markers, antibiotic selectable markers, targeting sequences, peptide purification tags, or accessory genes, or any combination thereof.

The nucleic acids described herein include nucleobases. The original, classical, natural or unmodified bases are adenine, cytosine, guanine, thymine and uracil. Other nucleobases include, but are not limited to, purine, pyrimidine, modified nucleobases, 5-methylcytosine, pseudouridine, dihydrouridine, inosine, 7-methylguanosine, hypoxanthine, xanthine, 5, 6-dihydrouracil, 5-hydroxymethylcytosine, 5-bromouracil, isoguanine, isocytosine, aminoallyl bases, dye-labeled bases, fluorescent bases, or biotin-labeled bases.

The nucleic acid or nucleic acid molecule may comprise one or more sequences encoding different peptides, polypeptides or proteins. These one or more sequences may be linked adjacently in the same nucleic acid or nucleic acid molecule, or have additional nucleic acids between them, such as a linker, repeat sequence, or restriction enzyme site, or any other sequence of about, at least about, no more than, or no more than about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 90, 95, 100, 150, 200, or 300 bases long, or any length within a range defined by any two of the foregoing lengths. The term "downstream" on a nucleic acid as used herein has a simple and ordinary meaning as understood in the specification and refers to a sequence following the 3' end of the preceding sequence on the strand comprising the coding sequence (sense strand) if the nucleic acid is double stranded. The term "upstream" on a nucleic acid as used herein has a simple and ordinary meaning as understood in the specification and refers to a sequence preceding the 5' end of a subsequent sequence on the strand comprising the coding sequence (sense strand) if the nucleic acid is double stranded. The term "grouping" on nucleic acids as used herein has a simple and ordinary meaning as understood in the specification and refers to two or more sequences, such as linkers, repeats, or restriction enzyme sites, occurring directly or adjacent to additional nucleic acids therebetween, or about, at least about, no more than, or no more than about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 95, 100, 150, 200, or 300 bases long, or any length within a range defined by any two of the foregoing lengths, but generally does not have a sequence encoding a functional or catalytic polypeptide, protein, or protein domain between the two.

As used herein, the term "codon" has a common meaning as understood by those skilled in the art and refers to a sequence of three nucleotides (RNA or DNA) corresponding to a particular amino acid or termination signal. As non-limiting examples, such codons may include 61 naturally occurring codons corresponding to non-standard amino acids, 3 stop codons, start codons, and synthetic codons.

As used herein, the term "polynucleotide" has a common meaning as understood by those skilled in the art, and thus refers to a class of compounds that includes polydeoxynucleotides, polydeoxyribonucleotides, and polyribonucleotides. Thus, "polynucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or a mimetic thereof, including polynucleotides consisting of naturally occurring nucleobases, sugar and Phosphodiesterase (PO) internucleoside (backbone) linkages, and "modified" or substituted polynucleotides having non-naturally occurring portions of similar function.

The terms "peptide", "polypeptide" and "protein" as used herein have the simple and ordinary meaning as understood in the specification and refer to macromolecules consisting of amino acids linked by peptide bonds. The term refers to both short chains (i.e., peptides, oligopeptides, and oligomers) and long chains. Various functions of peptides, polypeptides and proteins are known in the art, including but not limited to enzymes, structures, transport, defense, hormones or signals. As a non-limiting example, peptides produced by codon translation may include 20 common amino acids, namely norleucine, ornithine, norvaline, homoserine, selenocysteine, pyrrolysine, a combination of any one of the non-coding amino acids, any one of the 140 amino acids found in proteins, and synthetic amino acids constructed in the laboratory, but are not always produced biologically by a ribosomal complex using a nucleic acid template, although chemical synthesis is also available. By manipulating nucleic acid templates, peptide, polypeptide, and protein mutations, such as substitutions, deletions, truncations, additions, duplications, or fusions of more than one peptide, polypeptide, or protein, can be made. These fusions of more than one peptide, polypeptide or protein may be linked adjacently in the same molecule, or with additional amino acid linkages therebetween, such as a linker, repeat sequence, epitope or tag, or any other sequence of about, at least about, no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200 or 300 bases long, or any length within a range defined by any two of the foregoing lengths. The term "downstream" on a polypeptide as used herein has a simple and ordinary meaning as understood in the specification and refers to a sequence following the C-terminus of the preceding sequence. The term "upstream" on a polypeptide as used herein has a simple and ordinary meaning as understood in the specification and refers to a sequence preceding the N-terminus of the following sequence. The protein may comprise amino acids other than those encoded by the 20 genes. Proteins include proteins modified by natural processes (e.g., processing and other post-translational modifications) and chemical modification techniques. The same type of modification may be present at several sites in a given protein to the same or different degrees, and a protein may contain many modifications. Modifications can occur at the peptide backbone, amino acid side chains, and amino or carboxyl termini. Examples of modifications include acetylation; acylation; ADP ribosylation; amidation; covalent attachment of flavins, heme moieties, nucleotides or nucleotide derivatives, lipids or lipid derivatives, carbohydrates or phosphatidylinositol; crosslinking; cyclizing; disulfide bond formation; demethylation to form covalent crosslinks; glycosylation; hydroxylation; iodination; methylation; myristoylation; oxidizing protein hydrolysis processing; phosphorylation; s-nitrosation; racemization; lipid attachment; sulfation, gamma-carboxylation of glutamic acid residues; or hydroxylation.

As used herein, the term "codon optimization" has the usual meaning as understood by those skilled in the art and refers to the use of molecular biological methods to optimize sequences for gene expression or protein production. For example, modification of codons that are less common with more common codons may affect the half-life of the mrna or alter its structure by introducing secondary structures that interfere with translation of the information. In some embodiments, codons may be optimized using computer software and algorithms that predict the optimal sequence for expression efficiency. The nucleic acid may also be optimally expressed depending on the cell type in which it is contained. Suitable host cells may include, as non-limiting examples, prokaryotic cells such as e.coli (e.coli), pseudomonas aeruginosa (p.aeromonas), bacillus subtilis (b.subtius) or vibrio natrii (v.nategens), or eukaryotic cells such as s.cerevisiae (s.cerevisiae), plant cells, insect cells, nematode cells, amphibian cells, fish cells or mammalian cells including human cells, such as but not limited to T cells. AU or a portion of the gene may be optimized. In some embodiments, the desired expression regulation is achieved by optimizing substantially the entire gene. In other embodiments, the desired regulation will be achieved by optimizing some but not all of the genes.

As used herein, the term "promoter" has the usual meaning as understood by those skilled in the art and refers to a DNA sequence that regulates protein binding of 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 or Ptac. Typically, the promoter is located in the 5' region of the polynucleotide to be transcribed. More typically, a promoter is defined as the region upstream of a first exon. The promoter may be of any length. For example, short promoters, such as EF1a core promoters, are between 100 and 300 base pairs in length, while long promoters, such as EF1a/HTLV, are between 400 and 1000 base pairs in length. In some embodiments, the promoter is naturally occurring. In other embodiments, the promoter is chemically synthesized according to common techniques. See, for example, beaucage et al (1981) Tet. Lett.22:1859 and U.S. Pat. No.4668777. Promoters can regulate constitutive transcription, where the gene product is expressed continuously, and inducible transcription, where expression of the gene product is affected by certain conditions (e.g., light, temperature, chemical concentration, protein concentration, conditions in an organism, cell, or organelle, etc.). Promoters may be eukaryotic or prokaryotic and may be modified to increase the expression rate or total copy number of their corresponding gene products. The promoter may also include at least one control element, such as an upstream element. Such elements include UAR and optionally other DNA sequences that affect transcription of the polynucleotide, e.g., synthetic upstream elements. The term "promoter control element" as used herein describes an element that affects the activity of a promoter. Promoter control elements include transcriptional regulatory sequence determinants such as, but not limited to, enhancers, scaffold/matrix attachment regions, TATA boxes, transcriptional initiation locus control regions, UARs, URRs, other transcription factor binding sites, and inverted repeats.

As used herein, the term "regulatory sequence" has the usual meaning as understood by a person skilled in the art and refers to any nucleotide sequence that affects transcription or translation initiation and rate, or stability and/or mobility of a transcript or polypeptide product. Regulatory sequences include, but are not limited to, promoters, promoter control elements, protein binding sequences, 5 'and 3' UTRs, transcription initiation sites, termination sequences, polyadenylation sequences, introns, certain sequences within amino acid coding sequences, such as secretion signals, protease cleavage sites, and the like.

As used herein, the term "operably linked" has a general meaning as understood by those skilled in the art and refers to a physical or functional linkage between two or more elements (e.g., polypeptide sequences or polynucleotide sequences) that allows them to operate in their intended manner. For example, an operative linkage between a polynucleotide of interest and a regulatory sequence (e.g., a promoter) is a functional linkage that allows expression of the polynucleotide of interest. In this sense, the term "operably linked" refers to the positioning of a regulatory region and a coding sequence to be transcribed such that the regulatory region effectively regulates the transcription or translation of the coding sequence of interest. In some embodiments disclosed herein, the term "operably linked" refers to a configuration in which a regulatory sequence is placed in position relative to a sequence encoding a polypeptide or functional RNA such that the control sequence directs or regulates expression or cellular localization of mRNA encoding the polypeptide, or functional RNA, and/or functional RNA. Thus, a promoter is operably linked to a nucleic acid sequence if it is capable of mediating transcription of the nucleic acid sequence. The operatively connected elements may be continuous or discontinuous. Furthermore, in the context of polypeptides, "operably linked" refers to a physical linkage (e.g., direct or indirect linkage) between amino acid sequences (e.g., different fragments, regions, or domains) to provide the described activity of the polypeptide. In the present invention, various fragments, regions or domains of the chimeric polypeptides of the invention may be operably linked to maintain the proper folding, processing, targeting, expression, binding and other functional properties of the chimeric polypeptides in a cell. Unless otherwise indicated, the various regions, domains and fragments of the chimeric polypeptides of the invention are operably linked to one another. The operably linked regions, domains, and fragments of the chimeric polypeptides of the invention can be contiguous or discontinuous (e.g., linked to one another by a linker). The DNA operably linked to the promoter is under the transcriptional initiation control of the promoter or is functionally associated therewith.

As used herein, the term "self-cleaving" peptide "or" self-cleaving "peptide" has the ordinary meaning understood by those skilled in the art and refers to a peptide sequence that undergoes cleavage of a peptide bond between two constituent amino acids, resulting in separation of the two proteins flanking the sequence. This cleavage is believed to be the result of a ribosomal "jump" formed by the peptide bond between the C-terminal proline and glycine in the 2A peptide sequence. Along with the study of viruses, polycistronic mRNA regulatory elements were discovered. In 1988, two independent laboratories studying poliovirus and encephalomyocarditis virus reported Internal Ribosome Entry Site (IRES) elements.

The term "Internal Ribosome Entry Site (IRES)" as used herein has a simple and ordinary meaning as understood in the specification and refers to an element that recruits the 40S subunit to facilitate translation of downstream mRNA. Similarly, the self-cleaving peptides of the 2A family, P2A (SEQ ID NO: 3), E2A, F2A and T2A (SEQ ID NO: 4), are found in porcine teschovirus-1, equine rhinitis A, foot-and-mouth disease and Lespedeza-motor grader virus, respectively. Both 2A and IRES elements allow multiple peptides to be produced from a single strand of mrna.

As used herein, the term "selectable marker" or "selectable marker" has a common meaning as understood by those of skill in the art and refers to a gene encoding a polypeptide that provides a phenotype to a cell containing the gene such that the phenotype allows for positive or negative selection or screening of cells containing the selectable marker gene. The selectable marker gene may be used to distinguish between transformed cells and untransformed cells, or may be used to identify cells that have undergone recombination or other types of genetic modification. In some embodiments, the selectable marker is co-expressed with a codon-optimized sequence corresponding to the EGFR806CAR construct (SEQ ID NO: 1). Under such conditions, the presence of the marker allows selection of cells containing the codon-optimized sequence. Non-limiting examples of selection markers include DHFRdm (SEQ ID NO: 5), DHFR, HER2T, MDR1, MRP1, O6-MGMT, cytidine deaminase, glutathione transferase Yc or aldehyde dehydrogenase.

As used herein, the term "intracellular signaling domain" has a general meaning as understood by those of skill in the art and refers to a portion of a protein that faces the interior of a cell that, when activated, positively or negatively regulates one or more signaling pathways in a host cell. Non-limiting examples of signaling pathways include proliferation, cytokine release, survival, cytotoxicity, phagocytosis, and metabolism. In some embodiments, the signal domain comprises a primary signal domain. In some embodiments, the signaling domain comprises a primary signaling domain and a secondary signaling domain. In some embodiments, the protein having the intracellular signaling domain is a transmembrane receptor, such as, but not limited to, an EFGR, EGFR variant EGFR806CAR construct, or a codon optimized EGFR variant epidermal growth factor receptor construct. Non-limiting examples of intracellular signaling domains include 41BB, CD3 ζ, OX40, CD27, or CD28. In some embodiments, the sequence comprises two intracellular signaling domains, such as, but not limited to, 41BB and CD3 ζ (SEQ ID NO: 7).

As used herein, the term "transmembrane domain" has a general meaning as understood by those skilled in the art and refers to a portion of a protein that, when included, is incorporated into a cell or membrane region of an organelle. In some embodiments, the membrane is the plasma membrane of a cell. Thus, a "transmembrane" protein is a protein that comprises a transmembrane domain. Most transmembrane domains form an alpha helix. The transmembrane domain of a protein will span the entire membrane. The protein may comprise multiple transmembrane domains, each of which will independently transmembrane. Other domains of proteins are referred to as "extracellular" or "intracellular", depending on their position relative to the cytoplasm and outside the cell. Non-limiting examples of transmembrane domains include CD28tm (SEQ ID NO: 8), CD8 αtm, CD4tm, CD3 ζtm, or T Cell Receptor (TCR) associated ζ chains.

As used herein, the term "hinge" or "spacer" is used equivalently and has its ordinary meaning as understood by those skilled in the art, and refers to a domain whose length, position, and structure provide flexibility to a protein. In some cases, the spacer of the antigen recognition domain can be modified to reduce activation-induced cell death. The hinge region is present in IgG, igA and IgD immunoglobulin classes such as, but not limited to, igG4 (SEQ ID NO: 9).

Cells, T cells and CAR-T cells

The terms "individual," "subject," or "patient" as used herein have their ordinary meaning as understood by those of skill in the art and thus include human or non-human mammals. The term "mammal" is used in a general biological sense. Thus, it includes, but is not limited to, primates, including apes (chimpanzees, apes, monkeys) and humans, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, or pigs.

The term "cell" as used herein has a simple and ordinary meaning as understood from the specification and may refer to any cell type. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

The term "immune cell" as used herein has a simple and ordinary meaning as understood in the specification and refers to any cell involved in an adaptive or innate immune response. Immune cells develop from stem cells in the bone marrow and become a different type of white blood cell. Non-limiting examples of immune cells include memory cells, precursor T cells, precursor B cells, hematopoietic stem cells, myeloid progenitor cells, lymphoid progenitor cells, T helper cells, thymocytes, regulatory T cells, effector T cells, cytotoxic T cells, gamma/delta T cells, B cells, plasma cells, natural killer cells, antigen presenting cells, dendritic cells, macrophages, tissue cells, astrocytes, myeloid cells, monocytes and granulocytes, such as basophils, eosinophils, neutrophils or mast cells.

The term "T cell" is used in its usual biological sense. Thus, T cells are lymphocytes that participate in the adaptive immune system and contain T cell receptors on the cell surface. Non-limiting examples of T cells include cd4+ T cells, cd8+ cytotoxic T cells, naive cd8+ T cells, cd8+ memory T cells, central memory cd8+ T cells, regulatory cd8+ T cytoplasm, IPS-derived cd8+ T cells, effector memory cd8+ T cells, mixed cd8+ T cells, cd4+ T helper cells, naive cd4+ T cells, IPS-derived cd4+ T cells, effector memory cd4+ T cells, or mixed cd4+ T cells.

The term "B cell" is used in its usual biological sense. Thus, B cells are lymphocytes that participate in the adaptive immune system and secrete antibodies. B cells can produce various types of antibodies and surface markers such as, but not limited to, CD19, CD20, CDld, CD5, CD19, CD20, CD21, CD22, CD23/Fc epsilon RII, CD24, CD25/IL-2RαCD27/TNFRSF7, CD32, CD34, CD35, CD38, CD40 (TNFRSF 5), 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, B/CD 45R, clq R1/CD93, CD84/SLAMF5, BAFF R TNFRSF13C, B220/CD45R, B7-1/CD80, B7-2/CD86, TNFSF7, FRSF5, ENPP-1, HVEM/TNSF 14, BLFRDM 1/PRDM1, PRP 4/CD 148 or EMMIN-147.

The term "antibody" as used herein refers to an immunoglobulin molecule capable of specifically binding to a particular epitope on an antigen. The antibody may be an intact immunoglobulin derived from natural sources or recombinant sources, and may be an immunoreactive portion of an intact immunoglobulin. Antibodies useful in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, humanized monoclonal antibodies, intracellular antibodies ("in vivo antibodies"), fv, fab and F (ab) 2, and single chain antibodies (scFv), camelid antibodies or humanized antibodies (Harlow et al, 1999,Using antibodies:ALaboratory Manual,Cold Spring Harbor LaboratoryPress,NY;Harrow et al, 1989,antibodies:A Laboratory Manual,Cold SpringHarbor,New York;Houston et al, 1988,Proc.Natl.Acad.Sci.USA 85:5879-5883; bird et al, 1988,Science 242:423-426).

The term "antigen" as used herein has a simple and ordinary meaning as understood in the specification, and refers to a compound, composition or substance capable of stimulating an antibody or T cell response in an animal, including a composition that is injected or absorbed into the animal. The antigen reacts with the products of specific humoral or cellular immunity, including products induced by heterologous immunogens. The term "antigen" includes all relevant epitopes. Non-limiting examples of antigens include EGFR, EGFRvIII, HER2, MSLN, PSMA, CEA, GD2, IL13RαA2, GPC3, CAIX, L1-CAM, CA125, CD133, FAP, CTAG1B, MUC1, or FR- αAA.

The term "T cell receptor" (or "TCR") as used herein has the ordinary meaning as understood in the specification and refers to a transmembrane protein found on the surface of T cells that is capable of recognizing an antigen. TCRs are described using the international Immunogenetics (IMGT) TCR nomenclature and are linked to the IMGT public database of TCR sequences. Natural T cell receptors consist of two polypeptide chains (most commonly an alpha chain and a beta chain, or a gamma chain and a delta chain). Broadly, each strand includes a variable region, a linking region, and a constant region. Each variable region comprises three CDRs (complementarity determining regions) embedded in a framework sequence, one of which is a hypervariable region designated CDR 3. The linking region of the TCR is similarly defined by unique IMGT TRAJ and TRBJ nomenclature, and the constant region is defined by IMGT TRAC and TRBC nomenclature. Unique sequences defined by IMGT nomenclature are well known and are accessible to researchers in the TCR arts. For example, they may be found in IMGT public databases. "T cell receptor Profile", (2001) LeFranc and LeFranc, academic Press, ISBN 0-12-441352-8 also disclose sequences defined by the IMGT nomenclature, but due to their publication dates and subsequent time lags, information therein sometimes needs to be confirmed by reference to the IMGT database.

The term "chimeric antigen receptor" (or "CAR") T cell has a simple and ordinary meaning as understood in the specification, and refers to a T cell genetically engineered to produce an artificial T cell receptor for immunotherapy. Non-limiting examples of artificial T cell receptors include EGFR806CAR constructs or codon optimized EGFR806CAR constructs. The chimeric antigen receptor recognizes cell surface tumor-associated antigens independently of human leukocyte antigens and employs one or more signaling molecules to activate genetically modified T cells for killing, proliferation and cytokine production (Jena et al, 2010). The term "Chimeric Antigen Receptor (CAR)" as used herein may refer to an artificial T cell receptor, chimeric T cell receptor, or chimeric immune receptor, and includes engineered receptors that specifically transplant an artificial onto a particular immune effector cell. Adoptive transfer of CAR-expressing T cells has shown promise in a number of clinical trials. Clinical grade transgenic T cells can now be made using modular methods. In certain embodiments, for example, the CAR directs the specificity of the cell directly to a tumor-associated antigen. Non-limiting examples of tumor-associated antigens include EGFR, EGFRvIII, HER2, MSLN, PSMA, CEA, GD2, IL13Rα2, GPC3, CAIX, L1-CAM, CA125, CD133, FAP, CTAG1B, MUC1, or FR- α. In some embodiments, the CAR comprises an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising a tumor-associated antigen binding region. In certain instances, the CAR comprises a domain for additional co-stimulatory signaling, such as cd3ζ, fcR, CD27, CD28, CD137, DAP10, and/or OX40. In some cases, the molecule can be co-expressed with the CAR, including co-stimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products that conditionally ablate T cells upon addition of a prodrug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.

CAR T cells can also express truncated forms of the epidermal growth factor receptor (EGFRt) on the T cell surface (SEQ ID NO: 6). Targeting of cetuximab with the IgG1 monoclonal antibody to EGFRt has been shown to eliminate CD19 CART cells early and late after adoptive transfer in mice, resulting in complete and permanent recovery of normal functioning B cells without tumor recurrence. EGFRt can be incorporated into many clinical applications to regulate survival of genetically engineered cells. See, for example, paszkiewicz et al (2016) J Clin Invest 126 (11): 4262-4272.

Targeting of cancer and CAR-T treatment thereof

Further disclosed herein is a method of administering the novel polynucleotides and/or cells expressing the polynucleotides as a cancer treatment. The term "cancer" is used in its usual biological sense. Thus, it may include cancers of any cell type, such as, but not limited to glioblastoma, astrocytoma, meningioma, craniopharyngeoma, medulloblastoma and other brain cancers, leukemia, skin cancer, adrenal cancer, anal cancer, cholangiocarcinoma, bladder cancer, bone cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, eye cancer, gall bladder cancer, gastrointestinal cancer, hodgkin's lymphoma, hematological tumor, kaposi's sarcoma, cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, lymphoma, mesothelioma, melanoma, multiple myeloma, neuroblastoma, nasopharyngeal cancer, ovarian cancer, osteosarcoma, pancreatic cancer, pituitary cancer, retinoblastoma, salivary gland cancer, gastric cancer, small intestine cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulval cancer, waldenstrom's macroglobulinemia, wilms tumor, solid tumor or liquid tumor. The term "solid tumor" as used herein has its ordinary and plain meaning as understood from the specification and refers to an abnormal mass of tissue that does not contain areas of fluid or cysts. Non-limiting examples of solid tumors include sarcomas, carcinomas, or lymphomas. Many cancer tissues may form solid tumors such as, but not limited to, breast, brain, lung, liver, spleen, colon, kidney, pancreas, prostate, uterus, skin, head, neck, sarcoma, neuroblastoma, or ovarian cancer.

The term "anticancer agent" as used herein has a common meaning as understood according to the specification, and refers to a small molecule, compound, protein or other drug for treating, inhibiting or preventing cancer. Non-limiting examples of common classes of anticancer agents that may be used with any one or more of the alternatives described herein include alkylating agents, anti-EGFR antibodies, anti-Her-2 antibodies, antimetabolites, vinblastine, platinum-based formulations, anthracyclines, topoisomerase inhibitors, taxanes, antibiotics, immunomodulators: immune cell antibodies, interferons, interleukins, HSP90 inhibitors, antiandrogens, antiestrogens, antihypercalcemia agents, apoptosis inducers, aurora kinase inhibitors, bruton's tyrosine kinase inhibitors, calcineurin inhibitors, caM kinase II inhibitors, CD45 tyrosine phosphatase inhibitors, 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 strand breaks, E3 ligase inhibitors, EGF pathway inhibitors, farnesyl transferase inhibitors, flk-1 kinase inhibitors, glycogen synthase kinase-3 (GSK 3) inhibitors Histone Deacetylase (HDAC) inhibitors, I- κb- α kinase inhibitors, imidazotetrazinones, 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 extension inhibitors, serine/threonine kinase inhibitors, sterol biosynthesis inhibitors, VEGF pathway inhibitors, chemotherapeutics, alisretinate (alitretin), hexamethylenemine (altretamine), aminopterin, aminolevulinic acid, an Yading (amacrine), asparaginase, atrasentan, betamethadine, carboquone, decarbonylcchicine (demecolcine), ethylpropoxicam, elsamitrucin, etoxazole, hydroxyamino formamide, aldehyde hydrogen folic acid (leucovorin), lonidamine, amikaptone, massachusetin, methylaminolevulinate, propiazone, o-chlorobenzone, olanemens, omaxine, asparaginase, phenam sodium, timutene, siagen, poisson, or betadine.

The term "administration" or "administering" as used herein has a simple and ordinary meaning as understood in the specification and means providing or administering an agent, such as a composition disclosed herein, to a subject by any effective route. Exemplary routes of administration include, but are not limited to, oral, injection (e.g., intracranial, subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal, intranasal, vaginal, intraocular, or inhalation routes.

The term "effective amount" or "effective dose" as used herein has the ordinary meaning as understood by those skilled in the art and refers to the amount of a composition or compound that produces an observable biological effect. The actual dosage level of the active ingredient in the active compositions of the inventive subject matter may vary so as to administer the amount of the active composition or compound effective to achieve the desired response for the particular subject and/or application. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the composition, the formulation, the route of administration, the combination with other drugs or treatments, the severity of the disease being treated, and the physical condition and prior medical history of the subject being treated. In some embodiments, a minimum dose is administered and the dose is increased without limiting toxicity to a minimum effective amount of the dose. Determination and adjustment of effective dosages, as well as an assessment of when and how such adjustment is made, are contemplated herein.

As used herein, the term "pharmaceutically acceptable" has the ordinary meaning as understood by those skilled in the art and refers to carriers, diluents, excipients and/or stabilizers that are non-toxic or have an acceptable level of toxicity to the cells or mammals to which they are exposed at the dosages and concentrations employed. The terms "diluent", "excipient" and/or "carrier" as used herein have their ordinary meaning as understood by those skilled in the art and include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic or absorption delaying agents, and the like, which are compatible with administration to human, mouse, rat, cat, dog, or other vertebrate hosts.

Various pharmaceutically acceptable carriers, diluents, excipients, or combinations thereof may be incorporated into the pharmaceutical composition. In one embodiment, the pharmaceutically acceptable diluents, excipients and/or carriers are diluents, excipients and/or carriers approved by a government regulatory agency or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans, and non-human mammals, such as cats, dogs, non-human primates, or mice. Pharmaceutically acceptable diluents, excipients and/or carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water, saline solutions, and aqueous dextrose and glycerol solutions can be employed as liquid diluents, excipients, and/or carriers, particularly for injectable solutions. Suitable pharmaceutical diluents and/or excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water or ethanol. A non-limiting example of a pharmaceutically acceptable carrier is an aqueous pH buffered solution. The pharmaceutically acceptable carrier may further comprise one or more of the following: antioxidants (e.g., ascorbic acid), low molecular weight (less than about 10 residues) polypeptides, proteins (e.g., serum albumin, gelatin, immunoglobulins), hydrophilic polymers (e.g., polyvinylpyrrolidone), amino acids, carbohydrates (e.g., grape) Sugar, mannose or dextrin), chelating agent (such as EDTA), sugar alcohol such as mannitol or sorbitol, salt-forming counter ion (such as sodium), nonionic surfactant (such as sodium)Polyethylene glycol (PEG), HEG,>) Nanoparticles, nanolipid vesicles, micelles, lipid nanoparticles or liposomes. Suitable drug carriers also include any molecule or solvent that affects delivery, uptake, and metabolism of a drug, protein, or molecule. The composition may also contain minor amounts of wetting agents, fillers, emulsifiers or pH buffers, if desired. These compositions may take the form of solutions, suspensions, emulsions, sustained release formulations and the like. The formulation should be suitable for the mode of administration.

Certain sequences

Certain sequences useful for certain embodiments provided herein are listed in table 1 below.

TABLE 1

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Certain polynucleotides

Some embodiments of the methods and compositions provided herein include a polynucleotide comprising a sequence encoding a Chimeric Antigen Receptor (CAR) capable of specifically binding EGFR. In some embodiments, the sequence encoding the CAR is codon optimized for expression in a human cell. In some embodiments, the sequence encoding the CAR that is codon optimized for expression in a human cell is operably linked to a promoter comprising an E1a promoter (e.g., E1a minimal promoter). The CAR-encoding sequence that is codon optimized for expression in a human cell is operably linked to a promoter comprising an E1a promoter (e.g., E1a minimal promoter) and HTLV elements.

Some embodiments include a polynucleotide comprising a human codon optimized sequence encoding a polypeptide comprising an EGFR806CAR scFv. In some embodiments, the human codon optimized sequence includes the sequence set forth in SEQ ID NO. 1. Some embodiments further comprise a promoter operably linked to the human codon optimization 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 set forth in SEQ ID NO. 2.

Some embodiments further comprise at least one sequence encoding a self-cleaving peptide or an IRES, preferably wherein the sequence encoding the self-cleaving peptide or the IRES is codon optimized for expression in humans. In some embodiments, the self-cleaving peptide is a 2A self-cleaving peptide, such as 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.

Some embodiments further comprise sequences encoding one or more selectable markers, wherein the sequences encoding the one or more selectable markers are preferably codon optimized for expression in humans. In some embodiments, the one or more selectable markers comprises DHFRdm. In some embodiments, the sequence encoding the one or more selectable markers includes the sequence set forth in SEQ ID NO. 5.

Some embodiments further comprise a sequence encoding a truncated EGFR polypeptide (EGFRt), preferably wherein said sequence encoding EGFRt is codon optimized for expression in humans. In some embodiments, the sequence encoding the EGFRt includes the sequence set forth in SEQ ID NO. 6.

Some embodiments further comprise a sequence encoding one or more intracellular signaling domains, preferably wherein the sequence encoding one or more intracellular signaling domains is codon optimized for expression in humans. 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 set forth in SEQ ID NO. 7.

Some embodiments also include sequences encoding transmembrane domains, preferably wherein the sequences encoding the transmembrane domains are codon optimized for expression in humans. In some embodiments, the transmembrane domain comprises CD28tm. In some embodiments, the sequence encoding the transmembrane domain comprises the sequence set forth in SEQ ID NO. 8.

Some embodiments further comprise a sequence encoding a spacer, preferably wherein the sequence encoding the spacer is codon optimized for expression in humans. In some embodiments, the spacer comprises a portion of IgG4, such as a hinge region of IgG 4. In some embodiments, the sequence encoding the spacer is set forth in SEQ ID NO. 9.

In some embodiments, the polynucleotide has a sequence as set forth in SEQ ID NO. 10.

Certain cells

Some embodiments of the methods and compositions provided herein include cells comprising any one of the polynucleotides provided herein. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a precursor T cell or a hematopoietic stem cell. In some embodiments, the cell is a T cell, B cell, natural killer cell, antigen presenting cell, dendritic cell, macrophage or granulocyte, e.g., a basophil, eosinophil, neutrophil or mast cell. In some embodiments, the cell is a cd4+ T cell or a cd8+ T cell. In some embodiments, the cell is a cd8+ cytotoxic T cell selected from the group consisting of naive cd8+ T cells, cd8+ memory T cells, central memory cd8+ T cells, regulatory cd8+ T cells, IPS-derived cd8+ T cells, effector memory cd8+ T cells, and mixed cd8+ T cells. In some embodiments, the cell is a cd4+ T helper cell selected from the group consisting of naive cd4+ T cells, cd4+ memory T cells, central memory cd4+ T cells, regulatory cd4+ T cells, IPS-derived cd4+ T cells, effector memory cd4+ T cells, and mixed cd4+ T cells. In some embodiments, the cells are allogeneic to the subject, or autologous to the subject. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vivo. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

In some embodiments, the cells also express antibodies or binding fragments thereof specific for a B cell-specific cell surface molecule or scFv, such as CD19, CD20, CDld, CD5, CD19, D20, CD21, CD22, CD 23/Fepsilon RII, CD24, CD25/IL-2RαCD27/TNFRSF7, CD32, CD34, CD35, CD38, CD40 (TNFRSF 5), 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, B/CD 45R, clq R1/CD93, CD84/SLAMF5, BAFF TNFRSF13C, B/CD 45R, B-1/CD 80, B7-2/CD86, TNF 7, TNFRSF5, ENPP-1, IMPP-14/UMP 1, HVFDM 1, or PRDEDM 1/PRDM 1.

Certain therapies

Some embodiments of the methods and compositions provided herein include therapies that inhibit, ameliorate or treat cancer in a subject in need thereof. Some embodiments include methods of inhibiting amelioration or treatment of cancer in a subject (preferably a human) in need thereof, comprising administering to the subject any one of the polynucleotides provided herein or any cell provided herein containing such polynucleotides.

Some embodiments include the use of any one of the polynucleotides provided herein, or any one of the cells provided herein containing such polynucleotides, as a medicament or in the preparation of such a medicament.

In some embodiments, the cancer is leukemia, lymphoma, hematological tumor, liquid tumor, or solid tumor. In some embodiments, the solid tumor is selected from the group consisting of breast cancer, brain cancer, lung cancer, liver cancer, stomach cancer, spleen cancer, colon cancer, kidney cancer, pancreatic cancer, prostate cancer, uterine cancer, skin cancer, head cancer, neck cancer, sarcoma, neuroblastoma, and ovarian cancer. In some embodiments, the cancer is glioblastoma. In some embodiments, administration is by intracranial injection.

As disclosed herein, the inventors found that integration of the human codon optimized EGFR806CAR construct sequence into T cells can significantly enhance expression and bring therapeutic benefits to cancer treatment. Specific examples of these findings are described in the examples below.

Examples

Example 1-design and insertion of human codon optimized EGFR806CAR constructs into T cells

As disclosed herein, the inventors designed three new sequences for EGFR806CAR constructs to test expression in T cells and the effectiveness of cancer treatment. These constructs include the EGFR806CAR construct (fig. 1A), also referred to herein as the "HIV7.2 construct", controlled by a short EF1A promoter (253 nucleotides); and an EGFR806CAR construct (fig. 1B), also referred to herein as an "HIV7.3 construct", controlled by long EF1a and HTLV promoters (544 nucleotides). The third construct is controlled by the long EF1a and HTLV promoters, and the entire open reading frame is codon optimized for expression in humans (FIG. 1C) (SEQ ID NO: 10), also referred to herein as the "coHIV7.2 construct". Codon optimization was performed using the GeneArt online algorithm.

The open reading frame comprises a leader sequence, an EGFR806CAR construct, the hinge region of IgG4, the transmembrane domain CD28tm, the signal domains 41BB and cd3ζ, self-cleaving peptides P2A and T2A, the drug selection marker DHFRdm, and the surface marker EGFRt. This entire region is codon optimized in SEQ ID NO. 10 for expression in humans.

As disclosed herein, the inventors inserted three constructs into human primary T cells (fig. 2). The inventors stimulated polyclonal expansion of primary human T cells with 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 consisting of CD4 and CD8 cells in a 1:1 ratio. The "mock cells" to which only protamine sulfate was added are hereinafter referred to as negative controls. Three days total after stimulation, the inventors began selecting Methotrexate (MTX). The inventors began Fluorescence Activated Cell Sorting (FACS) flow cytometry analysis for a total of 7 days after stimulation. After a total of 12 days post-stimulation, the inventors continued FACS analysis, followed by rapid expansion and freezing of the cells for future in vivo studies. The inventors performed post-amplification assays including FACS analysis, western blot analysis, cytokine release, chromium release and ddPCR for a total of 26 days after stimulation.

Example 2-enhanced expression of human codon optimized EGFR806CAR constructs in T cells

As disclosed herein, the inventors established expression of EGFR806CAR constructs from all three sequence variants and a mock control in human primary T cells. Primary human T cells were stained with anti-EGFR biotin and streptavidin APC 6 days after transduction and 4 days after Methotrexate (MTX) selection (fig. 3). Surprisingly, the inventors found that long promoters enhance expression, and that human codon-optimized variants further significantly enhance expression in T cells. This trend of significantly enhanced expression of truncated EGFR (EGFRt) continued 11 days after transduction (fig. 4A). CAR expression by staining with egfrvlll-his and anti-his APC (fig. 4B) or the proteins L-biotin and streptavidin-BV 405 (fig. 4C) was also tested. Both egfrvlll (CAR antigen) and L protein bind to 806 CAR. Similar to EGFRt expression, CAR expression was significantly higher in T cells containing human codon optimized sequences. Human codon-optimized sequences showed significantly higher EGFRt and CAR expression even 7 days after rapid amplification (fig. 5A, 5B).

Also disclosed herein are the expression and protein processing of the CARs tested simultaneously with flow cytometry using western blot analysis by the inventors (fig. 6A). Protein lysates were collected and western blotted using standard methods. Western blots were visualized using an anti-CD 3 ζ antibody. The molecular weight of the CAR is about 50kD and the endogenous zeta is about 15kD. Positive control was 806CAR expressing H9 cells (> 99% purified by flow). The western blot results were identical to those shown by flow cytometry; expression is slightly enhanced by the introduction of a long promoter and significantly enhanced by the introduction of a long promoter and codon optimized open reading frame.

As a control, the inventors also assessed whether high expression from the human codon optimized EGFR806CAR construct was caused by high gene copy number in the cells. They tested this using drop-digital (dd) PCR under standard methods (fig. 9). ddPCR was performed using WPRE primers targeting the lentiviral backbone region integrated with the gene of interest (806 CAR DHFRdm EGFRt) in the genome. As shown, the average copy number per cell in the human codon optimized EGFR806CAR construct was unexpectedly low compared to the long and short promoter sequences. This suggests that the effects shown are from high gene expression, not genetic copy number.

In additional experiments, human T cells from the donor were transduced with the constructs shown in fig. 1A, 1B and 1C and selected with methotrexate. On day 14, cells were stained with 806CAR antigen egfrvlll-His, followed by secondary staining with anti-His PE. Staining levels were determined using FACS analysis (fig. 5C). Figure 5D depicts a bar graph of Median Fluorescence Intensity (MFI) quantification of PE dyes in cd8+ cells. Selected cells were also stained for EGFRt markers using erbitux-biotin and a second reagent, streptavidin-PE. Staining levels were determined using FACS analysis (fig. 5E). Figure 5F depicts bar graph MFI quantification of PE dye in cd8+ cells. Cell surface expression of 806CAR was significantly increased in cells containing the long promoter codon optimized construct (cohiv 7.3) compared to cells containing the long promoter construct (HIV 7.3) or the short promoter construct (HIV 7.2).

Expression of CAR and protein processing was concurrent with flow cytometry using western blot analysis (fig. 6B). The density of each CAR molecular band on western blots was quantified and normalized by the endogenous cd3ζ signal, which was plotted as a bar graph (fig. 6C). The gene copy number of the constructs in cells was tested under standard methods using droplet digital (dd) PCR (fig. 6D, fig. 6E). Fig. 6E depicts the normalized CD3 ζ intensity from fig. 6C, and again normalized by the average copy number per cell. Cells containing the long promoter construct (HIV 7.3) and the human codon optimized construct (cohiv 7.3) have similar copy numbers, which are about half the number of cells containing the short promoter construct (HIV 7.2). From FACS analysis and western blot analysis, the human codon optimized construct (cohiv 7.3) has the expression efficiencies of three constructs. Constructs with long promoters (EF 1a (L)) have higher transcriptional activity than constructs with short promoters (EF 1a (S)). In addition, codon optimization improves gene expression.

Example 3-human codon optimized EGFR806CAR construct increased cytokine release

As disclosed herein, the inventors analyzed whether enhanced expression from a human codon optimized EGFR806CAR construct resulted in enhanced cytokine release. It was tested using cytokine release assay (BioPlex) (fig. 7A, 7B, 7C). The K562 parent and K562/OKT3 served as negative and positive controls, respectively. K563 is an antigen presenting cell and OKT3 is an anti-TCR antibody expressed on K562 as a positive control. The K562/EGFRvIII line is the target line for 806CART cells. As shown in the figures, IL2, TNF-a and IFN-g all showed enhanced release with human codon optimized EGFR806CAR constructs. This consistently shows that the higher the EGFR806CAR expression, the greater the cytokine release.

In additional experiments, human T cells from the donor were transduced with the constructs shown in fig. 1A, 1B and 1C and selected with methotrexate. In cytokine release assays for IL2, TNFs, and IFNg, transduced effector cells are challenged with target cells expressing CAR targets. The K562 parent and K562/OKT3 are negative and positive controls, respectively. The K562/EGFRvIII line is the target cell line for 806CAR T cells. As shown in fig. 7D, cells containing the cohiv7.3 construct had the highest cytokine release upon ag+ tumor stimulation.

Example 4-human codon optimized EGFR806CAR construct works best in real tumor lines

As disclosed herein, the inventors analyzed whether enhanced expression from human codon optimized EGFR806CAR constructs was associated with cytotoxicity (fig. 8A). Cytotoxicity was assessed using a chromium release assay under standard conditions. As with the cytokine assay, the K562 parental line and K562/OKT3 were used as negative and positive controls, respectively. The K562/EGFRvIII line is an engineered target line for 806CAR T cells, expressing exogenous EGFRvIII. The ratio of effector cells to target cells varies from 30:1 to 1:1. All three CAR T cells showed high cytotoxicity using egfrvlll expressing K562 cells and different cytotoxicity using Be2 and U87 tumor lines. The data disclosed herein provide evidence of the benefit of using codon optimized EGFR806CAR constructed T cells to treat low antigen expressing cells, which are typically true tumor lines, rather than artificial tumor lines such as K562/egfrvlll.

In additional experiments, human T cells from the donor were transduced with the constructs shown in fig. 1A, 1B and 1C and selected with methotrexate. The transduced effector cells are challenged with target cells expressing CAR targets in a cytotoxicity assay (chromium release assay). As shown in fig. 8B, all 806CAR T cells showed effective killing of target ag+ tumor cells. Cells containing coHIV5.2 had the highest cytolytic activity against the natural tumour lines Be2 and U87 compared to cells containing HIV7.3 or HIV 7.2.

In additional experiments, human T cells from the donor were transduced with the constructs shown in fig. 1A, 1B and 1C and selected with methotrexate. Transduced effector cells were challenged twice at 0 and 96 hours with different target tumor cells expressing mCherry markers at a 2:1 ratio of E to T. mCherry signals were collected by Incucyte. The K562 cell line was a negative control, K562/OKTs was a positive control, and K562/1EGFRVIII and Be2 were CAR specific target cell lines. As shown in FIG. 8C, cells containing the coHIV7.3 construct significantly inhibited the growth of K562/EGFRVIII and Be2 cells after each tumor challenge. Cells containing the HIV7.3 construct or HIV7.2 construct were unable to inhibit the growth of K562/egfrvviii tumor cells after the second challenge. Cells containing HIV7.3 constructs or HIV7.3 constructs did not inhibit Be2 (low tumor antigen cell line) tumor cell growth.

Example 5-human codon optimized EGFR806CAR construct has significant therapeutic benefit in vivo

As disclosed herein, T cells containing the human codon optimized EGFR806CAR construct were tested for performance in vivo. Sequences containing short or long promoter and human codon optimized constructs were transduced into T cells prior to study in the intracranial NSG mouse model (fig. 10A). T cells were used at low doses (non-therapeutic) to enable detection of differences between test groups. Each test group contained 5 mice. U87 glioma cells (806 CAR target) expressing GFP fluc (GFP and firefly luciferase fusion protein) were injected intracranially (i.c.). After one week, T cells were injected intraperitoneally. Bioluminescence images were taken at least once a week and signal quantification was displayed. As shown in the figures, mice with human codon-optimized EGFR806CAR constructs had significantly lower bioluminescence after T cell injection, indicating reduced tumor formation (fig. 10B). Similarly, these mice had higher tumor-associated mortality survival over time compared to negative controls and mice given EGFR806CAR constructs with short promoters (fig. 10C). Taken together, this data shows that the human codon optimized EGFR806CAR construct with long promoter (SEQ ID NO: 10) has higher expression, enhanced cytokine release, higher performance in real tumor cells and higher performance in vivo.

In additional experiments, human T cells from the donor were transduced with the constructs shown in fig. 1A, 1B and 1C and selected with methotrexate. Transduced cells were studied in an intracranial NSG mouse model. T cells were used at low doses (non-therapeutic) to enable detection of differences between test groups. Each test group contained 5 mice. U87 glioma cells (806 CAR target) expressing GFP fluc (GFP and firefly luciferase fusion protein) were injected intracranially (i.c.). After one week, T cells were injected intraperitoneally. Bioluminescence images were taken at least once a week and the signal was quantified (fig. 10D). Kaplan-Meier analysis was performed (FIG. 10E). All mice injected with cells containing the coHIV7.3 construct survived for 90 days without symptoms. The remaining mice were euthanized for tumor-related symptoms.

Sequence listing

<110> Seattle Children Hospital

Michael jensen

Jiawei (Jiawei)

<120> anti-EGFR chimeric antigen receptor

<130> SCRI.348WO

<150> 63122839

<151> 2020-12-08

<150> 63234090

<151> 2021-08-17

<160> 10

<170> FastSEQ for Windows Version 4.0

<210> 1

<211> 726

<212> DNA

<213> artificial sequence

<220>

<223> codon optimized version of sequence present in Chile

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ttccccggca acaagctgga atggatgggc tacatcagct acagcggcaa cacccggtac 180

aaccccagcc tgaagtcccg gatctccatc accagagaca ccagcaagaa ccagttcttc 240

ctgcagctga acagcgtgac catcgaggac accgccacct actactgtgt gacagccggc 300

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<213> Homo sapiens (Homo sapiens)

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<220>

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<220>

<223> codon optimized version of the sequence present in the Leptospira Minus virus 2A

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<223> codon optimized version of sequence present in Chile

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<213> artificial sequence

<220>

<223> codon optimized version of sequence present in Chile

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<220>

<223> codon optimized version of sequence present in Chile

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<212> DNA

<213> artificial sequence

<220>

<223> codon optimized version of sequence present in Chile

<400> 9

gagtctaagt acggccctcc ttgtcctcca tgtcct 36

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<213> artificial sequence

<220>

<223> a sequence comprising a EFa/HTLV promoter and a sequence encoding a polypeptide,

the polypeptide comprises codon optimized human EGFR806scFV, igG4,

CD28tm, 41BB, CD3 ζ, P2A, DHFRdm, T2A and EGFRt

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ggatctgcga tcgctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg 60

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tcagctacag cggcaacacc cggtacaacc ccagcctgaa gtcccggatc tccatcacca 840

gagacaccag caagaaccag ttcttcctgc agctgaacag cgtgaccatc gaggacaccg 900

ccacctacta ctgtgtgaca gccggcagag gcttccctta ttggggacag ggaaccctgg 960

tcacagtgtc tgccggaagc acatctggct ctggcaaacc tggatctggc gagggctcta 1020

ccaagggcga catcctgatg acacagagcc ccagcagcat gtctgtgtcc ctgggcgata 1080

ccgtgtccat cacctgtcac agcagccagg acatcaacag caacatcggc tggctgcagc 1140

agaggcctgg caagtctttt aagggcctga tctaccacgg caccaacctg gatgatgagg 1200

tgcccagcag attttccggc tctggaagcg gagccgacta ctccctgaca atcagcagcc 1260

tggaaagcga ggacttcgcc gattactact gcgtgcagta cgcccagttt ccttggacct 1320

ttggcggagg cacaaagctg gaaatcaagc gcgagtctaa gtacggccct ccttgtcctc 1380

catgtcctat gttctgggtg ctcgtggttg ttggcggagt gctggcctgt tatagcctgc 1440

ttgtgaccgt ggccttcatc atcttttggg tcaagcgggg cagaaagaag ctgctgtaca 1500

tcttcaagca gcccttcatg cggcccgtgc agaccacaca agaggaagat ggctgctcct 1560

gcagattccc cgaggaagaa gaaggcggct gcgagctgag agtgaagttc agcagatccg 1620

ccgacgctcc tgcctatcag cagggacaga accagctgta caacgagctg aacctgggga 1680

gaagagaaga gtacgacgtg ctggacaagc ggagaggcag agatcctgag atgggcggca 1740

agcccagacg gaagaatcct caagagggcc tgtataatga gctgcagaaa gacaagatgg 1800

ccgaggccta cagcgagatc ggaatgaagg gcgagcgcag aagaggcaag ggacacgatg 1860

gactgtacca gggactgagc accgccacaa aggacaccta tgacgccctg cacatgcagg 1920

cccttccacc tagaggaagc ggcgccacaa atttcagcct gctgaaacag gccggcgacg 1980

tggaagagaa ccctggacct atggtcggaa gcctgaactg catcgtggcc gtgtctcaga 2040

acatgggcat cggcaagaac ggcgacttcc cttggcctcc tctgagaaac gagagccggt 2100

acttccagcg gatgaccacc acaagcagcg tggaaggcaa gcagaacctg gtcatcatgg 2160

gcaagaaaac ctggttcagc atccctgaga agaacagacc cctgaagggc agaatcaacc 2220

tggtgctgag cagagagctg aaagagcctc ctcagggcgc ccactttctg agcagatctc 2280

tggacgatgc cctgaagctg accgagcaac ctgagctggc caacaaggtg gacatggtct 2340

ggatcgttgg cggcagcagc gtgtacaaag aagccatgaa tcaccccggc cacctgaaac 2400

tgttcgtgac cagaatcatg caggacttcg agagcgacac attcttccca gagatcgacc 2460

tggaaaagta caaactgctg cctgagtacc ccggcgtgct gagcgacgtg caagaagaga 2520

aaggcatcaa gtacaagttc gaggtgtacg agaagaacga cggcggaggc gaaggcagag 2580

gttctctgct tacatgcgga gatgtggaag aaaatcccgg gcctatgctg ctgctcgtga 2640

caagcctgct cctgtgtgaa ctccctcatc cagcttttct gctcattccc cggaaagtgt 2700

gcaacggcat cggaatcggc gagttcaagg acagcctgag catcaacgcc accaacatca 2760

agcacttcaa gaactgcacc agcatcagcg gcgacctgca cattctgcct gtggccttta 2820

gaggcgacag cttcacccac acacctccac tggatcccca agagctggat atcctgaaaa 2880

ccgtgaaaga gatcaccgga tttctgttga tccaggcttg gcccgagaac cggacagatc 2940

tgcacgcctt cgagaacctc gagatcatca gaggccggac caagcagcac ggccagtttt 3000

ctctggccgt ggtgtccctg aatatcacct ctctgggcct gcgcagcctg aaagaaatct 3060

ccgatggcga cgtgatcatc agcggaaaca agaacctgtg ctacgccaac accatcaact 3120

ggaagaagct gttcggcacc tccggccaga aaacaaagat catctccaac cggggcgaga 3180

actcctgcaa ggctacaggc caagtgtgcc acgctctgtg tagccctgaa ggctgttggg 3240

gacccgagcc tagagattgc gtgtcctgca gaaacgtgtc ccggggcaga gaatgcgtgg 3300

acaagtgcaa tctgctcgag ggcgagccac gcgagttcgt ggaaaacagc gagtgcatcc 3360

agtgtcaccc cgagtgcctg cctcaggcca tgaacatcac atgcaccgga agaggccccg 3420

acaactgtat ccagtgcgcc cactatatcg acggccctca ctgcgtgaaa acctgtcctg 3480

ctggcgtgat gggagagaac aacaccctcg tgtggaagta cgccgatgcc ggacatgtgt 3540

gccacctgtg tcaccctaat tgcacctacg gctgtacagg cccaggactg gaaggctgcc 3600

ctacaaacgg acctaagatc cccagcattg ccaccggcat ggttggagcc ctgctgcttc 3660

tgctggtggt ggcccttgga atcggactgt ttatgtag 3698

Claims

1. A polynucleotide comprising a human codon optimized sequence encoding an anti-EGFR Chimeric Antigen Receptor (CAR).

2. The polynucleotide of claim 1, wherein the human codon optimized sequence comprises the sequence set forth in SEQ ID No. 1.

3. The polynucleotide of claim 1 or 2, further comprising a promoter operably linked to the human codon optimization sequence.

4. A polynucleotide according to claim 3, wherein the promoter comprises an EF1a sequence, or an EF1a/HTLV sequence, preferably a human EF1a sequence or a human EF1a/HTLV sequence.

5. The polynucleotide according to claim 4 wherein the promoter comprises the sequence set forth in SEQ ID NO. 2.

6. The polynucleotide of any one of claims 1-5, further comprising at least one sequence encoding a self-cleaving peptide or IRES, preferably wherein the sequence encoding a self-cleaving peptide or IRES is codon optimized for expression in a human.

7. The polynucleotide of claim 6, wherein the self-cleaving peptide is a 2A self-cleaving peptide such as P2A or T2A or both.

8. The polynucleotide of claim 7, wherein the sequence encoding the self-cleaving peptide comprises the sequence of SEQ ID NO. 3 or SEQ ID NO. 4.

9. The polynucleotide according to any one of claims 1-8, further comprising a sequence encoding one or more selectable markers, wherein the sequence encoding the one or more selectable markers is preferably codon optimized for expression in humans.

10. The polynucleotide of claim 9, wherein the one or more selectable markers comprise DHFRdm.

11. The polynucleotide of claim 9, wherein the sequence encoding the one or more selectable markers comprises the sequence set forth in SEQ ID No. 5.

12. The polynucleotide of any one of claims 1-11, further comprising a sequence encoding EGFRt, preferably wherein the sequence encoding EGFRt is codon optimized for expression in humans.

13. The polynucleotide of claim 12, wherein the sequence encoding EGFRt comprises the sequence of SEQ ID No. 6.

14. The polynucleotide according to any one of claims 1-13, further comprising a sequence encoding one or more intracellular signal domains, preferably wherein the sequence encoding the one or more intracellular signal domains is codon optimized for expression in a human.

15. The polynucleotide of claim 14, wherein the intracellular signaling domain comprises 41BB or cd3ζ or both.

16. The polynucleotide of claim 14, wherein the sequence encoding the one or more intracellular signaling domains comprises the sequence set forth in SEQ ID No. 7.

17. The polynucleotide of any one of claims 1-16, further comprising a sequence encoding a transmembrane domain, preferably wherein the sequence encoding the transmembrane domain is codon optimized for expression in a human.

18. The polynucleotide of claim 17, wherein the transmembrane domain comprises CD28tm.

19. The polynucleotide of claim 17, wherein the sequence encoding the transmembrane domain comprises the sequence set forth in SEQ ID No. 8.

20. The polynucleotide of any one of claims 1-19, further comprising a sequence encoding a spacer, preferably wherein the sequence encoding the spacer is codon optimized for expression in humans.

21. The polynucleotide of claim 20, wherein the spacer comprises a portion of IgG4, such as a hinge region of IgG 4.

22. The polynucleotide according to claim 20 wherein the sequence encoding the spacer is set forth in SEQ ID No. 9.

23. The polynucleotide according to claim 1, wherein the sequence of the polynucleotide is shown in SEQ ID No. 10.

24. An isolated cell comprising the polynucleotide of any one of claims 1-23.

25. The isolated cell of claim 24, wherein the cell is an immune cell.

26. The isolated cell of claim 25, wherein the cell is a precursor T cell or a hematopoietic stem cell.

27. The isolated cell of claim 26, wherein the cell is a T cell, B cell, natural killer cell, antigen presenting cell, dendritic cell, macrophage or granulocyte such as basophil, eosinophil, neutrophil or mast cell.

28. The isolated cell of claim 27, wherein the cell is a cd4+ T cell or a cd8+ T cell.

29. The isolated cell of claim 28, wherein the cell is a cd8+ cytotoxic T cell selected from the group consisting of naive cd8+ T cells, cd8+ memory T cells, central memory cd8+ T cells, regulatory cd8+ T cells, IPS-derived cd8+ T cells, effector memory cd8+ T cells, and mixed cd8+ T cells.

30. The isolated cell of claim 28, wherein the cell is a cd4+ T helper cell selected from the group consisting of naive cd4+ T cells, cd4+ memory T cells, central memory cd4+ T cells, regulatory cd4+ T cells, IPS-derived cd4+ T cells, effector memory cd4+ T cells, and mixed cd4+ T cells.

31. The isolated cell of any one of claims 24-30, wherein the cell is allogeneic to a subject or autologous to a subject.

32. The isolated cell of any one of claims 24-31, wherein the cell is ex vivo.

33. The isolated cell of any one of claims 24-31, wherein the cell is in vivo.

34. The isolated cell of any one of claims 24-33, wherein the cell is a mammalian cell.

35. The isolated cell of any one of claims 24-34, wherein the cell is a human cell.

36. An isolated cell comprising the polynucleotide of any one of claims 1-23; and wherein the isolated cell further expresses an antibody or binding fragment thereof specific for a B cell-specific cell surface molecule or scFv, such as CD19, CD20, CDld, CD5, CD19, CD20, CD21, CD22, CD 23/fcyrii, CD24, CD25/IL-2rαcd27/TNFRSF7, CD32, CD34, CD35, CD38, CD40 (TNFRSF 5), CD44, CD45, CD45.1, CD45.2, CD54 (ICAM-1), CD69, CD72, CD79, CD80, CD 84/slacf 5, LFA-1, CALLA, BCMA, B Cell Receptor (BCR), igM, igD, B/CD 45R, clqR/CD 93, CD 84/slacf 5, BAFFRTNFRSF13C, B/CD 45R, B7-1/CD80, B7-2/CD86, TNFSF7, TNFRSF5, ENPP-1, hvfsf 14, bldm 1/TNFRSF 1, impdm 1/prp 1, prp 1/147, or prp 147.

37. The isolated cell of claim 36, wherein the cell is an immune cell.

38. The isolated cell of claim 37, wherein the cell is a precursor T cell or a hematopoietic stem cell.

39. The isolated cell according to claim 38, wherein the cell is a T cell, B cell, natural killer cell, antigen presenting cell, dendritic cell, macrophage or granulocyte, such as a basophil, eosinophil, neutrophil or mast cell.

40. The isolated cell of claim 39, wherein the cell is a cd4+ T cell or a cd8+ T cell.

41. The isolated cell of claim 40, wherein the cell is a cd4+ T helper cell selected from the group consisting of naive cd4+ T cells, cd4+ memory T cells, central memory cd4+ T cells, regulatory cd4+ T cells, IPS-derived cd4+ T cells, effector memory cd4+ T cells, and mixed cd4+ T cells.

42. The isolated cell of any one of claims 36-41, wherein the cell is allogeneic to the subject or autologous to the subject.

43. The isolated cell of any one of claims 36-42, wherein the cell is ex vivo.

44. The isolated cell of any one of claims 36-42, wherein the cell is in vivo.

45. The isolated cell of any one of claims 36-42, wherein the cell is a mammalian cell.

46. The isolated cell of any one of claims 36-42, wherein the cell is a human cell.

47. A method of inhibiting or treating cancer in a subject, preferably a human, in need thereof, comprising administering to the subject the polynucleotide of any one of claims 1-23 or the cell of claims 24-46.

48. The method of claim 47, wherein the administering is by intracranial injection.

49. The method of any one of claims 47 or 48, wherein the cancer is glioblastoma.

50. The method of any one of claims 47-48, wherein the cancer is a solid tumor.

51. The method of claim 50, wherein the solid tumor is selected from the group consisting of breast cancer, brain cancer, liver cancer, stomach cancer, spleen cancer, colon cancer, kidney cancer, pancreatic cancer, prostate cancer, uterine cancer, skin cancer, head cancer, neck cancer, sarcoma, neuroblastoma, and ovarian cancer.

52. Use of the polynucleotide of any one of claims 1-23 or the cell of any one of claims 24-46 as a medicament.

53. A polynucleotide according to any one of claims 1 to 23 or a cell according to any one of claims 24 to 46 for use in the treatment or inhibition of cancer such as glioblastoma, leukemia, lymphoma, hematological, liquid or solid tumors.

54. A method of inhibiting or treating cancer in a subject, preferably a human, in need thereof, comprising administering to the subject the polynucleotide of any one of claims 1-23 or the cell of claims 24-46 in combination with an effective amount of at least one additional anti-cancer agent to provide a combination therapy with an enhanced therapeutic effect.

55. The method of claim 54, wherein the cancer is glioblastoma.

56. The method of claim 54, wherein the cancer is a solid tumor.

57. The method of claim 54, wherein the cancer is a lymphoma, hematological tumor, or liquid tumor.

58. The method of claim 54, wherein the anti-cancer agent is delivered with a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof.