Reduction of cytokine release syndrome in immunotherapy

Abstract

The present invention discloses a recombinant nucleic acid molecule encoded by at least ORF (100, 100a) to reduce cytokine storm during immunotherapy. The recombinant nucleic acid molecule comprises at least one first hairpin ring structure, at least one second hairpin ring structure and a first promoter (120). The first hairpin loop structure is formed from one or more first short hairpin RNA (130a) sequences. The first hairpin ring structure modulates the amount of interleukin 6 (IL6) cytokine during immunotherapy. The second hairpin loop structure is formed from one or more second short hairpin RNA (130b) sequences. The second hairpin ring structure modulates the amount of granulocyte monocyte colony stimulating factor (GMCSF) cytokine during immunotherapy and is disposed upstream or downstream of the first hairpin ring structure. The first promoter (120) is disposed upstream of the first hairpin ring structure and the second hairpin ring structure.

Description

[0001] priority

[0002] This application claims priority to Indian Provisional Application No. 202221053835 filed on October 20, 2022, the contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to artificially modified immune effector cells for reducing cytokine release syndrome. More specifically, the present disclosure relates to artificially modified immune effector cells that reduce the levels of interleukin 6 (IL6) and granulocyte monocyte colony stimulating factor (GMCSF) released during cancer immunotherapy. Background Art

[0004] Cancer is a disease in which abnormal cells divide uncontrollably and destroy body tissues. The body's natural immune cells preferably identify advanced tumors as "hot" or "cold," depending on the presence or absence of immune cells at the tumor site. In the case of hot tumors, although immune cells (including cytotoxic T lymphocytes, or T cells) are abundant at the tumor site, they are inactive due to inhibitory signals generated by the tumor microenvironment. In the case of cold tumors, it is the chemotactic signaling of T cells that is suppressed.

[0005] In order to enable immune cells to actively target cancer cells, immunotherapy based on chimeric antigen receptor (CAR) T cells has been introduced. CAR T cell-based immunotherapy is a type of adoptive cell therapy (ACT) in which immune cells are engineered (genetically manipulated) to specifically target and kill cancer cells. In other words, the CAR construct of the CAR T cell is designed to target a specific tumor-associated antigen (TAA).

[0006] Conventional CAR T cell-based therapies target a single tumor antigen that is only effective against B cell malignancies. Therefore, conventional CAR T cell-based therapies have limited application against other blood cell-related malignancies (low CD19), where "off-target effects" are a major problem.

[0007] Although most adverse events of CAR T cell immunotherapy are tolerable and acceptable, CAR T cell immunotherapy is not without significant side effects. The latest conventionally available CAR T cell-based immunotherapy (based on second-generation CAR design) often shows severe systemic toxicity, including the release of large amounts of cytokines. Cytokines are signaling molecules that regulate the function of the immune system. The release of large amounts of cytokines, known as cytokine release syndrome (CRS), causes immune-related complications and / or neurotoxicity. Neurotoxicity includes symptoms such as confusion, delirium, and seizures, which further leads to the rapid release of cytokines into the central nervous system. In CRS, the immune system overreacts and releases large amounts of cytokines, resulting in a cytokine “storm” in patients immediately after administration of CAR T cell-based immunotherapy. The released cytokines can cause fever, hypotension, and dyspnea. Interleukin 6 (IL6) and granulocyte monocyte colony-stimulating factor (GMCSF) are cytokines that are upregulated during CRS and are several key pro-inflammatory molecules that cause CRS.

[0008] For example, Figure 1 depicts the immune response of conventional CAR T cells. Conventional CAR T cells induce the release of high levels of cytokines, such as GMCSF, IL6, etc. The released GMCSF recruits pro-inflammatory immune cells, such as monocytes, neutrophils, basophils, and macrophages. These pro-inflammatory immune cells induce (upregulate) the release of high levels of pro-inflammatory cytokines, such as IL6, interleukin 1 (IL1), nitric oxide (NO), etc. These high levels of pro-inflammatory cytokines ultimately lead to CRS.

[0009] Although genome engineering approaches have been used to completely knock out genes involved in CRS, these approaches have limitations. For example, complete gene knockout may have adverse effects on CAR T cell survival and may also induce off-target effects.

[0010] Furthermore, due to the nature of CAR T cells as living therapeutic agents, engineered CAR T cells may transform into cancer cells due to uncontrolled proliferation of CAR T cells.

[0011] Therefore, CRS, along with the inability to control the proliferation of engineered CAR T cells, constitutes a major challenge in treating cancer patients with CAR T cell-based immunotherapy. In addition, patients who have been infused with conventional CAR T cells have been diagnosed with relapse (i.e., the recurrence of cancer cells) because conventional CAR T cells do not persist for long periods of time.

[0012] In view of the above discussion, a novel CAR T cell-based immunotherapy is needed to overcome the problems associated with conventional CAR T cell-based immunotherapy. Summary of the Invention

[0013] Specific embodiments of the present disclosure are described below with reference to the accompanying drawings. However, it should be understood that the disclosed embodiments are merely examples of the present disclosure and that the present disclosure can be embodied in various forms. Well-known functions or configurations are not described in detail to avoid obscuring the present disclosure with unnecessary detail. Therefore, the specific structural and functional details disclosed herein should not be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching those skilled in the art to use the present disclosure in various ways in almost any appropriately detailed structure.

[0014] In an exemplary embodiment, the present disclosure relates to a recombinant nucleic acid molecule that is at least encoded by an ORF to reduce a cytokine storm during immunotherapy. The recombinant nucleic acid molecule comprises at least one first hairpin loop structure, at least one second hairpin loop structure, and a first promoter. The first hairpin loop structure is formed by one or more first short hairpin RNA sequences. The first hairpin loop structure regulates the amount of interleukin 6 (IL6) cytokine during immunotherapy. The second hairpin loop structure is formed by one or more second short hairpin RNA sequences. The second hairpin loop structure regulates the amount of granulocyte monocyte colony stimulating factor (GMCSF) cytokine during immunotherapy. The second hairpin loop structure is arranged upstream or downstream of the first hairpin loop structure. The first promoter is arranged upstream of the first hairpin loop structure and the second hairpin loop structure.

[0015] In an exemplary embodiment, the present disclosure relates to a recombinant nucleic acid molecule that is at least encoded by an ORF to reduce cytokine storm during immunotherapy. The recombinant nucleic acid molecule comprises at least one first hairpin loop structure, at least one second hairpin loop structure, a first promoter, one or more chimeric antigen receptor genes, a second promoter, and one or more long terminal repeats. The first hairpin loop structure is formed by one or more first short hairpin RNA sequences. The first hairpin loop structure regulates the amount of interleukin 6 (IL6) cytokine during immunotherapy. The second hairpin loop structure is formed by one or more second short hairpin RNA sequences. The second hairpin loop structure regulates the amount of granulocyte monocyte colony stimulating factor (GMCSF) cytokine during immunotherapy. The second hairpin loop structure is arranged upstream or downstream of the first hairpin loop structure. The first promoter is arranged upstream of the first hairpin loop structure and the second hairpin loop structure. The chimeric antigen receptor gene comprises one or more single-chain variable fragment domains, one or more hinge domains, one or more costimulatory domains, one or more signaling domains, one or more safety switch domains, and one or more transmembrane domains. The single-chain variable fragment domain is configured to bind one or more tumor-associated antigens (TAA). The hinge domain is coupled to the single-chain variable fragment domain. The signaling domain is coupled to the costimulatory domain. The safety switch domain is coupled to the signaling domain. The transmembrane domain is operationally coupled to the hinge domain to the costimulatory domain. The second promoter is located upstream of the chimeric antigen receptor gene. The long terminal repeat sequence is located upstream of the first promoter and downstream of the chimeric antigen receptor gene.

[0016] In an exemplary embodiment, the present disclosure relates to a recombinant nucleic acid molecule that is at least encoded by an ORF to reduce cytokine storm during immunotherapy. The recombinant nucleic acid molecule comprises at least one first hairpin loop structure, at least one second hairpin loop structure, a first promoter, a poly A tail sequence, one or more chimeric antigen receptor genes, a second promoter, and one or more long terminal repeats. The first hairpin loop structure is formed by one or more first short hairpin RNA sequences. The first hairpin loop structure regulates the amount of interleukin 6 (IL6) cytokines during immunotherapy. The second hairpin loop structure is formed by one or more second short hairpin RNA sequences. The second hairpin loop structure regulates the amount of granulocyte monocyte colony stimulating factor (GMCSF) cytokines during immunotherapy. The second hairpin loop structure is arranged upstream or downstream of the first hairpin loop structure. The first promoter is arranged upstream of the first hairpin loop structure and the second hairpin loop structure. The poly A tail sequence is arranged downstream of the first hairpin loop structure and the second hairpin loop structure. The chimeric antigen receptor gene comprises one or more single-chain variable fragment domains, one or more hinge domains, one or more costimulatory domains, one or more signaling domains, one or more safety switch domains and one or more transmembrane domains. The single-chain variable fragment domain is configured to bind to one or more tumor-associated antigens (TAA). The hinge domain is coupled to the single-chain variable fragment domain. The signaling domain is coupled to the costimulatory domain. The safety switch domain is coupled to the signaling domain. The transmembrane domain is operationally coupled to the hinge domain to the costimulatory domain. The second promoter is located upstream of the chimeric antigen receptor gene. The long terminal repeat sequence is located upstream of the first promoter and downstream of the chimeric antigen receptor gene.

[0017] In an exemplary embodiment, the present disclosure relates to a transcript of a recombinant nucleic acid molecule, the transcript of the recombinant nucleic acid molecule being encoded by at least an ORF to reduce cytokine storm during immunotherapy. The transcript comprises a messenger RNA of a first hairpin loop structure or a second hairpin loop structure transcribed from the recombinant nucleic acid molecule. The messenger RNA of the first hairpin loop structure or the second hairpin loop structure is configured to bind to the messenger RNA of a cytokine.

[0018] In an exemplary embodiment, the present disclosure relates to a vector comprising at least one of a recombinant nucleic acid molecule linked to at least one of a plasmid, a cosmid, a viral vector, and a bacteriophage, wherein the recombinant nucleic acid molecule is encoded by at least an ORF to reduce cytokine storm during immunotherapy.

[0019] In exemplary embodiments, the present disclosure relates to an engineered immune cell comprising at least one of the recombinant nucleic acid molecules encoded by at least an ORF to reduce cytokine storm during immunotherapy.

[0020] In an exemplary embodiment, the present disclosure relates to a composition comprising engineered immune cells suspended in a nutrient medium. The engineered immune cells comprise at least one recombinant nucleic acid molecule encoded by at least one ORF to reduce cytokine storm during immunotherapy.

[0021] In an exemplary embodiment, the present disclosure relates to a method for preparing engineered immune cells. The method includes isolating a plurality of T cells from a population of peripheral blood mononuclear cells, replicating at least one recombinant nucleic acid molecule in a vector, the at least one recombinant nucleic acid molecule encoding at least an ORF for reducing cytokine storm during immunotherapy, and delivering the vector to the plurality of T cells.

[0022] From the detailed description that is carried out below with reference to the accompanying drawings, the aforementioned features and other features and advantages of the present invention will become more apparent. In the accompanying drawings, similar reference numerals indicate the same or similar parts in several views. These features will be described at a sufficiently detailed level to allow those skilled in the art to implement the present invention. In addition, it should be understood that other features may be used and structural changes may be made without departing from the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The above summary of the invention and the detailed description of the following illustrative embodiments will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present disclosure, exemplary configurations of the present disclosure are shown in the accompanying drawings. However, the present disclosure is not limited to the specific methods and means disclosed herein. In addition, those skilled in the art will appreciate that the drawings are not drawn to scale.

[0024] FIG1 depicts a schematic diagram of CRS induction (prior art) according to one or more exemplary embodiments of the present disclosure.

[0025] Figure 2 An ORF 100 is depicted according to one or more exemplary embodiments of the present disclosure.

[0026] Figure 2a Depicted is an ORF 100a according to one or more exemplary embodiments of the present disclosure.

[0027] Figure 3 Depicted is a CAR construct 200 according to one or more exemplary embodiments of the present disclosure.

[0028] Figure 3aDepicted is a CAR construct 300 according to one or more exemplary embodiments of the present disclosure.

[0029] Figure 4 Depicted is a graphical representation of CRS reduction according to one or more exemplary embodiments of the present disclosure.

[0030] Figure 5 Depicted is a method 400 of preparing CAR T cells according to one or more exemplary embodiments of the present disclosure.

[0031] Figures 6 to 8 Experimental observations according to one or more exemplary embodiments of the present disclosure are depicted. DETAILED DESCRIPTION

[0032] Before describing the present invention in detail, the definitions of certain words or phrases used throughout this patent document will be defined: the terms "include" and "comprising" and their derivatives mean including but not limited to; the term "or" is inclusive, meaning and / or; the phrases "coupled with" and "associated with" and their derivatives may mean including, included in, interconnected with, containing, contained in, connected to or connected with, coupled to or coupled with, communicable with, cooperating with, interleaved, juxtaposed, proximate, incorporated into or combined with, having the property of, etc.; definitions of certain words and phrases are provided throughout this patent document, and those skilled in the art will understand that these definitions apply to prior, as well as future uses of these defined words and phrases in many, if not most, instances.

[0033] References throughout this specification to "one embodiment," "embodiment," or similar language mean that the specific features, structures, or characteristics described in conjunction with the embodiment are included in at least one embodiment. Therefore, unless expressly stated otherwise, the phrases "in one embodiment," "in an embodiment," and similar language throughout this specification may, but do not necessarily, refer to the same embodiment, but rather mean "one or more but not all embodiments." Unless expressly stated otherwise, the terms "comprise," "comprising," "having," and variations thereof mean "including but not limited to." Unless expressly stated otherwise, an enumerated list of items does not mean that any or all items are mutually exclusive and / or mutually inclusive. Unless expressly stated otherwise, the terms "a," "an," and "the" also refer to "one or more."

[0034] As used herein, the term "activated" refers to a state in which cells have been sufficiently stimulated to induce detectable cell proliferation and / or differentiation into effector T cells (or activated T cells). Activation can also be associated with induced cytokine production and detectable effector function.

[0035] The term "activated T cells" refers in particular to T cells (or CAR T cells) that express a chimeric antigen receptor (CAR) construct and / or are capable of binding to a tumor-associated antigen (TAA).

[0036] The term "antibody" is used in the broadest sense and refers to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as bispecific antibodies) and antibody fragments, so long as they exhibit the desired biological activity or function. The antibodies in the present disclosure can exist in various forms, including, for example, polyclonal antibodies; monoclonal antibodies; Fv, Fab, Fab' and F(ab')2 fragments; as well as single-chain antibodies and humanized antibodies.

[0037] The term "antibody fragment" refers to a portion of a full-length antibody, such as the antigen-binding or variable region of an antibody. Other examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0038] The term "Fv" refers to the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment consists of a dimer of a heavy chain variable region domain and a light chain variable region domain in tight non-covalent association. The folding of these two domains produces six hypervariable loops (3 loops each for the H chain and the L chain), which contribute amino acid residues for antigen binding and give the antibody antigen binding specificity. However, even a single variable domain (or half of an Fv comprising only three complementary determining regions (CDRs) for antigen specificity) has the ability to recognize and bind to antigens, although the affinity is lower than that of the entire binding site (dimer).

[0039] As used herein, an "antibody heavy chain" refers to the larger of the two types of polypeptide chains found in all antibody molecules in their naturally occurring conformation. As used herein, an "antibody light chain" refers to the smaller of the two types of polypeptide chains found in all antibody molecules in their naturally occurring conformation. Kappa and lambda light chains refer to the two major antibody light chain isotypes.

[0040] The term "synthetic antibody" refers to an antibody produced using recombinant DNA technology, such as an antibody expressed by a bacteriophage. The term also includes antibodies produced by synthesizing a DNA molecule encoding the antibody and expressing the DNA molecule to obtain the antibody or the amino acids encoding the antibody. Synthetic DNA is obtained using techniques available and well known in the art.

[0041] The term "antigen" refers to a molecule that elicits an immune response, which may be related to antibody production or activation of specific immune competent cells, or both. Antigens include any macromolecule, including all proteins or peptides, or molecules derived from recombinant or genomic DNA. For example, DNA comprises a nucleotide sequence or partial nucleotide sequence encoding a protein or peptide that elicits an immune response, and thus encodes the term "antigen" as used herein. An antigen need not be encoded solely by the full-length nucleotide sequence of a gene. Antigens can be produced, synthesized, or derived from a biological sample, including a tissue sample, a tumor sample, a cell, or a biological fluid.

[0042] As used herein, the term "anti-tumor effect" refers to a biological effect associated with a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, a reduction in tumor cell proliferation, a reduction in tumor cell survival, an increase in the life expectancy of a subject with tumor cells, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" can also be manifested by the ability of peptides, polynucleotides, cells, and antibodies to prevent tumorigenesis in the first place.

[0043] The term "autoantigen" refers to an endogenous antigen that is mistakenly recognized as foreign by the immune system. Autoantigens include cellular proteins, phosphoproteins, cell surface proteins, cellular lipids, nucleic acids, glycoproteins, including cell surface receptors.

[0044] The term "autologous" is used to describe material derived from a subject that is subsequently reintroduced into the same subject.

[0045] The term "allogeneic" is used to describe a transplant that originates from a different subject of the same species. For example, the donor subject may or may not be related to the recipient subject, but the donor subject has similar immune system markers to the recipient subject.

[0046] The term "xenogeneic" is used to describe a transplant that originates from a subject of a different species. For example, the donor subject is from a different species than the recipient subject, and the donor and recipient subjects may be genetically and immunologically incompatible.

[0047] The term "cancer" is used to refer to a disease characterized by the rapid, uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body through the bloodstream and lymphatic system. Examples of various cancers include breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc.

[0048] The terms "complementary" and "complementarity" refer to polynucleotides (i.e., nucleotide sequences) that are related by the base pairing rules. For example, the sequence "AGT" is complementary to the sequence "TCA." Complementarity can be "partial," where only some of the bases of the nucleic acids match according to the base pairing rules, or there can be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands significantly affects the efficiency and strength of hybridization between nucleic acid strands.

[0049] The term "corresponding to" or "corresponding to..." refers to (a) a polynucleotide having a nucleotide sequence that is substantially identical or complementary to all or part of a reference polynucleotide sequence, or encoding an amino acid sequence that is identical to the amino acid sequence in a peptide or protein; or (b) a peptide or polypeptide having an amino acid sequence that is substantially identical to the amino acid sequence in a reference peptide or protein.

[0050] The term "costimulatory ligand" refers to a molecule on an antigen presenting cell (e.g., APC, dendritic cell, B cell, etc.) that specifically binds to a cognate costimulatory molecule on a T cell, thereby providing a signal that mediates T cell responses, including proliferation, activation, differentiation, and at least one of other cellular responses, in addition to the primary signal provided by the binding of, for example, a TCR / CD3 complex to an MHC molecule loaded with a peptide. Costimulatory ligands also particularly include agonists or antibodies that specifically bind to costimulatory molecules present on T cells (such as CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3), and ligands that specifically bind to CD83.

[0051] The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the T cell, such as proliferation. Costimulatory molecules include MHC class I molecules, BTLA, and Toll-like receptors.

[0052] The term "co-stimulatory signal" refers to a signal that, in combination with a primary signal (such as TCR / CD3 ligation), results in T cell proliferation and / or upregulation or downregulation of key molecules.

[0053] The term "coding" refers to the intrinsic properties and the biological properties resulting therefrom of a specific nucleotide sequence in a polynucleotide (e.g., a gene, cDNA, or mRNA) that is used as a template for synthesizing other polymers and macromolecules with a determined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a determined amino acid sequence in a biological process. Therefore, if transcription and translation of the mRNA corresponding to the gene produce a protein in a cell or other biological system, the gene encodes a protein. Coding strands (whose nucleotide sequence is identical to the mRNA sequence (except that "T" is replaced by "U") and are generally provided in sequence tables) and non-coding strands (used as templates for gene or cDNA transcription) can both be referred to as proteins or other products encoding the gene or cDNA.

[0054] The term "exogenous" refers to a molecule that does not naturally occur in wild-type cells or organisms but is generally introduced into cells by molecular biology techniques. Examples of exogenous polynucleotides include vectors, plasmids and / or artificial nucleic acid constructs that encode the desired protein. With respect to polynucleotides and proteins, the terms "endogenous" or "natural" refer to naturally occurring polynucleotides or amino acid sequences that can be found in a given wild-type cell or organism. In addition, a specific polynucleotide sequence that is isolated from a first organism and transferred to a second organism by molecular biology techniques is generally considered to be an "exogenous" polynucleotide or amino acid sequence with respect to the second organism. In specific embodiments, a polynucleotide sequence can be "introduced" into a microorganism that already contains such a polynucleotide sequence by molecular biology techniques, for example, to produce one or more additional copies of other naturally occurring polynucleotide sequences, thereby promoting overexpression of the encoded polypeptide.

[0055] The term "expression" refers to the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0056] The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control (regulatory) sequence operably linked to a nucleotide sequence to be expressed. An expression vector includes sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate recombinant polynucleotides.

[0057] In the context of this disclosure, the following abbreviations for common nucleic acid bases are used: "A" refers to adenine, "C" refers to cytosine, "G" refers to guanine, "T" refers to thymine, and "U" refers to uracil.

[0058] Unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" may also include introns, to the extent that a nucleotide sequence encoding a protein may contain introns in some forms.

[0059] The term "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in their ability to infect non-dividing cells; they can deliver large amounts of genetic information into the host cell's DNA, making them one of the most efficient gene delivery vectors. Furthermore, the use of lentiviruses enables integration of the genetic information into the host chromosome, resulting in stably transduced genetic information. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide a means to achieve significant levels of gene transfer in vivo.

[0060] The term "modulate" refers to mediating a detectable increase or decrease in the level of a response in a subject compared to the level of the response in a subject not receiving the treatment or compound, and / or compared to the level of the response in an otherwise identical but untreated subject. The term encompasses interfering with and / or influencing a natural signal or response, thereby mediating a beneficial therapeutic response in a subject (preferably a human).

[0061] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to promote translation.

[0062] The term "under transcriptional control" means that the promoter is operably linked to a polynucleotide and is in the correct location and orientation relative to the polynucleotide to control (regulate) transcription initiation by RNA polymerase and expression of the polynucleotide.

[0063] The term "overexpressed" tumor antigen or "overexpression" of a tumor antigen is intended to refer to abnormal expression levels of a tumor antigen in cells from a disease area (e.g., a solid tumor within a particular tissue or organ of a patient) relative to the expression level in normal cells from that tissue or organ. Patients with solid tumors or hematologic malignancies characterized by overexpression of a tumor antigen can be identified by standard assays known in the art.

[0064] The terms "patient," "subject," and "individual," and the like, are used interchangeably herein and refer to any person or animal amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject, or individual is a human or animal. In embodiments, the term "subject" is intended to include living organisms (e.g., mammals) in which an immune response can be elicited. Examples of subjects include humans and animals such as dogs, cats, mice, rats, and transgenic species thereof.

[0065] A subject in need or in need of treatment includes a subject having a disease, disorder, or condition in need of treatment. A subject in need thereof also includes a subject in need of treatment to prevent a disease, disorder, or condition.

[0066] The term "polynucleotide" or "nucleic acid" refers to mRNA, RNA, cRNA, rRNA, cDNA, or DNA. The term generally refers to a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides, or modified forms of either type of nucleotide. The term includes all forms of nucleic acids, including single-stranded and double-stranded forms of nucleic acids.

[0067] The terms "polynucleotide variant" and "variant" and the like refer to polynucleotides that show substantial sequence identity to a reference polynucleotide sequence or polynucleotides that hybridize to a reference sequence under stringent conditions as defined below. These terms also encompass polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion, or substitution of at least one nucleotide. Thus, the terms "polynucleotide variant" and "variant" include polynucleotides in which one or more nucleotides have been added or deleted or replaced by different nucleotides. In this regard, it is well understood in the art that certain changes, including mutations, additions, deletions, and substitutions, can be made to a reference polynucleotide such that the altered polynucleotide retains the biological function or activity of the reference polynucleotide or has increased activity (i.e., optimized) relative to the reference polynucleotide. Polynucleotide variants include, for example, polynucleotides having at least 50% (and at least 51% to at least 99% and all integer percentages therebetween, such as 90%, 95%, or 98%) sequence identity to a reference polynucleotide sequence as described herein. The terms "polynucleotide variant" and "variant" also include naturally occurring allelic variants and orthologs.

[0068] The terms "polypeptide," "polypeptide fragment," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues and variants and synthetic analogs thereof. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analog of a corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers. In certain aspects, polypeptides may include enzymatic polypeptides or "enzymes," which generally catalyze (i.e., increase) the rate of various chemical reactions.

[0069] The term "polypeptide variant" refers to a polypeptide that is distinguished from a reference polypeptide sequence by the addition, deletion, or substitution of at least one amino acid residue. In certain embodiments, a polypeptide variant is distinguished from a reference polypeptide by one or more substitutions, which may be conservative or non-conservative. In certain embodiments, a polypeptide variant comprises conservative substitutions, and in this regard, it is well understood in the art that some amino acids may be changed to other amino acids having broadly similar properties without altering the nature of the polypeptide's activity. Polypeptide variants also encompass polypeptides in which one or more amino acids have been added or deleted or replaced by different amino acid residues.

[0070] The term "promoter" refers to a DNA sequence that is recognized by the cell's synthetic machinery or introduced synthetic machinery and is required to initiate specific transcription of a polynucleotide sequence. The term "expression control (regulatory) sequence" refers to a DNA sequence necessary for expression of an operably linked coding sequence in a particular host organism. For example, control sequences suitable for prokaryotes include a promoter, an optional operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0071] The term "binding", "binding to..." or "interacting with..." refers to a molecule that recognizes and adheres to a second molecule in a sample or organism but does not substantially recognize or adhere to other structurally unrelated molecules in the sample. The term "specific binding" as used herein with respect to antibodies refers to an antibody that recognizes a specific antigen but does not substantially recognize or bind to other molecules in the sample. For example, an antibody that specifically binds to an antigen from one species can also bind to the antigen from one or more species. However, this cross-species reactivity itself does not change the classification of the antibody as a specific antibody. In another example, an antibody that specifically binds to an antigen can also bind to different allelic forms of the antigen. However, this cross-reactivity itself does not change the classification of the antibody as a specific antibody. In some cases, the term "specific binding" or "specific binding" can be used to refer to the interaction of an antibody, protein or peptide with a second chemical substance, meaning that the interaction depends on the presence of a specific structure (e.g., antigenic determinant or epitope) on the chemical substance; for example, an antibody recognizes and binds to a specific protein structure, rather than any protein. If the antibody is specific for epitope "A", then in a reaction containing labeled "A" and the antibody, the presence of molecules containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody.

[0072] The term "stimulation" refers to a primary response induced by binding of a stimulatory molecule (e.g., a TCR / CD3 complex) to its cognate ligand, thereby mediating a signal transduction event (such as signal transduction via the TCR / CD3 complex). Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-β and / or reorganization of cytoskeletal structure.

[0073] The term "stimulatory molecule" refers to a cognate stimulatory ligand present on an antigen presenting cell that specifically binds to a molecule on a T cell.

[0074] The term "stimulatory ligand" refers to a ligand that, when present on an antigen-presenting cell (e.g., APC, dendritic cell, B cell, etc.), can specifically bind to a cognate binding partner (referred to herein as a "stimulatory molecule") on a cell (e.g., a T cell), thereby mediating a primary response of the T cell (including activation, initiation of an immune response, proliferation, and the like).

[0075] The term "transfected" or "transformed" or "transduced" refers to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acid. Such cells include the primary subject cell and its progeny.

[0076] A "chimeric antigen receptor" (CAR) molecule is a recombinant polypeptide comprising at least an extracellular domain, a transmembrane domain, and a cytoplasmic domain or an intracellular domain.

[0077] Although the operations of the exemplary embodiments of the disclosed methods may be described in a particular sequential order for ease of presentation, it should be understood that the disclosed embodiments may encompass orders of operations other than the particular sequential order disclosed. For example, the operations described sequentially may be rearranged or performed simultaneously in some cases. Further, the descriptions and disclosures provided in conjunction with a particular embodiment are not limited to that embodiment and may apply to any embodiment disclosed herein. In addition, for simplicity, the accompanying drawings may not illustrate the various ways in which the disclosed systems, methods, and apparatus may be used in combination with other systems, methods, and apparatus.

[0078] Furthermore, the described features, advantages, and characteristics of the embodiments may be combined in any suitable manner. Those skilled in the relevant art will recognize that an embodiment may be practiced without one or more of the specific features or advantages of a particular embodiment. In other cases, additional features and advantages may be recognized in certain embodiments that may not be present in all embodiments. These features and advantages of the embodiments will become more fully apparent from the following description and the assigned claims, or may be learned through practice of the embodiments as set forth below.

[0079] The present disclosure discloses a chimeric antigen receptor T cell (CAR T cell). CAR T cells are artificially modified by genetic manipulation. CAR T cells can be derived from autologous or allogeneic sources. CAR T cells can be used to target different cell pathologies, including but not limited to B cell malignancies (blood cancers), solid tumors, etc. In embodiments, CAR T cells are directed against relapsed or refractory B cell acute lymphoblastic leukemia (ALL) or non-Hodgkin lymphoma (NHL).

[0080] CAR T cells can be genetically engineered to contain one or more sequences that express one or more chimeric antigen receptors (CARs or CAR constructs). CAR has a predefined specificity for one or more tumor-associated antigens (TAA), including but not limited to cluster of differentiation 19 (CD19), cluster of differentiation 7 (CD7), cluster of differentiation 20 (CD20), cluster of differentiation 22 (CD22), cluster of differentiation 123 (CD123), cluster of differentiation 133 (CD133), cluster of differentiation 30 (CD30), cluster of differentiation 138 (CD138), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), fibroblast activation protein alpha (FAP), mucin 1 (MUC1), disialoganglioside GD2 (GD2), carcinoembryonic antigen (CEA), prostate-specific membrane antigen (PSMA), human epidermal growth factor receptor 2 (HER2), New York esophageal squamous cell carcinoma-1 (NY-ESO-1), melanoma-associated antigen 3 (MAGEA-A3), human telomerase reverse transcriptase (hTERT), etc. In an exemplary embodiment, the CAR T cell includes a CAR specific for CD19.

[0081] Further, CAR T cells include at least two short hairpin RNA (shRNA) sequences to reduce the cytokine storm known as cytokine release syndrome (CRS) during immunotherapy. During CRS, rapid release of cytokines was observed. Interleukin 6 (IL6) and granulocyte monocyte colony stimulating factor (or granulocyte macrophage colony stimulating factor) (GMCSF) are some cytokines released during immunotherapy. The shRNA sequence can inhibit cytokine production and / or cytokine signaling by RNA interference (RNAi). In an exemplary embodiment, the transcript of the shRNA sequence is combined with the mRNA of the cytokine to inactivate the mRNA functionality and / or degrade the mRNA. In an alternative embodiment, the transcript of the shRNA sequence is combined with the mRNA of the cytokine to prevent its translation. In another exemplary embodiment, shRNA is combined with one or more receptors of the cytokine, thereby blocking the downstream signaling pathway of the cytokine. In another exemplary embodiment, the shRNA sequence expresses an anti-IL6 (aIL6) that binds to and neutralizes IL6 (exemplary cytokine), thereby at least partially blocking the activity of IL6.

[0082] In an exemplary embodiment, the CAR T cells of the present disclosure include an IL-6 receptor (IL6-R) conjugated to a synthetic notch (syn-notch) receptor and a GAL-VP64 domain. During CRS, the released IL6 binds to the IL6-R of the CAR T cells and induces syn-notch, followed by cutting and releasing the GAL-VP64 domain. The GAL-VP64 domain migrates to the nucleus of the CAR T cells and induces the expression of one of the thirty-six shRNA sequences specific for interleukin 6 (or IL6). The expression of the shRNA sequence results in the production of aIL6. aIL6 binds to and neutralizes IL6. IL6 is a key molecule involved in inducing CRS and causing premature apoptosis of CART cells. By neutralizing IL6, the CAR T cells disclosed herein have a lower risk of CRS, thereby having the ability to safely and effectively remove cancer cells.

[0083] In another exemplary embodiment, the CAR T cells of the present disclosure produce transcripts of at least one of the twenty shRNAs that are specific to granulocyte monocyte colony stimulating factor (or GMCSF), which contributes to cytokine release syndrome (CRS) during immunotherapy. In an exemplary embodiment, the shRNA sequence regulates (i.e., reduces) the amount of GMCSF produced by selectively binding to GMCSF mRNA and rendering it nonfunctional. GMCSF is a key molecule involved in initiating CRS and causing premature apoptosis of CAR T cells. By regulating the amount of GM-CSF produced, CAR T cells as disclosed herein have a lower risk of CRS and therefore persist for a longer period of time, thereby having the ability to safely and effectively remove cancer cells.

[0084] In an exemplary embodiment, the CAR T cells of the present disclosure can produce at least one shRNA specific for each of GMCSF mRNA and IL6 mRNA, thereby reducing the concentration levels of GMCSF and IL6 molecules, thereby reducing the CRS burden.

[0085] Further, CAR comprises a safety switch (SS) domain for regulating the proliferation of CAR T cells, thereby inhibiting the transformation of CART cells into cancer cells (e.g., during the manufacture of CAR T cells in the patient's body or after infusion). The SS domain may include but is not limited to inducible caspase 9 (iCaspase 9), truncated epidermal growth factor receptor (EGFRt), RQR8, or a combination thereof.

[0086] Therefore, for the reasons described above, the CAR T cells disclosed herein are programmed to elicit an artificial immune response against cancer cells without any undesirable side effects of CRS. Further, based on the patient's (or subject's) condition and requirements, the CAR T cells of the present disclosure can be selectively depleted to prevent the malignant transformation of the CAR T cells.

[0087] Referring now to the accompanying drawings, Figure 2 The antisense (or template) strand of an open reading frame (ORF) 100 of a CAR T cell (not shown) is depicted. Thus, ORF 100 may include a sense (or coding) strand (not shown) that is complementary to the antisense strand. The antisense strand of ORF 100 extends from the 3' end to the 5' end. Thus, in terms of orientation, the 3' end of the antisense strand of ORF 100 is upstream and the 5' end is downstream. The recombinant nucleotide sequence molecule is encoded by at least ORF 100.

[0088] At least a portion of ORF 100 (or nucleotide sequence molecule) can be introduced into one or more natural immune cells to produce engineered immune cells by genetic manipulation. ORF 100 (or part thereof) expresses one or more CARs (i.e., proteins and / or polypeptides) and / or one or more shRNA transcripts (as described below). Natural immune cells may include but are not limited to T lymphocytes (T cells), natural killer (NK) cells, γδT cells, etc. In an exemplary embodiment, ORF 100 is introduced into natural T cells by gene editing technology based on clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins (CRISPR-Cas). Additionally or alternatively, ORF 100 is introduced into natural T cells using lentiviral vectors, adeno-associated viruses (AAV), etc. The vector comprising ORF 100 can be selected from plasmids, cosmids, viral vectors, or phages. Plasmids can be eukaryotic expression plasmids. Viral vectors can be derived from lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, and / or Sendai viruses. In an exemplary embodiment, lentivirus (LV) is used to introduce ORF 100 into T cells. Lentiviral (LV) vectors provide an effective means for modifying eukaryotic cells, stably transferring, and expressing genes in host cells with low immunogenicity. LV vectors have a greater gene-carrying capacity and can transduce proliferating as well as non-proliferating cells. Further, LV vector-based gene delivery systems are the most effective methods for transducing difficult-to-transfect T cells. In an exemplary embodiment, clinical-grade viral vectors are commercially available from Lentigen and Sirion-Biotech.

[0089] ORF 100 may include one or more regions including one or more long terminal repeats (LTR) 110, one or more promoters, at least two hairpin loop structures formed by one or more first short hairpin RNA (shRNA) 130a sequences and / or one or more second shRNA 130b sequences, one or more genes, etc. One or more promoters may include a first promoter 120 and a second promoter 140. One or more genes may include, but are not limited to, multiple CAR genes 150. In an exemplary embodiment, ORF 100 includes a first hairpin loop structure formed by a first shRNA 130a sequence and a second hairpin loop structure formed by at least one second shRNA 130b sequence. Additionally or optionally, ORF100 may include one or more nucleotide sequences, such as genes for fluorescent protein, c-myc, HA, V5, etc. In an exemplary embodiment, the first and second hairpin loop structures regulate the amounts of IL6 and GMCSF cytokines, respectively, during immunotherapy. The transcripts (i.e., messenger RNA) of the first and second hairpin loop structures bind to the messenger RNA (mRNA) of the cytokine. In an exemplary embodiment, transcripts of the first and second hairpin loop structures bind to mRNAs of IL6 and GMCSF, respectively.

[0090] LTR 110 can be located on both sides of ORF 100, that is, LTR 110 can be set at the 5' end and 3' end of ORF 100. In other words, LTR 110 can be located upstream of the first promoter 120 and downstream of the CAR gene 150. In an exemplary embodiment, the LTR 110 at the 5' end of ORF 100 is encoded by SEQ ID NO.1. LTR 110 helps to integrate ORF 100 into the natural DNA of T cells while genetically manipulating the T cells. The integration of ORF 100 in natural DNA converts natural T cells into CAR T cells (i.e., exemplary engineered immune cells).

[0091] The transcription of ORF 100 in CAR-T cells can be controlled by one or more promoters, that is, the production of transcripts of one or more genes is controlled by one or more promoters. The promoter can be set upstream of one or more genes of ORF 100. One or more RNA polymerases can be bound to the promoter for transcribing ORF 100. RNA polymerases can include but are not limited to eukaryotic RNA polymerase II (Pol II), eukaryotic RNA polymerase III (Pol III), etc. After binding to the promoter, RNA polymerase can partially or completely transcribe ORF 100 into one or more messenger RNA (mRNA) (or transcripts). The mRNA can be further processed to provide one or more CARs (protein structures) and the first shRNA 130a and the second shRNA 130b transcripts (and their proteins / polypeptides). The processing of mRNA can include splicing, translating mRNA into at least one of one or more amino acid sequences (i.e., polypeptides) and post-translational modifications. Post-translational modifications can include but are not limited to folding (forming proteins) and / or glycosylation of the amino acid sequence.

[0092] like Figure 2 As shown, if the first shRNA 130a is set upstream of the second shRNA 130b, the first promoter 120 can be set upstream of the first shRNA 130a (i.e., the first hairpin loop structure). Alternatively, if the first shRNA 130a is set downstream of the second shRNA 130b, the first promoter 120 can be set upstream of the second shRNA 130b (i.e., the second hairpin loop structure) (not shown). The first promoter 120 can be selected from the full-length cytomegalovirus (CMV) promoter (encoded by SEQ ID NO. 2), the attenuated CMV promoter (encoded by SEQ ID NO. 3), the human UG (hU6) promoter (encoded by SEQ ID NO. 4), the mouse U6 (mU6) promoter, the chicken 7SK (ch7SK) promoter, the H1 promoter, the SNORD promoter, and the like. In an exemplary embodiment, the first promoter 120 comprises a GAL-VP64 recognition domain, which activates the first promoter 120 when the GAL-VP64 domain binds to the GAL-VP64 recognition domain.

[0093] The first promoter 120 transcribes the first shRNA 130a and the second shRNA 130b to produce one or more short RNA transcripts, each having a hairpin loop structure. The first shRNA 130a can be encoded by at least one of SEQ ID NO. 5 to SEQ ID NO. 40 (SEQ ID NO. 30 to SEQ ID NO. 40 are the sequences of the positive strand). The second shRNA 130b can be encoded by at least one of SEQ ID NO. 41 to SEQ ID NO. 60. The expression of the first shRNA 130a and the second shRNA 130b can be regulated by the first promoter 120.

[0094] The transcripts of the first shRNA 130a and the second shRNA 130b may include specific binding affinity for one or more molecules. CAR T cells include multiple IL6 receptors (IL6-R) conjugated to synthetic notch (syn-notch) receptors and GAL-VP64 domains. In an exemplary embodiment, during CRS, the released IL6 binds to the IL6-R of the CAR T cells and induces syn-notch, followed by cutting and releasing the GAL-VP64 domain. The GAL-VP64 domain migrates to the nucleus of the CAR T cells and induces the expression of one of the thirty-six first shRNA 130a sequences. The expression of the first shRNA 130a sequence results in the production of aIL6. aIL6 binds to and neutralizes IL6, thereby reducing the burden of CRS.

[0095] CAR T cells include multiple GMCSF gene sequences (not shown) that produce proinflammatory cytokines (such as GMCSF) that contribute to CRS. The second shRNA 130b can inhibit cytokine production and / or cytokine signaling by RNA interference (RNAi). In an exemplary embodiment, the transcript of the second shRNA 130b is combined with the mRNA corresponding to the cytokine (i.e., GMCSF) to functionally inactivate and / or degrade the mRNA.

[0096] In an exemplary embodiment, the first shRNA 130a (i.e., the transcript of the first shRNA 130a) expressed by the CAR T cell selectively binds to IL6 mRNA, thereby regulating IL6 cytokine levels (via RNAi). When the transcript of the first shRNA 130a binds to IL6 mRNA, the level (concentration) of the IL6 molecule decreases, resulting in a reduced burden of CRS.

[0097] In an exemplary embodiment, the second shRNA 130b (i.e., the transcript of the second shRNA 130b) expressed by the CAR T cells selectively binds to GMCSF mRNA, thereby regulating the level of the GMCSF cytokine (via RNAi). When the transcript of the second shRNA 130b binds to the GMCSF mRNA, the level (concentration) of the GMCSF molecule is reduced, resulting in a reduced burden of CRS.

[0098] Figure 2a Depicts ORF 100a of the present disclosure. ORF 100a is structurally identical to ORF 100, but adds an optional poly A tail 160 sequence, i.e., ORF 100a includes one or more LTRs 110, at least two hairpin loop structures formed by one or more first shRNA sequences 130a and / or one or more second shRNA sequences 130b, a first promoter 120, a second promoter 140, multiple CAR genes 150, a poly A tail 160 sequence, etc. In an exemplary embodiment, ORF 100a includes a first hairpin loop structure formed by a first shRNA 130a sequence and a second hairpin loop structure formed by at least one second shRNA 130b sequence. The recombinant nucleotide sequence molecule is at least encoded by ORF 100a. In an exemplary embodiment, the first and second hairpin loop structures regulate the amount of IL6 and GMCSF cytokines, respectively, during immunotherapy. The transcripts (i.e., messenger RNA) of the first and second hairpin loop structures bind to the messenger RNA (mRNA) of the cytokine. In an exemplary embodiment, transcripts of the first and second hairpin loop structures bind to mRNAs of IL6 and GMCSF, respectively.

[0099] The poly A tail 160 sequence can be downstream of the first shRNA 130a and the second shRNA 130b sequence. The expression of the poly A tail 160 sequence can be regulated by the first promoter 120. In an exemplary embodiment, Figure 2a As shown, the poly A tail 160 sequence is downstream of the hairpin loop structure formed by the second shRNA 130b sequence. The transcript of the poly A tail 160 sequence can contain multiple adenosine nucleotides. In exemplary embodiments, the poly A tail 160 sequence is derived from Simian Virus 40 (SV40) or bovine growth hormone (bGH). The SV40 poly A tail 160 sequence is encoded by SEQ ID NO. 61. The poly A tail 160 contributes to mRNA stability by providing protection against enzymatic cleavage, a process that can lead to mRNA degradation. This further ensures that the intended regulatory effects of the IL6 and GMCSF components are maintained over time without excessive degradation or interference.

[0100] Now refer to Figure 2, the second promoter 140 can be set upstream of the CAR gene 150. In other words, the CAR gene 150 can be set downstream of the second promoter 140. The second promoter 140 can be selected from the elongation factor 1 (EF1) full-length promoter (encoded by SEQ ID NO.62), the EF1α core promoter (encoded by SEQ ID NO.63), etc. Therefore, the expression of the CAR gene 150 can be regulated by the second promoter 140.

[0101] The CAR gene 150 comprises a sequence encoding one or more domains of the CAR 200, such as Figure 3 The one or more domains of CAR 200 include, but are not limited to, one or more single-chain variable fragment (scFV) domains 210, one or more hinge domains 220, one or more transmembrane domains 230, one or more co-stimulatory (CSTM) domains 240, one or more signaling domains 250, one or more safety switch (SS) domains 260, and the like.

[0102] In an exemplary embodiment, Figure 3 As shown, CAR 200 includes a scFv domain 210, a hinge domain 220, a transmembrane domain 230, a CSTM domain 240, a signaling domain 250, and an SS domain 260. Additionally or optionally, CAR 200 may include one or more peptide sequences disposed between the domains of CAR 200, such as a signal peptide, a linker peptide, and the like.

[0103] As with any antibody, the scFV domain 210 of CAR 200 can include a light chain 210a and a heavy chain 210b. In an embodiment, the scFV domain 210 of the CD19 antigen (TAA) can be encoded by SEQ ID NO.64, such that the scFV domain 210 comprises a polypeptide sequence defined by SEQ ID NO.65. In an alternative embodiment, the scFV domain 210 of the CD19 antigen (TAA) can be encoded by SEQ ID NO.66, such that the scFV domain 210 comprises a polypeptide sequence defined by SEQ ID NO.67. The light chain 210a and heavy chain 210b of the scFV domain 210 can be coupled to each other via a connecting protein 210c. The scFV domain 210 can have binding specificity (or affinity) for one or more TAAs, including but not limited to CD19, CD7, CD20, CD22, CD123, CD133, CD30, CD138, EGFR, EGFRvIII, FAP, MUC1, GD2, CEA, PSMA, HER2, NY-ESO-1, MAGEA-A3, hTERT, etc. In an exemplary embodiment, CAR 200 comprises one scFV domain 210 specific for the CD19 antigen of B lymphocytes (B cells). Alternatively, CAR 200 can comprise two or three scFV domains (not shown) for the same TAA or different TAAs.

[0104] Hinge domain 220 operably couples scFv domain 210 to transmembrane domain 230. In an exemplary embodiment, hinge domain 220 comprises a cluster of differentiation 8 (CD8) domain. Hinge domain 220 helps provide flexibility to scFv domain 210 relative to transmembrane domain 230. In an embodiment, hinge domain 220 is encoded by SEQ ID NO. 68, such that hinge domain 220 comprises a polypeptide sequence defined by SEQ ID NO. 69.

[0105] The transmembrane domain 230 can be disposed across a bi-lipid cell membrane (or membrane) 270. The membrane 270 separates the extracellular region 270a from the intracellular region 270b. In an exemplary embodiment, the transmembrane domain 230 includes a CD8 domain encoded by SEQ ID NO. 70, such that the transmembrane domain 230 comprises a polypeptide sequence defined by SEQ ID NO. 71. The transmembrane domain 230 facilitates operative coupling of the domain of the CAR 200 present in the extracellular region 270a to the domain of the CAR 200 present in the intracellular region 270b. In an exemplary embodiment, as Figure 3As shown, the scFv domain 210 and the hinge domain 220 are the only domains present in the extracellular region 270a.

[0106] The CSTM domain 240 can be coupled to the transmembrane domain 230. The CSTM domain 240 can be selected from cluster of differentiation 28 (CD28), inducible T cell co-stimulator (ICOS), OX40, 4-1BB, DAP10, DAP12, 2B4, and the like. The CD28 CSTM domain 240 can be encoded by SEQ ID NO. 72, such that the CD28 CSTM domain 240 includes the polypeptide sequence defined by SEQ ID NO. 73. The ICOS CSTM domain 240 can be encoded by SEQ ID NO. 74, such that the ICOS CSTM domain 240 includes the polypeptide sequence defined by SEQ ID NO. 75. The OX40 CSTM domain 240 can be encoded by SEQ ID NO. 76, such that the OX40 CSTM domain 240 includes the polypeptide sequence defined by SEQ ID NO. 77. The 4-1BB CSTM domain 240 can be encoded by SEQ ID NO. 78, such that the 4-1BB CSTM domain 240 comprises the polypeptide sequence defined by SEQ ID NO. 79. The DAP10 CSTM domain 240 can be encoded by SEQ ID NO. 80, such that the DAP10 CSTM domain 240 comprises the polypeptide sequence defined by SEQ ID NO. 81. The DAP12 CSTM domain 240 can be encoded by SEQ ID NO. 82, such that the DAP12 CSTM domain 240 comprises the polypeptide sequence defined by SEQ ID NO. 83. The 2B4 CSTM domain 240 can be encoded by SEQ ID NO. 84, such that the 2B4 CSTM domain 240 comprises the polypeptide sequence defined by SEQ ID NO. 85. In some embodiments, the CSTM domain 240 comprises 4-1BB. The CSTM domain 240 contributes to enhancing cell-mediated immune responses.

[0107] In an exemplary embodiment, CAR T cells are activated by subjecting CAR T cells to costimulatory molecules (e.g., CD3 and / or CD28). Costimulatory molecules stimulate CSTM domains 240, which in turn lead to CAR T cell activation, proliferation, and / or differentiation into effector T cells (thereby enabling CAR T cells to express CAR constructs). CD3 / CD28 activates the signal received by analog T cells during natural antigen recognition, therefore, providing a safe and effective method for activating CAR T cells in vitro and in vivo synthetically. The CAR T cells (or effector T cells) activated by CD3 / CD28 are then used to target and kill cancer cells.

[0108] The signaling domain 250 can be coupled to the CSTM domain 240. In an exemplary embodiment, the signaling domain 250 is a cluster of differentiation 3 zeta (CD3 zeta) domain encoded by SEQ ID NO. 86, such that the CD3 zeta signaling domain 250 comprises a polypeptide sequence defined by SEQ ID NO. 87. Following binding of the TAA to the scFv domain 210, the signaling domain 250 facilitates the delivery of activation signals to the CAR T cell.

[0109] The SS domain 260 can be coupled to the signaling domain 250. The SS domain 260 can be selected from iCaspase 9, EGFRt, RQR8, and the like. In an exemplary embodiment, the SS domain 260 includes iCaspase 9 encoded by SEQ ID NO. 88, such that the iCaspase 9 SS domain 260 comprises a polypeptide sequence defined by SEQ ID NO. 89. In another exemplary embodiment, the SS domain 260 includes RQR8 encoded by SEQ ID NO. 90, such that the RQR8 SS domain 260 comprises a polypeptide sequence defined by SEQ ID NO. 91. The SS domain 260 can control cell proliferation of CAR T cells, thereby preventing the transformation of CAR T cells into cancer cells. In other words, in the presence of a predefined inducer of iCaspase 9, EGFRt, and / or RQR8, the SS domain 260 allows for selective elimination of CAR T cells.

[0110] In an exemplary embodiment, the SS domain 260 of inducible caspase 9 (iCaspase 9) encodes a caspase recruitment domain (CARD; GenBank NM001 229) linked to two 12-kDa human FK506 binding proteins (FKBP12; GenBank AH002 818) containing the F36V mutation. Administration of a chemical inducer of dimerization (CID) leads to dimerization of inducible caspase 9 molecules, thereby causing their activation. Caspase 9 dimers then activate downstream effector caspases, such as caspase 3, and ultimately induce apoptosis.

[0111] In another exemplary embodiment, RQR8 SS domain 260 encodes a multi-epitopic molecule containing a CD34 epitope and two CD20 mimotopes. When the CD20 mimotope is bound to the FDA-approved CD20 antibody rituximab, the host cell undergoes apoptosis.

[0112] Figure 3aAnother embodiment of CAR 300 is depicted. Similar to CAR 200, CAR 300 comprises one or more scFv domains 310, one or more hinge domains 320, one or more transmembrane domains 330, one or more CSTM domains 340, one or more signaling domains 350, one or more SS domains 360, etc. The transmembrane domain 330 can be arranged across the membrane 370 so that the scFv domain 310 and the hinge domain 320 are arranged outside the CAR T cell, i.e., in the extracellular region 370a. The remaining domains of CAR 300 can be arranged inside the CAR T cell, i.e., in the intracellular region 370b.

[0113] In an exemplary embodiment, Figure 3a As shown, CAR 300 comprises a scFv domain 310, a hinge domain 320, a transmembrane domain 330, a first CSTM domain 340a and a second CSTM domain 340b, a signaling domain 350 and a SS domain 360.

[0114] Similar to the scFV domain 210 of CAR 200, the scFV domain 310 of CAR 300 can include a light chain 310a and a heavy chain 310b. The light chain 310a and the heavy chain 310b of the scFV domain 310 can be coupled to each other via a connecting protein 310c.

[0115] Similar to the CSTM domain 240 of CAR 200, the first CSTM domain 340a of CAR 300 can be selected from CD28, ICOS and / or OX40. The second CSTM domain 340b can be coupled to the first CSTM domain 340a. The second CSTM domain 340b can be selected from 4-1BB, DAP10, DAP12 and / or 2B4.

[0116] In an exemplary embodiment, the first CSTM domain 340a and the second CSTM domain 340b are CD28 and 4-1BB, respectively. Compared to the single CSTM domain 240 in CAR 200, the first CSTM domain 340a and the second CSTM domain 340b of CAR 300 help enhance the persistence of CAR T cells.

[0117] like Figure 4 As shown, the above-mentioned CAR T cells produce negligible amounts of cytokines, such as GMCSF and IL6. Therefore, the CAR T cells do not recruit any proinflammatory cells, thereby downregulating proinflammatory cytokines. The reduction in proinflammatory cytokine levels alleviates CRS.

[0118] Although the first shRNA 130a and the second shRNA 130b are described in the present disclosure with the example of CAR 200 / 300, the first shRNA 130a and the second shRNA 130b can be used with any functionally equivalent CAR construct and this is also within the scope of the teachings of the present disclosure.

[0119] You can follow Figure 5 The method 400 depicted in the foregoing is used to prepare the CAR T cells of the present invention for each cancer patient. Alternatively, the CAR T cells can be prepared from an allogeneic T cell line for all cancer patients.

[0120] Method 400 begins at step 401 by isolating peripheral blood mononuclear cells (PBMCs) by a ficoll-based method or an automated PBMC isolation instrument (as in the case of leukapheresis). In an exemplary embodiment, PBMCs are isolated by drawing blood from a cancer patient in need of CAR T cell-based immunotherapy.

[0121] At step 403, a plurality of T cells may be isolated from PBMCs. The isolated T cell population may comprise a plurality of CD4 + and CD8 + T cells. The isolated T cell population may comprise at least 40% CD4 + In an exemplary embodiment, the isolated T cell population comprises 40% CD4 + T cells and 60% CD8 + T cells.

[0122] At optional step 405, the T cells are activated by subjecting them to costimulatory molecules (e.g., CD3 and / or CD28). In an exemplary embodiment, the costimulatory molecules help the T cells become competent to receive ORF 100 via a lentiviral vector (described below).

[0123] At step 407, the ORF 100 of the present invention is prepared for delivery into an isolated T cell population. The ORF 100 can be linked and replicated in a predefined vector (or plasmid), including but not limited to pHR, pTRPE lentiviral vectors, etc. In an exemplary embodiment, the ORF 100 is replicated and packaged within a lentiviral particle.

[0124] At step 409, ORF 100 is delivered in the isolated T cell population via a predefined gene delivery method. The predefined gene delivery method can be one of transformation, transfection or transduction. In an exemplary embodiment, the isolated T cell population is transduced by a lentiviral-based gene delivery system (such as a third-generation self-inactivation (SIN) lentiviral system). In an exemplary embodiment of the present disclosure, ORF 100 is delivered to T cells via third-generation lentiviral particles, i.e., T cells are transduced with third-generation lentiviral particles. The resulting CAR T cells target and bind to specific cancer cells, thereby allowing CAR T cells to recognize and attack B cells (i.e., cancer cells) expressing CD19.

[0125] At step 4011, CAR T cells (or engineered immune cells) are expanded in a predefined nutrient medium for a predefined time to increase the number of CAR T cells. In an exemplary embodiment, the CAR T cells are expanded for 7 to 9 days to increase the number of CAR T cells by 20-40 times. The engineered immune cells suspended in the predefined nutrient medium can define a composition.

[0126] In an exemplary embodiment, the nutrient medium comprises AIM V medium with or without antibiotics, human serum albumin, phenol red, and L-glutamine. In another embodiment, the nutrient medium comprises CTS AIM V medium without antibiotics, phenol red, and serum. The nutrient medium can be periodically replaced with fresh nutrient medium after a predefined period of time. In an exemplary embodiment, the nutrient medium is replaced every 48 hours.

[0127] At step 413, the transduced T cells expressing CAR 200, 300 are selected and isolated by subjecting the transduced T cells to in vitro and / or in vivo testing techniques. Additionally or alternatively, the transduced T cells expressing CAR 200, 300 are selected by immunoassay-based selection or by cell sorting techniques.

[0128] The selected transduced T cells expressing CAR 200, 300 (i.e., CAR T cells) are refrigerated for storage and transportation. The frozen CAR T cells are thawed before infusion into the patient.

[0129] Alternatively, the selected T cells (ie, CAR T cells) can be infused directly into the patient, as determined by the physician.

[0130] Although method 400 is described with an exemplary sequence of steps, the steps of method 400 may be rearranged in practicing method 400 and remain within the teachings of the present disclosure.

[0131] Sequences that share at least 95% identity with the sequences disclosed in this disclosure are within the scope of the teachings of this disclosure.

[0132] The present disclosure is further described by reference to the following exemplary embodiments and examples. These exemplary embodiments and examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise indicated. Therefore, the present disclosure should in no way be construed as being limited to the following exemplary embodiments and examples, but rather should be construed to encompass any and all variations that become apparent as a result of the teachings provided herein.

[0133] Example 1: CAR T cell preparation method (present invention):

[0134] HEK293T cells packaged with lentivirus obtained from the American Type Culture Collection (ATCC) were inoculated with approximately 3.8×10 6 10 cells / plate were seeded in complete Dulbecco's Modified Eagle's Medium (DMEM) in a 10 cm tissue culture plate. The tissue culture plate containing HEK293T cells was then incubated at 37°C, 5% CO₂ for approximately 24 hours. After approximately 24 hours, the old medium was aspirated and replaced with 10 mL of fresh complete DMEM plus 25 μM chloroquine diphosphate. The tissue culture plate containing HEK293T cells was further incubated at 37°C, 5% CO₂ for approximately 6 hours.

[0135] Using polyethyleneimine (PEI, non-viral vector) as a transfection reagent, a mixture of slow plasmids and CAR plasmids (comprising ORF 100 of the present disclosure) was inserted into HEK293T cells. HEK293T cells were incubated at 37 ° C, 5% CO2 for another 36 hours, and then the culture medium containing viral particles was harvested at different time intervals (up to 72 hours). The harvested culture medium was centrifuged at 1000 rpm for 5 minutes to precipitate any remaining HEK293T cells. The supernatant was then filtered using a 0.45 μm PES filter. The filtrate (supernatant) containing viral particles was collected and refrigerated to avoid titer loss. The viral particles have a slow viral plasmid integrated with the CAR plasmid (comprising ORF 100 of the present disclosure).

[0136] Through a process called leukapheresis, 10 million PBMCs are collected from every 20 mL of patient blood. T cells are then isolated from the collected PBMCs using the RosetteSe human CD4+ T cell enrichment assay and the RosetteSe human CD8+ T cell enrichment assay. The isolated T cell population has 40% CD4+ T cells and 60% CD8+ T cells. The isolated T cells are cryopreserved in RPMI-1640 containing 20% ​​human AB serum and 10% DMSO.

[0137] The isolated T cells were cultured in a human T cell culture medium consisting of X-VIVO 15 (Lonza), 5% human AB serum, and 10 mM neutralized N-acetyl L-cysteine ​​(Sigma-Aldrich). IL-2 was also used for cell proliferation at 30 units / mL.

[0138] After 24 hours of culture, isolated T cells were stimulated (activated) with human T activator CD3 / CD28 immunomagnetic beads (Life Technologies) at a cell:bead ratio of 1:3.

[0139] At 48 hours, primary T cells were exposed to the supernatant (containing viral particles) for transduction. On day 4 after T cell stimulation, the immunomagnetic beads were removed and T cells were expanded until day 9. T cells were sorted using FACS ARIA II. Thus, T cells that exhibited basal CAR expression (i.e., CAR T cells) were isolated.

[0140] The isolated CAR T cells were expanded in a nutrient medium consisting of CTS AIM V medium without antibiotics, phenol red, or serum at 37°C and 5% CO2. The nutrient medium was replaced with fresh nutrient medium every 48 hours. It was observed that the number of CAR T cells increased 20-fold to 40-fold after approximately 7-9 days of expansion. The CAR T cells were frozen for storage and transportation.

[0141] Example 2: Infusion of CAR T cells (present invention):

[0142] CAR T cells (as obtained in Example 1) were thawed. Approximately 1×10 T cells per kg of patient body weight were then administered in an outpatient setting. 6 Individual CAR T cells are infused intravenously into the patient. CAR T cells rapidly migrate to the bone marrow and lymphoid tissues. After migration, they selectively bind to cancer cells expressing CD19. Binding between the CAR T cells and cancer cells triggers CAR T cell activation and proliferation, followed by cancer cell destruction through mechanisms such as cytokine release and cytotoxicity.

[0143] CAR T cells persist in the patient's body and continue to attack cancer cells expressing CD19, providing long-term therapeutic effects for the patient. The CAR T cells of the present disclosure have been observed to provide response rates of up to 90% in patients with acute lymphoblastic leukemia and up to 50%-60% in patients with non-Hodgkin's lymphoma. CAR T cells specifically target cancer cells expressing CD19, reducing the risk of harm to healthy cells and lowering the risk of false positive interactions. Unlike traditional chemotherapy and radiotherapy, CAR T cell therapy has minimal toxicity to healthy cells.

[0144] Example 3: IL6 and GMCSF knockdown (present invention):

[0145] The effects of the best performing first shRNA 130a sequence and second shRNA 130b sequence described in the present disclosure on the regulation of IL6 and GMCSF levels were evaluated. Screening was performed in T cells. Figure 6 As shown, the bar graph depicts the expression of IL6 and GMCSF in T cells that were activated (Act-T cells) for 24 hours to induce IL6 and GMCSF expression or not activated (control). The effects of non-targeting shRNA (Scram), the first shRNA 130a targeting IL6, and the second shRNA 130b targeting GMCSF in knocking down the IL6 gene and GMCSF gene were evaluated in activated T cells. T cells were transfected with the corresponding first shRNA 130a and second shRNA 130b, and mRNA was extracted 24 hours after transfection. RT-qPCR was used to measure IL6 and GMCSF mRNA specific primers. Statistical analysis was performed using a non-parametric t-test, revealing significance levels of **** (P<0.0001), ** (P<0.01), * (P<0.05), and ns (not significant) when comparing T cells expressing GMCSF / IL6 shRNA with T cells expressing Scram (see Figure 6 ). It was observed that the first shRNA 130a and the second shRNA 130b of the present disclosure significantly reduced the expression of IL6 and GMCSF genes via RNAi.

[0146] Example 4: IL6 and GMCSF production (present invention):

[0147] Protein fractions were collected from the corresponding T cells as described in Example 3. The collected protein fractions were subjected to enzyme-linked immunosorbent assay (ELISA) to determine the amount of IL6 and GMCSF cytokines present in each protein fraction. Figure 7 As shown, the bar graph depicts the presence of IL6 and GMCSF cytokines in the corresponding protein fractions. Statistical analysis was performed using a nonparametric t-test, revealing significance levels of **** (P < 0.0001), ** (P < 0.01), and ns (not significant) when comparing T cells expressing GMCSF / IL6 shRNA with T cells expressing Scram (see Figure 7 ).

[0148] Example 5: IL6 production in CAR T cells (present invention):

[0149] The effects of the first shRNA 130a and the second shRNA 130b sequences as described in Examples 3 and 4 on the regulation of IL6 and GMCSF levels were evaluated. Screening was performed in CAR T cells. Figure 8 As shown, the bar graph depicts the production of IL6 and GMCSF in non-activated T cells (control). Evaluation of non-targeting shRNA in activated CAR T cells (CART Scram ) and the first shRNA 130a and the second shRNA 130b targeting IL6 and GMCSF (CART shGMCSF / shIL6 ) in knocking down IL6 and GMCSF genes. con ) were also evaluated after activation. The assays and analyses were similar to those described in Example 4 above. Figure 8 As shown, the bar graph depicts the presence of IL6 and GMCSF cytokines in the corresponding protein fractions. Statistical analysis was performed using a nonparametric t-test, revealing a significance level of **** (P < 0.0001) when comparing T cells expressing IL6 shRNA with T cells expressing Scram (see Figure 8 ).

[0150] The scope of the present invention is limited only by the appended patent claims. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and that actual parameters, dimensions, materials, and / or configurations will depend on one or more specific applications in which the teachings of the present invention are used.

Claims

1. A recombinant nucleic acid molecule, encoded by at least ORF (100, 100a) for reducing cytokine storm during immunotherapy, the recombinant nucleic acid molecule comprising: a. at least one first hairpin loop structure, wherein the at least one first hairpin loop structure is formed by one or more first short hairpin RNA (130a) sequences, wherein the at least one first hairpin loop structure regulates the amount of interleukin 6 (IL6) cytokine during immunotherapy; b. at least one second hairpin loop structure, the at least one second hairpin loop structure being formed by one or more second short hairpin RNA (130b) sequences, the at least one second hairpin loop structure regulating the amount of granulocyte monocyte colony-stimulating factor (GMCSF) cytokine during immunotherapy, the at least one second hairpin loop structure being arranged upstream or downstream of the at least one first hairpin loop structure; and c. A first promoter (120), which is arranged upstream of the at least one first hairpin loop structure and the at least one second hairpin loop structure.

2. The recombinant nucleic acid molecule according to claim 1, characterized in that The first short hairpin RNA (130a) is encoded by at least one of SEQ ID NO. 5 to SEQ ID NO. 40 or a nucleotide sequence having at least 95% identity thereto.

3. The recombinant nucleic acid molecule according to claim 1, characterized in that The second short hairpin RNA (130b) is encoded by at least one of SEQ ID NO. 41 to SEQ ID NO. 60, or a nucleotide sequence having at least 95% identity thereto.

4. The recombinant nucleic acid molecule according to claim 1, characterized in that The first promoter (120) comprises at least one of the following: a full-length cytomegalovirus (CMV) promoter encoded by SEQ ID NO.2, an attenuated CMV promoter encoded by SEQ ID NO.3, a human UG (hU6) promoter encoded by SEQ ID NO.4, a mouse U6 (mU6) promoter, a chicken 7SK (ch7SK) promoter, an Hl promoter, and a SNORD promoter.

5. The recombinant nucleic acid molecule according to claim 1, characterized in that The poly A tail (160) sequence is arranged downstream of the first hairpin loop structure and the second hairpin loop structure.

6. The recombinant nucleic acid molecule according to claim 1, characterized in that The recombinant nucleic acid molecule comprises one or more chimeric antigen receptor genes (150) positioned downstream of a second promoter (140).

7. The recombinant nucleic acid molecule according to claim 1, characterized in that The messenger RNA of the first hairpin loop structure or the second hairpin loop structure transcribed from the recombinant nucleic acid molecule is configured to bind to the messenger RNA of the cytokine.

8. A recombinant nucleic acid molecule, encoded by at least ORF (100), for reducing cytokine storm during immunotherapy, the recombinant nucleic acid molecule comprising: a. at least one first hairpin loop structure, wherein the at least one first hairpin loop structure is formed by one or more first short hairpin RNA (130a) sequences, wherein the at least one first hairpin loop structure regulates the amount of interleukin 6 (IL6) cytokine during immunotherapy; b. at least one second hairpin loop structure, the at least one second hairpin loop structure being formed by one or more second short hairpin RNA (130b) sequences, the at least one second hairpin loop structure regulating the amount of granulocyte monocyte colony-stimulating factor (GMCSF) cytokine during immunotherapy, the at least one second hairpin loop structure being arranged upstream or downstream of the at least one first hairpin loop structure; c. a first promoter (120), wherein the first promoter is disposed upstream of the at least one hairpin loop structure and the at least one second hairpin loop structure; d. One or more chimeric antigen receptor genes (150), wherein the one or more chimeric antigen receptor genes express: i. one or more single-chain variable fragment domains (210), wherein the one or more single-chain variable fragment domains are configured to bind to one or more tumor-associated antigens (TAA), ii. one or more hinge domains (220), said one or more hinge domains being coupled to said one or more single-chain variable fragment domains (210), iii. one or more costimulatory domains (240), iv. one or more signaling domains (250), said one or more signaling domains coupled to said one or more costimulatory domains (240), v. one or more safety switch domains (260), said one or more safety switch domains coupled to said one or more signaling domains (250), and vi. one or more transmembrane domains (230) operably coupling the hinge domain (220) to the co-stimulatory domain (240); e. a second promoter (140) disposed upstream of the one or more chimeric antigen receptor genes (150), and f. One or more long terminal repeat sequences (110), wherein the one or more long terminal repeat sequences are arranged upstream of the first promoter (120) and downstream of the one or more chimeric antigen receptor genes (150).

9. The recombinant nucleic acid molecule according to claim 8, characterized in that The first short hairpin RNA (130a) is encoded by at least one of SEQ ID NO. 5 to SEQ ID NO. 40 or a nucleotide sequence having at least 95% identity thereto.

10. The recombinant nucleic acid molecule according to claim 8, characterized in that The second short hairpin RNA (130b) is encoded by at least one of SEQ ID NO. 41 to SEQ ID NO. 60, or a nucleotide sequence having at least 95% identity thereto.

11. The recombinant nucleic acid molecule according to claim 8, characterized in that The first promoter 120 comprises at least one of the following: a full-length cytomegalovirus (CMV) promoter encoded by SEQ ID NO. 2, an attenuated CMV promoter encoded by SEQ ID NO. 3, a human UG (hU6) promoter encoded by SEQ ID NO. 4, a mouse U6 (mU6) promoter, a chicken 7SK (ch7SK) promoter, an H1 promoter, and a SNORD promoter.

12. The recombinant nucleic acid molecule according to claim 8, characterized in that The one or more single-chain variable fragment domains (210) have binding affinity for at least one of the following: cluster of differentiation 19 (CD19), cluster of differentiation 7 (CD7), cluster of differentiation 20 (CD20), cluster of differentiation 22 (CD22), cluster of differentiation 123 (CD123), cluster of differentiation 133 (CD133), cluster of differentiation 30 (CD30), cluster of differentiation 138 (CD138), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), fibroblast activation protein alpha (FAP), mucin 1 (MUC1), disialoganglioside GD2 (GD2), carcinoembryonic antigen (CEA), prostate specific membrane antigen (PSMA), human epidermal growth factor receptor 2 (HER2), New York esophageal squamous cell carcinoma-1 (NY-ESO-1), melanoma associated antigen 3 (MAGEA-A3) and human telomerase reverse transcriptase (hTERT).

13. The recombinant nucleic acid molecule according to claim 8, characterized in that The single-chain variable fragment domain (210) for cluster of differentiation 19 (CD19) is encoded by at least one of SEQ ID NO. 64 and SEQ ID NO.

66.

14. The recombinant nucleic acid molecule according to claim 8, characterized in that The one or more hinge domains (220) at least partially comprise cluster of differentiation 8 (CD8) encoded by SEQ ID NO.

68.

15. The recombinant nucleic acid molecule according to claim 8, characterized in that The one or more co-stimulatory domains (240) include at least one of the following: cluster of differentiation 28 (CD28) encoded by SEQ ID NO.72, inducible T cell co-stimulatory molecule (ICOS) encoded by SEQ ID NO.74, OX40 encoded by SEQ ID NO.76, 4-1BB encoded by SEQ ID NO.78, DAP10 encoded by SEQ ID NO.80, DAP12 encoded by SEQ ID NO.82 and 2B4 encoded by SEQ ID NO.

84.

16. The recombinant nucleic acid molecule according to claim 8, characterized in that The one or more signaling domains (250) at least partially comprise cluster of differentiation 3 zeta (CD3 zeta) encoded by SEQ ID NO.

86.

17. The recombinant nucleic acid molecule according to claim 8, characterized in that The one or more safety switch domains (260) comprise at least one of inducible caspase 9 (iCaspase 9) encoded by SEQ ID NO.88, truncated epidermal growth factor receptor (EGFRt), and RQR8 encoded by SEQ ID NO.

90.

18. The recombinant nucleic acid molecule according to claim 8, characterized in that The one or more transmembrane domains (230) at least partially comprise cluster of differentiation 8 (CD8) encoded by SEQ ID NO.

70.

19. The recombinant nucleic acid molecule according to claim 8, characterized in that The second promoter (140) is at least one of the elongation factor 1 (EF1) full-length promoter encoded by SEQ ID NO.62 and the EF1α core promoter encoded by SEQ ID NO.

63.

20. The recombinant nucleic acid molecule according to claim 8, characterized in that The first promoter (120) and the second promoter (140) are configured to bind to at least one of eukaryotic RNA polymerase II (Pol II) and eukaryotic RNA polymerase III (Pol III).

21. The recombinant nucleic acid molecule according to claim 8, characterized in that The one or more long terminal repeat sequences (110) are encoded by SEQ ID NO.

1.

22. The recombinant nucleic acid molecule according to claim 8, characterized in that The one or more costimulatory domains (240) comprise: a. a first co-stimulatory domain (340a), comprising at least one of cluster of differentiation 28 (CD28), inducible T cell co-stimulatory molecule (ICOS) and / or OX40; and b. a second co-stimulatory domain (340b), comprising at least one of 4-1BB, DAP10, DAP12 and / or 2B4.

23. The recombinant nucleic acid molecule according to claim 8, characterized in that The messenger RNA of the first hairpin loop structure or the second hairpin loop structure transcribed from the recombinant nucleic acid molecule is configured to bind to the messenger RNA of the cytokine.

24. A recombinant nucleic acid molecule, encoded by at least ORF (100a), for reducing cytokine storm during immunotherapy, the recombinant nucleic acid molecule comprising: a. at least one first hairpin loop structure, wherein the at least one first hairpin loop structure is formed by one or more first short hairpin RNA (130a) sequences, wherein the at least one first hairpin loop structure regulates the amount of interleukin 6 (IL6) cytokine during immunotherapy; b. at least one second hairpin loop structure, the at least one second hairpin loop structure being formed by one or more second short hairpin RNA (130b) sequences, the at least one second hairpin loop structure regulating the amount of granulocyte monocyte colony-stimulating factor (GMCSF) cytokine during immunotherapy, the at least one second hairpin loop structure being arranged upstream or downstream of the at least one first hairpin loop structure; c. a first promoter (120), wherein the first promoter is disposed upstream of the at least one first hairpin loop structure and the at least one second hairpin loop structure; d. a poly A tail (160) sequence, wherein the poly A tail sequence is disposed downstream of the at least one first hairpin loop structure and the at least one second hairpin loop structure; e. One or more chimeric antigen receptor genes (150), wherein the one or more chimeric antigen receptor genes express: i. one or more single-chain variable fragment domains (210), wherein the one or more single-chain variable fragment domains are configured to bind to one or more tumor-associated antigens (TAA), ii. one or more hinge domains (220), said one or more hinge domains being coupled to said one or more single-chain variable fragment domains (210), iii. one or more costimulatory domains (240), iv. one or more signaling domains (250), said one or more signaling domains coupled to said one or more costimulatory domains (240), v. one or more safety switch domains (260), said one or more safety switch domains coupled to said one or more signaling domains (250), and vi. one or more transmembrane domains (230) operably coupling the hinge domain (220) to the co-stimulatory domain (240); f. a second promoter (140) disposed upstream of the one or more chimeric antigen receptor genes (150), and g. One or more long terminal repeat sequences (110), wherein the one or more long terminal repeat sequences are arranged upstream of the first promoter (120) and downstream of the one or more chimeric antigen receptor genes (150).

25. The recombinant nucleic acid molecule according to claim 24, characterized in that The first short hairpin RNA (130a) is encoded by at least one of SEQ ID NO. 5 to SEQ ID NO. 40, or a nucleotide sequence having at least 95% identity thereto.

26. The recombinant nucleic acid molecule according to claim 24, characterized in that The second short hairpin RNA (130b) is encoded by at least one of SEQ ID NO. 41 to SEQ ID NO. 60, or a nucleotide sequence having at least 95% identity thereto.

27. The recombinant nucleic acid molecule according to claim 24, characterized in that The first promoter 120 comprises at least one of the following: a full-length cytomegalovirus (CMV) promoter encoded by SEQ ID NO. 2, an attenuated CMV promoter encoded by SEQ ID NO. 3, a human UG (hU6) promoter encoded by SEQ ID NO. 4, a mouse U6 (mU6) promoter, a chicken 7SK (ch7SK) promoter, an H1 promoter, and a SNORD promoter.

28. The recombinant nucleic acid molecule according to claim 24, characterized in that The poly A tail (160) sequence is derived from Simian Virus 40 (SV40) or bovine growth hormone beta globulin (bGH) encoded by SEQ ID NO.

61.

29. The recombinant nucleic acid molecule according to claim 24, characterized in that The one or more single-chain variable fragment domains (210) have binding affinity for at least one of the following: cluster of differentiation 19 (CD19), cluster of differentiation 7 (CD7), cluster of differentiation 20 (CD20), cluster of differentiation 22 (CD22), cluster of differentiation 123 (CD123), cluster of differentiation 133 (CD133), cluster of differentiation 30 (CD30), cluster of differentiation 138 (CD138), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), fibroblast activation protein alpha (FAP), mucin 1 (MUC1), disialoganglioside GD2 (GD2), carcinoembryonic antigen (CEA), prostate specific membrane antigen (PSMA), human epidermal growth factor receptor 2 (HER2), New York esophageal squamous cell carcinoma-1 (NY-ESO-1), melanoma associated antigen 3 (MAGEA-A3) and human telomerase reverse transcriptase (hTERT).

30. The recombinant nucleic acid molecule according to claim 24, characterized in that The single-chain variable fragment domain (210) for cluster of differentiation 19 (CD19) is encoded by at least one of SEQ ID NO. 64 and SEQ ID NO.

66.

31. The recombinant nucleic acid molecule according to claim 24, characterized in that The one or more hinge domains (220) at least partially comprise cluster of differentiation 8 (CD8) encoded by SEQ ID NO.

68.

32. The recombinant nucleic acid molecule according to claim 24, characterized in that The one or more co-stimulatory domains (240) include at least one of the following: cluster of differentiation 28 (CD28) encoded by SEQ ID NO.72, inducible T cell co-stimulatory molecule (ICOS) encoded by SEQ ID NO.74, OX40 encoded by SEQ ID NO.76, 4-1BB encoded by SEQ ID NO.78, DAP10 encoded by SEQ ID NO.80, DAP12 encoded by SEQ ID NO.82 and 2B4 encoded by SEQ ID NO.

84.

33. The recombinant nucleic acid molecule according to claim 24, characterized in that The one or more signaling domains (250) at least partially comprise cluster of differentiation 3 zeta (CD3 zeta) encoded by SEQ ID NO.

86.

34. The recombinant nucleic acid molecule according to claim 24, characterized in that The one or more safety switch domains (260) comprise at least one of inducible caspase 9 (iCaspase 9) encoded by SEQ ID NO.88, truncated epidermal growth factor receptor (EGFRt), and RQR8 encoded by SEQ ID NO.

90.

35. The recombinant nucleic acid molecule according to claim 24, characterized in that The one or more transmembrane domains (230) at least partially comprise cluster of differentiation 8 (CD8) encoded by SEQ ID NO.

70.

36. The recombinant nucleic acid molecule according to claim 24, characterized in that The second promoter (140) is at least one of the elongation factor 1 (EF1) full-length promoter encoded by SEQ ID NO.62 and the EF1α core promoter encoded by SEQ ID NO.

63.

37. The recombinant nucleic acid molecule according to claim 24, wherein The first promoter (120) and the second promoter (140) are configured to bind to at least one of eukaryotic RNA polymerase II (Pol II) and eukaryotic RNA polymerase III (Pol III).

38. The recombinant nucleic acid molecule according to claim 24, characterized in that The one or more long terminal repeat sequences (110) are encoded by SEQ ID NO.

1.

39. The recombinant nucleic acid molecule according to claim 24, characterized in that The one or more costimulatory domains (240) comprise: a. a first co-stimulatory domain (340a), comprising at least one of cluster of differentiation 28 (CD28), inducible T cell co-stimulatory molecule (ICOS) and / or OX40; and b. a second co-stimulatory domain (340b), comprising at least one of 4-1BB, DAP10, DAP12 and / or 2B4.

40. The recombinant nucleic acid molecule according to claim 24, characterized in that The messenger RNA of the first hairpin loop structure or the second hairpin loop structure transcribed from the recombinant nucleic acid molecule is configured to bind to the messenger RNA of the cytokine.

41. A transcript of a recombinant nucleic acid molecule encoded by at least ORF (100, 100a), said transcript comprising: a. The messenger RNA of the first hairpin loop structure or the second hairpin loop structure, wherein the messenger RNA of the first hairpin loop structure or the second hairpin loop structure is transcribed from the recombinant nucleic acid molecule according to any one of claims 1 to 40, and the messenger RNA of the first hairpin loop structure or the second hairpin loop structure is configured to bind to the messenger RNA of the cytokine.

42. A vector comprising: a. At least one of the recombinant nucleic acid molecules according to any one of claims 1 to 40, which is linked to at least one of a plasmid, a cosmid, a viral vector and a bacteriophage.

43. The carrier according to claim 42, characterized in that The viral vector is derived from at least one of a lentivirus, a retrovirus, an adenovirus, an adeno-associated virus, and a Sendai virus.

44. An engineered immune cell, comprising: a. At least one of the recombinant nucleic acid molecules according to any one of claims 1 to 40.

45. The engineered immune cell according to claim 44, characterized in that The recombinant nucleic acid molecule is linked to at least one of a plasmid, a cosmid, a viral vector and a bacteriophage.

46. ​​The engineered immune cell according to claim 44, characterized in that The recombinant nucleic acid molecule is linked to a viral vector derived from at least one of a lentivirus, a retrovirus, an adenovirus, an adeno-associated virus, and a Sendai virus.

47. A composition comprising: a. An engineered immune cell comprising at least one of the recombinant nucleic acid molecules according to any one of claims 1 to 40, b. Engineered immune cells suspended in a nutrient medium.

48. The composition according to claim 47, characterized in that The recombinant nucleic acid molecule is linked to at least one of a plasmid, a cosmid, a viral vector and a bacteriophage.

49. The composition according to claim 47, characterized in that The recombinant nucleic acid molecule is linked to a viral vector derived from at least one of a lentivirus, a retrovirus, an adenovirus, an adeno-associated virus, and a Sendai virus.

50. A method (400) for preparing an engineered immune cell, the method comprising: a. Isolate multiple T cells from a peripheral blood mononuclear cell population; b. replicating at least one recombinant nucleic acid molecule according to any one of claims 1 to 40 in a vector; c. delivering the vector into the plurality of T cells.

51. The method (400) of claim 50, wherein: Isolating the plurality of T cells comprises isolating a plurality of CD4 + and CD8 + T cells.

52. The method (400) of claim 50, wherein: After isolating the plurality of T cells, the plurality of T cells are contacted with a costimulatory molecule comprising at least one of cluster of differentiation 3 (CD3) and cluster of differentiation 28 (CD28).

53. The method (400) of claim 50, wherein: Replicating the recombinant nucleic acid molecule includes ligating the recombinant nucleic acid molecule to at least one of a plasmid, a cosmid, a viral vector, and a bacteriophage.

54. The method (400) of claim 50, wherein: Replicating the recombinant nucleic acid molecule includes linking the recombinant nucleic acid molecule to a viral vector derived from at least one of a lentivirus, a retrovirus, an adenovirus, an adeno-associated virus, and a Sendai virus.

55. The method (400) of claim 50, wherein: Delivering the vector into the plurality of T cells includes transforming, transfecting, or transducing the plurality of T cells with the vector.

56. The method (400) of claim 50, wherein: After delivering the vector into the plurality of T cells, the T cells are expanded in a predefined nutrient medium for a predefined period of time.

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