Mitigation of cytokine release syndrome in immunotherapy

Abstract

The present invention discloses a recombinant nucleic acid molecule encoded by at least ORFs (100, 100a) for reducing 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 (120). The first and second hairpin loop structures regulate the amounts of granulocyte-monocyte colony-stimulating factor (GMCSF) and interleukin-6 (IL6) cytokines, respectively. The first hairpin loop structure is formed by at least two first short hairpin RNA (130a) sequences and at least one microRNA 30 (130) sequence. The second hairpin loop structure is formed by at least two second short hairpin RNA (130b) sequences and at least one microRNA 30 (130) sequence. The first promoter (120) is located upstream of the first and second hairpin loop structures.

Description

[Technical Field]

[0001] Priority This application claims the benefit of Indian Provisional Application No. 202221053839, filed on October 20, 2022, the contents of which are incorporated herein by reference.

[0002] The present disclosure relates to artificially modified immune effector cells that reduce 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 technology]

[0003] Cancer is a disease in which abnormal cells divide uncontrollably and destroy body tissues. Preferably, the body's natural immune cells recognize advanced tumors as either "hot" or "cold" depending on whether immune cells are present or absent at the tumor site. In the case of hot tumors, immune cells (including cytotoxic T lymphocytes or T cells) are abundant at the tumor site, but are not active due to the inhibitory signal produced by the tumor microenvironment. In the case of cold tumors, it is the chemotactic signaling of T cells that is inhibited.

[0004] Chimeric antigen receptor (CAR) T cell-based immunotherapy has been introduced to enable immune cells to actively target cancerous cells. CAR T cell-based immunotherapy is an adoptive cell therapy (ACT) in which immune cells are engineered (genetically modified) to specifically target and kill cancer cells. In other words, the CAR construct of CAR T cells is designed to target specific tumor-associated antigens (TAAs).

[0005] Conventional CAR T cell-based therapies target a single tumor antigen, which is effective only against B cell malignancies. Therefore, conventional CAR T cell-based therapies have limited applicability to other hematopoietic malignancies (low CD19), where "off-target effects" are a major concern.

[0006] Although most adverse events associated with CAR T-cell immunotherapy are tolerable and tolerable, CAR T-cell immunotherapy is not without significant side effects. The most current CAR T-cell-based immunotherapies available to date (based on second-generation CAR designs) often exhibit severe systemic toxicity, including the release of large amounts of cytokines. Cytokines are signaling molecules that regulate immune system function. The release of large amounts of cytokines, known as cytokine release syndrome (CRS), can lead to immune-related complications and / or neurotoxicity. Neurotoxicity includes symptoms such as confusion, drowsiness, and seizures, which are further caused by the rapid release of cytokines into the central nervous system. In CRS, the immune system overreacts, releasing a barrage of cytokines, causing patients to experience a cytokine "storm" shortly after receiving CAR T-cell-based immunotherapy. The released cytokines cause fever, hypotension, and shortness of breath. Interleukin-6 (IL6) and granulocyte-monocyte colony-stimulating factor (GMCSF), cytokines upregulated during CRS, are just a few of the key pro-inflammatory molecules contributing to CRS.

[0007] For example, Figure 1 shows 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 the release (upregulation) of high levels of pro-inflammatory cytokines, such as IL6, interleukin 1 (IL1), and nitric oxide (NO). These high levels of pro-inflammatory cytokines ultimately lead to CRS.

[0008] Genome engineering approaches have been used to completely knock out genes involved in CRS, but they have limitations: for example, complete gene knockout can have undesirable effects on CAR T cell survival and can also induce off-target effects.

[0009] Furthermore, due to the nature of CAR T cells as biotherapeutics, engineered CAR T cells can transform into cancerous cells due to uncontrolled proliferation of CAR T cells.

[0010] Therefore, CRS, combined with the inability to control the proliferation of engineered CAR T cells, poses a major challenge in treating cancer patients with CAR T cell-based immunotherapy. Furthermore, because conventional CAR T cells do not persist for long periods, patients infused with conventional CAR T cells have been diagnosed with relapse (i.e., reoccurrence of cancerous cells).

[0011] In light of the foregoing discussion, there is a need for novel CAR T cell-based immunotherapies that overcome the problems associated with conventional CAR T cell-based immunotherapies. Summary of the Invention

[0012] Specific embodiments of the present disclosure will be described below with reference to the accompanying drawings. However, it should be understood that the disclosed embodiments are merely examples of the present disclosure, which may be embodied in various forms. To avoid obscuring the present disclosure in unnecessary detail, well-known functions or structures are not described in detail. Therefore, the specific structural and functional details disclosed herein should not be construed as limiting, but merely as a basis for the claims and as a representative basis for teaching those skilled in the art how to use the present disclosure in various ways in substantially any appropriately detailed structure.

[0013] In an exemplary embodiment, the present disclosure relates to a recombinant nucleic acid molecule encoded by at least an ORF for reducing cytokine storm during immunotherapy. The recombinant nucleic acid molecule includes at least one first hairpin loop structure, at least one second hairpin loop structure, and a first promoter. The first and second hairpin loop structures regulate the amounts of granulocyte-monocyte colony-stimulating factor (GMCSF) and interleukin-6 (IL6) cytokines, respectively, during immunotherapy. The first hairpin loop structure is formed by at least two first short hairpin RNA sequences and at least one microRNA 30 sequence. The first short hairpin RNA sequence forms the stem of the first hairpin loop structure, and the microRNA 30 sequence forms the loop of the second hairpin loop structure. The second hairpin loop structure is located either upstream or downstream of the first hairpin loop structure. The second hairpin loop structure is formed by at least two second short hairpin RNA sequences and at least one microRNA 30 sequence. The second short hairpin RNA sequence forms the stem of the second hairpin loop structure, and the microRNA 30 sequence forms the loop of the second hairpin loop structure. The first promoter is located upstream of the first and second hairpin loop structures.

[0014] In an exemplary embodiment, the present disclosure relates to a recombinant nucleic acid molecule encoded by at least an ORF for reducing cytokine storm during immunotherapy. The recombinant nucleic acid molecule includes 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 repeat sequences. The first and second hairpin loop structures regulate the amounts of granulocyte-monocyte colony-stimulating factor (GMCSF) cytokine and interleukin-6 (IL6) cytokine, respectively, during immunotherapy. The first hairpin loop structure is formed by at least two first short hairpin RNA sequences and at least one microRNA 30 sequence. The first short hairpin RNA sequence forms the stem of the first hairpin loop structure, and the microRNA 30 sequence forms the loop of the second hairpin loop structure. The second hairpin loop structure is located either upstream or downstream of the first hairpin loop structure. The second hairpin loop structure is formed by at least two second short hairpin RNA sequences and at least one microRNA 30 sequence. The second short hairpin RNA sequence forms the stem of the second hairpin loop structure, and the microRNA 30 sequence forms the loop of the second hairpin loop structure. A first promoter is placed upstream of the first and second hairpin loop structures. One or more chimeric antigen receptor genes express 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 domains are configured to bind to one or more tumor-associated antigens. The hinge domain is linked to the one or more single-chain variable fragment domains. The signaling domain is linked to one or more costimulatory domains. The safety switch domain is linked to the signaling domain. The transmembrane domain operably links the hinge domain to the costimulatory domain. A second promoter is placed upstream of the chimeric antigen receptor gene. The long terminal repeat sequence is positioned upstream of the first promoter and downstream of the chimeric antigen receptor gene.

[0015] In an exemplary embodiment, the present disclosure relates to a recombinant nucleic acid molecule encoded by at least an ORF for reducing 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 repeat sequences. The first and second hairpin loop structures regulate the amounts of granulocyte-monocyte colony-stimulating factor (GMCSF) cytokine and interleukin-6 (IL6) cytokine, respectively, during immunotherapy. The first hairpin loop structure is formed by at least two first short hairpin RNA sequences and at least one microRNA 30 sequence. The first short hairpin RNA sequence forms the stem of the first hairpin loop structure, and the microRNA 30 sequence forms the loop of the second hairpin loop structure. The second hairpin loop structure is located either upstream or downstream of the first hairpin loop structure. The second hairpin loop structure is formed by at least two second short hairpin RNA sequences and at least one microRNA 30 sequence. The second short hairpin RNA sequence forms the stem of the second hairpin loop structure, and the microRNA 30 sequence forms the loop of the second hairpin loop structure. A first promoter is placed upstream of the first and second hairpin loop structures. One or more chimeric antigen receptor genes express 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 domains are configured to bind to one or more tumor-associated antigens. The hinge domain is linked to the one or more single-chain variable fragment domains. The signaling domain is linked to one or more costimulatory domains. The safety switch domain is linked to the signaling domain. The transmembrane domain operably links the hinge domain to the costimulatory domain. A second promoter is placed upstream of the chimeric antigen receptor gene. The long terminal repeat sequence is positioned upstream of the first promoter and downstream of the chimeric antigen receptor gene.

[0016] In an exemplary embodiment, the present disclosure relates to a transcript of a recombinant nucleic acid molecule encoded by at least an ORF for reducing cytokine storm during immunotherapy. The transcript includes a messenger RNA with a first hairpin loop structure and / or a second hairpin loop structure transcribed from the recombinant nucleic acid molecule. The messenger RNA with the hairpin loop structure is configured to bind to the messenger RNA of a cytokine.

[0017] In an exemplary embodiment, the present disclosure relates to a vector comprising at least one recombinant nucleic acid molecule ligated to at least one of a plasmid, a cosmid, a viral vector, and a phage, wherein the recombinant nucleic acid molecule is encoded by at least an ORF for reducing cytokine storm during immunotherapy.

[0018] In an exemplary embodiment, the present disclosure relates to a genetically engineered immune cell comprising at least one recombinant nucleic acid molecule encoded by at least an ORF for reducing cytokine storm during immunotherapy.

[0019] In an exemplary embodiment, the present disclosure relates to a composition comprising engineered immune cells suspended in a nutrient medium, the engineered immune cells comprising at least one recombinant nucleic acid molecule encoded by at least an ORF for reducing cytokine storm during immunotherapy.

[0020] In an exemplary embodiment, the present disclosure relates to a method for preparing engineered immune cells, comprising isolating a plurality of T cells from a population of peripheral blood mononuclear cells, replicating in a vector at least one recombinant nucleic acid molecule encoded by at least an ORF for reducing cytokine storm during immunotherapy, and delivering the vector into the plurality of T cells.

[0021] The foregoing and other features and advantages of the present invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying drawings, in which like reference numerals designate the same or similar parts throughout the several views. These features are described at a level of detail sufficient to enable one skilled in the art to practice the invention. It is also understood that other features may be used and structural changes may be made without departing from the scope of the present invention.

[0022] The foregoing summary, as well as the following detailed description of exemplary embodiments, will be better understood when read in conjunction with the assigned drawings. For the purpose of illustrating the present disclosure, there are shown in the drawings exemplary configurations of the present disclosure. However, the present disclosure is not limited to the particular methods and instrumentalities disclosed therein. Moreover, those skilled in the art will understand that the drawings are not to scale. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 shows a diagram of CRS guidance (prior art), in accordance with one or more exemplary embodiments of the present disclosure.

[0024] [Figure 2] FIG. 2 depicts ORF 100 according to one or more exemplary embodiments of the present disclosure.

[0025] FIG. 2a shows ORF 100a according to one or more exemplary embodiments of the present disclosure.

[0026] [Figure 3] FIG. 3 shows a CAR construct 200 according to one or more exemplary embodiments of the present disclosure.

[0027] FIG. 3a shows a CAR construct 300 according to one or more exemplary embodiments of the present disclosure.

[0028] [Figure 4]FIG. 4 shows an illustration of CRS reduction in accordance with one or more exemplary embodiments of the present disclosure.

[0029] [Figure 5] FIG. 5 illustrates a method 400 for generating CAR-T cells according to one or more exemplary embodiments of the present disclosure.

[0030] [Figure 6] FIG. 6 illustrates experimental observations according to one or more exemplary embodiments of the present disclosure. [Figure 7] FIG. 7 illustrates experimental observations according to one or more exemplary embodiments of the present disclosure. [Figure 8] FIG. 8 illustrates experimental observations according to one or more exemplary embodiments of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0031] Detailed Description of the Drawings Before describing the present invention in detail, definitions of certain words or phrases used throughout this patent document are defined: the terms "include" and "comprise," and their derivatives, mean open-ended inclusion; the term "or" is inclusive and / or; the phrases "coupled with" and "associated with," and their derivatives may mean include, contained within, interconnect, contain, be contained within, connect (to or with), couple to or with, be in communication (with), cooperate (with), interleave, juxtapose, be adjacent (to), be bound to or with, have the characteristic of, etc.; definitions of certain words and phrases are provided throughout this patent document, and those skilled in the art will understand that such definitions apply to previous and future uses of many, if not most, of such defined words and phrases.

[0032] References throughout this specification to "one embodiment," "an embodiment," or similar language mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment. Thus, appearances of "in one embodiment," "in an embodiment," and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment and mean "one or more, but not all, embodiments" unless otherwise specified. The terms "including," "comprising," "having," and variations thereof mean "including but not limited to," unless otherwise specified. An enumerated list of items does not imply that any or all of the items are mutually exclusive and / or mutually inclusive unless explicitly stated otherwise. The terms "a," "an," and "the" also refer to "one or more" unless otherwise specified.

[0033] As used herein, the term "activation" refers to a state of cells that have been stimulated sufficiently 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.

[0034] The term "activated T cells" refers, inter alia, 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).

[0035] The term "antibody" is used in the broadest sense and refers to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity or function. Antibodies in this disclosure can exist in a variety of forms, including, for example, polyclonal antibodies; monoclonal antibodies; Fv, Fab, Fab', and F(ab')2 fragments; and single-chain antibodies and humanized antibodies.

[0036] The term "antibody fragment" refers to a portion of a full-length antibody, such as the antigen-binding or variable region of the antibody. Other examples of antibody fragments include Fab, Fab', F(ab'), and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0037] The term "Fv" refers to the minimum antibody fragment that contains a complete antigen-recognition and binding site. This fragment consists of a dimer of one heavy-chain variable region domain and one light-chain variable region domain in tight, noncovalent association. The folding of these two domains results in six hypervariable loops (three loops from each H and L chain) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific complementarity-determining regions (CDRs)) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site (dimer).

[0038] As used herein, "antibody heavy chain" refers to the larger of the two polypeptide chains present in all antibody molecules in their naturally occurring conformation. As used herein, "antibody light chain" refers to the smaller of the two polypeptide chains present in all antibody molecules in their naturally occurring conformation. K and A light chains refer to the two major antibody light chain isotypes.

[0039] 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 an antibody produced by synthesizing a DNA molecule encoding the antibody and expressing the DNA molecule to obtain the antibody or to obtain the amino acids that encode the antibody. Synthetic DNA is available and can be obtained using techniques well known in the art.

[0040] The term "antigen" refers to a molecule that elicits an immune response, which may include antibody production, activation of specific immunocompetent cells, or both. Antigens include any macromolecule, including whole proteins or peptides, or molecules derived from recombinant or genomic DNA. For example, DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein or peptide that elicits an immune response and thus encodes an "antigen," as the term is used herein. An antigen need not be encoded solely by the full-length nucleotide sequence of a gene. Antigens can be generated, synthesized, or derived from biological samples, including tissue samples, tumor samples, cells, or biological fluids.

[0041] 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 viability, an increase in the life expectancy of a subject bearing tumor cells, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" may also be manifested by the ability of peptides, polynucleotides, cells, and antibodies to prevent the development of tumors in the first place.

[0042] 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, and glycoproteins (including cell surface receptors).

[0043] The term "autologous" is used to describe material originating from a subject that is subsequently reintroduced into the same subject.

[0044] The term "allogeneic" is used to describe a graft derived from a different subject of the same species. As an 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.

[0045] The term "xenogeneic" is used to describe a graft derived from a subject of a different species. In one example, the donor subject may be from a different species than the recipient subject, and the donor and recipient subjects may be genetically and immunologically incompatible.

[0046] The term "cancer" is used to refer to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Various cancers include, for example, 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.

[0047] The terms "complementary" and "complementarity" refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence "AGT" is complementary to the sequence "TCA." Complementarity can be "partial," in which 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 has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.

[0048] The term "corresponds to" or "corresponding to" refers to (a) a polynucleotide having a nucleotide sequence substantially identical to or complementary to all or a portion of a reference polynucleotide sequence, or encoding an amino acid sequence identical to an amino acid sequence in a peptide or protein, or (b) a peptide or polypeptide having an amino acid sequence substantially identical to a sequence of amino acids in a reference peptide or protein.

[0049] The term "costimulatory ligand" refers to a molecule on an antigen-presenting cell (e.g., an APC, a dendritic cell, a B cell, etc.) that specifically binds to a cognate costimulatory molecule on a T cell, thereby providing a signal that mediates a T cell response, including at least one of proliferation, activation, differentiation, and other cellular responses, in addition to the primary signal provided, for example, by binding of the TCR / CD3 complex to a peptide-loaded MHC molecule. Costimulatory ligands also 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.

[0050] The term "costimulatory molecule" refers to the cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as proliferation. Costimulatory molecules include MHC class I molecules, BTLA, and Toll-like receptors.

[0051] The term "costimulatory signal" refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell proliferation and / or up-regulation or down-regulation of key molecules.

[0052] The term "encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence (except that "T" is replaced by "U") and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0053] The term "exogenous" refers to a molecule that does not naturally occur in a wild-type cell or organism but is typically introduced into a cell by molecular biological techniques. Examples of exogenous polynucleotides include vectors, plasmids, and / or artificial nucleic acid constructs that encode a desired protein. With respect to polynucleotides and proteins, the terms "endogenous" or "natural" refer to a naturally occurring polynucleotide or amino acid sequence that can be found in a given wild-type cell or organism. Additionally, a particular polynucleotide sequence isolated from a first organism and transferred into a second organism by molecular biological techniques is typically considered an "exogenous" polynucleotide or amino acid sequence with respect to the second organism. In certain embodiments, a polynucleotide sequence can be "introduced" into a microorganism already containing such a polynucleotide sequence by molecular biological techniques, for example, to generate one or more additional copies of the naturally occurring polynucleotide sequence, thereby facilitating overexpression of the encoded polypeptide.

[0054] The term "expression" refers to the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0055] The term "expression vector" refers to a vector containing a recombinant polynucleotide comprising expression control (regulatory) sequences operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied 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 the recombinant polynucleotide.

[0056] In the context of the present disclosure, the following abbreviations for commonly occurring nucleobases are used: "A" refers to adenine, "C" refers to cytosine, "G" refers to guanine, "T" refers to thymine, and "U" refers to uracil.

[0057] Unless otherwise specified, 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 that encodes a protein may, in some versions, contain intron(s).

[0058] The term "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells, and because they can deliver significant amounts of genetic information into the DNA of host cells, they are one of the most efficient gene delivery vectors. Furthermore, the use of lentiviruses allows for the integration of genetic information into host chromosomes, resulting in stably transduced genetic information. HIV, SIV, and FIV are all examples of lentiviruses. Lentivirus-derived vectors provide a means to achieve significant levels of gene transfer in vivo.

[0059] 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 the subject in the absence of a treatment or compound, and / or compared to the level of the response in an otherwise identical but untreated subject. This term encompasses perturbing and / or affecting a natural signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0060] 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 sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.

[0061] The term "under transcriptional control" refers to a promoter operably linked to, and in the correct position and orientation relative to, a polynucleotide so as to control (regulate) the initiation of transcription by RNA polymerase and the expression of the polynucleotide.

[0062] The term "overexpressed" tumor antigen or "overexpression" of a tumor antigen is intended to indicate an abnormal level of expression of the tumor antigen in cells from a diseased area, such as a solid tumor, within a particular tissue or organ of a patient, compared to the expression level in normal cells from that tissue or organ. Patients with solid tumors or hematological tumors characterized by overexpression of tumor antigens can be determined by standard assays known in the art.

[0063] Terms such as "patient," "subject," and "individual" are used interchangeably herein and refer to any human or animal suitable for the methods described herein. In certain non-limiting embodiments, a patient, subject, or individual is a human or an animal. In embodiments, the term "subject" is intended to include a living organism (e.g., a mammal) 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.

[0064] A subject in need or in need of treatment includes a subject having a disease, condition, or disorder in need of treatment. Subjects in need also include subjects in need of treatment for the prevention of a disease, condition, or disorder.

[0065] The term "polynucleotide" or "nucleic acid" refers to mRNA, RNA, cRNA, rRNA, cDNA, or DNA. The term refers to a polymeric form of nucleotides, typically 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 acid, including single- and double-stranded forms of nucleic acids.

[0066] Terms such as "polynucleotide variant" and "variant" refer to a polynucleotide that exhibits substantial sequence identity with a reference polynucleotide sequence or hybridizes to a reference sequence under stringent conditions, as defined below. These terms also encompass polynucleotides that differ 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 with different nucleotides. In this regard, it is well understood in the art that certain modifications, including mutations, additions, deletions, and substitutions, can be made to a reference polynucleotide such that the modified polynucleotide retains the biological function or activity of the reference polynucleotide or has increased (i.e., optimized) activity compared to the reference polynucleotide. Polynucleotide variants include, for example, polynucleotides having at least 50% sequence identity (and from at least 51% to at least 99% and all integer percentages therebetween, e.g., 90%, 95%, or 98%) to a reference polynucleotide sequence described herein. The terms "polynucleotide variant" and "variant" also include naturally occurring allelic variants and orthologues.

[0067] The terms "polypeptide," "polypeptide fragment," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogs thereof. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-natural amino acids, such as chemical analogs of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. In certain embodiments, polypeptides can include enzymatic polypeptides or "enzymes," which typically catalyze various chemical reactions (i.e.,).

[0068] 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 can be conservative or non-conservative. In certain embodiments, a polypeptide variant includes conservative substitutions, and in this regard, it is well understood in the art that some amino acids can be changed to other amino acids with broadly similar properties without changing the nature of the activity of the polypeptide. Polypeptide variants also encompass polypeptides in which one or more amino acids are added or deleted or replaced with different amino acid residues.

[0069] The term "promoter" refers to a DNA sequence recognized by a cell's or introduced synthetic machinery necessary to initiate the specific transcription of a polynucleotide sequence. The term "expression control (regulatory) sequence" refers to a DNA sequence necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences that are suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0070] The terms "bind," "binds," or "interacts with" refer to a molecule that recognizes and attaches to a second molecule in a sample or organism but does not substantially recognize or attach to other structurally unrelated molecules in the sample. As used herein with respect to antibodies, the term "specifically binds" 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 may also bind to that antigen from one or more species. However, such cross-species reactivity does not in itself change the specific classification of the antibody. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity does not in itself change the specific classification of the antibody. In some examples, the terms "specific binding" or "specifically binds" can be used in reference to the interaction of an antibody, protein, or peptide with a second chemical species, meaning that the interaction is dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the chemical species. For example, an antibody recognizes and binds to a specific protein structure rather than just any protein. If an antibody is specific for epitope "A," then in a reaction containing labeled "A" and the antibody, the presence of a molecule containing epitope A (or free unlabeled A) will reduce the amount of labeled A bound to the antibody.

[0071] The term "stimulation" refers to a primary response induced by the 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 through the TCR / CD3 complex. Stimulation can mediate changes in the expression of specific molecules, such as downregulation of TGF-β and / or rearrangement of cytoskeletal structure.

[0072] The term "stimulatory molecule" refers to the cognate stimulatory ligand present on an antigen-presenting cell that specifically binds to a molecule on a T cell.

[0073] The term "stimulatory ligand" refers to a ligand that, when present on an antigen-presenting cell (e.g., an APC, a dendritic cell, a B cell, etc.), is capable of specifically binding 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 by the T cell, including activation, initiation of an immune response, proliferation, and similar processes.

[0074] The terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0075] A "chimeric antigen receptor" (CAR) molecule is a recombinant polypeptide that includes at least an extracellular domain, a transmembrane domain, and a cytoplasmic or intracellular domain.

[0076] Although the operations of exemplary embodiments of the disclosed methods may be described in a particular order for convenient presentation, it should be understood that the disclosed embodiments may encompass orders of operations other than the particular order disclosed. For example, operations described sequentially may in some cases be reordered or performed simultaneously. Moreover, descriptions and disclosures provided in connection with one particular embodiment are not limited to that embodiment but may apply to any embodiment disclosed herein. Moreover, for simplicity, the accompanying figures may not show the various ways in which the disclosed systems, methods, and apparatus can be used in combination with other systems, methods, and apparatus.

[0077] Furthermore, the described features, advantages, and characteristics of the embodiments may be combined in any suitable manner. Those skilled in the art will recognize that embodiments may be practiced without one or more of the specific features or advantages of a particular embodiment. In other instances, 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 claims, or may be learned by practice of the embodiments described below.

[0078] The present disclosure discloses chimeric antigen receptor T cell(s) (CAR T cells). CAR T cells are artificially engineered through genetic manipulation. CAR T cells can be derived from either autologous or allogeneic sources. CAR T cells can be used to target various cellular disorders, including, but not limited to, B-cell malignancies (blood cancers), solid tumors, and the like. In one embodiment, the CAR T cells are directed against relapsed or refractory B-cell acute lymphoblastic leukemia (ALL) or non-Hodgkin's lymphoma (NHL).

[0079] CAR T cells can be engineered to contain one or more sequences that express one or more chimeric antigen receptors (CARs or CAR constructs). CARs have predetermined 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), and human telomerase reverse transcriptase (hTERT). In an exemplary embodiment, the CAR T cells comprise a CAR specific for CD19.

[0080] Furthermore, the CAR T cells contain at least two short hairpin RNA (shRNA) sequences along with a microRNA 30 (miR30) sequence to reduce cytokine storms, known as cytokine release syndrome (CRS), during immunotherapy. Rapid cytokine release is observed during CRS. Interleukin 6 (IL6) and granulocyte-monocyte colony-stimulating factor (or granulocyte-macrophage colony-stimulating factor) (GMCSF) are some of the cytokines released during immunotherapy. The shRNA sequence can inhibit cytokine production and / or cytokine signaling through RNA interference (RNAi). In an exemplary embodiment, the shRNA transcript binds to the cytokine mRNA and functionally inactivates and / or degrades the mRNA. In an alternative embodiment, the shRNA sequence transcript binds to the cytokine mRNA and prevents its translation. In another exemplary embodiment, the shRNA binds to one or more receptors for the cytokine, thereby blocking the downstream signaling pathway of the cytokine. In yet another exemplary embodiment, the shRNA sequence expresses anti-IL6 (aIL6) which binds to and neutralizes IL6 (an exemplary cytokine), thus at least partially blocking the activity of IL6.

[0081] In an exemplary embodiment, the CAR T cells of the present disclosure comprise an IL-6 receptor (IL6-R) conjugated with a synthetic Notch (syn-notch) receptor and a GAL-VP64 domain. During CRS, released IL6 binds to the IL6-R of the CAR T cell and induces syn-notch, followed by cleavage and release of the GAL-VP64 domain. The GAL-VP64 domain translocates to the nucleus of the CAR T cell and induces the expression of one of 36 shRNA sequences specific for interleukin 6 (or IL6). This expression of the shRNA sequence(s) results in the production of aIL6, which binds to and neutralizes IL6. IL6 is a key molecule involved in the induction of CRS and leads to early CAR T cell apoptosis. By neutralizing IL6, CAR T cells as disclosed herein have a lower risk of CRS and thus have the ability to safely and effectively eliminate cancerous cells.

[0082] In another exemplary embodiment, the CAR-T cells of the present disclosure produce a transcript of at least one of 20 shRNAs specific for granulocyte-monocyte colony-stimulating factor (or GMCSF), which contributes to cytokine release syndrome (CRS) during immunotherapy. In an exemplary embodiment, the shRNA sequence selectively binds to GMCSF mRNA and renders it nonfunctional, thereby regulating (i.e., reducing) the amount of GMCSF produced. GMCSF is a key molecule involved in inducing CRS and leading to early CAR T cell apoptosis. By regulating the amount of GM-CSF produced, the CAR T cells disclosed herein have a lower risk of CRS, are therefore longer-lasting, and have the ability to safely and effectively eliminate cancerous cells.

[0083] In an exemplary embodiment, the CAR T cells of the present disclosure produce at least one shRNA specific for each of GMCSF mRNA and IL6 mRNA, thereby reducing the concentration levels of GMCSF and IL6 molecules, and thereby reducing the CRS burden.

[0084] Additionally, the CAR contains a safety switch (SS) domain to regulate CAR T cell proliferation and thereby inhibit transformation of the CAR T cells into cancerous cells (e.g., during manufacturing or after infusion of the CAR T cells in a patient). SS domains may include, but are not limited to, inducible caspase 9 (iCaspase 9), cleaved epidermal growth factor receptor (EGFRt), RQR8, or a combination thereof.

[0085] Therefore, the CAR T cells as disclosed herein are programmed to induce an artificial immune response against cancerous cells without any of the undesirable side effects of CRS. Furthermore, based on the condition and requirements of the patient (or subject), the CAR T cells of the present disclosure can be selectively depleted to prevent malignant transformation of the CAR T cells.

[0086] Referring now to the figures, Figure 2 shows the antisense (or template) strand of open reading frame (ORF) 100 of a CAR T cell (not shown). 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 direction, 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.

[0087] At least a portion of ORF 100 (or a recombinant nucleotide sequence molecule) can be introduced into one or more natural immune cells via genetic engineering to produce engineered immune cells. ORF 100 (or a portion thereof) expresses one or more CARs (i.e., proteins and / or polypeptides) and / or one or more shRNA transcripts (described below). Natural immune cells may include, but are not limited to, T lymphocytes (T cells), natural killer (NK) cells, gamma delta (γδ) T cells, etc. In an exemplary embodiment, ORF 100 is introduced into natural T cells via clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas)-based gene editing technology. Additionally or alternatively, ORF 100 is introduced into natural T cells using a lentiviral vector, adeno-associated virus (AAV), etc. The vector containing ORF 100 can be selected from a plasmid, cosmid, viral vector, or phage. The plasmid can be a eukaryotic expression plasmid. Viral vectors can be derived from lentivirus, retrovirus, adenovirus, adeno-associated virus, and / or Sendai virus. In an exemplary embodiment, lentivirus (LV) is used to introduce ORF 100 into T cells. Lentiviral (LV) vectors offer an effective means for eukaryotic cells, stable transfer, and gene expression modification in host cells with lower immunogenicity. LV vectors have a larger gene carrying capacity and can transduce proliferating and non-proliferating cells. Furthermore, LV vector-based gene delivery systems are the most efficient method for transducing hard-to-transfect T cells. In an exemplary embodiment, clinical-grade viral vectors were commercially obtained from Lentigen and Sirion-Biotech.

[0088] ORF 100 may include one or more regions including one or more long terminal repeats (LTRs) 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 together with at least one microRNA 30 (miR30) 130 sequence, one or more genes, etc. The one or more promoters may include a first promoter 120 and a second promoter 140. The 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 together with at least one microRNA 30 (miR30) 130 sequence, and a second hairpin loop structure formed by at least one second shRNA 130b sequence together with at least one microRNA 30 (miR30) 130 sequence. Additionally or optionally, ORF 100 may include one or more nucleotide sequences, such as genes for fluorescent proteins, c-myc, 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 RNAs) of the first and second hairpin loop structures bind to cytokine messenger RNAs (mRNAs). In an exemplary embodiment, the transcripts of the first and second hairpin loop structures bind to IL6 and GMCSF mRNAs, respectively.

[0089] LTR 110 may be adjacent to ORF 100, i.e., LTR 110 may be located at the 5' and 3' ends of ORF 100. In other words, LTR 110 is located upstream of first promoter 120 and downstream of CAR gene 150. In an exemplary embodiment, LTR 110 at the 5' end of ORF 100 is encoded by SEQ ID NO: 1. LTR 110 aids in the integration of ORF 100 into the native DNA of a T cell while genetically engineering the T cell. Integration of ORF 100 into the native DNA transforms the native T cell into a CAR T cell (i.e., an exemplary engineered immune cell).

[0090] Transcription of ORF 100 in CAR-T cells can be controlled by one or more promoters, i.e., the production of transcripts of one or more genes is controlled by one or more promoters. The promoter may be located upstream of one or more genes of ORF 100. One or more RNA polymerases can bind to the promoter for transcription of 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, the RNA polymerase can partially or completely transcribe ORF 100 into one or more messenger RNAs (mRNAs) (or transcripts). The mRNA can be further processed to provide one or more CARs (protein structures) and the first and second shRNA 130a, 130b transcripts (and their proteins / polypeptides). Processing of mRNA may include at least one of splicing, translation of mRNA into one or more amino acid sequences (i.e., polypeptides), and post-translational modifications, etc. Post-translational modifications may include, but are not limited to, folding (into a protein) and / or glycosylation of the amino acid sequence, etc.

[0091] As shown in FIG. 2, when the first shRNA 130a is positioned upstream of the second shRNA 130b, the first promoter 120 may be positioned upstream of the first shRNA 130a (i.e., at the first hairpin loop structure(s)). Alternatively, although not shown, when the first shRNA 130a is positioned downstream of the second shRNA 130b, the first promoter 120 may be positioned upstream of the second shRNA 130b. The first promoter 120 may be selected from 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, an SNORD promoter, etc. In an exemplary embodiment, first promoter 120 includes a GAL-VP64 recognition domain that activates first promoter 120 when the GAL-VP64 domain binds to the GAL-VP64 recognition domain.

[0092] The first promoter 120 transcribes the first shRNA 130a and the second shRNA 130b to generate 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 NOs: 5 to 40. The second shRNA 130b can be encoded by at least one of SEQ ID NOs: 41 to 60.

[0093] In an exemplary embodiment, as shown in FIG. 2, the hairpin loop structure of ORF 100 is provided with two first and second shRNA sequences 130a, 130b, respectively, that complement each other and form the stem of the hairpin loop structure.

[0094] The transcripts of the first shRNA 130a and the second shRNA 130b may contain specific binding affinities for one or more molecules. CAR-T cells contain multiple IL6 receptors (IL6-R) conjugated with a synthetic Notch receptor (syn-notch) and a GAL-VP64 domain. In an exemplary embodiment, during CRS, released IL6 binds to the IL6-R of the CAR T cell and induces syn-notch, followed by cleavage and release of the GAL-VP64 domain. The GAL-VP64 domain translocates to the nucleus of the CAR T cell and induces the expression of one of the 36 first shRNA 130a sequences. The expression of the first shRNA 130a sequence(s) results in the production of aIL6. aIL6 binds to and neutralizes IL6, thereby reducing the burden of CRS.

[0095] The CAR T cells contain multiple GMCSF gene sequences (not shown) that produce pro-inflammatory cytokines (such as GMCSF) that contribute to CRS. The second shRNA 130b can inhibit cytokine production and / or cytokine signaling through RNA interference (RNAi). In an exemplary embodiment, the transcript of second shRNA 130b binds to the mRNA corresponding to the cytokine (i.e., GMCSF) and functionally inactivates and / or degrades 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 cells selectively binds to IL6 mRNA, thereby regulating IL6 cytokine levels (via RNAi). Binding of the transcript of the first shRNA 130a to IL6 mRNA reduces the level (concentration) of IL6 molecules, reducing the 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 GMCSF cytokine levels (via RNAi). Binding of the transcript of the second shRNA 130b to GMCSF mRNA reduces the level (concentration) of the GMCSF molecule, reducing the burden of CRS.

[0098] The miR30 130 is a short RNA sequence that acts as a regulatory element for the transcription of the first and second shRNA 130a and 130b sequences, i.e., the miR30 130 at least partially regulates the expression of the first and second shRNA 130a and 130b sequences. The miR30 130 may be adjacent to and / or located between the first and second shRNA 130a and 130b sequences, respectively. In an exemplary embodiment, the miR30 130 is encoded by SEQ ID NO: 61. In an exemplary embodiment, as shown in FIG. 2, the miR30 130 sequence forms the loop of the hairpin loop structure formed by the first and second shRNA 130a and 130b sequences. The expression of the first shRNA 130a and the second shRNA 130b can be regulated by miR30 130 and / or the first promoter 120.

[0099] While excessive GMCSF production (such as during CRS) is harmful, low levels of GMCSF promote improved antitumor activity of CAR T cells. miR30 130 helps maintain low levels of GMCSF by regulating the expression of the second shRNA 130b sequence. Therefore, the miR30 130 sequence helps reduce the risk of potential CRS induction while preserving the beneficial antitumor activity of GMCSF.

[0100] In an exemplary embodiment, the expression of the miR30 130 sequence is regulated by the host cell (i.e., CAR T cell). Because the expression of the first and second shRNA 130a, 130b sequences and the miR30 130 sequence are both controlled by the first promoter 120, the host cell can regulate the expression of the first and second shRNA 130a, 130b sequences together with the miR30 130 sequence. Regulation of the expression of the first and second shRNA 130a, 130b sequences by the host cell prevents overexpression of the first and second shRNA 130a, 130b sequences. The first and second shRNA 130a, 130b, together with the miR30 130, enable strict control of the levels of GMCSF and IL6. Thus, the CAR T cells of the present disclosure have more precise and sustained suppression of GMCSF and IL6 production.

[0101] Furthermore, proper balancing (modulation) of GMCSF expression resulted in the suppression of exhaustion markers on the CAR T cells of the present disclosure. This suppression of exhaustion markers promotes a less exhausted, more functional CAR-T cell population and enhances their long-term anti-tumor activity and persistence. Because CAR T cells must survive and function in a harsh tumor microenvironment, this enhanced persistence is important for long-term, sustained anti-tumor efficacy. Thus, CAR-T cell survival and persistence are enhanced while reducing the risk of cytokine-related toxicity.

[0102] The first and second shRNAs 130a, 130b and miR30 130 promote better infiltration of CAR T cells into tumor sites. Improved tumor penetration is essential for CAR T cells to effectively recognize and target cancer cells.

[0103] In an exemplary embodiment, the combination of a first shRNA 130a, a second shRNA 130b, and miR30 130 provides an advanced strategy for enhancing CAR T cell infiltration into the tumor microenvironment. This innovative approach centers on the inhibition of GMCSF expression, which plays a pivotal role in promoting immune responses and inflammation. By utilizing the second shRNA 130b, GM-CSF expression is specifically targeted and downregulated. This serves as a fundamental step in modulating the tumor microenvironment, as GM-CSF is known to contribute to the recruitment and activation of various immune cells, including neutrophils, macrophages, and dendritic cells. Furthermore, the incorporation of miR30 130 further enhances GM-CSF inhibition. miR30 130 acts as a post-transcriptional regulator that fine-tunes gene expression. By suppressing GM-CSF mRNA translation, miR30 130 adds an extra layer of control to ensure minimal GMCSF production. This dual approach of the first and second shRNAs 130a, 130b and miR30 130 acts synergistically to reduce GMCSF levels, thereby mitigating immune interference that would otherwise hinder CAR T cell infiltration into tumor sites. Thus, the first and second shRNAs 130a, 130b and miR30 130 promote better infiltration of CAR T cells into tumor sites. Improved tumor penetration is essential for CAR T cells to effectively recognize and target cancer cells.

[0104] 2a shows ORF 100a of the present disclosure. ORF 100a is structurally the same as ORF 100 with the addition of 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 130a sequences together with miR30 130 sequences, and / or one or more second shRNA 130b sequences together with miR30 130 sequences, 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 the first shRNA 130a sequence together with miR30 130 sequences, and a second hairpin loop structure formed by at least one second shRNA 130b sequence together with miR30 130 sequences. The recombinant nucleotide sequence molecule is encoded by at least 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 RNAs) of the first and second hairpin loop structures bind to cytokine messenger RNA (mRNA). In an exemplary embodiment, the transcripts of the first and second hairpin loop structures bind to IL6 and GMCSF mRNA, respectively.

[0105] The polyA tail 160 sequence may be downstream of the first and second shRNA 130a, 130b sequences and / or the miR30 130 sequence. Expression of the polyA tail sequence 160 may be regulated by the first promoter 120. In an exemplary embodiment, as shown in FIG. 2a, the polyA tail 160 sequence is downstream of the hairpin loop structure formed by the second shRNA 130b sequence and the miR30 130 sequence. The transcript of the polyA tail 160 sequence may contain multiple adenosine nucleotides. In an exemplary embodiment, the polyA tail 160 sequence is derived from either Simian Virus 40 (SV40) or bovine growth hormone (bGH). The SV40 polyA tail 160 sequence is encoded by SEQ ID NO: 62. The polyA tail 160 aids in mRNA stability by providing protection against enzymatic cleavage, a process that can lead to mRNA degradation. It further ensures that the intended regulatory effects of the IL6, GMCSF and miR30 130 components are maintained over time without undue degradation or interference.

[0106] 2, the second promoter 140 can be positioned upstream of the CAR gene 150. In other words, the CAR gene 150 can be positioned 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: 63), the EF1 alpha core promoter (encoded by SEQ ID NO: 64), and the like. Thus, the expression of the CAR gene 150 can be regulated by the second promoter 140.

[0107] CAR gene 150 comprises a sequence encoding one or more domains of CAR 200, as shown in 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 costimulatory (CSTM) domains 240, one or more signaling domains 250, one or more safety switch (SS) domains 260, etc.

[0108] In an exemplary embodiment, as shown in Figure 3, CAR 200 comprises one scFV domain 210, one hinge domain 220, one transmembrane domain 230, one CSTM domain 240, one signaling domain 250 and one SS domain 260. Additionally or optionally, CAR 200 may comprise one or more peptide sequences disposed between the domains of CAR 200, such as a signal peptide, a linker peptide, etc.

[0109] As with any antibody, scFV domain 210 of CAR 200 can comprise a light chain 210a and a heavy chain 210b. In one embodiment, scFV domain 210 against CD19 antigen (TAA) can be encoded by SEQ ID NO: 65, such that scFV domain 210 comprises the polypeptide sequence defined by SEQ ID NO: 66. In an alternative embodiment, scFV domain 210 against CD19 antigen (TAA) can be encoded by SEQ ID NO: 67, such that scFV domain 210 comprises the polypeptide sequence defined by SEQ ID NO: 68. Light chain 210a and heavy chain 210b of scFV domain 210 can be linked to each other via a linker protein 210c. scFV domain 210 may 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 may comprise two or three scFV domains (not shown) for the same or different TAAs.

[0110] Hinge domain 220 operably links 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 serves to provide flexibility to scFV domain 210 relative to transmembrane domain 230. In one embodiment, hinge domain 220 is encoded by SEQ ID NO:69, such that hinge domain 220 comprises the polypeptide sequence defined by SEQ ID NO:5770.

[0111] Transmembrane domain 230 may be disposed across lipid bilayer cell membrane (or membrane) 270. Membrane 270 separates extracellular region 270a and intracellular region 270b. In an exemplary embodiment, transmembrane domain 230 comprises a CD8 domain encoded by SEQ ID NO:71, such that transmembrane domain 230 comprises the polypeptide sequence defined by SEQ ID NO:72. Transmembrane domain 230 helps operably link the domains of CAR 200 present in extracellular region 270a to the domains of CAR 200 present in intracellular region 270b. In an exemplary embodiment, as shown in FIG. 3, scFV domain 210 and hinge domain 220 are the only domains present in extracellular region 270a.

[0112] The CSTM domain 240 can be linked 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, etc. The CD28 CSTM domain 240 can be encoded by SEQ ID NO:73, such that the CD28 CSTM domain 240 comprises the polypeptide sequence defined by SEQ ID NO:74. The ICOS CSTM domain 240 can be encoded by SEQ ID NO:75, such that the ICOS CSTM domain 240 comprises the polypeptide sequence defined by SEQ ID NO:76. The OX40 CSTM domain 240 can be encoded by SEQ ID NO:77, such that the OX40 CSTM domain 240 comprises the polypeptide sequence defined by SEQ ID NO:78. The 4-1BB CSTM domain 240 can be encoded by SEQ ID NO:79, such that the 4-1BB CSTM domain 240 comprises the polypeptide sequence defined by SEQ ID NO:80. The DAP10 CSTM domain 240 can be encoded by SEQ ID NO: 81, such that the DAP10 CSTM domain 240 comprises the polypeptide sequence defined by SEQ ID NO: 82. The DAP12 CSTM domain 240 can be encoded by SEQ ID NO: 83, such that the DAP12 CSTM domain 240 comprises the polypeptide sequence defined by SEQ ID NO: 84. The 2B4 CSTM domain 240 can be encoded by SEQ ID NO: 85, such that the 2B4 CSTM domain 240 comprises the polypeptide sequence defined by SEQ ID NO: 86. In one embodiment, the CSTM domain 240 comprises 4-IBB. The CSTM domain 240 serves to enhance cell-mediated immune responses.

[0113] In an exemplary embodiment, CAR T cells are activated by exposing them to costimulatory molecules (e.g., CD3 and / or CD28). The costimulatory molecules stimulate the CSTM domain 240, which in turn leads to the activation, proliferation, and / or differentiation of the CAR T cells into effector T cells (thus enabling the CAR T cells to express the CAR construct). CD3 / CD28 activation mimics the signals T cells receive during natural antigen recognition, thus providing a safe and effective method for synthetically activating CAR T cells in vitro and in vivo. The CD3 / CD28-activated CAR T cells (or effector T cells) are then used to target and kill cancerous cells.

[0114] Signaling domain 250 can be linked to CSTM domain 240. In an exemplary embodiment, signaling domain 250 is a cluster of differentiation 3 zeta (CD3ζ) domain encoded by SEQ ID NO: 87, such that CD3ζ signaling domain 250 comprises the polypeptide sequence defined by SEQ ID NO: 88. Signaling domain 250 helps transmit an activation signal to the CAR T cell after the TAA binds to scFV domain 210.

[0115] The SS domain 260 can be linked 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 comprises iCaspase 9 encoded by SEQ ID NO: 89, such that the iCaspase 9 SS domain 260 comprises the polypeptide sequence defined by SEQ ID NO: 90. In another exemplary embodiment, the SS domain 260 comprises RQR8 encoded by SEQ ID NO: 91, such that the RQR8 SS domain 260 comprises the polypeptide sequence defined by SEQ ID NO: 92. The SS domain 260 can control the cell proliferation of CAR T cells, thereby preventing the transformation of CAR T cells into cancerous cells. In other words, in the presence of a predetermined inducer of iCaspase 9, EGFRt, and / or RQR8, the SS domain 260 enables the selective depletion of CAR T cells.

[0116] In an exemplary embodiment, the inducible caspase-9 (iCaspase-9) SS domain 260 encodes a caspase recruitment domain (CARD; GenBank NM001 229) linked to two 12 kDa human FK506-binding protein (FKBP12; GenBank AH002 818) containing the F36V mutation. Administration of a chemical inducer of dimerization (CID) results in the dimerization of inducible caspase-9 molecules, leading to their activation. The caspase-9 dimer then activates downstream effector caspases, such as caspase-3, ultimately inducing cell apoptosis.

[0117] In another exemplary embodiment, the RQR8 SS domain 260 encodes a multi-epitope molecule carrying a CD34 epitope and two CD20 mimetics, which, upon binding to the FDA-approved CD20 antibody rituximab, cause host cells to undergo cell apoptosis.

[0118] 3a shows another embodiment of CAR 300. 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 domains 330 may be positioned across the membrane 370 such that the scFV domains 310 and hinge domains 320 are located on the outside of the CAR T cell, i.e., in the extracellular region 370a. The remaining domains of CAR 300 may be located inside the CAR T cell, i.e., in the intracellular region 370b.

[0119] In an exemplary embodiment, as shown in FIG. 3 a, CAR 300 comprises one scFV domain 310, one hinge domain 320, one transmembrane domain 330, first and second CSTM domains 340a and 340b, one signaling domain 350, and one SS domain 360.

[0120] Similar to scFV domain 210 of CAR 200, scFV domain 310 of CAR 300 may comprise a light chain 310a and a heavy chain 310b. Light chain 310a and heavy chain 310b of scFV domain 310 may be linked to each other via a linker protein 310c.

[0121] Similar to CSTM domain 240 of CAR 200, first CSTM domain 340a of CAR 300 can be selected from CD28, ICOS, and / or OX40. Second CSTM domain 340b can be linked to first CSTM domain 340a. Second CSTM domain 340b can be selected from 4-IBB, DAP10, DAP12, and / or 2B4.

[0122] In an exemplary embodiment, the first CSTM domain 340a and the second CSTM domain 340b are CD28 and 4-IBB, respectively. Compared to the single CSTM domain 240 of CAR 200, the first CSTM domain 340a and the second CSTM domain 340b of CAR 300 serve to enhance the persistence of CAR T cells.

[0123] The above-mentioned CAR-T cells produce only small amounts of cytokines, such as GMCSF and IL6, as shown in Figure 4. Therefore, the CAR T cells do not recruit any pro-inflammatory cells, thereby downregulating pro-inflammatory cytokines. Attenuating the levels of pro-inflammatory cytokines alleviates CRS.

[0124] Although the first and second shRNAs 130a, 130b and miR30 130 are described in this disclosure using the example of CAR 200 / 300, the first and second shRNAs 130a, 130b and miR30 130 may be used with any functionally equivalent CAR construct, which is within the teachings of this disclosure.

[0125] CAR T cells of the present invention can be prepared for each cancer patient according to method 400 shown in Figure 5. Alternatively, CAR T cells can be prepared from allogeneic T cell lines for all cancer patients.

[0126] Method 400 begins at step 401 by isolating peripheral blood mononuclear cells (PBMCs) by either a Ficoll-based method (as in leukapheresis) or an automated PBMC isolation instrument. In an exemplary embodiment, PBMCs are isolated by drawing blood from a cancer patient in need of CAR-T cell-based immunotherapy.

[0127] In step 403, a plurality of T cells may be isolated from the PBMCs. The isolated T cell population may comprise a plurality of CD4 + and CD8 +The isolated T cell population may comprise at least 40% CD4 + In an exemplary embodiment, the isolated T cell population may comprise 40% CD4 + T cells and 60% CD8 + Contains T cells.

[0128] In 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 render the T cells competent to receive ORF 100 via a lentiviral vector (described below).

[0129] In step 407, ORF 100 of the present invention is prepared for delivery into the isolated T cell population. ORF 100 can be ligated and replicated into a predetermined vector (or plasmid), including, but not limited to, pHR, pTRPE lentiviral vectors, etc. In an exemplary embodiment, ORF 100 is replicated and packaged within lentiviral particles.

[0130] In step 409, ORF 100 is delivered into the isolated T cell population by a predetermined gene delivery method. The predetermined method for gene delivery can be one of transformation, transfection, or transduction. In an exemplary embodiment, the isolated T cell population is transduced by a lentiviral gene delivery system, such as a third-generation self-inactivating (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., the T cells are transduced with a third-generation lentivirus. The resulting CAR T cells target and bind specific cancer cells, thereby enabling the CAR T cells to recognize and attack CD19-expressing B cells (i.e., cancerous cells).

[0131] In step 411, the CAR T cells (or engineered immune cells) are grown in a defined nutrient medium for a defined period of time to expand the number of CAR T cells. In an exemplary embodiment, the CAR T cells are expanded for 7-9 days to increase the number of CAR T cells by 20-40 fold. The engineered immune cells suspended in a predefined nutrient medium may define a composition.

[0132] In an exemplary embodiment, the nutrient medium comprises AIM V medium with or without antibiotics, human serum albumin, phenol red, and L-glutamine. In an alternative embodiment, the nutrient medium comprises CTS AIM V medium without antibiotics, phenol red, and serum. The nutrient medium may be periodically replaced with fresh nutrient medium after a predetermined period of time. In an exemplary embodiment, the nutrient medium is replaced every 48 hours.

[0133] In step 413, 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, transduced T cells expressing CAR 200, 300 are selected by immunoassay-based selection or cell sorting techniques.

[0134] Selected transduced T cells expressing CAR 200, 300 (i.e., CAR T cells) are refrigerated for storage and transport. Frozen CAR T cells are thawed prior to infusion into patients.

[0135] Alternatively, the selected T cells (i.e., CAR T cells) can be infused directly into the patient as determined by the physician.

[0136] Although method 400 is described with an exemplary series of steps, the steps of method 400 may be rearranged while performing method 400 and this is within the teachings of the present disclosure.

[0137] Sequences sharing at least 95% identity with the sequences disclosed in this disclosure are within the teachings of this disclosure.

[0138] The present disclosure will be further described with 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 specified. 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 evident as a result of the teachings provided herein.

[0139] Example 1: Method for preparing CAR T cells (the present invention): Lentivirus-packaging HEK293T cells, obtained from the American Type Culture Collection (ATCC), were plated in complete Dulbecco's modified Eagle's medium (DMEM) in 10 cm tissue culture plates at approximately 3.8 × 10 per plate. 6 The tissue culture plates containing HEK293T cells were 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 DMEM complete and 25 μM chloroquine diphosphate. The tissue culture plates containing HEK293T cells were incubated at 37°C, 5% CO2 for approximately 6 more hours.

[0140] A mixture of lentiplasmid and CAR plasmid (containing ORF 100 of the present disclosure) was inserted into HEK293T cells using polyethyleneimine (PEI, a non-viral vector) as a transfection reagent. After incubating HEK293T cells at 37°C and 5% CO2 for approximately 36 hours, the medium containing the viral particles was collected at different intervals for up to 72 hours. The collected medium was centrifuged at 1000 rpm for 5 minutes to pellet the remaining HEK293T cells. The supernatant was then filtered using a 0.45 μm PES filter. The filtrate (supernatant) containing the viral particles was collected and refrigerated to avoid loss of titer. The viral particles contained the lentiplasmid integrated into the CAR plasmid (containing ORF 100 of the present disclosure).

[0141] Ten million PBMCs were collected per 20 mL of patient blood through a process called leukapheresis. T cells were 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 contained 40% CD4+ T cells and 60% CD8+ T cells. The isolated T cells were cryopreserved in RPMI-1640 containing 20% ​​human AB serum and 10% DMSO.

[0142] The isolated T cells were cultured in human T cell medium consisting of X-VIVO 15 (Lonza), 5% human AB serum, and 10 mM neutralized N-acetyl-L-cysteine ​​(Sigma-Aldrich). IL-2 was used for cell proliferation at 30 units / mL.

[0143] After 24 hours of culture, isolated T cells were stimulated (activated) with Human T-Activator CD3 / CD28 Dynabeads (Life Technologies) at a cell:bead ratio of 1:3.

[0144] At 48 hours, primary T cells were exposed to the supernatant (containing viral particles) for transduction. On day 4 after T cell stimulation, the Dynabeads were removed, and T cells were expanded until day 9. T cells were sorted by FACs ARIA II. Thus, T cells showing basal CAR expression (i.e., CAR T cells) were isolated.

[0145] The isolated CAR T cells were expanded in nutrient medium containing antibiotic-free CTS AIM V medium, phenol red, and serum at 37°C and 5% CO2. The nutrient medium was replaced with fresh nutrient medium every 48 hours. After approximately 7–9 days of CAR T cell expansion, a 20–40-fold increase in CAR T cell numbers was observed. CAR T cells were then frozen for storage and transport.

[0146] Example 2: Infusion of CAR T cells (of the present invention): CAR T cells (obtained in Example 1) were thawed. Approximately 1 x 10 CAR T cells per kilogram of patient body weight were then intravenously infused into the patient in an outpatient setting. The CAR T cells rapidly migrated to the bone marrow and lymphoid tissues. After migration, the CAR T cells selectively bound to cancer cells expressing CD19. Binding between the CAR T cells and cancerous cells led to the activation and proliferation of the CAR T cells, followed by the death of the cancer cells through mechanisms such as cytokine release and cytotoxicity.

[0147] CAR T cells persist in patients and continue to attack CD19-expressing cancerous cells, providing long-term therapeutic benefit to patients. The disclosed CAR T cells 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 CD19-expressing cancer cells, reducing the risk of harm to healthy cells and the risk of false-positive interactions. Unlike conventional chemotherapy and radiation therapy, CAR T cell therapy is minimally toxic to healthy cells.

[0148] Example 3: IL6 and GMCSF knockdown (invention): The best-performing first and second shRNA 130a and 130b sequences described in this disclosure were evaluated for their effects on regulating IL6 and GMCSF levels. Screening was performed in T cells. The bar graphs show IL6 and GMCSF expression in T cells (Act-T cells) activated for 24 hours to induce IL6 expression or inactivation (control), as shown in Figure 6. A non-targeting shRNA (mir30-Scram) and the first and second shRNAs 130a and 130b targeting IL6 and GMCSF were evaluated in activated T cells for their effects on knocking down the IL6 and GMCSF genes. T cells were transfected with the first and second shRNAs 130a and 130b, respectively, and mRNA was prepared 24 hours post-transfection. Assays were performed by RT-qPCR using IL6 and GMCSF mRNA-specific primers. Statistical analysis was performed using a non-parametric t-test, with significance levels indicated as ****(P<0.0001), **(P<0.01), *(P<0.05) when comparing GMCSF / IL6 mir30 shRNA-expressing T cells with mir30 Scram-expressing T cells (see Figure 6). The first and second shRNA 130a, 130b sequences of the present disclosure were observed to significantly reduce IL6 gene expression via RNAi.

[0149] Example 4: Production of IL6 and GMCSF (invention): Protein fractions were collected from each T cell 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. The bar graph shows the IL6 and GMCSF cytokines present in each protein fraction, as shown in Figure 7. Statistical analysis was performed using a nonparametric t-test, with significance levels indicated as **** (P<0.0001), *** (P<0.001), and ns (not significant) when comparing T cells expressing mir30 GMCSF / IL6 shRNA with those expressing Scram (see Figure 7).

[0150] Example 5: IL6 and GMCSF production in CAR T cells (present invention): The best-performing first and second shRNA 130a, 130b sequences observed in Examples 3 and 4 were evaluated for their effect on regulating IL6 and GMCSF levels. Screening was performed on CAR T cells. The bar graph shows IL6 and GMCSF produced in non-operating T cells (control), as shown in Figure 8. Non-targeting shRNA (CAR T cells) miR30-Scram ) and the first and second shRNAs 130a, 130b targeting IL6 and GMCSF (CART miR30-shgmcsf / shIL6 ) were evaluated in activated CAR T cells for their effect on knocking down the IL6 gene. con ) were also assessed after their activation. Assays and analysis were similar to those described in Example 4 above. Bar graphs show the IL6 and GMCSF cytokines present in each protein fraction, as shown in Figure 8. Statistical analysis was performed using a non-parametric t-test showing significant differences at the **** confidence level (P<0.0001) when comparing IL6 and GMCSF mir30-shRNA-expressing T cells with those expressing mir30-Scram (see Figure 8).

[0151] The scope of the present invention is limited only by the appended claims. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application or 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 for regulating the amount of interleukin 6 (IL6) cytokine during immunotherapy, wherein the at least one first hairpin loop structure comprises: i. one or more first short hairpin RNA (130a) sequences that form the stem of said at least one first hairpin loop structure; and ii. at least one microRNA 30 (130) sequence that forms the loop of the at least one hairpin loop structure; and the at least one first hairpin loop structure formed by b. at least one second hairpin loop structure that regulates the amount of granulocyte monocyte colony-stimulating factor (GMCSF) cytokine during immunotherapy, wherein the at least one second hairpin loop structure is located either upstream or downstream of the at least one first hairpin loop structure, and the at least one second hairpin loop structure comprises: i. one or more second short hairpin RNA (130b) sequences that form the stem of said at least one second hairpin loop structure; and ii. at least one microRNA 30 (130) sequence that forms the loop of the at least one hairpin loop structure; and the at least one second hairpin loop structure formed by c. a first promoter (120) located upstream of the at least one first hairpin loop structure and the at least one second hairpin loop structure; A recombinant nucleic acid molecule comprising:

2. 2. The recombinant nucleic acid molecule of claim 1, wherein 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. 2. The recombinant nucleic acid molecule of claim 1, wherein the second short hairpin RNA (130b) is encoded by at least one of SEQ ID NOs: 41 to 60, or a nucleotide sequence having at least 95% identity thereto.

4. 2. The recombinant nucleic acid molecule of claim 1, wherein the first promoter 120 comprises at least one of 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.

5. 2. The recombinant nucleic acid molecule of claim 1, wherein a polyA tail (160) sequence is located downstream of the first hairpin loop structure and the second hairpin loop structure.

6. 2. The recombinant nucleic acid molecule of claim 1, wherein the recombinant nucleic acid molecule comprises one or more chimeric antigen receptor genes (150) arranged downstream of a second promoter (140).

7. 2. The recombinant nucleic acid molecule of claim 1, wherein 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 a 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 for regulating the amount of interleukin 6 (IL6) cytokine during immunotherapy, wherein the at least one first hairpin loop structure comprises: i. one or more first short hairpin RNA (130a) sequences that form the stem of said at least one first hairpin loop structure; and ii. at least one microRNA 30 (130) sequence that forms the loop of the at least one hairpin loop structure; and the at least one first hairpin loop structure formed by b. at least one second hairpin loop structure that regulates the amount of granulocyte monocyte colony-stimulating factor (GMCSF) cytokine during immunotherapy, wherein the at least one second hairpin loop structure is located either upstream or downstream of the at least one first hairpin loop structure, and the at least one second hairpin loop structure comprises: i. one or more second short hairpin RNA (130b) sequences that form the stem of said at least one second hairpin loop structure; and ii. at least one microRNA 30 (130) sequence that forms the loop of the at least one hairpin loop structure; and the at least one second hairpin loop structure formed by c. a first promoter (120) located upstream of the at least one hairpin loop structure and the at least one second hairpin loop structure; d. below i. one or more single-chain variable fragment domains (210) configured to bind to one or more tumor-associated antigens (TAA); ii. one or more hinge domains (220) linked 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) linked to said one or more costimulatory domains (240); v. one or more safety switch domains (260) linked to said one or more signaling domains (250); vi. one or more transmembrane domains (230) operably linking said hinge domain (220) to said costimulatory domain (240); one or more chimeric antigen receptor genes (150) expressing e. a second promoter (140) located upstream of the one or more chimeric antigen receptor genes (150); f. one or more long terminal repeat sequences (110) located upstream of the first promoter (120) and downstream of the one or more chimeric antigen receptor genes (150); A recombinant nucleic acid molecule comprising:

9. 9. The recombinant nucleic acid molecule of claim 8, wherein 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. 9. The recombinant nucleic acid molecule of claim 8, wherein the second short hairpin RNA (130b) is encoded by at least one of SEQ ID NOs: 41 to 60, or a nucleotide sequence having at least 95% identity thereto.

11. 9. The recombinant nucleic acid molecule of claim 8, wherein the first promoter 120 comprises at least one of 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. 9. The recombinant nucleic acid molecule of claim 8, wherein the one or more single-chain variable fragment domains (210) have binding affinity for at least one of 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. 9. The recombinant nucleic acid molecule of claim 8, wherein the single-chain variable fragment domain (210) against cluster of differentiation 19 (CD19) is encoded by at least one of SEQ ID NOs: 65 and 67.

14. 9. The recombinant nucleic acid molecule of claim 8, wherein the one or more hinge domains (220) comprise at least in part a cluster of differentiation 8 (CD8) encoded by SEQ ID NO:

69.

15. 9. The recombinant nucleic acid molecule of claim 8, wherein the one or more costimulatory domains (240) comprise at least one of cluster of differentiation 28 (CD28) encoded by SEQ ID NO:73, inducible T cell co-stimulator (ICOS) encoded by SEQ ID NO:75, OX40 encoded by SEQ ID NO:77, 4-1BB encoded by SEQ ID NO:79, DAP10 encoded by SEQ ID NO:81, DAP12 encoded by SEQ ID NO:83, and 2B4 encoded by SEQ ID NO:

85.

16. 9. The recombinant nucleic acid molecule of claim 8, wherein the one or more signaling domains (250) comprise at least in part cluster of differentiation 3 zeta (CD3ζ) encoded by SEQ ID NO:

87.

17. 9. The recombinant nucleic acid molecule of claim 8, wherein the one or more safety switch domains (260) comprise at least one of inducible caspase 9 (iCaspase9) encoded by SEQ ID NO: 89, truncated epidermal growth factor receptor (EGFRt), and RQR8 encoded by SEQ ID NO:

91.

18. 9. The recombinant nucleic acid molecule of claim 8, wherein the one or more transmembrane domains (230) comprise at least in part a cluster of differentiation 8 (CD8) domain encoded by SEQ ID NO:

71.

19. 9. The recombinant nucleic acid molecule of claim 8, wherein the second promoter (140) is at least one of the elongation factor 1 (EF1) full-length promoter encoded by SEQ ID NO:63, the EF1 alpha core promoter encoded by SEQ ID NO:

64.

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

21. 9. The recombinant nucleic acid molecule of claim 8, wherein the one or more long terminal repeat sequences (110) are encoded by SEQ ID NO:

1.

22. said one or more costimulatory domains (240) a. a first costimulatory domain (340a) comprising at least one of cluster of differentiation 28 (CD28), inducible T-cell co-stimulator (ICOS), and / or OX40; b. a second costimulatory domain (340b) comprising at least one of 4-IBB, DAP10, DAP12 and / or 2B4; 9. The recombinant nucleic acid molecule of claim 8, comprising:

23. 9. The recombinant nucleic acid molecule of claim 8, wherein 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 a 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 for regulating the amount of interleukin 6 (IL6) cytokine during immunotherapy, wherein the at least one first hairpin loop structure comprises: i. one or more first short hairpin RNA (130a) sequences that form the stem of said at least one first hairpin loop structure; and ii. at least one microRNA 30 (130) sequence that forms the loop of the at least one hairpin loop structure; and the at least one first hairpin loop structure formed by b. at least one second hairpin loop structure that regulates the amount of granulocyte monocyte colony-stimulating factor (GMCSF) cytokine during immunotherapy, wherein the at least one second hairpin loop structure is located either upstream or downstream of the at least one first hairpin loop structure, and the at least one second hairpin loop structure comprises: i. one or more second short hairpin RNA (130b) sequences that form the stem of said at least one second hairpin loop structure; and ii. at least one microRNA 30 (130) sequence that forms the loop of the at least one hairpin loop structure; and the at least one second hairpin loop structure formed by c. a first promoter (120) located 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 located downstream of the at least one first hairpin loop structure and the at least one second hairpin loop structure; e. below i. one or more single-chain variable fragment domains (210) configured to bind to one or more tumor-associated antigens (TAA); ii. one or more hinge domains (220) linked 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) linked to said one or more costimulatory domains (240); v. one or more safety switch domains (260) linked to said one or more signaling domains (250); vi. one or more transmembrane domains (230) operably linking said hinge domain (220) to said costimulatory domain (240); one or more chimeric antigen receptor genes (150) expressing f. a second promoter (140) located upstream of the one or more chimeric antigen receptor genes (150); g. one or more long terminal repeat sequences (110) located upstream of the first promoter (120) and downstream of the one or more chimeric antigen receptor genes (150); A recombinant nucleic acid molecule comprising:

25. 25. The recombinant nucleic acid molecule of claim 24, wherein 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. 25. The recombinant nucleic acid molecule of claim 24, wherein the second short hairpin RNA (130b) is encoded by at least one of SEQ ID NOs: 41 to 60, or a nucleotide sequence having at least 95% identity thereto.

27. 25. The recombinant nucleic acid molecule of claim 24, wherein the first promoter 120 comprises at least one of 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. 25. The recombinant nucleic acid molecule of claim 24, wherein the polyA tail (160) sequence is derived from either Simian Virus 40 (SV40) encoded by SEQ ID NO: 62 or bovine growth hormone beta globulin (bGH).

29. 25. The recombinant nucleic acid molecule of claim 24, wherein the one or more single-chain variable fragment domains (210) have binding affinity for at least one of 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. 25. The recombinant nucleic acid molecule of claim 24, wherein the single-chain variable fragment domain (210) against cluster of differentiation 19 (CD19) is encoded by at least one of SEQ ID NOs: 65 and 67.

31. 25. The recombinant nucleic acid molecule of claim 24, wherein the one or more hinge domains (220) comprise at least in part a cluster of differentiation 8 (CD8) encoded by SEQ ID NO:

69.

32. 25. The recombinant nucleic acid molecule of claim 24, wherein the one or more costimulatory domains (240) comprise at least one of cluster of differentiation 28 (CD28) encoded by SEQ ID NO:73, inducible T-cell co-stimulator (ICOS) encoded by SEQ ID NO:75, OX40 encoded by SEQ ID NO:77, 4-1BB encoded by SEQ ID NO:79, DAP10 encoded by SEQ ID NO:81, DAP12 encoded by SEQ ID NO:83, and 2B4 encoded by SEQ ID NO:

85.

33. 25. The recombinant nucleic acid molecule of claim 24, wherein the one or more signaling domains (250) comprise, at least in part, cluster of differentiation 3 zeta (CD3ζ) encoded by SEQ ID NO:

87.

34. 25. The recombinant nucleic acid molecule of claim 24, wherein the one or more safety switch domains (260) comprise at least one of inducible caspase 9 (iCaspase9) encoded by SEQ ID NO: 89, truncated epidermal growth factor receptor (EGFRt), and RQR8 encoded by SEQ ID NO:

91.

35. 25. The recombinant nucleic acid molecule of claim 24, wherein the one or more transmembrane domains (230) comprise at least in part the cluster of differentiation 8 (CD8) encoded by SEQ ID NO:

71.

36. 25. The recombinant nucleic acid molecule of claim 24, wherein the second promoter (140) is at least one of the elongation factor 1 (EF1) full-length promoter encoded by SEQ ID NO: 63, the EF1 alpha core promoter encoded by SEQ ID NO:

64.

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

38. 25. The recombinant nucleic acid molecule of claim 24, wherein the one or more long terminal repeat sequences (110) are encoded by SEQ ID NO:

1.

39. said one or more costimulatory domains (240) a. a first costimulatory domain (340a) comprising at least one of cluster of differentiation 28 (CD28), inducible T-cell co-stimulator (ICOS), and / or OX40; b. a second costimulatory domain (340b) comprising at least one of 4-IBB, DAP10, DAP12 and / or 2B4; 25. The recombinant nucleic acid molecule of claim 24, comprising:

40. 25. The recombinant nucleic acid molecule of claim 24, wherein 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 a cytokine.

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

42. A vector comprising: a. At least one recombinant nucleic acid molecule of any one of claims 1 to 40 ligated into at least one of a plasmid, a cosmid, a viral vector, and a phage. A vector comprising:

43. 43. The vector of claim 42, wherein 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. 1. An engineered immune cell, comprising: a. At least one recombinant nucleic acid molecule according to any one of claims 1 to 40. engineered immune cells, including

45. 45. The engineered immune cell of claim 44, wherein the recombinant nucleic acid molecule is ligated into at least one of a plasmid, a cosmid, a viral vector, and a phage.

46. 45. The engineered immune cell of claim 44, wherein the recombinant nucleic acid molecule is ligated into a viral vector derived from at least one of a lentivirus, a retrovirus, an adenovirus, an adeno-associated virus, and a Sendai virus.

47. 1. A composition comprising: a. an engineered immune cell comprising at least one recombinant nucleic acid molecule of any one of claims 1 to 40; b. The engineered immune cells suspended in a nutrient medium A composition comprising:

48. 48. The composition of claim 47, wherein the recombinant nucleic acid molecule(s) is ligated into at least one of a plasmid, a cosmid, a viral vector, and a phage.

49. 48. The composition of claim 47, wherein the recombinant nucleic acid molecule(s) is ligated into a viral vector derived from at least one of a lentivirus, a retrovirus, an adenovirus, an adeno-associated virus, and a Sendai virus.

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

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

52. 51. The method (400) of claim 50, wherein after isolating the plurality of T cells, the plurality of T cells is subjected to a costimulatory molecule comprising at least one of cluster of differentiation 3 (CD3) and cluster of differentiation 28 (CD28).

53. 51. The method (400) of claim 50, wherein replicating the recombinant nucleic acid molecule(s) comprises ligating the recombinant nucleic acid molecule(s) into at least one of a plasmid, a cosmid, a viral vector, and a phage.

54. 51. The method (400) of claim 50, wherein replicating the recombinant nucleic acid molecule(s) comprises ligating the recombinant nucleic acid molecule(s) into a viral vector derived from at least one of a lentivirus, a retrovirus, an adenovirus, an adeno-associated virus, and a Sendai virus.

55. 51. The method (400) of claim 50, wherein delivering the vector into the plurality of T cells comprises transforming, transfecting or transducing the plurality of T cells with the vector.

56. 51. The method (400) of claim 50, wherein after the vector is delivered into the plurality of T cells, the T cells are expanded in a predetermined nutrient medium for a predetermined period of time.

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