Reduction of cytokine release syndrome in immunotherapy

Abstract

The present invention discloses a recombinant nucleic acid molecule encoded by at least ORF (100, 100a) to reduce cytokine storm during immunotherapy. The recombinant nucleic acid molecule comprises at least one first hairpin ring structure, at least one second hairpin ring structure and a first promoter (120). The first and second hairpin ring structures modulate the amount of granulocyte monocyte colony stimulating factor (GMCSF) cytokine and interleukin 6 (IL6) cytokine, respectively. The first hairpin ring structure is formed from at least two first short hairpin RNA (130a) sequences and at least one microRNA30 (130) sequence. The second hairpin ring structure is formed from at least two second short hairpin RNA (130b) sequences and at least one microRNA30 (130) sequence. The first promoter (120) is disposed upstream of the first and second hairpin ring structures.

Description

[0001] Priority

[0002] This application claims priority to Indian Provisional Application No. 202221053839, filed on October 20, 2022, the content of which is incorporated herein by reference. Technical Field

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

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

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

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

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

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

[0009] Although genomic engineering methods have been used to completely knockout the genes involved in CRS, these methods have their limitations. For example, the complete knockout of genes may have an adverse effect on the survival of CAR T cells and may also induce off-target effects.

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

[0011] Therefore, CRS together with the inability to control the proliferation of engineered CAR T cells constitutes the main challenges in treating cancer patients with CAR T cell-based immunotherapy. Additionally, patients who receive conventional CAR T cell infusions have been diagnosed with recurrence (i.e., the recurrence of cancer cells) because conventional CAR T cells cannot survive for a long time.

[0012] In view of the above discussion, there is a need for a novel CAR T cell-based immunotherapy to overcome the problems associated with conventional CAR T cell-based immunotherapy. SUMMARY OF THE INVENTION

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

[0014] In an exemplary embodiment, the present disclosure relates to a recombinant nucleic acid molecule that is at least encoded by an ORF to reduce cytokine storm during immunotherapy. The recombinant nucleic acid molecule comprises at least one first hairpin loop structure, at least one second hairpin loop structure, and a first promoter. The first and second hairpin loop structures regulate the amounts of granulocyte-macrophage 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 microRNA30 sequence. The first short hairpin RNA sequences form the stem of the first hairpin loop structure, and the microRNA30 sequence forms the loop of the second hairpin loop structure. The second hairpin loop structure is disposed 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 microRNA30 sequence. The second short hairpin RNA sequences form the stem of the second hairpin loop structure, and the microRNA30 sequence forms the loop of the second hairpin loop structure. The first promoter is disposed upstream of the first and second hairpin loop structures.

[0015] In an exemplary embodiment, the present disclosure relates to a recombinant nucleic acid molecule that is at least encoded by an ORF to reduce cytokine storm during immunotherapy. The recombinant nucleic acid molecule comprises at least one first hairpin loop structure, at least one second hairpin loop structure, a first promoter, one or more chimeric antigen receptor genes, a second promoter, and one or more long terminal repeats. The first and second hairpin loop structures respectively regulate the amounts of granulocyte monocyte colony stimulating factor (GMCSF) cytokine and interleukin 6 (IL6) cytokine during immunotherapy. The first hairpin loop structure is formed by at least two first short hairpin RNA sequences and at least one microRNA30 sequence. The first short hairpin RNA sequences form the stem of the first hairpin loop structure, and the microRNA30 sequence forms the loop of the second hairpin loop structure. The second hairpin loop structure is disposed 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 microRNA30 sequence. The second short hairpin RNA sequences form the stem of the second hairpin loop structure, and the microRNA30 sequence forms the loop of the second hairpin loop structure. The first promoter is disposed upstream of the first and second hairpin loop structures. The one or more chimeric antigen receptor genes express one or more single-chain variable fragment domains, one or more hinge domains, one or more co-stimulatory domains, one or more signal transduction domains, one or more safety switch domains, and one or more transmembrane domains. The single-chain variable fragment domain is configured to bind to one or more tumor-associated antigens. The hinge domain is coupled to the one or more single-chain variable fragment domains. The signal transduction domain is coupled to the one or more co-stimulatory domains. The safety switch domain is coupled to the signal transduction domain. The transmembrane domain operatively couples the hinge domain to the co-stimulatory domain. The second promoter is located upstream of the chimeric antigen receptor gene. The long terminal repeat is located upstream of the first promoter and downstream of the chimeric antigen receptor gene.

[0016] In an exemplary embodiment, the present disclosure relates to a recombinant nucleic acid molecule that is at least encoded by an ORF to reduce cytokine storm during immunotherapy. The recombinant nucleic acid molecule comprises at least one first hairpin loop structure, at least one second hairpin loop structure, a first promoter, a poly-A tail sequence, one or more chimeric antigen receptor genes, a second promoter, and one or more long terminal repeat sequences. The first and second hairpin loop structures regulate the amounts of granulocyte-macrophage 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 microRNA30 sequence. The first short hairpin RNA sequences form the stem of the first hairpin loop structure, and the microRNA30 sequence forms the loop of the second hairpin loop structure. The second hairpin loop structure is disposed 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 microRNA30 sequence. The second short hairpin RNA sequences form the stem of the second hairpin loop structure, and the microRNA30 sequence forms the loop of the second hairpin loop structure. The first promoter is disposed upstream of the first and second hairpin loop structures. The one or more chimeric antigen receptor genes express one or more single-chain variable fragment domains, one or more hinge domains, one or more co-stimulatory domains, one or more signaling domains, one or more safety switch domains, and one or more transmembrane domains. The single-chain variable fragment domain is configured to bind to one or more tumor-associated antigens. The hinge domain is coupled to the one or more single-chain variable fragment domains. The signaling domain is coupled to the one or more co-stimulatory domains. The safety switch domain is coupled to the signaling domain. The transmembrane domain operably couples the hinge domain to the co-stimulatory domain. The second promoter is located upstream of the chimeric antigen receptor gene. The long terminal repeat sequence is located upstream of the first promoter and downstream of the chimeric antigen receptor gene.

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

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

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

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

[0021] In an exemplary embodiment, the present disclosure relates to a method of preparing engineered immune cells. The method includes isolating a plurality of T cells from a population of peripheral blood mononuclear cells. Replicating at least one recombinant nucleic acid molecule in a vector, the at least one recombinant nucleic acid molecule being encoded by an ORF to reduce cytokine storm during immunotherapy. And delivering the vector into the plurality of T cells.

[0022] The foregoing features and other features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the reference drawings. In the drawings, like reference numerals indicate identical or similar parts in several views. These features will be described in sufficient detail to enable those skilled in the art of the subject matter to practice the present invention. Additionally, it should be understood that other features may be used and structural changes may be made without departing from the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0024] Figure 1 A diagram depicting CRS induction (prior art) according to one or more exemplary embodiments of the present disclosure is shown.

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

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

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

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

[0029] Figure 4 Depicts an illustration of CRS reduction according to one or more exemplary embodiments of the present disclosure.

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

[0031] Figures 6 to 8 Depicts experimental observations according to one or more exemplary embodiments of the present disclosure. Detailed Description

[0032] Before describing the invention in detail, definitions of certain words and phrases used throughout this patent document will be defined: The terms "comprising" and "including" and their derivatives mean including but not limited to; the term "or" is inclusive and means and / or; the phrases "coupled to" and "associated with" and their derivatives may mean including, being included in, interconnected with, containing, being contained in, connected to or connecting with, coupled to or coupled with, capable of communicating with, cooperating with, interleaving, juxtaposing, approaching, bound to or binding with, having the attribute of, etc.; definitions of certain words and phrases are provided throughout this patent document, and those of ordinary skill in the art will understand that these definitions apply, in many if not most cases, to both the prior and future use of the defined words and phrases.

[0033] References throughout this specification to "one embodiment", "an embodiment", or similar language mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, appearances of the phrases "in one embodiment", "in an embodiment", and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment, but rather mean "one or more but not all embodiments". Unless expressly stated otherwise, the terms "comprising", "including", "having", and their variants mean "including but not limited to". Unless expressly stated otherwise, a list of items does not imply that any or all of the items are mutually exclusive and / or mutually inclusive. Unless expressly stated otherwise, the terms "a", "an", and "the" also refer to "one or more".

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

[0035] The term "activated T cell" particularly refers to a T cell (or CAR T cell) that expresses a chimeric antigen receptor (CAR) construct and / or is capable of binding to a tumor-associated antigen (TAA).

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

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

[0038] The term "Fv" refers to the smallest antibody fragment that contains the complete antigen recognition and binding site. This fragment consists of a dimer of one heavy-chain variable domain and one light-chain variable domain that are tightly non-covalently associated. The folding of these two domains gives rise to six hypervariable loops (3 loops each in the H chain and L chain), which contribute the amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv that includes only three complementarity-determining regions (CDRs) specific for the antigen) has the ability to recognize and bind the antigen, although with a lower affinity than the entire binding site (dimer).

[0039] As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains that exist in all antibody molecules in their naturally occurring conformation. As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains that exist in all antibody molecules in their naturally occurring conformation. K and A light chains refer to the two major antibody light-chain isotypes.

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

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

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

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

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

[0045] The term "allogeneic" is used to describe a graft that is derived from a different subject of the same species. For example, the donor subject can be related or unrelated to the recipient subject, but the donor subject has immune system markers similar to those of the recipient subject.

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

[0047] 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 through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc.

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

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

[0050] The term "costimulatory ligand" refers to a molecule on an antigen-presenting cell (e.g., APC, dendritic cell, B cell, etc.) that specifically binds to a cognate costimulatory molecule on a T cell, thereby providing a signal that mediates a T cell response, including at least one of proliferation, activation, differentiation, and other cellular responses, in addition to the primary signal provided by, for example, the binding of the TCR / CD3 complex to a peptide-loaded MHC molecule. Costimulatory ligands also specifically 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, as well as ligands that specifically bind CD83.

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

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

[0053] The term "encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide (e.g., a gene, cDNA, or mRNA) to serve as a template for the synthesis, in a biological process, of other polymers and macromolecules having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the resulting biological property. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces a 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 which is typically provided in a sequence listing) and the non-coding strand (which serves as the template for transcription of the gene or cDNA) can be said to encode the protein or other product of that gene or cDNA.

[0054] The term "exogenous" refers to a molecule that is not naturally present in a wild-type cell or organism but is generally introduced into a cell by molecular biology 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 "native" refer to naturally occurring polynucleotides or amino acid sequences that can be found in a given wild-type cell or organism. In addition, a specific polynucleotide sequence isolated from a first organism and transferred to a second organism by molecular biology techniques is generally considered to be an "exogenous" polynucleotide or amino acid sequence with respect to the second organism. In a particular embodiment, a polynucleotide sequence can be "introduced" into a microorganism that already contains such a polynucleotide sequence, e.g., to produce one or more additional copies of another naturally occurring polynucleotide sequence, thereby facilitating overexpression of the encoded polypeptide.

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

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

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

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

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

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

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

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

[0063] The term "overexpressed" tumor antigen or "overexpression" of a tumor antigen is intended to mean an abnormal level of expression of a tumor antigen in cells from a diseased area (e.g., a solid tumor within a particular tissue or organ of a patient) as compared to the level of expression in normal cells from that tissue or organ. Patients with solid tumors or hematological malignancies characterized by overexpression of a tumor antigen can be determined by standard assays known in the art.

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

[0065] Subjects in need or requiring treatment include subjects suffering from a disease, disorder, or condition that requires treatment. Subjects in need also include subjects requiring treatment to prevent a disease, disorder, or condition.

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

[0067] The terms "polynucleotide variant" and "variant", etc. refer to polynucleotides that exhibit substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize to the reference sequence under stringent conditions defined below. These terms also encompass polynucleotides that differ from the 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 a different nucleotide. In this regard, it is well understood in the art that certain alterations can be made to a reference polynucleotide, including mutations, additions, deletions, and substitutions, and that the polynucleotide so altered retains the biological function or activity of the reference polynucleotide or has increased activity (i.e., optimized) relative to the reference polynucleotide. Polynucleotide variants include, for example, polynucleotides having at least 50% (and at least 51% to at least 99% and all integer percentages therebetween, such as 90%, 95%, or 98%) sequence identity with the reference polynucleotide sequences described herein. The terms "polynucleotide variant" and "variant" also include naturally occurring allelic variants and orthologs.

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

[0069] The term "polypeptide variant" refers to a polypeptide that differs from a reference polypeptide sequence by the addition, deletion, or substitution of at least one amino acid residue. In certain embodiments, the polypeptide variant differs from the reference polypeptide by one or more substitutions, which may be conservative or non-conservative. In certain embodiments, the polypeptide variant comprises 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 altering the nature of the polypeptide activity. Polypeptide variants also encompass polypeptides in which one or more amino acids have been added or deleted or replaced with different amino acid residues.

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

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

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

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

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

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

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

[0077] Although, for convenience of presentation, the operations of the exemplary embodiments of the disclosed methods may be described in a particular order, it should be understood that the disclosed embodiments can encompass an order of operations other than the particular order disclosed. For example, operations described in sequence may in some cases be rearranged or performed concurrently. Further, the description and disclosure provided in connection with one particular embodiment are not limited to that embodiment and may be applied to any embodiment disclosed herein. Additionally, for simplicity, the figures may not show the various ways in which the disclosed systems, methods, and devices may be used in combination with other systems, methods, and devices.

[0078] Furthermore, the described features, advantages, and characteristics of the embodiments can be combined in any suitable manner. Those skilled in the relevant art will recognize that embodiments can 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 the appended claims, or may be learned by practice of the embodiments as set forth below.

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

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

[0081] Furthermore, the CAR T cells comprise at least two short hairpin RNA (shRNA) sequences together with a microRNA 30 (miR30) sequence to reduce a cytokine storm called cytokine release syndrome (CRS) during immunotherapy. During CRS, a rapid release of cytokines is observed. 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 sequences can inhibit cytokine production and / or cytokine signaling through RNA interference (RNAi). In an exemplary embodiment, the transcript of the shRNA binds to the mRNA of the cytokine to inactivate the mRNA functionally and / or degrade the mRNA. In an alternative embodiment, the transcript of the shRNA sequence binds to the mRNA of the cytokine to prevent its translation. In another exemplary embodiment, the shRNA binds to one or more receptors of the cytokine, thereby blocking the downstream signaling pathway of the cytokine. In yet another exemplary embodiment, the shRNA sequence expresses an anti-IL6 (aIL6) that binds and neutralizes IL6 (an exemplary cytokine), thereby at least partially blocking the activity of IL6.

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

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

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

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

[0086] Thus, for the reasons described above, the CAR T cells disclosed herein are programmed to initiate an artificial immune response against cancer cells without any undesirable side effects of CRS. Further, 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 the malignant transformation of CAR T cells.

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

[0088] At least a portion of ORF 100 (or recombinant nucleotide sequence molecule) can be introduced into one or more natural immune cells by genetic manipulation to generate engineered immune cells. ORF 100 (or a portion thereof) expresses one or more chimeric antigen receptors (CARs) (i.e., proteins and / or polypeptides) and / or one or more shRNA transcripts (as described below). Natural immune cells can include, but are not limited to, T lymphocytes (T cells), natural killer (NK) cells, gamma delta T cells, etc. In an exemplary embodiment, ORF 100 is introduced into natural T cells by a clustered regularly interspaced short palindromic repeats–CRISPR associated protein (CRISPR-Cas)-based gene editing technique. Additionally or alternatively, ORF 100 is introduced into natural T cells using a lentiviral vector, an adeno-associated virus (AAV), etc. The vector containing ORF 100 can be selected from plasmids, cosmids, viral vectors, or phages. The plasmid can be a eukaryotic expression plasmid. The viral vector 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. The lentivirus (LV) vector provides an effective means for modifying eukaryotic cells, stably transferring, and expressing genes in less immunogenic host cells. The LV vector has a greater gene accommodation capacity and can transduce both proliferating and non-proliferating cells. Further, the LV vector-based gene delivery system is the most effective method for transducing T cells that are difficult to transfect. In an exemplary embodiment, clinical-grade viral vectors are commercially available from Lentigen and Sirion-Biotech.

[0089] ORF 100 may comprise one or more regions, which include 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 microRNA30 (miR30) 130 sequence, one or more genes, etc. One or more promoters may include a first promoter 120 and a second promoter 140. One or more genes may include, but are not limited to, multiple CAR genes 150. In an exemplary embodiment, ORF 100 comprises a first hairpin loop structure formed by a first shRNA 130a sequence together with at least one microRNA30 (miR30) 130 sequence and a second hairpin loop structure formed by at least one second shRNA 130b sequence together with at least one microRNA30 (miR30) 130 sequence. Additionally or optionally, ORF 100 may comprise 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 of the first and second hairpin loop structures (i.e., messenger RNAs) bind to the messenger RNAs (mRNAs) of the cytokines. In an exemplary embodiment, the transcripts of the first and second hairpin loop structures bind to the mRNAs of IL6 and GMCSF, respectively.

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

[0091] Transcription of ORF 100 within 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 can be disposed upstream of one or more genes of ORF 100. One or more RNA polymerases can bind to the promoter for transcribing ORF 100. The RNA polymerase can include, but is 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 transcribe ORF 100 partially or completely 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 shRNA 130a and the second shRNA 130b transcripts (and their proteins / polypeptides). Processing of the mRNA can include at least one of splicing, translating the mRNA into one or more amino acid sequences (i.e., polypeptides), and post-translational modification, etc. Post-translational modification can include, but is not limited to, folding of the amino acid sequence (forming a protein) and / or glycosylation, etc.

[0092] As Figure 2 shown, if the first shRNA 130a is disposed upstream of the second shRNA 130b, the first promoter 120 can be disposed upstream of the first shRNA 130a (i.e., the first hairpin loop structure). Alternatively, if the first shRNA 130a is disposed downstream of the second shRNA 130b, the first promoter 120 can be disposed upstream of the second shRNA 130b (not shown). The first promoter 120 can 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 Hl promoter, an SNORD promoter, etc. In an exemplary embodiment, the first promoter 120 contains a GAL-VP64 recognition domain that activates the first promoter 120 when the GAL-VP64 domain binds to the GAL-VP64 recognition domain.

[0093] The first promoter 120 transcribes the first shRNA 130a and the second shRNA 130b to produce one or more short RNA transcripts each having a hairpin loop structure. The first shRNA 130a can be encoded by at least one of SEQ ID NO.5 - SEQ ID NO.40. The second shRNA 130b can be encoded by at least one of SEQ ID NO.41 - SEQ ID NO.60.

[0094] In an exemplary embodiment, as Figure 2 shown, the hairpin loop structure of ORF 100 is provided with two first shRNA130a and second shRNA 130b sequences, each sequence being such that they are complementary to each other and form the stem of the hairpin loop structure.

[0095] The transcripts of the first shRNA 130a and the second shRNA 130b may include specific binding affinities for one or more molecules. The CAR T cells comprise a plurality of IL6 receptors (IL6-R) conjugated to a synthetic notch (syn-notch) receptor and a GAL-VP64 domain. In an exemplary embodiment, during CRS, the released IL6 binds to the IL6-R of the CAR T cells and induces syn-notch, which subsequently cleaves and releases the GAL-VP64 domain. The GAL-VP64 domain migrates to the nucleus of the CAR T cells and induces the expression of one of the thirty-six first shRNA 130a sequences. The expression of the first shRNA 130a sequence results in the production of aIL6. The aIL6 binds and neutralizes IL6, thereby reducing the burden of CRS.

[0096] The CAR T cells comprise a plurality of 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 the second shRNA 130b binds to the mRNA corresponding to the cytokine (i.e., GMCSF) to inactivate the mRNA functionally and / or degrade the mRNA.

[0097] In an exemplary embodiment, the first shRNA 130a expressed by the CAR T cells (i.e., the transcript of the first shRNA 130a) selectively binds to the IL6 mRNA, thereby regulating the IL6 cytokine level (via RNAi). When the transcript of the first shRNA130a binds to the IL6 mRNA, the level (concentration) of the IL6 molecule decreases, resulting in a reduced burden of CRS.

[0098] In an exemplary embodiment, the second shRNA 130b expressed by the CAR T cells (i.e., the transcript of the second shRNA 130b) selectively binds to the GMCSF mRNA, thereby regulating the GMCSF cytokine level (via RNAi). When the transcript of the second shRNA 130b binds to the GMCSF mRNA, the level (concentration) of the GMCSF molecule decreases, resulting in a reduced burden of CRS.

[0099] miR30 130 is a short RNA sequence that serves as a regulatory element for the transcription of the first shRNA 130a and the second shRNA 130b sequences, i.e., miR30 130 at least partially regulates the expression of the first shRNA 130a and the second shRNA 130b sequences. miR30130 can be located on either side of the first shRNA 130a and the second shRNA 130b sequences and / or can be disposed between the first shRNA 130a and the second shRNA 130b sequences, respectively. In an exemplary embodiment, miR30 130 is encoded by SEQ ID No. 61. In an exemplary embodiment, as Figure 2 shown, the miR30 130 sequence forms the loop portion of the hairpin loop structure formed by the first shRNA 130a and the second shRNA 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.

[0100] Although excessive GMCSF production (such as during CRS) is harmful, low levels of GMCSF promote the improvement of the anti-tumor activity of CAR T cells. miR30 130 helps maintain low levels of GMCSF by regulating the expression of the second shRNA 130b sequence. Thus, the miR30 130 sequence helps retain the beneficial anti-tumor activity of GMCSF while mitigating the potential CRS induction risk.

[0101] In an exemplary embodiment, the expression of the miR30 130 sequence is regulated by a host cell (i.e., a CAR T cell). Since the expression of both the first shRNA 130a and the second shRNA 130b sequences and the miR30 130 sequence are controlled by the first promoter 120, the host cell is capable of regulating the expression of the first shRNA 130a and the second shRNA 130b sequences together with the miR30 130 sequence. The regulation of the expression of the first shRNA 130a and the second shRNA 130b sequences by the host cell prevents the overexpression of the first shRNA130a and the second shRNA 130b sequences. The first shRNA 130a and the second shRNA 130b together with miR30 130 allow for tight regulation of the levels of GMCSF and IL6. Thus, the CAR T cells of the present disclosure have a more precise and sustained inhibition of GMCSF and IL6 production.

[0102] Furthermore, proper balancing (regulation) of GMCSF expression results in inhibition of exhaustion markers on the CAR T cells of the present disclosure. Inhibition of said exhaustion markers promotes the formation of a less exhausted and more functional CAR T cell population, enhancing the long-term anti-tumor activity and persistence of the cell population. Said enhanced persistence is crucial for durable anti-tumor effects as CAR T cells need to survive and function in the hostile tumor microenvironment. Thus, CAR T cell survival and persistence are enhanced while reducing the risk of cytokine-related toxicities.

[0103] The first shRNA 130a, the second shRNA 130b, and miR30 130 promote better infiltration of CAR T cells into the tumor site. Improved tumor penetration is essential for the effective recognition and targeting of cancer cells by CAR T cells.

[0104] In an exemplary embodiment, the combination of the first shRNA 130a, the second shRNA 130b, and miR30 130 provides a precision strategy for enhancing CAR T cell infiltration into the tumor microenvironment. This innovative approach revolves around the inhibition of GMCSF expression, a cytokine that plays a key role in promoting immune responses and inflammation. By leveraging the second shRNA 130b, the expression of GM-CSF is specifically targeted and downregulated. This is 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. Additionally, the incorporation of miR30 130 further enhances GM-CSF inhibition. miR30 130 acts as a post-transcriptional regulator that fine-tunes gene expression. By inhibiting the translation of GM-CSF mRNA, miR30 130 adds an additional layer of control to ensure minimal GMCSF production. This dual approach of the first shRNA 130a, the second shRNA 130b, and miR30 130 works synergistically to reduce GMCSF levels, thereby alleviating immune interference that might otherwise impede CAR T cell infiltration into the tumor site. Thus, the first shRNA 130a, the second shRNA 130b, and miR30 130 promote better infiltration of CAR T cells into the tumor site. Improved tumor penetration is essential for the effective recognition and targeting of cancer cells by CAR T cells.

[0105] Figure 2aDepicts ORF 100a of the present disclosure. ORF 100a is structurally identical to ORF 100, but with the addition of an optional polyA tail 160 sequence, i.e., ORF 100a contains 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 polyA tail 160 sequence, etc. In an exemplary embodiment, ORF 100a contains a first hairpin loop structure formed by a first shRNA 130a sequence together with miR30 130 sequence and a second hairpin loop structure formed by at least one second shRNA 130b sequence together with miR30 130 sequence. The recombinant nucleotide sequence molecule is at least encoded by ORF 100a. In an exemplary embodiment, the first and second hairpin loop structures regulate the amounts of IL6 and GMCSF cytokines, respectively, during immunotherapy. The transcripts of the first and second hairpin loop structures (i.e., messenger RNAs) bind to the messenger RNAs (mRNAs) of the cytokines. In an exemplary embodiment, the transcripts of the first and second hairpin loop structures bind to the mRNAs of IL6 and GMCSF, respectively.

[0106] The polyA tail 160 sequence can be downstream of the first shRNA 130a and second shRNA 130b sequences and / or miR30 130 sequence. The expression of the polyA tail 160 sequence can be regulated by the first promoter 120. In an exemplary embodiment, as Figure 2a shown, the polyA tail 160 sequence is downstream of the hairpin loop structure formed by the second shRNA 130b sequence and miR30 130 sequence. The transcript of the polyA tail 160 sequence can contain multiple adenosine nucleotides. In an exemplary embodiment, the polyA tail 160 sequence is from 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 contributes to 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 excessive degradation or interference.

[0107] Now refer to Figure 2, the second promoter 140 can be disposed upstream of the CAR gene 150. In other words, the CAR gene 150 can be disposed 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 EF1α core promoter (encoded by SEQ ID NO. 64), etc. Thus, the expression of the CAR gene 150 can be regulated by the second promoter 140.

[0108] The CAR gene 150 comprises a sequence encoding one or more domains of the CAR 200, such as Figure 3 shown. One or more domains of the CAR 200 include, but are not limited to, one or more single-chain variable fragment (scFV) domains 210, one or more hinge domains 220, one or more transmembrane domains 230, one or more co-stimulatory (CSTM) domains 240, one or more signaling domains 250, one or more safety switch (SS) domains 260, etc.

[0109] In an exemplary embodiment, as Figure 3 shown, the CAR 200 includes an scFV domain 210, a hinge domain 220, a transmembrane domain 230, a CSTM domain 240, a signaling domain 250, and an SS domain 260. Additionally or optionally, the CAR 200 can include one or more peptide sequences, such as signal peptides, linker peptides, etc., disposed between the domains of the CAR 200.

[0110] As with any antibody, the scFV domain 210 of CAR 200 can include a light chain 210a and a heavy chain 210b. In an embodiment, the scFV domain 210 of the CD19 antigen (TAA) can be encoded by SEQ ID NO.65, such that the scFV domain 210 comprises the polypeptide sequence defined by SEQ ID NO.66. In an alternative embodiment, the scFV domain 210 of the CD19 antigen (TAA) can be encoded by SEQ ID NO.67, such that the scFV domain 210 comprises the polypeptide sequence defined by SEQ ID NO.68. The light chain 210a and heavy chain 210b of the scFV domain 210 can be coupled to each other via a linker protein 210c. The scFV domain 210 can have binding specificity (or affinity) for one or more TAAs, including but not limited to CD19, CD7, CD20, CD22, CD123, CD133, CD30, CD138, EGFR, EGFRvIII, FAP, MUC1, GD2, CEA, PSMA, HER2, NY-ESO-1, MAGEA-A3, hTERT, etc. In an exemplary embodiment, CAR 200 comprises one scFV domain 210 that is specific for the CD19 antigen of B lymphocytes (B cells). Alternatively, CAR 200 can comprise two or three scFV domains (not shown) for the same TAA or different TAAs.

[0111] The hinge domain 220 operably couples the scFV domain 210 to the transmembrane domain 230. In an exemplary embodiment, the hinge domain 220 includes a cluster of differentiation 8 (CD8) domain. The hinge domain 220 helps to provide flexibility to the scFV domain 210 relative to the transmembrane domain 230. In an embodiment, the hinge domain 220 is encoded by SEQ ID NO.69, such that the hinge domain 220 comprises the polypeptide sequence defined by SEQ ID NO.5770.

[0112] The transmembrane domain 230 can span the bilayer lipid cell membrane (or membrane) 270. The membrane 270 separates the extracellular region 270a from the intracellular region 270b. In an exemplary embodiment, the transmembrane domain 230 includes a CD8 domain encoded by SEQ ID NO.71, such that the transmembrane domain 230 comprises the polypeptide sequence defined by SEQ ID NO.72. The transmembrane domain 230 helps to operably couple the domains of CAR 200 present in the extracellular region 270a to the domains of CAR 200 present in the intracellular region 270b. In an exemplary embodiment, as Figure 3As shown, the scFV domain 210 and the hinge domain 220 are the only domains present in the extracellular region 270a.

[0113] The CSTM domain 240 can be coupled to the transmembrane domain 230. The CSTM domain 240 can be selected from Cluster of Differentiation 28 (CD28), Inducible T cell Costimulator (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 an embodiment, the CSTM domain 240 comprises 4-1BB. The CSTM domain 240 helps to enhance the cell-mediated immune response.

[0114] In an exemplary embodiment, the CAR T cells are activated by exposing the CAR T cells to a costimulatory molecule (e.g., CD3 and / or CD28). The costimulatory molecule stimulates the CSTM domain 240, which in turn results in 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 received by T cells during natural antigen recognition and thus provides a safe and effective method for synthetically activating CAR T cells both in vitro and in vivo. The CD3 / CD28-activated CAR T cells (or effector T cells) are then used to target and kill cancer cells.

[0115] The signaling domain 250 can be coupled to the CSTM domain 240. In an exemplary embodiment, the signaling domain 250 is the cluster of differentiation 3 zeta (CD3ζ) domain encoded by SEQ ID NO.87, such that the CD3ζ signaling domain 250 comprises the polypeptide sequence defined by SEQ ID NO.88. After the TAA binds to the scFV domain 210, the signaling domain 250 helps to transmit an activation signal to the CAR T cell.

[0116] The SS domain 260 can be coupled to the signaling domain 250. The SS domain 260 can be selected from iCaspase 9, EGFRt, RQR8, etc. In an exemplary embodiment, the SS domain 260 comprises iCaspase9 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 CART cells from transforming into cancer cells. In other words, in the presence of a predefined inducer of iCaspase 9, EGFRt, and / or RQR8, the SS domain 260 allows for the selective elimination of CAR T cells.

[0117] In an exemplary embodiment, the inducible caspase 9 (iCaspase9) SS domain 260 encodes a caspase recruitment domain (CARD; GenBank NM001 229) linked to two 12-kDa human FK506-binding proteins (FKBP12; GenBank AH002 818) containing the F36V mutation. Administration of a dimerization chemical inducer (CID) results in the dimerization of inducible caspase 9 molecules, leading to their activation. The caspase 9 dimer will then activate downstream effector caspases, such as caspase 3, and ultimately induce apoptosis.

[0118] In another exemplary embodiment, the RQR8 SS domain 260 encodes a multi-epitope molecule containing a CD34 epitope and two CD20 mimetic epitopes. When the CD20 mimetic epitopes bind to the FDA-approved CD20 antibody rituximab, the host cell undergoes apoptosis.

[0119] Figure 3aDepicts another embodiment of CAR 300. Similar to CAR 200, CAR 300 includes one or more scFV domains 310, one or more hinge domains 320, one or more transmembrane domains 330, one or more CSTM domains 340, one or more signaling domains 350, one or more SS domains 360, etc. The transmembrane domain 330 can be disposed across the membrane 370 such that the scFV domain 310 and the hinge domain 320 are disposed outside the CAR T cell, i.e., in the extracellular region 370a. The remaining domains of CAR 300 can be disposed inside the CAR T cell, i.e., in the intracellular region 370b.

[0120] In an exemplary embodiment, as Figure 3a shown, CAR 300 includes one scFV domain 310, one hinge domain 320, one transmembrane domain 330, a first CSTM domain 340a and a second CSTM domain 340b, one signaling domain 350, and one SS domain 360.

[0121] Similar to the scFV domain 210 of CAR 200, the scFV domain 310 of CAR 300 can include a light chain 310a and a heavy chain 310b. The light chain 310a and the heavy chain 310b of the scFV domain 310 can be coupled to each other via a linker protein 310c.

[0122] Similar to the CSTM domain 240 of CAR 200, the first CSTM domain 340a of CAR 300 can be selected from CD28, ICOS, and / or OX40. The second CSTM domain 340b can be coupled to the first CSTM domain 340a. The second CSTM domain 340b can be selected from 4-1BB, DAP10, DAP12, and / or 2B4.

[0123] In an exemplary embodiment, the first CSTM domain 340a and the second CSTM domain 340b are CD28 and 4-1BB, respectively. Compared with the single CSTM domain 240 in CAR 200, the first CSTM domain 340a and the second CSTM domain 340b of CAR 300 contribute to enhancing the persistence of CAR T cells.

[0124] As Figure 4 shown, the above CAR T cells produce negligible amounts of cytokines such as GMCSF and IL6. Therefore, the CAR T cells do not recruit any pro-inflammatory cells, thereby downregulating pro-inflammatory cytokines. The attenuation of pro-inflammatory cytokine levels alleviates CRS.

[0125] Although the first shRNA 130a, the second shRNA 130b, and miR30 130 are described in this disclosure in connection with examples of CAR 200 / 300, the first shRNA 130a, the second shRNA 130b, and miR30 130 can be used with any functionally equivalent CAR construct, and this is equally within the scope of the teachings of this disclosure.

[0126] The CAR T cells of the present invention can be prepared for each cancer patient by following Figure 5 the method 400 depicted therein. Alternatively, the CAR T cells can be prepared from an allogeneic T cell line for all cancer patients.

[0127] Method 400 begins at step 401 by separating peripheral blood mononuclear cells (PBMCs) by a ficoll-based method or an automated PBMC separation instrument (such as in the case of apheresis). In an exemplary embodiment, PBMCs are separated by drawing blood from a cancer patient in need of CAR T cell-based immunotherapy.

[0128] At step 403, a plurality of T cells can be separated from the PBMCs. The separated T cell population can contain a plurality of CD4 + and CD8 + T cells. The separated T cell population can contain at least 40% CD4 + T cells. In an exemplary embodiment, the separated T cell population contains 40% CD4 + T cells and 60% CD8 + T cells.

[0129] At optional step 405, the T cells are activated by subjecting them to co-stimulatory molecules (e.g., CD3 and / or CD28). In an exemplary embodiment, the co-stimulatory molecules help the T cells become capable of receiving ORF 100 via a lentiviral vector (described below).

[0130] At step 407, the ORF 100 of the present invention is prepared for delivery within the separated T cell population. ORF 100 can be ligated and replicated in a predefined vector (or plasmid), which includes but is not limited to pHR, pTRPE lentiviral vectors, etc. In an exemplary embodiment, ORF 100 is replicated and packaged within lentiviral particles.

[0131] At step 409, ORF 100 is delivered within the isolated T cell population via a predefined gene delivery method. The predefined gene delivery method can be one of transformation, transfection, or transduction. In an exemplary embodiment, the isolated T cell population is transduced by a lentivirus-based gene delivery system such as a third-generation self-inactivating (SIN) lentivirus 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 third-generation lentivirus. The resulting CAR T cells target and bind to specific cancer cells, thereby allowing the CAR T cells to recognize and attack B cells (i.e., cancer cells) expressing CD19.

[0132] At step 411, the CAR T cells (or engineered immune cells) are expanded in a predefined nutrient medium for a predefined time to increase the number of CAR T cells. In an exemplary embodiment, the CAR T cells are expanded for 7 to 9 days to increase the number of CAR T cells by 20 - 40 times. The engineered immune cells suspended in the predefined nutrient medium can define a composition.

[0133] In an exemplary embodiment, the nutrient medium comprises AIM V medium with or without antibiotics, human serum albumin, phenol red, and L-glutamine. In another embodiment, the nutrient medium comprises CTS AIM V medium without antibiotics, phenol red, and serum. The nutrient medium can be periodically replaced with fresh nutrient medium after a predefined time. In an exemplary embodiment, the nutrient medium is replaced every 48 hours.

[0134] At step 413, the transduced T cells expressing CAR 200, 300 are selected and isolated by subjecting the transduced T cells to in vitro and / or in vivo testing techniques. Additionally or alternatively, the transduced T cells expressing CAR 200, 300 are selected by immunoassay-based selection or by cell sorting techniques.

[0135] The selected transduced T cells expressing CAR 200, 300 (i.e., CAR T cells) are refrigerated for storage and transportation. The frozen CAR T cells are thawed before being infused into a patient.

[0136] Alternatively, as determined by the physician, the selected T cells (i.e., CAR T cells) can be directly infused into the patient.

[0137] Although method 400 is described with an exemplary sequence of steps, the steps of method 400 can be rearranged when practicing method 400, and this is also within the scope of the teachings of the present disclosure.

[0138] Sequences that share at least 95% identity with the sequences disclosed in this disclosure are within the scope of the teachings of this disclosure.

[0139] The present disclosure is further described by reference to the following exemplary embodiments and examples. These exemplary embodiments and examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise stated. Accordingly, the present disclosure should in no way be construed as limited to the following exemplary embodiments and examples, but rather should be construed to cover any and all variations that become apparent as a result of the teachings provided herein.

[0140] Example 1: CAR T Cell Preparation Method (the present invention):

[0141] Lentivirus-packaged HEK293T cells obtained from the American Type Culture Collection (ATCC) were seeded at approximately 3.8×10 6 cells / plate in complete Dulbecco's Modified Eagle's Medium (DMEM) in a 10 cm tissue culture plate. Then the tissue culture plate containing HEK293T cells was incubated at 37 °C, 5% CO2 for about 24 hours. After about 24 hours, the old medium was aspirated and replaced with 10 mL of fresh complete DMEM and 25 μM chloroquine diphosphate. The tissue culture plate containing HEK293T cells was further incubated at 37 °C, 5% CO2 for about 6 hours.

[0142] Using polyethyleneimine (PEI, a non-viral vector) as a transfection reagent, a mixture of a lentiviral plasmid and a CAR plasmid (containing ORF 100 of this disclosure) was inserted into HEK293T cells. The HEK293T cells were incubated at 37 °C, 5% CO2 for about 36 hours and then the medium containing viral particles was harvested at different time intervals (up to 72 hours). The harvested medium was centrifuged at 1000 rpm for 5 minutes to pellet any remaining HEK293T cells. Then the supernatant was filtered using a 0.45 μm PES filter. The filtrate (supernatant) containing viral particles was collected and refrigerated to avoid titer loss. The viral particles have a lentiviral plasmid integrated with the CAR plasmid (containing ORF 100 of this disclosure).

[0143] Via a process called leukapheresis, 10 million PBMCs were collected from every 20 mL of patient blood. Then T cells were isolated from the collected PBMCs using the RosetteSep Human CD4+ T Cell Enrichment Assay and the RosetteSep Human CD8+ T Cell Enrichment Assay. The isolated T cell population consisted of 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.

[0144] The isolated T cells were cultured in a human T cell medium consisting of X-VIVO 15 (Lonza), 5% human AB serum, and 10 mM neutralized N-acetyl-L-cysteine (Sigma-Aldrich). Further, IL-2 was used for cell proliferation and was used at 30 units / mL IL-2.

[0145] After 24 hours of culture, the isolated T cells were stimulated (activated) with human T activator CD3 / CD28 immunomagnetic beads (Life Technologies) at a cell:bead ratio of 1:3.

[0146] At 48 hours, the primary T cells were exposed to the supernatant (containing virus particles) for transduction. On the 4th day after T cell stimulation, the immunomagnetic beads were removed and the T cells were expanded until the 9th day. The T cells were sorted using FACs ARIA II. Thus, T cells showing basal CAR expression (i.e., CAR T cells) were isolated.

[0147] The isolated CAR T cells were expanded in a nutrient medium at 37 °C, 5% CO2, which included CTS AIM V medium without antibiotics, phenol red, and serum. The nutrient medium was replaced with fresh nutrient medium every 48 hours. It was observed that after expanding the CAR T cells for about 7 - 9 days, the number of CAR T cells increased by 20 - 40 fold. The CAR T cells were frozen for storage and transportation.

[0148] Example 2: Infusion of CAR T cells (the present invention):

[0149] The CAR T cells (obtained as in Example 1) were thawed. Then, approximately 1×10 6 CAR T cells per kg of patient body weight were 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. The binding between the CAR T cells and the cancer cells triggered the activation and proliferation of the CAR T cells, after which the cancer cells were killed by mechanisms such as cytokine release and cytotoxicity.

[0150] The CAR T cells persisted in the patient's body and continued to attack cancer cells expressing CD19, providing a long-term therapeutic effect for the patient. It was observed that the CAR T cells of the present disclosure provided a response rate of up to 90% in patients with acute lymphoblastic leukemia and up to 50% - 60% in patients with non-Hodgkin lymphoma. The CAR T cells specifically targeted cancer cells expressing CD19, reducing the risk of damage to healthy cells and the risk of false-positive interactions. Different from traditional chemotherapy and radiotherapy, CAR T cell therapy has minimal toxicity to healthy cells.

[0151] Example 3: IL6 and GMCSF knockdown (the present invention):

[0152] Evaluate the effect of the top-performing first shRNA 130a and second shRNA 130b sequences on the regulation of IL6 and GMCSF levels as described in the present disclosure. Screening was performed in T cells. As Figure 6 shown, the bar graph depicts IL6 and GMCSF expression in activated (Act-T cells) for 24 hours to induce IL6 expression or unactivated (control) T cells. Evaluate the effect of non-targeting shRNA (mir30-Scram) and the first shRNA 130a and second shRNA 130b targeting IL6 and GMCSF on knocking down the IL6 and GMCSF genes in activated T cells. Transfect T cells with the corresponding first shRNA 130a and second shRNA 130b, and prepare mRNA 24 hours after transfection. Determination was performed by RT-qPCR using IL6 and GMCSF mRNA-specific primers. Statistical analysis was performed using a non-parametric t-test, revealing significance levels expressed as **** (P<0.0001), ** (P<0.01), * (P<0.05) when comparing T cells expressing GMCSF / IL6 mir30 shRNA with T cells expressing mir30 Scram (see Figure 6 ). It was observed that the first shRNA 130a and second shRNA 130b sequences of the present disclosure significantly reduced the expression of the IL6 gene via RNAi.

[0153] Example 4: IL6 and GMCSF production (the present invention):

[0154] As described in Example 3, collect the protein fractions from the corresponding T cells. Perform an enzyme-linked immunosorbent assay (ELISA) on the collected protein fractions to determine the amounts of IL6 and GMCSF cytokines present in each protein fraction. As Figure 7 shown, the bar graph depicts the IL6 and GMCSF cytokines present in the corresponding protein fractions. Statistical analysis was performed using a non-parametric t-test, revealing significance levels expressed as **** (P<0.0001), *** (P<0.001) and ns (not significant) when comparing T cells expressing mir30 GMCSF / IL6 shRNA with T cells expressing Scram (see Figure 7 ).

[0155] Example 5: IL6 and GMCSF production in CAR T cells (the present invention):

[0156] Evaluate the effect of the top-performing first shRNA 130a and second shRNA 130b sequences, as observed in Examples 3 and 4, on the regulation of IL6 and GMCSF levels. Screening was performed in CAR T cells. As Figure 8 shown, the bar graphs depict IL6 and GMCSF produced in unactivated T cells (control). Non-targeting shRNA (CART miR30-Scram ) and the first shRNA 130a and second shRNA 130b targeting IL6 and GMCSF (CART miR30 -shgmcsf / shIL6 ) were evaluated for their role in knocking down the IL6 gene. Evaluation was also performed after activation of conventional CAR T cells (CART con ). The assays and analyses were similar to those described in Example 4 above. As Figure 8 shown, the bar graphs depict the IL6 and GMCSF cytokines present in the corresponding protein fractions. Statistical analysis was performed using a non-parametric t-test, which revealed a significant difference with a confidence level of **** (P<0.0001) when comparing T cells expressing IL6 and GMCSF mir30-shRNA with T cells expressing mir30-Scram (see Figure 8 ).

[0157] The scope of the present invention is limited only by the appended patent claims. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and the actual parameters, dimensions, materials, and / or configurations will depend on one or more specific applications in which the teachings of the present invention are used.

Claims

1. A recombinant nucleic acid molecule, said recombinant nucleic acid molecule being at least encoded by ORF(100,100a) to reduce cytokine storm during immunotherapy, said recombinant nucleic acid molecule comprising: a. At least one first hairpin loop structure, said at least one first hairpin loop structure regulating the amount of interleukin 6 (IL6) cytokine during immunotherapy, said at least one first hairpin loop structure being formed by: i. One or more first short hairpin RNA (130a) sequences, said one or more first short hairpin RNA sequences forming the stem of said at least one first hairpin loop structure, and ii. At least one microRNA30 (130) sequence, said at least one microRNA30 sequence forming the loop of said at least one hairpin loop structure; b. At least one second hairpin loop structure, said at least one second hairpin loop structure regulating the amount of granulocyte-macrophage colony-stimulating factor (GMCSF) cytokine during immunotherapy, said at least one second hairpin loop structure being disposed upstream or downstream of said at least one first hairpin loop structure, said at least one second hairpin loop structure being formed by: i. One or more second short hairpin RNA (130b) sequences, said one or more second short hairpin RNA sequences forming the stem of said at least one second hairpin loop structure, and ii. At least one microRNA30 (130) sequence, said at least one microRNA30 sequence forming the loop of said at least one hairpin loop structure; and c. A first promoter (120), said first promoter being disposed upstream of said at least one first hairpin loop structure and said at least one second hairpin loop structure.

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

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

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

5. The recombinant nucleic acid molecule according to claim 1, wherein A polyA tail (160) sequence is disposed downstream of said first hairpin loop structure and said second hairpin loop structure.

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

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

8. A recombinant nucleic acid molecule, said recombinant nucleic acid molecule being encoded by at least ORF(100) to reduce cytokine storm during immunotherapy, said recombinant nucleic acid molecule comprising: a. At least one first hairpin loop structure, said at least one first hairpin loop structure regulating the amount of interleukin 6 (IL6) cytokine during immunotherapy, said at least one first hairpin loop structure being formed by: i. One or more first short hairpin RNA (130a) sequences, said one or more first short hairpin RNA sequences forming the stem of said at least one first hairpin loop structure, and ii. At least one microRNA30 (130) sequence, said at least one microRNA30 sequence forming the loop of said at least one hairpin loop structure; b. At least one second hairpin loop structure, said at least one second hairpin loop structure regulating the amount of granulocyte-macrophage colony-stimulating factor (GMCSF) cytokine during immunotherapy, said at least one second hairpin loop structure being disposed upstream or downstream of said at least one first hairpin loop structure, said at least one second hairpin loop structure being formed by: i. One or more second short hairpin RNA (130b) sequences, said one or more second short hairpin RNA sequences forming the stem of said at least one second hairpin loop structure, and ii. At least one microRNA30 (130) sequence, said at least one microRNA30 sequence forming the loop of said at least one hairpin loop structure; c. A first promoter (120), said first promoter being disposed upstream of said at least one hairpin loop structure and said at least one second hairpin loop structure; d. One or more chimeric antigen receptor genes (150), said one or more chimeric antigen receptor genes expressing: i. One or more single-chain variable fragment domains (210), said one or more single-chain variable fragment domains being configured to bind to one or more tumor-associated antigens (TAA), ii. One or more hinge domains (220), said one or more hinge domains being coupled to said one or more single-chain variable fragment domains (210), iii. One or more co-stimulatory domains (240), iv. One or more signaling domains (250), said one or more signaling domains being coupled to said one or more co-stimulatory domains (240), v. One or more safety switch domains (260), said one or more safety switch domains being coupled to said one or more signaling domains (250), and vi. One or more transmembrane domains (230) operably coupling said hinge domain (220) to said co-stimulatory domain (240); e. A second promoter (140), said second promoter being disposed upstream of said one or more chimeric antigen receptor genes (150), and f. One or more long terminal repeats (110), the one or more long terminal repeats being disposed upstream of the first promoter (120) and downstream of the one or more chimeric antigen receptor genes (150).

9. The recombinant nucleic acid molecule according to claim 8, wherein The first short hairpin RNA (130a) is encoded by at least one of SEQ ID NOs: 5 - 40 or a nucleotide sequence having at least 95% identity therewith.

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

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

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

13. The recombinant nucleic acid molecule according to claim 8, wherein The single - chain variable fragment domain (210) for cluster of differentiation 19 (CD19) is encoded by at least one of SEQ ID NO.65 and SEQ ID NO.

67.

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

69.

15. The recombinant nucleic acid molecule according to claim 8, wherein, The one or more co - stimulatory domains (240) comprise at least one of the following: cluster of differentiation 28 (CD28) encoded by SEQ ID NO.73, inducible T - cell co - stimulator molecule (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. The recombinant nucleic acid molecule according to claim 8, wherein, The one or more signaling domains (250) at least partially comprise cluster of differentiation 3 zeta (CD3ζ) encoded by SEQ ID NO.

87.

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

91.

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

71.

19. The recombinant nucleic acid molecule according to claim 8, wherein, The second promoter (140) is at least one of the elongation factor 1 (EF1) full-length promoter encoded by SEQID NO.63 and the EF1α core promoter encoded by SEQ ID NO.

64.

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

21. The recombinant nucleic acid molecule according to claim 8, wherein The one or more long terminal repeats (110) are encoded by SEQ ID NO.

1.

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

23. The recombinant nucleic acid molecule according to claim 8, wherein The messenger RNA of the first hairpin structure or the second hairpin 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 that is at least encoded by an ORF (100a) to reduce cytokine storm during immunotherapy, the recombinant nucleic acid molecule comprising: a. At least one first hairpin structure that regulates the amount of interleukin 6 (IL6) cytokine during immunotherapy, the at least one first hairpin structure being formed by: i. One or more first short hairpin RNA (130a) sequences that form the stem of the at least one first hairpin structure, and ii. At least one microRNA30 (130) sequence that forms the loop of the at least one hairpin structure; b. At least one second hairpin structure that regulates the amount of granulocyte-macrophage colony-stimulating factor (GMCSF) cytokine during immunotherapy, the at least one second hairpin structure being disposed upstream or downstream of the at least one first hairpin structure, the at least one second hairpin structure being formed by: i. One or more second short hairpin RNA (130b) sequences, wherein the one or more second short hairpin RNA sequences form the stem of the at least one second hairpin loop structure, and ii. At least one microRNA30 (130) sequence, wherein the at least one microRNA30 sequence forms the loop of the at least one hairpin loop structure; c. A first promoter (120), which is disposed upstream of the at least one first hairpin loop structure and the at least one second hairpin loop structure; d. A poly-A tail (160) sequence, which is disposed downstream of the at least one first hairpin loop structure and the at least one second hairpin loop structure; e. One or more chimeric antigen receptor genes (150), wherein the one or more chimeric antigen receptor genes express: i. One or more single-chain variable fragment domains (210), which are configured to bind to one or more tumor-associated antigens (TAAs), ii. One or more hinge domains (220), which are coupled to the one or more single-chain variable fragment domains (210), iii. One or more co-stimulatory domains (240), iv. One or more signaling domains (250), which are coupled to the one or more co-stimulatory domains (240), v. One or more safety switch domains (260), which are coupled to the one or more signaling domains (250), and vi. One or more transmembrane domains (230) operably couple the hinge domain (220) to the co-stimulatory domain (240); f. A second promoter (140), which is disposed upstream of the one or more chimeric antigen receptor genes (150), and g. One or more long terminal repeat sequences (110), which are disposed upstream of the first promoter (120) and downstream of the one or more chimeric antigen receptor genes (150).

25. The recombinant nucleic acid molecule according to claim 24, wherein The first short hairpin RNA (130a) is encoded by at least one of SEQ ID NOs. 5-40 or a nucleotide sequence having at least 95% identity therewith.

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

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

28. The recombinant nucleic acid molecule according to claim 24, wherein The polyA tail (160) sequence is derived from Simian virus 40 (SV40) or bovine growth hormone beta globulin (bGH) encoded by SEQ ID NO.

62.

29. The recombinant nucleic acid molecule according to claim 24, wherein The one or more single-chain variable fragment domains (210) have binding affinity for at least one of the following: Cluster of Differentiation 19 (CD19), Cluster of Differentiation 7 (CD7), Cluster of Differentiation 20 (CD20), Cluster of Differentiation 22 (CD22), Cluster of Differentiation 123 (CD123), Cluster of Differentiation 133 (CD133), Cluster of Differentiation 30 (CD30), Cluster of Differentiation 138 (CD138), Epidermal Growth Factor Receptor (EGFR), Epidermal Growth Factor Receptor Variant III (EGFRvIII), Fibroblast Activation Protein alpha (FAP), Mucin 1 (MUC1), Disialoganglioside GD2 (GD2), Carcinoembryonic Antigen (CEA), Prostate-Specific Membrane Antigen (PSMA), Human Epidermal Growth Factor Receptor 2 (HER2), New York Esophageal Squamous Cell Carcinoma-1 (NY-ESO-1), Melanoma-Associated Antigen 3 (MAGEA-A3), and Human Telomerase Reverse Transcriptase (hTERT).

30. The recombinant nucleic acid molecule according to claim 24, wherein The single-chain variable fragment domain (210) for Cluster of Differentiation 19 (CD19) is encoded by at least one of SEQ ID NO.65 and SEQ ID NO.

67.

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

69.

32. The recombinant nucleic acid molecule according to claim 24, wherein The one or more co-stimulatory domains (240) comprise at least one of the following: Cluster of Differentiation 28 (CD28) encoded by SEQ ID NO.73, Inducible T-Cell Co-Stimulator Molecule (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. The recombinant nucleic acid molecule according to claim 24, wherein The one or more signaling domains (250) at least partially comprise Cluster of Differentiation 3 zeta (CD3ζ) encoded by SEQ ID NO.

87.

34. The recombinant nucleic acid molecule according to claim 24, wherein The one or more safety switch domains (260) comprise at least one of Inducible Caspase 9 (iCaspase 9) encoded by SEQ ID NO.89, Truncated Epidermal Growth Factor Receptor (EGFRt), and RQR8 encoded by SEQ ID NO.

91.

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

71.

36. The recombinant nucleic acid molecule according to claim 24, wherein The second promoter (140) is at least one of the full-length promoter of Elongation Factor 1 (EF1) encoded by SEQ ID NO.63 and the EF1α core promoter encoded by SEQ ID NO.

64.

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

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

1.

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

40. The recombinant nucleic acid molecule according to 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 an ORF (100, 100a), the transcript comprising: a. Messenger RNA of a first hairpin loop structure or a second hairpin loop structure, the messenger RNA of the first hairpin loop structure or the second hairpin loop structure being transcribed from the recombinant nucleic acid molecule according to any one of claims 1 to 40, the messenger RNA of the first hairpin loop structure or the second hairpin loop structure being configured to bind to the messenger RNA of a cytokine.

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

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

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

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

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

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

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

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

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

52. The method (400) according to claim 50, characterized in that, After isolating the plurality of T cells, contacting the plurality of T cells with a costimulatory molecule comprising at least one of cluster of differentiation 3 (CD3) and cluster of differentiation 28 (CD28).

53. The method (400) according to claim 50, characterized in that, Replicating the recombinant nucleic acid molecule comprises ligating the recombinant nucleic acid molecule to at least one of a plasmid, a cosmid, a viral vector, and a phage.

54. The method (400) according to claim 50, characterized in that, Replicating the recombinant nucleic acid molecule comprises ligating the recombinant nucleic acid molecule to a viral vector derived from at least one of a lentivirus, a retrovirus, an adenovirus, an adeno-associated virus, and a Sendai virus.

55. The method (400) according to claim 50, characterized in that, Delivering the vector into the plurality of T cells comprises transforming, transfecting, or transducing the plurality of T cells with the vector.

56. The method (400) according to claim 50, characterized in that, After delivering the vector into the plurality of T cells, amplifying the T cells in a predefined nutrient medium for a predefined period of time.

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