Reduction of cytokine release syndrome

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 hairpin ring structure and a first promoter (120). The at least one hairpin ring structure is formed from one or more short hairpin RNA (130) sequences. The at least one hairpin ring structure modulates the amount of interleukin 6 (IL6) cytokine during immunotherapy. The first promoter (120) is disposed upstream of the at least one hairpin ring structure.

Description

[0001] Priority

[0002] This application claims priority to Indian Provisional Application No. 202221053838, 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 level of interleukin 6 (IL6) 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 recognize 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, adoptive cell therapy (ACT) based on chimeric antigen receptor (CAR) T cells has been introduced. Adoptive cell therapy based on CAR T cells is a form of adoptive cell transfer 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 a major problem.

[0007] Although most of the 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) is a cytokine that is upregulated during CRS and is one of the 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 granulocyte-macrophage colony-stimulating factor (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 challenge in treating cancer patients with CAR T cell-based immunotherapy. Additionally, patients who receive conventional CAR T cell infusions have been diagnosed with relapse (i.e., 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] The following describes specific embodiments of the present disclosure 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 hairpin loop structure and a first promoter. The at least one hairpin loop structure is formed by one or more short hairpin RNA sequences. The at least one hairpin loop structure regulates the amount of interleukin 6 (IL6) cytokine during immunotherapy. The first promoter is disposed upstream of the at least one hairpin loop structure.

[0015] In an exemplary embodiment, the present disclosure relates to a recombinant nucleic acid molecule that is at least encoded by an ORF to reduce cytokine storm during immunotherapy. The recombinant nucleic acid molecule comprises at least one hairpin loop structure, a first promoter, one or more chimeric antigen receptor genes, a second promoter, and one or more long terminal repeat sequences. The at least one hairpin loop structure is formed by one or more short hairpin RNA sequences. The at least one hairpin loop structure regulates the amount of interleukin 6 (IL6) cytokine during immunotherapy. The first promoter is disposed upstream of the at least one hairpin loop structure. 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 (TAAs). The hinge domain is coupled to the one or more single-chain variable fragment domains. The signaling domain is coupled to the co-stimulatory domain. The safety switch domain is coupled to the signaling domain. The transmembrane domain operatively couples the hinge domain to the co-stimulatory domain. The second promoter is located upstream of the one or more chimeric antigen receptor genes. The long terminal repeat sequence is located upstream of the first promoter and downstream of the chimeric antigen receptor genes.

[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 hairpin loop structure, a first promoter, a polyA tail sequence, one or more chimeric antigen receptor genes, a second promoter, and one or more long terminal repeat sequences. The at least one hairpin loop structure is formed by one or more short hairpin RNA sequences. The at least one hairpin loop structure regulates the amount of interleukin 6 (IL6) cytokine during immunotherapy. The first promoter is disposed upstream of the at least one hairpin loop structure. The polyA tail sequence is located downstream of the hairpin loop structure. 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 (TAAs). The hinge domain is coupled to the one or more single-chain variable fragment domains, and the signaling domain is coupled to the co-stimulatory domain. The safety switch domain is coupled to the signaling domain. The transmembrane domain operatively couples the hinge domain to the co-stimulatory domain. The second promoter is located upstream of the one or more chimeric antigen receptor genes. The long terminal repeat sequence is located upstream of the first promoter and downstream of the chimeric antigen receptor genes.

[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 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 that comprises at least one of the recombinant nucleic acid molecules at least encoded by an ORF to reduce cytokine storm during immunotherapy.

[0020] In an exemplary embodiment, the present disclosure relates to a composition that comprises engineered immune cells suspended in a nutrient medium. The engineered immune cells comprise at least one of the recombinant nucleic acid molecules at least 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 at least to reduce cytokine storm during immunotherapy. And delivering the vector into the plurality of T cells.

[0022] From the following detailed description taken in conjunction with the reference drawings, the foregoing and other features and advantages of the present invention will become more apparent. In the drawings, like reference numerals indicate the same or similar parts in several views. The features will be described in sufficient detail to enable those skilled in the art of the subject matter to practice the present invention. Further, 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. Further, those skilled in the art will understand that the drawings are not drawn to scale.

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

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

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

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

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

[0029] Figure 4 A diagram of CRS reduction / attenuation according to one or more exemplary embodiments of the present disclosure is depicted.

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

[0031] Figures 6 to 8 depicts experimental observations in accordance with one or more exemplary embodiments of the present disclosure. DETAILED DESCRIPTION

[0032] Before describing the invention in detail, certain words or 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 property of, etc.; definitions for certain words and phrases are provided throughout this patent document, and one 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 words and phrases for which they are defined.

[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 cells" particularly refers to T cells (or CAR T cells) that express a chimeric antigen receptor (CAR) construct and / or are capable of binding to a tumor-associated antigen (TAA).

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

[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 from antibody fragments.

[0038] The term "Fv" refers to the smallest antibody fragment that contains a 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 from the H and L chains), 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 an Fv that only includes three complementarity-determining regions (CDRs) specific for the antigen) has the ability to recognize and bind 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 are present 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 are present 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 "anti-tumor 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. An "anti-tumor effect" can also be manifested by the ability of a peptide, polynucleotide, cell, and antibody 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 derived from a subject that is subsequently reintroduced into the same subject.

[0045] The term "allogeneic" is used to describe a graft 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 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, and the like.

[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 impact 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 to CD83.

[0051] The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response in 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, when combined 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 (such as a gene, cDNA or mRNA) to serve as a template for the synthesis of other polymers and macromolecules with a defined nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a defined amino acid sequence in a biological process and the resulting biological property. Thus, if the transcription and translation of the mRNA corresponding to the gene produces a protein in a cell or other biological system, the gene encodes the protein. Both the coding strand (whose nucleotide sequence is the same as the mRNA sequence (except that "T" is replaced by "U") and is usually provided in the sequence listing) and the non-coding strand (which serves as the template for gene or cDNA transcription) can be said to encode the protein or other product of the gene or cDNA.

[0054] The term "exogenous" refers to a molecule that does not naturally occur in a wild-type cell or organism but is generally introduced into the cell by molecular biology techniques. Examples of exogenous polynucleotides include vectors, plasmids and / or artificial nucleic acid constructs encoding a desired protein. With respect to polynucleotides and proteins, the terms "endogenous" or "native" refer to a naturally occurring polynucleotide or amino acid sequence 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 an "exogenous" polynucleotide or amino acid sequence with respect to the second organism. In a specific embodiment, a polynucleotide sequence can be "introduced" into a microorganism that already contains such a polynucleotide sequence, for example, to produce one or more additional copies of other naturally occurring polynucleotide sequences, 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 containing a recombinant polynucleotide that contains an expression control (regulatory) sequence operably linked to the 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 incorporating recombinant polynucleotides (e.g., lentiviruses, retroviruses, adenoviruses and adeno-associated viruses).

[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 one another 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 the 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 the host cell, and thus they are one of the most efficient methods of gene delivery vectors. In addition, the use of lentiviruses enables the integration of genetic information into the host chromosome, resulting in stably transduced genetic information. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide a means of achieving 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 it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if the presequence or secretory leader is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to promote 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 those having a disease, disorder or condition for which treatment is needed. Subjects in need also include those in need of treatment to prevent a disease, disorder or condition.

[0066] The term "polynucleotide" or "nucleic acid" refers to mRNA, RNA, cRNA, rRNA, cDNA or DNA. The term generally refers to 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 changes can be made to a reference polynucleotide, including mutations, additions, deletions and substitutions, and the polynucleotide so altered retains the biological function or activity of the reference polynucleotide or has increased activity (i.e., is 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, polypeptides can include enzymatic polypeptides or "enzymes", which generally catalyze (i.e., increase) the rate of various chemical reactions.

[0069] The term "polypeptide variant" refers to a polypeptide that 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 for the expression of an operably linked coding sequence in a particular host organism. For example, control sequences suitable for prokaryotes include a promoter, an optional operator sequence, and a ribosome binding site. It is known that eukaryotic cells utilize promoters, polyadenylation signals, and enhancers.

[0071] The terms "bind", "binds to", or "interacts with" refer to a molecule that recognizes and adheres to a second molecule in a sample or organism but that 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 by itself does not change 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 by itself does not change the classification of the antibody as a specific antibody. In some instances, the term "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 a molecule 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 a cell includes the primary subject cell and its 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 cover an order of operations other than the particular order disclosed. For example, the 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 can 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 can be used in combination with other systems, methods, and devices.

[0078] Moreover, 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 cases, additional features and advantages may be recognized in certain embodiments that may not be present in all embodiments. These features and advantages of the embodiments will become more fully apparent from the following description and the 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, and the like. In an embodiment, the CAR T cell targets 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 predetermined 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), New York esophageal squamous cell carcinoma-1 (NY-ESO-1), melanoma-associated antigen 3 (MAGEA-A3), human telomerase reverse transcriptase (hTERT), and the like. 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 one short hairpin RNA (shRNA) sequence to reduce a cytokine storm known as cytokine release syndrome (CRS) during immunotherapy. During CRS, a rapid release of cytokines is observed. Interleukin 6 (IL6) is one of the cytokines released during immunotherapy. The shRNA sequence can inhibit cytokine production and / or cytokine signaling through RNA interference (RNAi). In an exemplary embodiment, the shRNA sequence expresses an anti-IL6 (aIL6) that binds and neutralizes IL6 (the exemplary cytokine), thereby at least partially blocking the activity of IL6. In another exemplary embodiment, the transcript of the shRNA sequence binds to the mRNA of the cytokine to functionally inactivate the mRNA and / or degrade the mRNA. In yet another exemplary embodiment, the transcript of the shRNA sequence binds to the mRNA of the cytokine to prevent its translation.

[0082] In an exemplary embodiment, the CAR T cells of the present disclosure comprise an interleukin-6 receptor (IL6-R) conjugated to a synthetic notch (syn-notch) receptor and a GAL-VP64 domain. During CRS, the released IL6 binds to the IL6-R of the CAR T cells and induces syn-notch, 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 thirty-six shRNA sequences specific for interleukin 6 (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 CAR T cells. By neutralizing IL6, the CAR T cells disclosed herein have a lower risk of CRS and thus the ability to safely and effectively eliminate cancer cells.

[0083] Furthermore, the CAR comprises a safety switch (SS) domain for regulating the proliferation of CAR T cells, thereby inhibiting the transformation of CAR T 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.

[0084] Thus, for the above reasons, 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 disclosed herein can be selectively depleted to prevent the malignant transformation of CAR T cells.

[0085] Referring now to the drawings, Figure 2 depicts the antisense (or template) strand of the open reading frame (ORF) 100 of a CAR T cell (not shown). 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 encoded by at least the ORF 100.

[0086] At least a portion of the ORF 100 (or the recombinant nucleotide sequence molecule) can be introduced into one or more natural immune cells by genetic manipulation to produce engineered immune cells. The ORF 100 (or a portion thereof) expresses one or more CARs (i.e., proteins and / or polypeptides) and / or one or more shRNA 130 transcripts (described below). Natural immune cells can include, but are not limited to, T lymphocytes (T cells), natural killer (NK) cells, gamma-delta T cells, and the like. In an exemplary embodiment, the ORF100 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, the ORF 100 is introduced into natural T cells using a lentiviral vector, an adeno-associated virus (AAV), or the like. The vector containing the 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, a lentivirus (LV) is used to introduce the 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.

[0087] ORF 100 may comprise one or more regions, which include one or more long terminal repeats (LTRs) 110, one or more promoters, at least one hairpin loop structure, one or more genes, etc. The one or more promoters may include a first promoter 120 and a second promoter 140. The one or more genes may include, but are not limited to, multiple CAR genes 150. 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 hairpin loop structure regulates the amount of IL6 cytokine during immunotherapy. The transcript of the hairpin loop structure (i.e., messenger RNA) binds to the messenger RNA (mRNA) of the cytokine. In an exemplary embodiment, the transcript of the hairpin loop structure binds to the mRNA of IL6.

[0088] LTR 110 may be located on both sides of ORF 100, i.e., LTR 110 may be disposed at the 5' end and the 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 ORF100 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).

[0089] The 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 may be disposed upstream of one or more genes of ORF 100. One or more RNA polymerases may bind to the promoter for transcribing ORF 100. The RNA polymerase may 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 may transcribe ORF 100 partially or completely into one or more messenger RNAs (mRNAs) (or transcripts). The mRNA may be further processed to provide one or more CAR (protein structures) and shRNA 130 transcripts (and their proteins / polypeptides). The processing of mRNA may include at least one of splicing, translating the mRNA into one or more amino acid sequences (i.e., polypeptides), and post-translational modifications, etc. Post-translational modifications may include, but are not limited to, folding of the amino acid sequence (forming a protein) and / or glycosylation, etc.

[0090] As Figure 2As shown, the first promoter 120 can be disposed upstream of the shRNA 130 (i.e., the hairpin loop structure). The first promoter 120 can be selected from a full-length cytomegalovirus (CMV) promoter (encoded by SEQ ID NO.2), a 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, a SNORD promoter, and the like. In an exemplary embodiment, the first promoter 120 comprises a GAL-VP64 recognition domain that activates the first promoter 120 when the GAL-VP64 domain binds to the GAL-VP64 recognition domain.

[0091] The first promoter 120 transcribes the shRNA 130 to produce one or more short RNA transcripts having a hairpin loop structure. The shRNA 130 can be encoded by at least one of SEQ ID NOs. 5-40 (SEQ ID NOs. 30-40 are the sequences of the sense strand). The expression of the shRNA 130 can be regulated by the first promoter 120.

[0092] The transcript of the shRNA 130 can include specific binding affinity for one or more molecules. The CAR T cells comprise a plurality of IL6 receptors (IL6-R) conjugated with 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, followed by cleavage and release of the GAL-VP64 domain. The GAL-VP64 domain migrates to the nucleus of the CAR T cells and induces the expression of at least one of the thirty-six shRNA 130 sequences. The expression of the shRNA 130 sequence results in the production of aIL6. The aIL6 binds and neutralizes IL6, thereby reducing the burden of CRS.

[0093] In an alternative embodiment, the shRNA 130 (i.e., the transcript of the shRNA 130) expressed by the CAR T cells selectively binds to the IL6 mRNA, thereby regulating the IL6 cytokine level (via RNAi). When the transcript of the shRNA 130 binds to the IL6 mRNA, the level (concentration) of the IL6 molecule decreases, resulting in a reduced burden of CRS.

[0094] Figure 2aDepicts ORF 100a of the present disclosure. ORF 100a is structurally identical to ORF 100, but with an optional polyA tail 160 sequence added, i.e., ORF 100a includes one or more LTRs 110, one or more shRNA 130 sequences, a first promoter 120, a second promoter 140, multiple CAR genes 150, a polyA tail 160 sequence, etc. At least one hairpin loop structure can be formed by one or more short hairpin RNA (shRNA) 130 sequences. The recombinant nucleotide sequence molecule is at least encoded by ORF 100a. In an exemplary embodiment, the hairpin loop structure regulates the amount of IL6 cytokine during immunotherapy. The transcript of the hairpin loop structure (i.e., messenger RNA) binds to the messenger RNA (mRNA) of the cytokine. In an exemplary embodiment, the transcript of the hairpin loop structure binds to the mRNA of IL6.

[0095] The polyA tail sequence 160 can be disposed downstream of the shRNA 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 shRNA 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.41. 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 effect of the IL6 component is maintained over time without excessive degradation or interference.

[0096] Now referring 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.42), the EF1α core promoter (encoded by SEQ ID NO.43), etc. Thus, the expression of the CAR gene 150 can be regulated by the second promoter 140.

[0097] The CAR gene 150 contains a sequence encoding one or more domains of the CAR 200, such as Figure 3As shown. One or more domains of CAR 200 include, but are not limited to, one or more single-chain variable fragment (scFV) domains 210, one or more hinge domains 220, one or more transmembrane domains 230, one or more costimulatory (CSTM) domains 240, one or more signaling domains 250, one or more safety switch (SS) domains 260, and the like.

[0098] In an exemplary embodiment, as Figure 3 shown, CAR 200 includes one scFV domain 210, one hinge domain 220, one transmembrane domain 230, one CSTM domain 240, one signaling domain 250, and one SS domain 260. Additionally or optionally, CAR 200 may include one or more peptide sequences disposed between the domains of CAR 200, such as signal peptides, linker peptides, and the like.

[0099] Like any antibody, the scFV domain 210 of CAR 200 may include a light chain 210a and a heavy chain 210b. In an embodiment, the scFV domain 210 of the CD19 antigen (TAA) may be encoded by SEQ ID NO.44, such that the scFV domain 210 comprises the polypeptide sequence defined by SEQ ID NO.45. In an alternative embodiment, the scFV domain 210 of the CD19 antigen (TAA) may be encoded by SEQ ID NO.46, such that the scFV domain 210 comprises the polypeptide sequence defined by SEQ ID NO.47. The light chain 210a and the heavy chain 210b of the scFV domain 210 may be coupled to each other via a linker protein 210c. The scFV domain 210 may have binding specificity (or affinity) for one or more TAAs, including but not limited to CD19, CD7, CD20, CD22, CD123, CD133, CD30, CD138, EGFR, EGFRvIII, FAP, MUC1, GD2, CEA, PSMA, HER2, NY-ESO-1, MAGEA-A3, hTERT, and the like. 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 may comprise two or three scFV domains (not shown) for the same TAA or different TAAs.

[0100] The hinge domain 220 operably couples the scFV domain 210 to the transmembrane domain 230. In an exemplary embodiment, the hinge domain 220 comprises a cluster of differentiation 8 (CD8) domain. The hinge domain 220 contributes to providing 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. 48 such that the hinge domain 220 comprises the polypeptide sequence defined by SEQ ID NO. 49.

[0101] 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 comprises a CD8 domain encoded by SEQ ID NO. 50 such that the transmembrane domain 230 comprises the polypeptide sequence defined by SEQ ID NO. 51. The transmembrane domain 230 contributes to operably coupling the domains of the CAR 200 present in the extracellular region 270a to the domains of the CAR 200 present in the intracellular region 270b. In an exemplary embodiment, as Figure 3 shown, the scFV domain 210 and the hinge domain 220 are the only domains present in the extracellular region 270a.

[0102] 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.52, such that the CD28 CSTM domain 240 comprises the polypeptide sequence defined by SEQ ID NO.53. The ICOS CSTM domain 240 can be encoded by SEQ ID NO.54, such that the ICOS CSTM domain 240 comprises the polypeptide sequence defined by SEQ ID NO.55. The OX40 CSTM domain 240 can be encoded by SEQ ID NO.56, such that the OX40 CSTM domain 240 comprises the polypeptide sequence defined by SEQ ID NO.57. The 4-1BB CSTM domain 240 can be encoded by SEQ ID NO.58, such that the 4-1BB CSTM domain 240 comprises the polypeptide sequence defined by SEQ ID NO.59. The DAP10 CSTM domain 240 can be encoded by SEQ ID NO.60, such that the DAP10 CSTM domain 240 comprises the polypeptide sequence defined by SEQ ID NO.61. The DAP12 CSTM domain 240 can be encoded by SEQ ID NO.62, such that the DAP12 CSTM domain 240 comprises the polypeptide sequence defined by SEQ ID NO.63. The 2B4 CSTM domain 240 can be encoded by SEQ ID NO.64, such that the 2B4 CSTM domain 240 comprises the polypeptide sequence defined by SEQ ID NO.65. In an embodiment, the CSTM domain 240 comprises 4-1BB. The CSTM domain 240 helps to enhance the cell-mediated immune response.

[0103] In an exemplary embodiment, CAR T cells are activated by exposing them to costimulatory molecules (e.g., CD3 and / or CD28). The costimulatory molecules stimulate the CSTM domain 240, which in turn results in the activation, proliferation, and / or differentiation of 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.

[0104] 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.66, such that the CD3ζ signaling domain 250 comprises the polypeptide sequence defined by SEQ ID NO.67. After the TAA binds to the scFV domain 210, the signaling domain 250 facilitates the transmission of an activation signal to the CAR T cell.

[0105] 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.68, such that the iCaspase 9 SS domain 260 comprises the polypeptide sequence defined by SEQ ID NO.69. In another exemplary embodiment, the SS domain 260 comprises RQR8 encoded by SEQ ID NO.70, such that the RQR8 SS domain 260 comprises the polypeptide sequence defined by SEQ ID NO.71. 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.

[0106] 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.

[0107] In another exemplary embodiment, the RQR8 SS domain 260 encodes a multi-epitope molecule containing a CD34 epitope and two CD20 mimic epitopes. When the CD20 mimic epitopes bind to the FDA-approved CD20 antibody rituximab, the host cell undergoes apoptosis.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] As Figure 4 shown, the above CAR T cells produce a negligible amount of cytokines, such as 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.

[0114] Although shRNA 130 is described in this disclosure in connection with examples of CAR 200 / 300, shRNA 130 can be used with any functionally equivalent CAR construct, and this is equally within the scope of the teachings of this disclosure.

[0115] The CAR T cells of the present invention can be prepared for each cancer patient by following Figure 5 the method 400 depicted in. Alternatively, the CAR T cells can be prepared from allogeneic T cell lines for all cancer patients.

[0116] Method 400 begins at step 401, where peripheral blood mononuclear cells (PBMCs) are isolated by a ficoll-based method or an automated PBMC separation instrument (such as in the case of apheresis). In an exemplary embodiment, PBMCs are isolated by drawing blood from a cancer patient in need of CAR T cell-based immunotherapy.

[0117] At step 403, a plurality of T cells can be isolated from the PBMCs. The isolated T cell population can contain a plurality of CD4 + and CD8 + T cells. The isolated T cell population can contain at least 40% CD4 + T cells. In an exemplary embodiment, the isolated T cell population contains 40% CD4 + T cells and 60% CD8 + T cells.

[0118] At optional step 405, the T cells are activated by subjecting them to costimulatory molecules (e.g., CD3 and / or CD28). In an exemplary embodiment, the costimulatory molecules help the T cells become capable of receiving ORF 100 via a lentiviral vector (described below).

[0119] At step 407, the ORF 100 of the present invention is prepared for delivery within the isolated 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.

[0120] 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) lentiviral system. In an exemplary embodiment of the present disclosure, ORF 100 is delivered to T cells via third-generation lentiviral particles, i.e., T cells are transduced with third-generation 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.

[0121] 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 to 40 times. The engineered immune cells suspended in the predefined nutrient medium can define a composition.

[0122] 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.

[0123] 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.

[0124] In an optional step 415, the selected transduced T cells expressing CAR 200, 300 (i.e., CAR T cells) are refrigerated for storage and transportation. The frozen CAR T cells are thawed before infusion into a patient.

[0125] Alternatively, as determined by a physician, the selected T cells (i.e., CAR T cells) can be directly infused into a patient.

[0126] 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.

[0127] Sequences that share at least 95% identity with the sequences disclosed in the present disclosure are within the scope of the teachings of the present disclosure.

[0128] 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 specified. Accordingly, the present disclosure should in no way be construed as limited to the following exemplary embodiments and examples, but should be construed to cover any and all variations that become apparent as a result of the teachings provided herein.

[0129] Example 1: CAR T cell preparation method (the present invention):

[0130] 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 Medium (DMEM) in a 10 cm tissue culture plate. The tissue culture plate containing HEK293T cells was then incubated at 37 °C, 5% CO2 for approximately 24 hours. After approximately 24 hours, the old medium was aspirated and replaced with 10 mL of fresh complete DMEM and 25 μM chloroquine diphosphate. The tissue culture plate containing HEK293T cells was further incubated at 37 °C, 5% CO2 for approximately 6 hours.

[0131] 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 the present disclosure) was inserted into HEK293T cells. The HEK293T cells were incubated at 37 °C, 5% CO2 for an additional approximately 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. The supernatant was then filtered using a 0.45 μm PES filter. The filtrate (supernatant) containing viral particles was collected and stored refrigerated to avoid titer loss. The viral particles have a lentiviral plasmid integrated with the CAR plasmid (containing ORF 100 of the present disclosure).

[0132] Via a process called leukapheresis, 10 million PBMCs were collected from every 20 mL of patient blood. T cells were then 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.

[0133] The isolated T cells were cultured in human T cell medium consisting of X-VIVO 15 (Lonza), 5% human AB serum, and 10 mM neutralized N-acetyl-L-cysteine (Sigma-Aldrich). Further, IL-2 was used for cell proliferation and was used at 30 units / mL IL-2.

[0134] 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.

[0135] At 48 hours, the primary T cells were exposed to the supernatant (containing viral particles) for transduction. On day 4 after T cell stimulation, the immunomagnetic beads were removed and the T cells were expanded until day 9. The T cells were sorted using FACs ARIA II. Thus, T cells exhibiting basal CAR expression (i.e., CAR T cells) were isolated.

[0136] The isolated CAR T cells were expanded in a nutrient medium at 37 °C, 5% CO2, which consisted of 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 approximately 7 - 9 days of expanding the CAR T cells, the number of CAR T cells increased by 20 - 40-fold. The CAR T cells were frozen for storage and transportation.

[0137] Example 2: Infusion of CAR T cells (the present invention):

[0138] 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 through mechanisms such as cytokine release and cytotoxicity.

[0139] 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 a response rate of 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.

[0140] Example 3: IL6 knockdown (the present invention):

[0141] The effects of 36 shRNA 130 sequences on the regulation of IL6 levels as described in the present disclosure were evaluated. Screening was performed in T cells. As Figure 6 shown, the bar graph depicts IL6 expression in activated T cells (Act-T cells) that were activated for 24 hours to induce IL6 expression or unactivated (control). The effects of non-targeting shRNA (Scram) and various IL6-targeting shRNA 130 (having SEQ ID NO.5 to SEQ ID NO.40) on knocking down the IL6 gene were evaluated in activated T cells. T cells were transfected with the corresponding shRNA130, and mRNA was extracted 24 hours after transfection. Determination was performed by RT-qPCR using IL6 mRNA-specific primers. Statistical analysis was performed using a non-parametric t-test, and the significance levels of **** (P<0.0001), *** (P<0.001), and ** (P<0.01) were revealed when comparing T cells expressing IL6 shRNA with T cells expressing Scram (see Figure 6 ). It was observed that the shRNA 130 sequences of the present disclosure significantly reduced the expression of the IL6 gene via RNAi.

[0142] Example 4: IL6 production (the present invention):

[0143] As described in Example 3, protein fractions were collected from the corresponding T cells. The collected protein fractions were subjected to an enzyme-linked immunosorbent assay (ELISA) to determine the amount of IL6 cytokine present in each protein fraction. As Figure 7 shown, the bar graph depicts the IL6 cytokine present in the corresponding protein fractions. Statistical analysis was performed using a non-parametric t-test, and the significance level of **** (P<0.0001) was revealed when comparing T cells expressing IL6 shRNA with T cells expressing Scram (see Figure 7 ).

[0144] Example 5: IL6 production in CAR T cells (the present invention):

[0145] The effects of the best-performing shRNA 130 sequences on the regulation of IL6 levels as observed in Examples 3 and 4 were evaluated. Screening was performed in CAR T cells. As Figure 8 shown, the bar graph depicts the IL6 produced in unactivated T cells (control). The non-targeting shRNA (CART Scram ) and the IL6-targeting shRNA 130 (CART shIL6) Role in knocking down the IL6 gene. Also evaluated 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 graph depicts the IL6 cytokine present in the corresponding protein fractions. Statistical analysis was performed using a non-parametric t-test, which revealed a significance level of **** (P < 0.0001) when comparing T cells expressing IL6 shRNA with T cells expressing Scram (see Figure 8 ).

[0146] 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 of the teachings of the present invention.

Claims

1. A recombinant nucleic acid molecule, the recombinant nucleic acid molecule being at least encoded by ORF(100, 100a) to reduce cytokine storm during immunotherapy, the recombinant nucleic acid molecule comprising: a. At least one hairpin loop structure, the at least one hairpin loop structure being formed by one or more short hairpin RNA (130) sequences, the at least one hairpin loop structure regulating the amount of interleukin 6 (IL6) cytokine during immunotherapy; and b. A first promoter (120), the first promoter being disposed upstream of the at least one hairpin loop structure.

2. The recombinant nucleic acid molecule according to claim 1, wherein The short hairpin RNA (130) 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 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.

4. The recombinant nucleic acid molecule according to claim 1, wherein A polyA tail (160) sequence is disposed downstream of the hairpin loop structure.

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

6. The recombinant nucleic acid molecule according to claim 1, wherein The messenger RNA of the hairpin loop structure transcribed from the recombinant nucleic acid molecule is configured to bind to the messenger RNA of the cytokine.

7. A recombinant nucleic acid molecule, the recombinant nucleic acid molecule being at least encoded by ORF(100) to reduce cytokine storm during immunotherapy, the recombinant nucleic acid molecule comprising: a. At least one hairpin loop structure, the at least one hairpin loop structure being formed by one or more short hairpin RNA (130) sequences, the at least one hairpin loop structure regulating the amount of interleukin 6 (IL6) cytokine during immunotherapy; b. A first promoter (120), the first promoter being disposed upstream of the at least one hairpin loop structure; c. One or more chimeric antigen receptor genes (150), the one or more chimeric antigen receptor genes expressing: i. One or more single-chain variable fragment domains (210), the 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), the one or more hinge domains being 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), the one or more signaling domains being coupled to the one or more co-stimulatory domains (240), v. One or more safety switch domains (260), the one or more safety switch domains being 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); d. A second promoter (140), which is disposed upstream of the one or more chimeric antigen receptor genes (150), and e. One or more long terminal repeats (110), which are disposed upstream of the first promoter (120) and downstream of the one or more chimeric antigen receptor genes (150).

8. The recombinant nucleic acid molecule according to claim 7, wherein The short hairpin RNA (130) is encoded by at least one of SEQ ID NO. 5 - SEQ ID NO. 40 or a nucleotide sequence having at least 95% identity thereto.

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

10. The recombinant nucleic acid molecule according to claim 7, 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).

11. The recombinant nucleic acid molecule according to claim 7, wherein The single-chain variable fragment domain (210) for cluster of differentiation 19 (CD19) is encoded by at least one of SEQ ID NO. 44 and SEQ ID NO.

46.

12. The recombinant nucleic acid molecule according to claim 7, wherein The one or more hinge domains (220) at least partially comprise cluster of differentiation 8 (CD8) encoded by SEQ ID NO.

48.

13. The recombinant nucleic acid molecule according to claim 7, 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. 52, Inducible T-cell Co-stimulator molecule (ICOS) encoded by SEQ ID NO. 54, OX40 encoded by SEQ ID NO. 56, 4-1BB encoded by SEQ ID NO. 58, DAP10 encoded by SEQ ID NO. 60, DAP12 encoded by SEQ ID NO. 62, and 2B4 encoded by SEQ ID NO.

64.

14. The recombinant nucleic acid molecule according to claim 7, wherein, The one or more signaling domains (250) at least partially comprise Cluster of Differentiation 3ζ (CD3ζ) encoded by SEQ ID NO.

66.

15. The recombinant nucleic acid molecule according to claim 7, wherein, The one or more safety switch domains (260) comprise at least one of Inducible Caspase 9 (iCaspase 9) encoded by SEQ ID NO. 68, truncated Epidermal Growth Factor Receptor (EGFRt), and RQR8 encoded by SEQ ID NO.

70.

16. The recombinant nucleic acid molecule according to claim 7, wherein The one or more transmembrane domains 230 at least partially comprise Cluster of Differentiation 8 (CD8) encoded by SEQ ID NO.

50.

17. The recombinant nucleic acid molecule according to claim 7, wherein The second promoter (140) is at least one of the full-length promoter of Elongation Factor 1 (EF1) encoded by SEQ ID NO. 42 and the EF1α core promoter encoded by SEQ ID NO.

43.

18. The recombinant nucleic acid molecule according to claim 7, 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).

19. The recombinant nucleic acid molecule according to claim 7, wherein The one or more long terminal repeats (110) are encoded by SEQ ID NO.

1.

20. The recombinant nucleic acid molecule according to claim 7, 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 molecule (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.

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

22. A recombinant nucleic acid molecule, at least encoded by an ORF (100a) to reduce cytokine storm during immunotherapy, the recombinant nucleic acid molecule comprising: a. At least one hairpin loop structure formed by one or more short hairpin RNA (130) sequences, the at least one hairpin loop structure regulating the amount of interleukin 6 (IL6) cytokine during immunotherapy; b. The first promoter (120), which is disposed upstream of the at least one hairpin loop structure; c. A poly-A tail (160) sequence disposed downstream of the hairpin loop structure; d. One or more chimeric antigen receptor genes (150), wherein the one or more chimeric antigen receptor genes express: i. One or more single-chain variable fragment domains (210), 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); e. A second promoter (140), which is disposed upstream of the one or more chimeric antigen receptor genes (150), and f. 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).

23. The recombinant nucleic acid molecule according to claim 22, wherein The short hairpin RNA (130) is encoded by at least one of SEQ ID NO.5 - SEQ ID NO.40 or a nucleotide sequence having at least 95% identity thereto.

24. The recombinant nucleic acid molecule according to claim 22, 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.

25. The recombinant nucleic acid molecule according to claim 22, wherein The poly-A tail (160) sequence is derived from simian virus 40 (SV40) encoded by SEQ ID NO.41 or bovine growth hormone beta globulin (bGH).

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

27. The recombinant nucleic acid molecule according to claim 22, wherein, The single-chain variable fragment domain (210) for cluster of differentiation 19 (CD19) is encoded by at least one of SEQ ID NO.44 and SEQ ID NO.

46.

28. The recombinant nucleic acid molecule according to claim 22, wherein, The one or more hinge domains (220) at least partially comprise cluster of differentiation 8 (CD8) encoded by SEQ ID NO.

48.

29. The recombinant nucleic acid molecule according to claim 22, 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.52, inducible T cell co-stimulator molecule (ICOS) encoded by SEQ ID NO.54, OX40 encoded by SEQ ID NO.56, 4-1BB encoded by SEQ ID NO.58, DAP10 encoded by SEQ ID NO.60, DAP12 encoded by SEQ ID NO.62, and 2B4 encoded by SEQ ID NO.

64.

30. The recombinant nucleic acid molecule according to claim 22, wherein, The one or more signaling domains (250) at least partially comprise cluster of differentiation 3ζ (CD3ζ) encoded by SEQ ID NO.

66.

31. The recombinant nucleic acid molecule according to claim 22, wherein The one or more safety switch domains (260) comprise at least one of inducible caspase 9 (iCaspase 9) encoded by SEQ ID NO.68, truncated epidermal growth factor receptor (EGFRt), and RQR8 encoded by SEQ ID NO.

70.

32. The recombinant nucleic acid molecule according to claim 22, wherein, The one or more transmembrane domains 230 at least partially comprise cluster of differentiation 8 (CD8) encoded by SEQ ID NO.

50.

33. The recombinant nucleic acid molecule according to claim 22, wherein, The second promoter (140) is at least one of the elongation factor 1 (EF1) full-length promoter encoded by SEQ ID NO.42 and the EF1α core promoter encoded by SEQ ID NO.

43.

34. The recombinant nucleic acid molecule according to claim 22, 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).

35. The recombinant nucleic acid molecule according to claim 22, wherein The one or more long terminal repeats (110) are encoded by SEQ ID NO.

1.

36. The recombinant nucleic acid molecule according to claim 22, 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 molecule (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.

37. The recombinant nucleic acid molecule according to claim 22, wherein The messenger RNA of the hairpin loop structure transcribed from the recombinant nucleic acid molecule is configured to bind to the messenger RNA of a cytokine.

38. A transcript of a recombinant nucleic acid molecule encoded by at least an ORF (100, 100a), the transcript comprising: a. A messenger RNA of a hairpin loop structure, the messenger RNA of the hairpin loop structure being transcribed from the recombinant nucleic acid molecule according to any one of claims 1 to 37, the messenger RNA of the hairpin loop structure being configured to bind to the messenger RNA of a cytokine.

39. A vector, the vector comprising: a. At least one of the recombinant nucleic acid molecules according to any one of claims 1 to 37, 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.

40. The carrier according to claim 39, 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.

41. An engineered immune cell, the engineered immune cell comprising: a. At least one of the recombinant nucleic acid molecules according to any one of claims 1 to 37.

42. The engineered immune cell according to claim 41, wherein The recombinant nucleic acid molecule is linked to at least one of a plasmid, a cosmid, a viral vector, and a phage.

43. The engineered immune cell according to claim 41, 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.

44. A composition, the composition comprising: a. Engineered immune cells, the engineered immune cells comprising at least one of the recombinant nucleic acid molecules according to any one of claims 1 to 37, b. Engineered immune cells, the engineered immune cells being suspended in a nutrient medium.

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

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

47. A method (400) for preparing an engineered immune cell, the method comprising: a. Isolating a plurality of T cells from a population of peripheral blood mononuclear cells; b. Replicating at least one recombinant nucleic acid molecule according to any one of claims 1 to 37 in a vector; c. Delivering the vector into the plurality of T cells.

48. The method (400) according to claim 47, characterized in that, Isolating the plurality of T cells includes isolating a plurality of CD4 + and CD8 + T cells.

49. The method (400) according to claim 47, characterized in that, After isolating the plurality of T cells, the plurality of T cells are contacted with a co-stimulatory molecule comprising at least one of cluster of differentiation 3 (CD3) and cluster of differentiation 28 (CD28).

50. The method (400) according to claim 47, characterized in that, Replicating the recombinant nucleic acid molecule comprises ligating the recombinant nucleic acid molecule to at least one of a plasmid, cosmid, viral vector, and phage.

51. The method (400) according to claim 47, 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 lentivirus, retrovirus, adenovirus, adeno-associated virus, and Sendai virus.

52. The method (400) according to claim 47, 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.

53. The method (400) according to claim 47, characterized in that, After delivering the vector into the plurality of T cells, the T cells are expanded in a predefined nutrient medium for a predefined period of time.

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