GM-CSF inhibitors and their uses

Abstract

This application provides a GM-CSF inhibitor and its use, specifically relating to an isolated nucleic acid molecule for inhibiting the expression of the GM-CSF gene in cells. The nucleic acid molecule comprises an antisense oligonucleotide, wherein the antisense oligonucleotide comprises at least 15 consecutive nucleotides differing from the nucleotide sequence shown in SEQ ID NO:4 by no more than 3 nucleotides, and the antisense oligonucleotide comprises a complementary region complementary to at least a portion of the mRNA encoding GM-CSF, the complementary region being 17-21 nucleotides in length. This application also relates to the use of the nucleic acid molecule in the preparation of CAR-T cells. Compared to wild-type T cells, T cells incorporating the nucleic acid molecule of this application show downregulated GM-CSF expression without affecting CAR expression or the cytotoxic activity of CAR-T cells.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to a GM-CSF inhibitor for reducing cytokine release syndrome and its use. Background Technology

[0002] Chimeric antigen receptor T-cell (CAR-T) cell therapy has emerged as a novel and potentially revolutionary treatment for cancer. However, its widespread use is limited by potentially fatal toxic side effects, including cytokine release syndrome (CRS) and neurotoxicity during CAR-T therapy. Up to 50% of patients receiving CAR-T19 cell therapy have experienced grade 3 or higher CRS or neurotoxicity, and several deaths have been reported. Currently, there are no effective treatments to prevent CRS.

[0003] The development of CRS is directly related to the proliferation of T cells and the high production of T cell effector cytokines, such as interleukin-6 (IL-6), interferon-γ (IFN-γ), monocyte chelate protein 1 (MCP-1), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Furthermore, preclinical studies have shown that IL-6, a key cytokine in the development of CRS, is not produced by CAR-T cells; instead, it is primarily produced by monocytes and macrophages. These observations provide a strong rationale for investigating GM-CSF neutralization as a potential strategy to reduce CRS and neuroinflammation (NI) associated with CAR-T cell therapy. Summary of the Invention

[0004] The purpose of this application is to provide a feasible method for reducing GM-CSF expression in activated CAR-T cells, which may help prevent CRS and thus improve the therapeutic effect of CAR-T therapy.

[0005] The purpose of this application is also to provide a method for preparing CAR-T cells with reduced GM-CSF expression, which may help prevent the occurrence of CRS, has no effect on the expression of CAR molecules, and has almost no effect on the killing effect of CAR-T cells.

[0006] On the one hand, this application provides an isolated nucleic acid molecule for inhibiting the expression of the GM-CSF gene in cells, the nucleic acid molecule comprising an antisense oligonucleotide, wherein the antisense oligonucleotide comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence shown in SEQ ID NO: 4 by no more than 3 nucleotides.

[0007] In some embodiments, the antisense oligonucleotide includes a complementary region that is substantially complementary to at least a portion of the mRNA encoding GM-CSF.

[0008] In some embodiments, the length of the complementary region is less than 30 nucleotides.

[0009] In some embodiments, the length of the complementary region is 17-21 nucleotides.

[0010] In some embodiments, the length of the complementary region is 19 nucleotides.

[0011] In some embodiments, the nucleic acid molecule inhibits the expression of GM-CSF by at least 20% when it comes into contact with cells expressing GM-CSF.

[0012] In some embodiments, the nucleic acid molecule is dsRNA.

[0013] In some embodiments, the nucleic acid molecule is siRNA.

[0014] In some embodiments, the siRNA comprises a sense strand and an antisense strand, the antisense strand comprising a complementary region substantially complementary to at least a portion of the mRNA encoding GM-CSF, and the antisense strand comprising at least 15 consecutive nucleotides differing from the nucleotide sequence shown in SEQ ID NO: 4 by no more than 3 nucleotides; the sense strand and antisense strand complement each other to form a complementary region of 17 to 21 nucleotides in length.

[0015] The siRNA comprises a sense strand and an antisense strand. The sense strand comprises SEQ ID NO: 10 or a sequence that differs from it by no more than 3 nucleotides, and the antisense strand comprises SEQ ID NO: 4 or a sequence that differs from it by no more than 3 nucleotides.

[0016] In some implementations, the length of each chain is between 17 and 23 nucleotides.

[0017] In some embodiments, the siRNA comprises the sense strand of the nucleotide sequence shown in SEQ ID NO: 10 and the antisense strand of the nucleotide sequence shown in SEQ ID NO: 4.

[0018] In some implementations, no more than three nucleotides in each chain are replaced by other nucleotides, while essentially maintaining the ability to suppress GM-CSF expression in cultured cells.

[0019] On the other hand, this application provides a carrier containing the nucleic acid molecules described in this application.

[0020] Cells, including the nucleic acid molecules or vectors described in this application.

[0021] In some implementations, it includes immune effector cells.

[0022] In some embodiments, the immune effector cells include T cells, B cells, natural killer (NK) cells, mast cells, or phagocytes.

[0023] In some embodiments, the immune effector cells contain nucleic acid molecules encoding chimeric antigen receptors (CARs).

[0024] In some embodiments, the nucleic acid molecule encoding CAR includes mRNA.

[0025] In some embodiments, the immune effector cells include engineered immune effector cells.

[0026] In some embodiments, the engineered immune effector cells include CAR-T cells.

[0027] On the other hand, this application provides the use of the nucleic acid molecules, vectors, or cells described in this application in the preparation of drugs for treating tumors.

[0028] In some embodiments, the tumor includes a solid tumor or a hematoma.

[0029] In some embodiments, the tumor expresses tumor-associated antigens.

[0030] In some embodiments, the tumor-associated antigen includes CD19.

[0031] In some embodiments, the drug includes CAR-T cells.

[0032] In some embodiments, the drug comprises anti-CD19 CAR-T cells.

[0033] On the other hand, this application provides the use of the nucleic acid molecule, the vector, or the cell described in this application in the preparation of a medicament for treating immunotherapy-related toxicities caused by GM-CSF release during CAR-T cell therapy.

[0034] In some embodiments, the immunotherapy-related toxicity is selected from cytokine release syndrome, neurotoxicity, neuroinflammation, or a combination thereof.

[0035] On the other hand, this application provides a composition comprising:

[0036] a. The nucleic acid molecule described in this application, the vector described in this application, or the cell described in this application; and

[0037] b. Drug-acceptable carriers.

[0038] On the other hand, this application provides a method for inhibiting GM-CSF expression in cells, the method comprising: contacting cells with the nucleic acid molecules described in this application.

[0039] In some embodiments, the contact is performed in vivo or in vitro.

[0040] In some embodiments, the method includes:

[0041] a) Introducing the nucleic acid molecule according to this application into the cell; and

[0042] b) The cells produced in step a) are maintained for a period of time sufficient to allow for the degradation of the mRNA transcripts of the GM-CSF gene, thereby inhibiting the expression of the GM-CSF gene in the cells.

[0043] In some implementations, the GM-CSF expression is suppressed by at least about 20%.

[0044] On the other hand, this application provides a method for regulating T cell function, the method comprising introducing the nucleic acid molecule described in this application into T cells.

[0045] In some embodiments, the level of one or more cytokines and / or chemokines expressed by the T cells incorporating the nucleic acid molecules described in this application is lower than or equal to the level of one or more cytokines and / or chemokines expressed by wild-type T cells.

[0046] In some embodiments, the expression of GM-CSF in T cells incorporating the nucleic acid molecules described herein is suppressed by at least about 20% compared to wild-type T cells.

[0047] In some embodiments, the method further includes modifying the T cells to be specific by administering a nucleic acid molecule encoding a CAR to the T cells.

[0048] In some embodiments, the nucleic acid molecule encoding CAR includes mRNA.

[0049] In some embodiments, the CAR includes an anti-CD19 CAR.

[0050] In some embodiments, the mRNA encodes an anti-CD19 CAR, which comprises the nucleotide sequence shown in SEQ ID NO: 13.

[0051] In some embodiments, the anti-CD19 CAR comprises the amino acid sequence shown in SEQ ID NO: 14.

[0052] In some embodiments, the nucleic acid molecules described herein and / or the nucleic acid molecules encoding CARs are introduced into T cells by any of the following methods: sonication, electrical pulses, electroporation, osmotic shock, calcium phosphate precipitation, DEAE dextran transfection, lipid-mediated delivery, and passive delivery.

[0053] In some embodiments, the nucleic acid molecule described herein and the nucleic acid molecule encoding CAR are introduced into T cells simultaneously via electroporation.

[0054] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0055] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and accompanying drawings described in detail below. A brief description of the drawings is given below.

[0056] Figure 1 The image shows the intracellular staining results of GM-CSF cytokines in T cells electroporated with different GM-CSF siRNAs and control siRNA (NC) according to this application.

[0057] Figure 2 The results shown are ELISA test results of T cells releasing GM-CSF cytokines via electroporation using different GM-CSF siRNAs and control siRNA (NC) according to this application.

[0058] Figure 3 The image shows the intracellular staining results of GM-CSF cytokines in T cells electroporated with anti-CD19 FMC63 CAR mRNA and different amounts of GM-CSF siRNA-4 or control siRNA (NC) at different time points.

[0059] Figure 4The image shows FACS staining results of T cells electroporated with anti-CD19 FMC63 CAR mRNA and different amounts of GM-CSF siRNA-4 or control siRNA (NC) at different time points using CD19-Fc recombinant protein.

[0060] Figure 5 The figure shows the killing curves of CAR-T cells containing 5 μg of anti-CD19 FMC63 CAR mRNA and different amounts of GM-CSF siRNA-4 or control siRNA (NC), with an E / T ratio of 3:1.

[0061] Figure 6 The figure shows the killing curves of CAR-T cells containing 5 μg of anti-CD19 FMC63 CAR mRNA and different amounts of GM-CSF siRNA-4 or control siRNA (NC), where the E / T ratio is 1:1. Detailed Implementation

[0062] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0063] In this application, “granulocyte-macrophage colony-stimulating factor” (GM-CSF) generally refers to a naturally occurring small glycoprotein with an internal disulfide bond and a molecular weight of approximately 23 kDa. In some embodiments, GM-CSF refers to human GM-CSF. In some embodiments, GM-CSF refers to non-human GM-CSF. In humans, it is encoded by a gene located within the cytokine cluster on chromosome 5. The sequences of the human gene and protein are known. The protein has an N-terminal signaling sequence and a C-terminal receptor-binding domain (Rasko and Gough, *The Cytokine Handbook*, A. Thomson et al., Academic Press, New York (1994), pp. 349-369). Although the amino acid sequences are not similar, its three-dimensional structure is similar to that of interleukins. GM-CSF is produced by mesenchymal cells present in hematopoietic environments and peri-inflammatory sites in response to a large number of inflammatory mediators. GM-CSF can stimulate bone marrow cells to produce neutrophils, macrophages, and mixed granulocyte-macrophage colonies, and can also stimulate fetal hepatic progenitor cells to form eosinophil colonies. GM-CSF can also stimulate certain functional activities of mature granulocytes and macrophages. GM-CSF (a cytokine present in the bone marrow microenvironment) recruits inflammatory monocyte-derived dendritic cells, stimulates the secretion of high levels of IL-6 and CCL2 / MCP-1, and leads to a feedback loop that recruits more monocytes and inflammatory dendritic cells to inflammatory sites.

[0064] In this application, the term "immunotherapy-related toxicity" generally refers to a range of inflammatory symptoms caused by high levels of immune activation. Different types of toxicity are associated with different immunotherapy approaches. In some embodiments, immunotherapy-related toxicities include capillary leakage syndrome, cardiomyopathy, respiratory disorders, CAR-T cell-associated encephalopathy syndrome (CRES), neurotoxicity, colitis, seizures, cytokine release syndrome (CRS), cytokine storm, decreased left ventricular ejection fraction, diarrhea, disseminated intravascular coagulation, edema, encephalopathy, rash, gastrointestinal bleeding, gastrointestinal perforation, phagocytic lymphohistiocytosis (HLH), liver disease, hypertension, hypophysitis, immune-related adverse events, immune-mediated hepatitis, immunodeficiency, local ischemia, hepatotoxicity, macrophage activation syndrome (MAS), pleural effusion, pericardial effusion, pneumonia, polyarthritis, posterior reversible encephalopathy syndrome (PRES), pulmonary hypertension, thromboembolism, and elevated transaminases.

[0065] Reducing or eliminating CRS and neurotoxicity is of great value in immunotherapies, such as CAR-T cell therapy, and identifying what drives or exacerbates characteristic CAR-T inflammatory responses is crucial. While many cytokines, signaling molecules, and cell types are involved in this pathway, GM-CSF appears to be a central cytokine. Normally undetectable in human serum, it is essential for the cyclical positive feedback loop that drives inflammation to extremes of cytokine storms and endothelial cell activation. Neurotoxicity and the cytokine storm are not the result of simultaneous cytokine release, but rather a cascade of inflammatory responses triggered by GM-CSF, leading to the transport and recruitment of myeloid cells to tumor sites. These myeloid cells produce the cytokines observed in CRS and neurotoxicity, thus perpetuating the inflammatory cascade.

[0066] In this application, the terms "isolated" or "purified" generally refer to molecules (e.g., peptides, nucleic acids, etc.) that have been at least partially isolated from other molecules that are normally bound to them in their natural state. The term "isolated nucleic acid molecule" refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to 3' ends (e.g., the GM-CSF siRNA nucleic acid sequence provided in this application) or similar, which has been isolated from at least about 50% of peptides, lipids, carbohydrates, polynucleotides, or other materials that are naturally found along with the nucleic acid molecules when total nucleic acids are isolated from source cells. In some embodiments, the isolated nucleic acid molecule is substantially free of any other contaminating nucleic acid molecules or other molecules found in the natural environment of the nucleic acid that could interfere with its use in peptide production or its therapeutic, diagnostic, prophylactic, or research uses.

[0067] In this application, the terms "nucleic acid," "nucleic acid molecule," and "polynucleotide" are used interchangeably and generally refer to a polymeric form of nucleotides (deoxyribonucleotides or ribonucleotides or their analogues) of any length. Polynucleotides can have any three-dimensional structure and can perform any function. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, siRNA, miRNA, shRNA, dsRNA, and primers. Polynucleotides may be modified or substituted at one or more bases, sugars, and / or phosphate esters with any of the various modifications or substitutions described in this application or known in the art. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogues. If present, the nucleotide structure may be modified before or after polymer assembly. The nucleotide sequence may be blocked by non-nucleotide components. Polynucleotides can be modified post-polymerization, for example, by coupling with a labeled component. The term can refer to both double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiment of a polynucleotide in this application includes both double-stranded forms and each of two complementary single-stranded forms known or predicted to constitute a double-stranded form.

[0068] In this application, the term "oligonucleotide" generally refers to a polymer composed of multiple nucleotide residues (deoxyribonucleotides or ribonucleotides, or their associated structural variants or synthetic analogs) linked by phosphodiester bonds (or their associated structural variants or synthetic analogs). Therefore, while the term "oligonucleotide" generally refers to a nucleotide polymer in which the nucleotide residues and their linkages are naturally occurring, it should be understood that the scope of the term also includes various analogs, including but not limited to: peptide nucleic acids (PNAs), aminophosphates, thiophosphates, methylphosphonates, 2-O-methylribonucleic acid, etc. The exact size of the molecule may depend on the specific application. Oligonucleotides are generally short in length, typically containing about 10-30 nucleotide residues, but the term can also refer to molecules of any length, although the terms "polynucleotide" or "nucleic acid" are generally used for larger oligonucleotides. In some embodiments, the oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and / or unmodified deoxyribonucleosides (DNA) and / or one or more modified nucleotides. The term "modified oligonucleotide" generally refers to an oligonucleotide that contains at least one modified nucleoside and / or at least one modified nucleoside-to-nucleotide bond.

[0069] In this application, the term "antisense oligonucleotide" refers to a single-stranded oligonucleotide molecule having a nucleobase sequence complementary to a corresponding fragment of a target nucleic acid (e.g., a target genomic sequence, a precursor mRNA, or an mRNA molecule). In some embodiments, the antisense oligonucleotide is 12 to 30 nucleobases in length. In some embodiments, the antisense oligonucleotide is an unmodified or modified nucleic acid having a nucleotide sequence complementary to the sequence of a target nucleic acid (such as GM-CSF mRNA).

[0070] In this application, the term "dsRNA" refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands, referred to as having "sense" and "antisense" orientations relative to the target RNA (i.e., the IFN-γ gene). In some embodiments of this application, the double-stranded RNA (dsRNA) triggers the degradation of the target RNA (e.g., mRNA) through a post-transcriptional gene silencing mechanism (referred to herein as RNA interference or RNAi). The double-stranded structure can be of any length that allows for the specific degradation of the desired target RNA via the RISC pathway, and can be in the length range of about 19 to 36 base pairs, for example, about 19-30 base pairs, for example, about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs. Ranges and lengths between the above ranges and lengths are also included in this application.

[0071] In this application, the term "short interfering RNA (siRNA)" refers to small double-stranded RNAs that interfere with gene expression. siRNAs are the transmitters of RNA interference (the process of double-stranded RNA silencing homologous genes). siRNAs typically consist of two single-stranded RNAs, each about 15-25 nucleotides long, forming a double strand, which may contain single-stranded overhangs. Processing of the double-stranded RNA by an enzyme complex, such as a polymerase, produces cleavage of the double-stranded RNA, thereby generating siRNAs. The RNA interference (RNAi) silencing complex uses the antisense strand of the siRNA to guide mRNA cleavage, thereby promoting mRNA degradation. To use siRNAs, for example, to silence specific genes in mammalian cells, base-pair regions are selected to avoid opportunistic complementation to unrelated mRNAs. RNAi silencing complexes have been identified in the art, for example, by Fire et al., Nature 391:806-81 (1998) and McManus et al., Nat. Rev. Genet. 3(10):737-747 (2002).

[0072] In this application, the term "antisense strand" generally refers to the strand of siRNA that includes a region substantially complementary to the target sequence. When used herein, the term "complementary region" generally refers to a region on the antisense strand that is substantially complementary to a sequence (e.g., the target sequence) as defined herein. When the complementary region is not perfectly complementary to the target sequence, mismatches can occur within the molecule or in terminal regions. Typically, the most permissible mismatches are in terminal regions, for example, within 5, 4, 3, or 2 nucleotides at the 5' and / or 3' ends.

[0073] In this application, the term "sense strand" generally refers to a strand of siRNA that includes regions substantially complementary to the region referred to herein as the antisense strand. The "sense" strand is sometimes called the "sense" strand, the "passenger" strand, or the "antiguide" strand. With their sequence, the antisense strand targets the desired mRNA, while the sense strand targets a different target. Therefore, if the antisense strand is incorporated into a RISC, the correct target is targeted. Incorporation of the sense strand can lead to off-target effects. These off-target effects can be limited by using modifications on the sense strand or by using a 5' cap.

[0074] In this application, the term "complementary" when used to describe a first nucleotide sequence (such as a sense strand or target mRNA) in relation to a second nucleotide sequence (such as an antisense strand) refers to the ability of an oligonucleotide or polynucleotide containing the first nucleotide sequence to hybridize (form base pair hydrogen bonds) with an oligonucleotide or polynucleotide containing the second nucleotide sequence under certain conditions to form a double helix or double-stranded structure. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs, and include native or modified nucleotides or nucleotide analogs, provided that the above requirements regarding their hybridization ability are met. "Complementarity" does not necessarily require nucleobase complementarity on every nucleotide. Instead, some mismatches are tolerable.

[0075] In this application, the terms "target nucleic acid" or "target sequence" generally refer to a continuous portion of the nucleotide sequence of the mRNA molecule formed during GM-CSF gene transcription, including mRNA of the RNA processing product, which is the major transcription product. The target portion of the sequence should be at least long enough to serve as a substrate for cleavage directed by antisense oligonucleotides or siRNA at or near the location of that portion of the nucleotide sequence of the mRNA molecule formed during GM-CSF gene transcription. In one embodiment, the target sequence is located within the protein-coding region of GM-CSF. The target sequence may be about 19-36 nucleotides in length, for example, preferably about 19-30 nucleotides in length. Ranges and lengths between the above ranges and lengths also include portions of this application.

[0076] In this application, the terms “reduction” and “reduction” are used interchangeably and generally refer to any change less than the original. “Reduction” and “reduction” are relative terms and need to be compared between before and after the measurement. “Reduction” and “reduction” include complete depletion.

[0077] In some embodiments, the term "reduction" refers to an overall reduction, detectable by standard methods known in the art (such as those described herein), of the expression level / amount of a gene, gene product (e.g., protein), or biomarker in a first sample compared to the expression level / amount of the corresponding gene, gene product (e.g., protein), or biomarker in a second sample by approximately 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the term "reduction" refers to a reduction in the expression level / amount of a gene or biomarker in the first sample, wherein the reduction is at least about 0.9-fold, 0.8-fold, 0.7-fold, 0.6-fold, 0.5-fold, 0.4-fold, 0.3-fold, 0.2-fold, 0.1-fold, 0.05-fold, or 0.01-fold in the expression level / amount of the corresponding gene or biomarker in the second sample. In some embodiments, the first sample is a sample obtained from a subject, and the second sample is a reference sample.

[0078] In this application, the term "pharmaceutically acceptable" generally refers to one or more non-toxic substances that do not interfere with the effectiveness of the biological activity of the active ingredient. Such formulations typically contain salts, excipients, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. These pharmaceutically acceptable formulations may also typically contain compatible solid or liquid fillers, diluents, or encapsulation materials suitable for human administration.

[0079] In this application, the term "prevention and / or treatment" includes not only the prevention and / or treatment of disease, but also generally includes preventing the onset of disease, slowing or reversing the progression of disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing and / or alleviating one or more symptoms associated with the disease, reducing the severity and / or duration of the disease and / or any symptoms associated with it and / or preventing a further increase in the severity of the disease and / or any symptoms associated with it, preventing, reducing or reversing any physiological damage caused by the disease, and any pharmacological effects that are generally beneficial to the patient being treated. The CAR-T cell or pharmaceutical composition of this application constitutes a viable therapeutic agent without achieving a complete cure or eradication of any symptom or manifestation of the disease. As recognized in the relevant art, a drug used as a therapeutic agent may reduce the severity of a given disease state, but does not need to eliminate every manifestation of the disease to be considered a useful therapeutic agent. Similarly, a treatment administered prophylactically constitutes a viable preventive agent without completely and effectively preventing the onset of the condition. Simply reducing the impact of the disease on the subject (e.g., by reducing the number or severity of their symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or reducing the likelihood of the disease occurring or worsening, is sufficient.

[0080] In this application, the terms “disease” or “symptom” are used interchangeably and generally refer to any deviation of a subject from a normal state, such as any change in the state of the body or certain organs that impairs or disrupts the performance of function, and / or causes symptoms such as discomfort, dysfunction, pain or even death in a person who is ill or in contact with such a person.

[0081] In this application, the term "tumor" generally refers to all neoplasmic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cellular proliferative disorder," "proliferative lesion," and "tumor" are not mutually exclusive when used herein. In this application, the tumor may include solid tumors and / or hematologic malignancies.

[0082] In this application, the term "administration" generally refers to the introduction of the pharmaceutical preparation of this application into the body of a subject by any route of introduction or delivery. Any method known to those skilled in the art for contacting cells, organs, or tissues with the drug may be employed. Administration may include, but is not limited to, intravenous, intra-arterial, intranasal, intraperitoneal, intramuscular, subcutaneous transdermal, or oral administration. The daily dose may be divided into one, two, or more doses in suitable forms to be administered at one, two, or more times during a certain time period.

[0083] In this application, the term "contact" generally refers to two or more substances of different types coming into contact with each other in any order, in any manner, and for any duration. Contact can occur in vivo, ex vivo, or in vitro.

[0084] In this application, the terms "effective amount" or "effective dose" generally refer to an amount sufficient to achieve or at least partially achieve the desired effect. A "therapeutic effective amount" or "therapeutic effective dose" of a drug or therapeutic agent generally refers to any amount of drug that, when used alone or in combination with another therapeutic agent, promotes disease remission (proven by a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or prevention of damage or disability due to the disease). A "preventive effective amount" or "preventive effective dose" of a drug generally refers to an amount of drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of disease development or relapse, inhibits the development or relapse of the disease. The ability of a therapeutic agent or preventive agent to promote disease remission or inhibit the development or relapse of the disease can be assessed using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, predicting efficacy in humans in animal model systems, or by determining the activity of the agent in an in vitro assay.

[0085] In this application, the term "subject" generally refers to a human or non-human animal (including mammals) requiring diagnosis, prognosis, improvement, prevention, and / or treatment of a disease, such as humans, non-human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), livestock (dogs and cats), farm animals (poultry such as chickens and ducks, horses, cattle, goats, sheep, pigs), and laboratory animals (mice, rats, rabbits, guinea pigs). Human subjects include fetuses, newborns, infants, adolescents, and adult subjects. Subjects include animal disease models.

[0086] In this application, the terms “comprising,” “including,” “having,” “may,” “containing,” and variations thereof are generally intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional actions or structures. The term “composed of” generally indicates that no other components can exist (or similarly, features, integers, steps, etc.). Unless the context clearly specifies otherwise, the singular forms such as “a,” “an,” “the” in English, and “a,” “a,” “the,” and “the” in Chinese generally include the plural form of the things referred to.

[0087] In this application, the term "about" generally means approximately, in the region of, roughly, or around. When the term "about" is used to refer to a range of values, the cutoff value or specific value is used to indicate that the stated value may differ from the listed value by up to 10%. Therefore, the term "about" can be used to cover variations of ±10% or less, ±5% or less, ±1% or less, ±0.5% or less, or ±0.1% or less from a specific value.

[0088] It should be understood that the term "at least" preceding a number or series of numbers includes the number adjacent to the term "at least," and all subsequent numbers or integers logically included, as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 19 nucleotides in a nucleic acid molecule of 21 nucleotides" means that 19, 20, or 21 nucleotides have the indicated property. When "at least" appears before a series of numbers or a range, it should be understood that "at least" can modify each number in that series or range.

[0089] It should be understood that "not more than" or "less than" as used herein refers to the value or integer adjacent to the phrase and logically lower, such as to zero, as the context suggests. For example, a double strand with "not more than 3 nucleotides" overhangs has 3, 2, 1, or 0 nucleotide overhangs. When "not more than" appears before a series of numbers or ranges, it should be understood that "not more than" can modify each number in that series or range. The ranges used herein include both upper and lower limits.

[0090] On one hand, this application provides an isolated nucleic acid molecule for inhibiting the expression of the GM-CSF gene in cells, the nucleic acid molecule comprising an antisense oligonucleotide, wherein the antisense oligonucleotide comprises at least 15 consecutive nucleotides selected from any one of the following sequences differing by no more than 3, 2 or 1 nucleotides: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

[0091] In some embodiments, the antisense oligonucleotide includes a complementary region that is substantially complementary or fully complementary to at least a portion of the mRNA encoding GM-CSF.

[0092] In some embodiments, the length of the complementary region is less than 30 nucleotides. For example, the complementary region may be 15 to 25 nucleotides in length, or, for example, 17 to 21 nucleotides in length.

[0093] For example, the complementary region is 19 nucleotides in length.

[0094] In some embodiments, the nucleic acid molecule inhibits the expression of GM-CSF by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% when it comes into contact with cells expressing the GM-CSF.

[0095] In some embodiments, the nucleic acid molecule is an antisense oligonucleotide.

[0096] In some embodiments, the nucleic acid molecule is dsRNA.

[0097] In some embodiments, the nucleic acid molecule is siRNA.

[0098] The siRNA described in this application may further include one or more single-stranded nucleotide overhangs, for example, 1, 2, or 3 nucleotides. The overhang may be on the sense strand, antisense strand, or any combination thereof. Furthermore, the overhanging nucleotides may be present at the 5'-end, 3'-end, or both ends of the antisense or sense strand of the siRNA. The overhang may be caused by one strand being longer than the other, or by two strands of equal length interleaved. The overhang may form a mismatch with the target mRNA, or it may be complementary to the targeted gene sequence, or it may be another sequence.

[0099] In some embodiments, the siRNA comprises a sense strand and an antisense strand, the antisense strand comprising a complementary region substantially complementary to at least a portion of the mRNA encoding GM-CSF, and the antisense strand comprising at least 15, 16, 17, 18, or 19 consecutive nucleotides selected from any one of the following sequences differing by no more than 3 nucleotides: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; the sense and antisense strands complement each other to form a complementary region of 17 to 21 nucleotides in length. For example, the sense and antisense strands complement each other to form a complementary region of 17, 18, or 19 nucleotides in length.

[0100] For example, the antisense strand contains any one of the following sequences that differ by no more than 3 nucleotides: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6; and the sense and antisense strands are complementary to form a complementary region of 17, 18 or 19 nucleotides in length.

[0101] In some embodiments, the siRNA is a 19-nucleotide-long, blunt-ended form.

[0102] For example, the siRNA comprises a sense strand and an antisense strand. The sense strand may comprise any one of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, or a sequence differing from it by no more than 3 nucleotides. The antisense strand may comprise any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, or a sequence differing from it by no more than 3 nucleotides.

[0103] In some implementations, the justice chain and the antisense chain may be selected from any one or more combinations of the following:

[0104] A sense strand containing the nucleotide sequence shown in SEQ ID NO: 7, and an antisense strand containing the nucleotide sequence shown in SEQ ID NO: 1;

[0105] A sense strand containing the nucleotide sequence shown in SEQ ID NO: 8, and an antisense strand containing the nucleotide sequence shown in SEQ ID NO: 2;

[0106] A sense strand containing the nucleotide sequence shown in SEQ ID NO: 9, and an antisense strand containing the nucleotide sequence shown in SEQ ID NO: 3;

[0107] A sense strand containing the nucleotide sequence shown in SEQ ID NO: 10, and an antisense strand containing the nucleotide sequence shown in SEQ ID NO: 4;

[0108] A sense strand containing the nucleotide sequence shown in SEQ ID NO: 11, and an antisense strand containing the nucleotide sequence shown in SEQ ID NO: 5; and

[0109] The sense strand contains the nucleotide sequence shown in SEQ ID NO: 12, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 6.

[0110] In some implementations, the length of each chain is between 17 and 23 nucleotides.

[0111] In some implementations, no more than three nucleotides in each chain are replaced by other nucleotides, while essentially maintaining the ability to suppress GM-CSF expression in cultured cells.

[0112] In some embodiments, the siRNA contains at least one modified nucleotide.

[0113] In some embodiments, one or more nucleotides on the sense strand and / or antisense strand are modified to form modified nucleotides.

[0114] In some embodiments, all nucleotides of the sense strand and all nucleotides of the antisense strand contain modifications.

[0115] On the other hand, this application provides a carrier containing the nucleic acid molecules described in this application.

[0116] In some embodiments, the vector includes a viral vector, which can be used to introduce the nucleic acid molecules of this application into cells. Such viral vectors include, for example, recombinant retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex virus-1. Retroviral vectors and adeno-associated virus vectors are generally understood as recombinant gene delivery systems selected for in vivo transfer of exogenous genes, particularly for entry into the human body. Alternatively, they can be used to introduce exogenous genes into T cells in vitro. These vectors efficiently deliver genes into T cells, and the transferred nucleic acids are stably integrated into the host cell's chromosomal DNA.

[0117] On the other hand, this application provides a cell that includes the nucleic acid molecule or the vector described in this application.

[0118] Methods for introducing nucleic acids into cells (such as T cells) are well known and routine practices in the art, including transformation, transfection, electroporation, nuclear injection, or fusion with vectors such as liposomes, micelles, ghost cells, and protoplasts. Host T cells can be isolated and / or purified. T cells can also be in vivo transformed cells to induce transient or permanent expression of peptides in vivo. The T cells can also be isolated cells transformed in vitro and introduced post-transformation, for example, to generate peptides for therapeutic purposes in vivo.

[0119] The antisense oligonucleotides or siRNAs of the present invention can be introduced into T cells using transfection methods well known in the art. These methods include sonication, electroporation, electroporation, osmotic shock, calcium phosphate precipitation and DEAE-dextran transfection, lipid-mediated delivery, passive delivery, etc. The term "transfection" encompasses a variety of techniques that can be used to introduce nucleic acids into mammalian cells, including electroporation, calcium phosphate precipitation, DEAE-dextran treatment, lipid transfection, microinjection, and viral infection. Suitable methods for transfecting mammalian cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press (1989)) and other laboratory textbooks.

[0120] In some implementations, it includes immune effector cells, such as T cells, B cells, natural killer (NK) cells, mast cells, or phagocytes.

[0121] In some embodiments, the immune effector cells include engineered immune effector cells, such as CAR-T cells.

[0122] On the other hand, a kit is provided that contains the nucleic acid molecules of this application, and optionally packaging inserts, packaging labels, instructions or other labels.

[0123] In this application, the term "chimeric antigen receptor" or "CAR" generally refers to a group of peptides, typically of two types in the simplest embodiment, which, when present in immune effector cells, provide cell-to-target cell specificity (typically cancer cells) and generate intracellular signaling. In some embodiments, the CAR comprises at least one extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain"), which includes functional signaling domains derived from stimulatory and / or costimulatory molecules as defined below. In some embodiments, the group of peptides is in the same polypeptide chain (e.g., comprising a chimeric fusion protein). In some embodiments, the group of peptides is discontinuous, e.g., in different polypeptide chains. In some aspects, the group of peptides includes a dimerization switch that can couple the peptides to each other in the presence of a dimerizing molecule, e.g., coupling the antigen-binding domain to the intracellular signaling domain. On one hand, the stimulatory molecule of the CAR is a ζ chain associated with the T-cell receptor complex. On another aspect, the cytoplasmic signaling domain includes a primary signaling domain (e.g., the primary signaling domain of CD3-ζ). In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one co-stimulatory molecule as defined below. In one aspect, the co-stimulatory molecule is selected from 4-1BB (i.e., CD137), CD27, ICOS, and / or CD28. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from the stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain, the intracellular signaling domain comprising a functional signaling domain derived from the co-stimulatory molecule and a functional signaling domain derived from the stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain, the intracellular signaling domain comprising a functional signaling domain derived from one or more co-stimulatory molecules and a functional signaling domain derived from the stimulatory molecule. On one hand, the CAR comprises a chimeric fusion protein containing an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the intracellular signal transduction domain comprises at least two functional signal transduction domains derived from one or more co-stimulatory molecules and a functional signal transduction domain derived from a stimulatory molecule. In another aspect, the CAR comprises an optional leader sequence at the N-terminus (N-ter) of the CAR fusion protein. In yet another aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (e.g., scFv) during cellular processing and localizes the CAR to the cell membrane.

[0124] On the other hand, this application provides a CAR-T cell, wherein the CAR-T cell is a CAR-T cell modified with the nucleic acid molecule described in this application.

[0125] The term "CAR-T" or "CAR-T cell" generally refers to a T cell capable of expressing a CAR (also known as a "chimeric antigen receptor"). The CAR typically refers to a fusion protein containing an extracellular domain capable of binding antigens and at least one intracellular domain. The CAR is a core component of chimeric antigen receptor T cells (CAR-T) and may include a targeting portion (e.g., a portion that binds to tumor-associated antigens (TAAs),) a hinge region, a transmembrane region, and an intracellular domain. In one embodiment, the CAR is Anti-CD19 FMC63, the sequence information of which is shown in Table 1.

[0126] Table 1 Anti-CD19 FMC63 CAR Sequence

[0127] SEQ IDNO. Name Sequence 13 Anti-CD19 FMC63 CAR nucleotide sequence atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggaccaagctggagatcacaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggccaaggaacctcagtcaccgtctcctcaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa 14 Anti-CD19 FMC63 CAR amino acid sequence MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 15 Anti-CD19 FMC63 scFv nucleotide sequence gacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggaccaagctggagatcacaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggccaaggaacctcagtcaccgtctcctca 16 Amino acid sequence of Anti-CD19 FMC63scFv DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS 17 CD8 signal peptide nucleotide sequence atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccg 18 CD8 signal peptide amino acid sequence MALPVTALLLPLALLLHAARP 19 CD8 Hinge nucleotide sequence accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgat 20 CD8 Hinge amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD 21 4-1BB costimulatory domain nucleotide sequence aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg 22 4-1BB costimulatory domain amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 23 CD3-zeta nucleotide sequence agagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa 24 CD3-zeta amino acid sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 25 Anti-CD19 FMC63LFR1 DIQMTQTTSSLSASLGDRVTISC 26 Anti-CD19 FMC63LCDR1 RASQDISKYLN 27 Anti-CD19 FMC63LFR2 WYQQKPDGTVKLLIY 28 Anti-CD19 FMC63LCDR2 HTSRLHS 29 Anti-CD19 FMC63LFR3 GVPSRFSGSGSGTDYSLTISNLEQEDIATYFC 30 Anti-CD19 FMC63LCDR3 QQGNTLPYT 31 Anti-CD19 FMC63LFR4 FGGGTKLEIT 32 Anti-CD19 FMC63VL DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT 33 Anti-CD19 FMC63HFR1 EVKLQESGPGLVAPSQSLSVTCTVSGVSLP 34 Anti-CD19 FMC63HCDR1 DYGVS 35 Anti-CD19 FMC63HFR2 WIRQPPRKGLEWLG 36 Anti-CD19 FMC63HCDR2 VIWGSETTYYNSALKS 37 Anti-CD19 FMC63HFR३ RLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAK 38 Anti-CD19 FMC63HCDR3 HYYYGGSYAMDY 39 Anti-CD19 FMC63HFR4 WGQGTSVTVSS 40 Anti-CD19 FMC63VH EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

[0128] In some embodiments, the CAR-T cells are CD19 CAR-T cells; in other embodiments, the CAR-T cells are BMCA CAR-T cells; in other embodiments, the CAR-T cells are dual CD19 / CD22 CAR-T cells. In other embodiments, the CAR-T cells are dual CD19 / CD20 CAR-T cells.

[0129] On the other hand, this application provides a composition comprising:

[0130] a. The nucleic acid molecule described in this application, the vector described in this application, the cell described in this application, or the CAR-T cell described in this application; and

[0131] b. Drug-acceptable carriers.

[0132] Pharmaceutically acceptable carriers are generally defined as substances suitable for administration to a subject that are biologically harmless or do not cause other adverse effects. Such carriers are typically inert components of the drug. Generally, the carrier is administered to the subject along with the active ingredient without causing any undesirable biological effects or interacting in a harmful manner with any other components of the pharmaceutical composition contained therein. Suitable drug carriers are described in Martin, Remington's Pharmaceutical Sciences, 18th ed., Mark Publishers, Easton, PA, (1990), the contents of which are incorporated herein by reference.

[0133] A more specific form of this application provides pharmaceutical compositions comprising a therapeutically effective amount of antisense polynucleotides and pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers. Such compositions include various buffer contents (e.g., phosphates, Tris-HCl, acetates), pH and ionic strength agents, and additives such as detergents and solubilizers (e.g., Tween 80, polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol), and fillers (e.g., lactose, mannitol). These substances can be incorporated into particulate formulations of polymeric compounds, such as, but not limited to, polylactic acid or polyglycolic acid, or into liposomes. Hyaluronic acid may also be used. Such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the disclosed compositions. The compositions can be prepared in liquid form or as dry powders, such as lyophilized forms. It should be understood that the pharmaceutical compositions provided in this disclosure can be administered in any manner known in the art. For example, the pharmaceutical composition for administration can be administered by injection, oral administration, or via the lungs or nose.

[0134] On the other hand, this application provides the use of the nucleic acid molecules, vectors, cells, or CAR-T cells described in this application in the preparation of drugs for treating tumors.

[0135] In some embodiments, the tumor includes a solid tumor or a hematoma.

[0136] In some embodiments, the tumor expresses tumor-associated antigens.

[0137] In some embodiments, the tumor expresses CD19, CD22, CD20 and / or BCMA.

[0138] In some embodiments, the tumors include acute lymphoblastic leukemia (ALL), acute myeloid leukemia, B-cell prolymphoblastic leukemia, B-cell acute lymphoblastic leukemia (BALL), blastic plasmacytic dendritic cell vegetations, Burkitt lymphoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloid leukemia, chronic or acute leukemia, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), hairy cell leukemia, Hodgkin's disease, malignant lymphoproliferative disorders, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma, myelodysplastic syndromes, non-Hodgkin's lymphoma (NHL), plasmacytosis, and other related conditions. Plasmoblastic lymphoma, plasmacytoid dendritic cell vegetations, plasmacytoma (including plasmacytic cachexia; solitary myeloma; solitary plasmacytoma; extramedullary plasmacytoma; and multiple plasmacytoma), POEMS syndrome (also known as Crow-Fukase syndrome; Takatsuki disease; and PEP syndrome), primary mediastinal large B-cell lymphoma (PMBC), small cell or large cell follicular lymphoma, splenic marginal zone lymphoma (SMZL), systemic amyloid light chain amyloidosis, T-cell acute lymphoblastic leukemia (TALL), T-cell lymphoma, transformed follicular lymphoma or Warschitz's macroglobulinemia, mantle cell lymphoma (MCL), transformed follicular lymphoma (TFL), primary mediastinal B-cell lymphoma (PMBCL), multiple myeloma, pilocellular lymphoma / leukemia or a combination thereof.

[0139] In some embodiments, the tumor includes acute lymphoblastic leukemia (BALL), chronic B-cell lymphoblastic leukemia (BCLL), B-cell Hodgkin's lymphoma (BHL), and non-Hodgkin's lymphoma (BNHL).

[0140] On the other hand, this application provides the use of the nucleic acid molecule, the vector, the cell, or the CAR-T cell described in this application in the preparation of a drug for immunotherapy-related toxicity caused by GM-CSF release during CAR-T cell therapy.

[0141] On the other hand, immunotherapy-related toxicities include brain disease, injury, or dysfunction. Specifically, brain disease, injury, or dysfunction includes CAR-T cell-related NT or CAR-T cell-related encephalopathy syndrome (CRES). Inhibiting or reducing the incidence of brain disease, injury, or dysfunction includes alleviating headache, delirium, anxiety, tremor, seizure activity, confusion, altered arousal, hallucinations, speech disorders, ataxia, apraxia, facial nerve palsy, motor weakness, seizures, non-convulsive EEG seizures, altered level of consciousness, coma, endothelial activation, vascular leakage, intravascular coagulation, or any combination thereof. On the other hand, immunotherapy-related toxicities include CAR-T-induced cytokine release syndrome (CRS). Inhibiting or reducing the incidence of CRS includes reducing or suppressing, but not limited to, high fever, myalgia, nausea, hypotension, hypoxia, or shock, or combinations thereof. In this regard, immunotherapy-related toxicities are life-threatening.

[0142] In some embodiments, the immunotherapy-related toxicity is selected from cytokine release syndrome, neurotoxicity, neuroinflammation, or a combination thereof.

[0143] On the other hand, this application provides a method for inhibiting GM-CSF expression in cells, the method comprising: contacting cells with the nucleic acid molecules described in this application.

[0144] In some embodiments, the contact is performed in vivo or in vitro.

[0145] In some embodiments, the method includes:

[0146] a) Introducing the nucleic acid molecule according to this application into the cell; and

[0147] b) The cells produced in step a) are maintained for a period of time sufficient to allow for the degradation of the mRNA transcripts of the GM-CSF gene, thereby inhibiting the expression of the GM-CSF gene in the cells.

[0148] In some implementations, the GM-CSF expression is suppressed by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0149] On the other hand, this application provides a method for regulating T cell function, the method comprising administering the nucleic acid molecule described in this application to the T cell.

[0150] In some embodiments, the level of one or more cytokines and / or chemokines expressed by the T cells incorporating the nucleic acid molecules described in this application is lower than or equal to the level of one or more cytokines and / or chemokines expressed by wild-type T cells.

[0151] In some embodiments, the expression of GM-CSF in T cells incorporating the nucleic acid molecules described herein is suppressed by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to wild-type T cells.

[0152] In some embodiments, the method further includes modifying the specificity of the T cells by administering a nucleic acid molecule encoding a chimeric antigen receptor (CAR) gene to the T cells.

[0153] In some embodiments, the nucleic acid molecule encoding the chimeric antigen receptor (CAR) gene includes mRNA.

[0154] In some implementations, the CAR includes a CD19 CAR.

[0155] In some embodiments, the CD19 CAR contains the amino acid sequence shown in SEQ ID NO: 14.

[0156] In some embodiments, the mRNA encodes a CD19 CAR, which comprises the nucleotide sequence shown in SEQ ID NO: 13.

[0157] In some embodiments, the nucleic acid molecules described herein and / or the nucleic acid molecules encoding the CAR gene are introduced into T cells by any of the following methods: sonication, electrical pulse, electroporation, osmotic shock, calcium phosphate precipitation, DEAE dextran transfection, lipid-mediated delivery, and passive delivery.

[0158] In some embodiments, the nucleic acid molecule described herein and the nucleic acid molecule encoding the CAR gene are introduced into T cells simultaneously via electroporation.

[0159] On the other hand, this application provides an immunotherapy comprising administering to a subject in need an effective amount of chimeric antigen receptor-expressing T cells (CAR-T cells), T cell receptor-modified T cells (TCR-T), tumor-infiltrating lymphocytes (TIL), chimeric antigen receptor-modified natural killer cells (CAR-NK), or dendritic cells, or any combination thereof, modified with the nucleic acid molecules described in this application.

[0160] A cytokine storm involves an immune response consisting of a positive feedback loop between cytokines and leukocytes, in which the levels of various cytokines are elevated. The term "cytokine storm" is used interchangeably with the terms "cytokine cascade" and "hypercytemia," which have all the same properties and meanings. In some embodiments, a cytokine storm is characterized by IL-2 release and lymphocyte proliferation. Cytokine storms can lead to potentially life-threatening complications, including cardiac dysfunction, adult respiratory distress syndrome, neurotoxicity, kidney and / or liver failure, and disseminated intravascular coagulation. Elevated GM-CSF levels can serve as both a predictive biomarker for CRS and an indicator of its severity. More than a key component of the inflammatory cascade, GM-CSF is a key initiator responsible for both CRS and NT.

[0161] As mentioned above, CAR-T cell therapy is currently limited by the risks of life-threatening neurotoxicity and CRS. Despite aggressive management, all CAR-T responders experience some degree of CRS. Up to 50% of patients treated with CD19 CAR-T have at least grade 3 CRS or neurotoxicity. GM-CSF levels and T-cell expansion are the factors most associated with grade 3 or higher CRS and neurotoxicity.

[0162] On the other hand, this application provides a method for treating and / or preventing immunotherapy-related toxicities in a subject, the method comprising administering CAR-T cells to a subject in need, the CAR-T cells being modified with the nucleic acid molecules described in this application to reduce the expression of the GM-CSF gene (e.g., gene silencing).

[0163] In some implementations, antisense oligonucleotides or siRNA have been transfected into T cells (such as CAR-T cells), and the method involves administering the transfected T cells to a subject. Antisense oligonucleotide transfection or siRNA can be introduced into T cells via electroporation.

[0164] In some embodiments, the immunotherapy-related toxicity is selected from cytokine release syndrome, neurotoxicity, neuroinflammation, or a combination thereof.

[0165] In some embodiments, the GM-CSF includes human GM-CSF (hGM-CSF).

[0166] In some embodiments, the levels of one or more cytokines and / or chemokines expressed by the CAR-T cells modified with the nucleic acid molecules described in this application are lower than or equal to the levels of one or more cytokines and / or chemokines expressed by wild-type CAR-T cells.

[0167] In some embodiments, the CAR-T cells are CD19 CAR-T cells.

[0168] In some embodiments, the subject is said to have acute lymphoblastic leukemia (BALL), chronic B-cell leukemia (BCLL), B-cell Hodgkin's lymphoma (BHL), non-Hodgkin's lymphoma (BNHL), diffuse large B-cell lymphoma (DLBCL), or primary mediastinal large B-cell lymphoma.

[0169] In some implementations, it also includes the application of a GM-CSF antagonist.

[0170] In some embodiments, administration of GM-CSF-silenced or gene-knockout CAR-T cells is performed in conjunction with a recombinant GM-CSF antagonist, which further improves expansion, persistence, tolerance to senescence, and tolerance to non-responsiveness. In some embodiments, the GM-CSF antagonist is an anti-GM-CSF antibody.

[0171] Without being limited by any theory, the embodiments described below are merely for illustrating the nucleic acid molecules, cells, and uses of this application, and are not intended to limit the scope of the invention. Example

[0172] Example 1: In vitro transcription (IVT) of CD19 FMC63 CAR mRNA

[0173] 1. The pDA-FMC63 CAR plasmid was linearized by digesting it with the Spe1 enzyme;

[0174] 2. The linearized vector was purified using a PCR Cleanup kit (Qiagen) and eluted with RNase-free water;

[0175] 3. The concentration of DNA was measured using a nanodropper and examined by running an agarose DNA gel.

[0176] 4. Perform IVT according to the manufacturer's protocol (Thermofisher, Cat No: AMB13455); specifically, add 1 μg template DNA, NTP / ARCA buffer, T7 buffer, GTP, T7 enzyme and RNase-free H2O in a volume of 20 μl to a 0.2 ml PCR tube and incubate at 37°C for 4 hours.

[0177] 5.4 hours later, 2 μL of DNase I was added to each reaction and incubated at 37°C for 15 minutes;

[0178] 6. Then follow the manufacturer's recommendations for the tailgating procedure;

[0179] 7. Purify IVT mRNA using the RNasy kit (Qiagen);

[0180] 8. Measure RNA concentration using a nanodropper and examine it by running a PAGE gel.

[0181] Example 2: Electroporation of mRNA and siRNA into A549-GFP or T cells

[0182] 1. Collect A549-GFP tumor cells and T cells, and wash them three times with Opti-MEM medium;

[0183] 2. Resuspend the cell particles in Opti-MEM medium and adjust the cell concentration to 1×10e7 / ml;

[0184] 3. Add 5 μg CD19 mRNA, 10 μg anti-CD19 FMC63 CAR mRNA, and GM-CSF siRNA (1.5 μM, 4.5 μM, 7.5 μM) or control siRNA (4.5 μM, 7.5 μM) to a 1.5 ml EP tube, then add 100 μl of T cells or A549 cells and mix well.

[0185] 4. Set the parameters on the BTX ECM 830 machine: a) For T cells: 500 voltage, 0.7 ms; b) For A549 tumor cells: 300 voltage, 0.5 ms;

[0186] 5. Add 100 μL of cells mixed with RNA to the BTX electroporation cup, and gently tap to avoid creating air bubbles;

[0187] 6. Perform electroporation, then transfer the cells to preheated culture medium and culture at 37°C to obtain anti-CD19 CAR-T cells or 549-GFP cells overexpressing CD19.

[0188] Example 3 Intracellular staining of GM-CSF cytokines in CAR-T cells

[0189] 1. Before staining, the CAR-T cells obtained in Example 2 were treated with 50 ng / ml PMA, 1 ug / ml Ionomycin and GolgiStop (BD Biosciences, 1500-fold dilution) for 6 hours using siRNA-4 electroporation.

[0190] 2.6 hours later, the CAR-T cells were transferred to a 96-well plate and rotated at 4°C and 1500 rpm for 3 minutes.

[0191] 3. Resuspend the cell particles in 100 μL / well of 1× fixation / permeabilization buffer and incubate at 4°C for 30 minutes (the fixation / permeabilization buffer is a mixture of fixation / permeabilization concentrate (Thermofisher, Cat. No. 00-5123-43) and fixation / permeabilization dilution (Thermofisher, Cat. No. 00-5223-56) in a 1:3 ratio).

[0192] 4. Add 100 μL of 1X permeabilization buffer (Thermofisher, Cat. No. 00-8333-56) to each fixed / permeabilized cell well and rotate at 4°C and 1800 rpm for 3 minutes;

[0193] 5. Wash the cells again with 200 μL of 1X permeability buffer and rotate at 4°C and 1800 rpm for 5 minutes.

[0194] 6. Dilute the anti-GM-CSF mobile antibody in 1X permeability buffer and stain CAR-T cells at 4°C for 30 minutes;

[0195] 7. Add 150 μL / well of 1X permeation buffer and rotate at 4°C and 1800 rpm for 5 minutes;

[0196] 8. Wash the cells again with 200 μL of 1X permeability buffer;

[0197] 9. Resuspend the cells in 200 μL FACS for flow cytometry analysis.

[0198] The results are as follows Figure 1 As shown, GM-CSF expression was downregulated in T cells electroporated with different GM-CSF siRNAs according to this application, with GM-CSF siRNA-4 showing the best knockout efficiency.

[0199] Intracellular staining of T cells electroporated with anti-CD19 FMC63 CAR mRNA and different amounts of GM-CSF siRNA-4 (1.5 uM, 4.5 uM, 7.5 uM) or control siRNA (NC) at different time points was performed. Data were analyzed by flow cytometry.

[0200] The results are as follows Figure 3 As shown, the inhibitory effect of GM-CSF siRNA-4 can last for at least 3 days.

[0201] Example 4: ELISA test for GM-CSF cytokine release from CAR-T cells

[0202] 1. Before collecting the supernatant, CAR-T cells were treated with 50 ng / ml PMA and 1 ug / ml Ionomycin in 96-well plates for 6 hours;

[0203] After 2.6 hours, spin-press at 4°C and 1500 rpm for 3 minutes;

[0204] 3. Then transfer the supernatant to a new 96-well plate;

[0205] 4. Detect GM-CSF cytokine levels using ELISA, as recommended by the manufacturer (Biolegend, Cat:432004).

[0206] The results are as follows Figure 2 As shown, among T cells electroporated with different GM-CSF siRNAs of this application, the knockout efficiency of GM-CSF siRNA-4 was the best.

[0207] Example 5: FACS staining of anti-CD19 CAR in CAR-T cells

[0208] 1. Collect T cells on days 1, 2, and 3 after T cell electroporation;

[0209] 2. Dilute CD19-Fc recombinant protein with FACS buffer to a final concentration of 2.5 μg / ml; stain CAR-T cells at 4°C for 30 minutes;

[0210] 3. After 30 minutes, wash the CAR-T cells three times with FACS buffer;

[0211] 4. Dilute the PE anti-human IgG Fc secondary antibody with FACS buffer at a dilution ratio of 1:200; stain the CAR-T cells at 4°C for 30 minutes.

[0212] 5. After 30 minutes, wash the cells twice with FACS buffer, then resuspend the cells in 200 μL of FACS for flow cytometry analysis;

[0213] The results are as follows Figure 4 As shown, electroporated siRNA-4 has no effect on the expression of CAR molecules.

[0214] Example 6: In vitro cytotoxicity assay of anti-CD19 CAR-T cells

[0215] 1. Twelve hours before co-culture, A549-EGFP cells electroporated with 5 μg CD19 mRNA were seeded into flat-bottomed 96-well plates at 3000 cells / 100 μl / well.

[0216] 2. Dilute CAR-T cells to an appropriate concentration and seed 100 μL / well of cells into tumor cells with different E / T ratios, such as 3:1 and 1:1;

[0217] 3. Place the co-culture plate into the IncuCyte S3 machine and set the scan parameters;

[0218] 4. After scanning for 4 days, analyze the total green object integrated intensity (TGOII, GCU x µm² / Well) and calculate the kill efficiency.

[0219] result Figures 5-6 As shown, anti-CD19 CAR-T cells electroporated with different amounts of siRNA-4 were able to kill tumor cells, and their killing effect was not affected compared with that of anti-CD19 CAR-T cells alone. sequence list <110> Shanghai U-Tee Biopharmaceutical Co., Ltd. <120> GM-CSF inhibitors and their uses <130> 0260-PA-004 <160> 40 <170> PatentIn version 3.5 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <223> GM-CSF siRNA-1 antisense strand <400> 1 acauuucuga gaugacuuc 19 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <223> GM-CSF siRNA-2 antisense strand <400> 2 aaggugauaa ucuggguug 19 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <223> GM-CSF siRNA-3 antisense strand <400> 3 agugucucua cucagguuc 19 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <223> GM-CSF siRNA-4 antisense strand <400> 4 ucucuacuca gguucagga 19 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <223> GM-CSF siRNA-5 antisense strand <400> 5 cagugucucu acucagguu 19 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <223> GM-CSF siRNA-6 antisense strand <400> 6 cucuacucag guucaggag 19 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <223> GM-CSF siRNA-1 Justice Chain <400> 7 gaagucaucu cagaaaugu 19 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <223> GM-CSF siRNA-2 Justice Chain <400> 8 caacccagau uaucaccuu 19 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <223> GM-CSF siRNA-3 Justice Chain <400> 9 gaaccugagu agagacacu 19 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <223> GM-CSF siRNA-4 Justice Chain <400> 10 uccugaaccu gaguagaga 19 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <223> GM-CSF siRNA-5 Justice Chain <400> 11 aaccugagua gagacacug 19 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <223> GM-CSF siRNA-6 Justice Chain <400> 12 cuccugaacc ugaguagag 19 <210> 13 <211> 1461 <212> DNA <213> Artificial Sequence <223> Anti-CD19 CAR nucleotide sequence <400> 13 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccggacatcc agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc 120 accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa 180 ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca 240 tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag 300 [[ID=1​​​​​​​​​​​gttttcttaa aaatgaacag tctgcaact gatgacacag ccatttacta ctgtgccaaa 720 cattattact acggtggtag ctagctatg gactactggg gccaaggaac ctcagtcacc 780 gtctcctcaa ccacgacgcc agcgccgcga ccacacac cggcgcccac catcgcgtcg 840 cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 900 agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 960 gtccttctcc tgtcactggt tatcacctt tactgcaac ggggcagaaa gaaactcctg 1020 tatatattca aaaaccatt tatgagacca gtashaacta ctcagagga agatggctgt 1080 agctgccgat ttccagaga agagaagga ggatgtgaac tgagagtgaa gttcagcagg 1140 agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta 1200 ggacgaagg aggagtacga tgttttggac agagacgtg gccgggaccc tgagatgggg 1260 ggaaagccga gaggagaa cccctcaggaa ggcctgtaca atgaacctgca gaagataag 1320 atggcggagg cctacagtga gattgggatg aaggcgagc gccggagggg caggggcac 1380 gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440 caggccctgc cccctcgcta a 1461 <210> 14 <211> 486 <212> PRT <213> Artificial Sequence <223> Anti-CD19 CAR Amino Acid Sequence <400> 14 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu 20 25 30 Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln 35 40 45 Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr 50 55 60 Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile 85 90 95 Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly 100 105 110 Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu 130 135 140 Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser 145 150 155 160 Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly 165 170 175 Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly 180 185 190 Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser 195 200 205 Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys 210 215 220 Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys 225 230 235 240 His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly 245 250 255 Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro 260 265 270 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 275 280 285 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 290 295 300 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 305 310 315 320 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 325 330 335 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 340 345 350 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 355 360 365 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 370 375 380 Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 385 390 395 400 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 405 410 415 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 420 425 430 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 435 440 445 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 450 455 460 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 465,470,475,480 Gln Ala Leu Pro Pro Arg 485 <210> 15 <211> 726 <212> DNA <213> Artificial Sequence <223> Anti‐CD19 FMC63 scFv <400> 15 gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60 atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120 gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180 aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240 gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300 gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 360 ggactgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 420 tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 480 cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 540 tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 600 ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 660 tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc 720 tcctca 726 <210> 16 <211> 242 <212> PRT <213> Artificial Sequence <223> Anti‐CD19 FMC63 scFv <400> 16 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr and Ser Cys Arg Ala Ser Gln Asp and Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu 115 120 125 Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys 130 135 140 Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg 145 150 155 160 Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser 165 170 175 Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile 180 185 190 Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln 195 200 205 Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly 210 215 220 Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val 225 230 235 240 Ser Ser <210> 17 <211> 63 <212> DNA <213> Artificial Sequence <223> CD8 signal peptide nucleotide sequence <400> 17 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccg 63 <210> 18 <211> 21 <212> PRT ​​​​​​​​​​​​​​​​​​​​​ <223> CD8 Hinge nucleotide sequence <400> 19 accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60 tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120 gacttcgcct gtgat 135 <210> 20 <211> 45 <212> PRT <213> Artificial Sequence <223> CD8 Hinge amino acid sequence <400> 20 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> twenty one <211> 126 <212> DNA <213> Artificial Sequence <223> 4-1BB costimulatory domain nucleotide sequence <400> twenty one aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60 actactcaag aggagatgg ctgtagctgc cgatttccag aagaagaga aggagatgt gaactg 126 <210> 22 <211> 42 <212> PRT <213> Artificial Sequence <223> 4‐1BB costimulatory domain cycle <400> 22 Light Arg Gly Arg Light Light Leu Leu Tyr Ile Phe Light Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Gly Cys Glu Leu 35 40 <210> 23 <211> 339 <212> DNA <213> Artificial Sequence <223> CD3‐zeta cycle cycle <400> 23 agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 120. tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagatagat ggcggaggcc tacagtgaga ttgggatga aggcgagcgc 240 cggaggggca aggggcacga tggctttac cagggtctca gtacagccac caggacacc 300 tacgacgccc ttcacatgca ggccctgccc cctcgctaa 339 <210> 24 <211> 112 <212> PRT <213> Artificial Sequence <223> CD3‑Zeta™ <400> 24 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 25 <211> 23 <212> PRT <213> Artificial Sequence <223> Anti‑CD19 FMC63 LFR1 <400> 25 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys 20 <210> 26 <211> 11 <212> PRT <213> Artificial Sequence <223> Anti‑CD19 FMC63 LCDR1 <400> 26 Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn 1 5 10 <210> 27 <211> 15 <212> PRT <213> Artificial Sequence <223> Anti‑CD19 FMC63 LFR2 <400> 27 Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr 1 5 10 15 <210> 28 <211> 7 <212> PRT <213> Artificial Sequence <223> Anti‑CD19 FMC63 LCDR2 <400> 28 His Thr Ser Arg Leu His Ser 1 5 <210> 29 <211> 32 <212> PRT <213> Artificial Sequence <223> Anti‑CD19 FMC63 LFR3 <400> 29 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser 1 5 10 15 Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys 20 25 30 <210> 30 <211> 9 <212> PRT <213> Artificial Sequence <223> Anti‑CD19 FMC63 LCDR3 <400> 30 Gln Gln Gly Asn Thr Leu Pro Tyr Thr 1 5 <210> 31 <211> 10 <212> PRT <213> Artificial Sequence <223> Anti‑CD19 FMC63 LFR4 <400> 31 Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr 1 5 10 <210> 32 <211> 107 <212> PRT <213> Artificial Sequence <223> Anti‑CD19 FMC63 VL <400> 32 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr 100 105 <210> 33 <211> 30 <212> PRT <213> Artificial Sequence <223> Anti‑CD19 FMC63 HFR1 <400> 33 Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro 20 25 30 <210> 34 <211> 5 <212> PRT <213> Artificial Sequence <223> Anti‑CD19 FMC63 HCDR1 <400> 34 Asp Tyr Gly Val Ser 1 5 <210> 35 <211> 14 <212> PRT <213> Artificial Sequence <223> Anti‑CD19 FMC63 HFR2 <400> 35 Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly 1 5 10 <210> 36 <211> 16 <212> PRT <213> Artificial Sequence <223> Anti‑CD19 FMC63 HCDR2 <400> 36 Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser 1 5 10 15 <210> 37 <211> 32 <212> PRT <213> Artificial Sequence <223> Anti‑CD19 FMC63 HFR3 <400> 37 Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys 1 5 10 15 Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys 20 25 30 <210> 38 <211> 12 <212> PRT <213> Artificial Sequence <223> Anti‑CD19 FMC63 HCDR3 <400> 38 His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr 1 5 10 <210> 39 <211> 11 <212> PRT <213> Artificial Sequence <223> Anti‑CD19 FMC63 HFR4 <400> 39 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 1 5 10 <210> 40 <211> 120 <212> PRT <213> Artificial Sequence <223> Anti‑CD19 FMC63 VH <400> 40 Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120

Claims

1. An isolated nucleic acid molecule, wherein the nucleic acid molecule is siRNA, which is used to inhibit the expression of the GM-CSF gene in anti-CD19 CAR-T cells, wherein the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO: 10, and the nucleotide sequence of the antisense strand of the siRNA is shown in SEQ ID NO:

4.

2. A vector comprising the nucleic acid molecule of claim 1.

3. Anti-CD19 CAR-T cells, comprising the nucleic acid molecule of claim 1 or the vector of claim 2.

4. The use of the anti-CD19 CAR-T cells according to claim 3 in the preparation of drugs for treating lung cancer.

5. A composition comprising: a. The nucleic acid molecule of claim 1, the vector of claim 2, or the anti-CD19 CAR-T cells of claim 3; and b. Drug-acceptable carriers.

6. A method for in vitro modulation of T cell function, the method comprising introducing the nucleic acid molecule of claim 1 into a T cell, and modifying the specificity of the T cell by introducing a nucleic acid molecule encoding an anti-CD19 CAR into the T cell.

7. The method of claim 6, wherein the nucleic acid molecule encoding anti-CD19 CAR comprises mRNA.

8. The method of claim 7, wherein the mRNA encodes anti-CD19 CAR, and the nucleotide sequence encoding anti-CD19 CAR is shown in SEQ ID NO:

13.

9. The method of claim 8, wherein the nucleic acid molecule of claim 1 and / or the nucleic acid molecule encoding CAR is introduced into T cells by any of the following methods: sonication, electroporation, osmotic shock, calcium phosphate precipitation, DEAE dextran transfection, and lipid-mediated delivery.

10. The method according to claim 9, wherein the nucleic acid molecule of claim 1 and the nucleic acid molecule encoding CAR are simultaneously introduced into T cells by electroporation.

Images