Use of cytokine antagonist in preparation of drug for preventing and / or treating crs

Abstract

The present invention relates to the field of cell therapies, and in particular to use of a cytokine antagonist in the preparation of a drug for preventing and / or treating death from CRS. Disclosed is use of a cytokine antagonist in the preparation of a drug for preventing and / or treating death from CRS, the cytokine antagonist being a TNFα antagonist.

Description

Use of a cytokine antagonist for the preparation of a medicament for preventing and / or treating CRS TECHNICAL FIELD

[0001] The present application relates to the field of cell therapy, in particular to the use of a cytokine antagonist for the preparation of a medicament for preventing and / or treating CRS lethality. BACKGROUND

[0002] One of the major side effects of CAR-T cell therapy is Cytokine Release Syndrome (CRS). CRS can be induced by many different stimuli, not only from drugs, but also from bacterial and viral infections. CRS has also been described as a Cytokine Storm, which stems from the overproduction of cytokines, not only from T cell stimulation, but also from the stimulation of Bystander immune cells such as monocytes and macrophages and non-immune cells such as endothelial cells. Cytokines that can be elevated in CRS include: IL-6, TNFa, IFNy, IL-8, IL-10, MCP-1, MIP-lbeta and GM-CSF. The result of CRS is a massive systemic inflammatory response that can extend to the central nervous system, leading to fatal neurotoxicity.

[0003] Furthermore, a severe complication of CRS is the development of neurotoxicity, such as immune effector cell-associated neurotoxicity syndrome (ICANS), which can lead to psychiatric disorders and death. CRS-induced neurotoxicity is associated with disruption of the blood-brain barrier by endothelial activation leading to CAR-T cell and high levels of cytokines into the brain.

[0004] The U.S. Food and Drug Administration recently approved an antibody against IL-6, tocilizumab, for the treatment of CAR-T cell-induced severe or life-threatening CRS (Le RQ. et al., FDA approval summary: Tocilizumab for treatment of chimeric antigen receptor T cell-induced severe or life-threatening cytokine release syndrome. The Oncologist 23:943-947 (2018)). However, tocilizumab, an IL-6 antibody, has been shown to have little effect in patients who received treatment for CRS-induced neurotoxicity (Gust J. et al., Endothelial activation and blood-brain barrier disruption in neurotoxicity after adoptive immunotherapy with CD19 CAR-T cells. Cancer Discov. 121:1404-1419 (2017)).

[0005] Therefore, there is an unmet need for a method or a related drug that can effectively prevent and / or treat CAR-T cell-induced CRS lethality. SUMMARY

[0006] The inventors of the present application first discovered that TNFα (Tumor Necrosis Factor alpha, “TNFα”) is the cytokine that causes CAR-T cell-induced CRS lethality; whereas the cytokine IL-6, which has been extensively studied in clinical trials, does not cause CAR-T cell-induced CRS lethality.

[0007] The inventors of the present application first discovered that TNFα antagonists, such as TNFα antibodies, can effectively prevent, treat, neutralize, reduce, or alleviate CAR-T cell-induced CRS lethality.

[0008] The first aspect of the present application provides the use of a cytokine antagonist, which is a TNFα antagonist, in the preparation of a medicament for preventing and / or treating CRS lethality.

[0009] In some embodiments of the present application, the TNFα antagonist includes a small molecule compound, an antibody, a nucleic acid, a peptide segment, a polypeptide, a fusion protein, a peptidomimetics, a carbohydrate, a glycomimetics, a lipid, and an aptamer.

[0010] Preferably, the TNFα antagonist includes an anti-TNFα antibody.

[0011] In the present application, the TNFα antagonist is not particularly limited as long as it can bind to TNFα and inhibit or block the activity of TNFα, including but not limited to Etanercept, Infliximab, Adalimumab, Golimumab, and Certolizumab Pegol, etc.

[0012] In some embodiments of the present application, the CRS occurs after the subject receives a cell therapy, which is selected from at least one of a CAR-T cell therapy, a CAR-NK cell therapy, and a CAR-M (Macrophage) cell therapy.

[0013] Preferably, the cell therapy includes a CAR-T cell therapy.

[0014] In some embodiments of the application, the therapeutically effective amount of the TNFα antagonist is administered to the subject at least one of before, concurrently with, within the first hour, within the second hour, within the third hour, within the fourth hour, within the fifth hour, within the sixth hour, within the seventh hour, within the eighth hour, within the ninth hour, within the tenth hour, within the eleventh hour, within the twelfth hour, within the thirteenth hour, within the fourteenth hour, within the fifteenth hour, within the sixteenth hour, within the seventeenth hour, within the eighteenth hour, within the nineteenth hour, within the twentieth hour, within the twenty-first hour, within the twenty-second hour, within the twenty-third hour, within the twenty-fourth hour (within the first day), within the second day, within the third day, within the fourth day, within the fifth day, within the sixth day, within the seventh day, within the eighth day, within the ninth day, within the tenth day, within the eleventh day, within the twelfth day, within the thirteenth day, within the fourteenth day, within the fifteenth day, within the sixteenth day, within the seventeenth day, within the eighteenth day, within the nineteenth day, within the twentieth day, within the twenty-first day, within the twenty-second day, within the twenty-third day, within the twenty-fourth day, within the twenty-fifth day, within the twenty-sixth day, within the twenty-seventh day, within the twenty-eighth day, within the twenty-ninth day, within the thirtieth day, within the thirty-first day, within the first month, within the second month, within the third month, within the fourth month, within the fifth month, within the sixth month, within the seventh month, within the eighth month, within the ninth month, within the tenth month, within the eleventh month, and within the twelfth month of the CAR-T cell therapy.

[0015] Preferably, the therapeutically effective amount is between 100 μg / kg and 10 mg / kg (Kilogram, "kg").

[0016] More preferably, the therapeutically effective amount is 1 mg / kg.

[0017] In some embodiments of the application, the CAR-T cell therapy comprises an ex vivo CAR-T cell therapy and an in vivo CAR-T cell therapy.

[0018] In some embodiments of the application, when the subject receives an ex vivo CAR-T cell therapy, the therapeutically effective amount of the TNFα antagonist depends on the amount of CAR-T cells administered to the subject.

[0019] In some embodiments of the application, when the subject receives an in vivo CAR-T cell therapy, the therapeutically effective amount of the TNFα antagonist depends on the amount of vector carrying a CAR gene administered to the subject.

[0020] In some embodiments of the application, the TNFα antagonist is a TNFα antibody, and the therapeutically effective amount is between 100 μg / kg and 10 mg / kg; preferably, the therapeutically effective amount is 1 mg / kg.

[0021] In the present application, the CRS lethality includes various conditions of CRS lethality induced after a subject receives a cell therapy, such as a CAR-T cell therapy, including but not limited to conditions of CRS-induced neurotoxicity lethality, etc.

[0022] In some embodiments of the present application, the CRS lethality includes CRS lethality of grade ≥3.

[0023] In some embodiments of the present application, the CRS lethality includes CRS-induced neurotoxicity lethality.

[0024] Preferably, the CRS-induced neurotoxicity lethality includes immune effector cell-associated neurotoxicity syndrome (ICANS) lethality.

[0025] In some embodiments of the present application, the CRS lethality includes CRS-induced ICANS lethality.

[0026] In some embodiments of the present application, the CRS lethality includes CRS-induced ICANS lethality of grade ≥3.

[0027] The second aspect of the present application also provides a composition comprising the TNFα antagonist provided by the first aspect of the present application.

[0028] Preferably, the composition can further comprise a pharmaceutically acceptable excipient or carrier.

[0029] The third aspect of the present application also provides a method for preventing and / or treating CRS lethality, the cytokine release syndrome occurring after a subject receives a cell therapy, the method comprising administering to the subject a therapeutically effective amount of a TNFα antagonist.

[0030] In some embodiments of the present application, the TNFα antagonist is selected from at least one of a small molecule compound, an antibody, a nucleic acid, a peptide segment, a polypeptide, a fusion protein, a peptidomimetic, a carbohydrate, a glycomimetic, a lipid, and an aptamer.

[0031] Preferably, the TNFα antagonist comprises an anti-TNFα antibody.

[0032] In some embodiments of the present application, the CRS occurs after a subject receives a cell therapy, the cell therapy being selected from at least one of a CAR-T cell therapy, a CAR-NK cell therapy, and a CAR-M cell therapy.

[0033] Preferably, the cell therapy comprises a CAR-T cell therapy.

[0034] In some embodiments of the application, the therapeutically effective amount of the TNFα antagonist is administered to the subject at least one of before, concurrently with, within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8 hours, within 9 hours, within 10 hours, within 11 hours, within 12 hours, within 13 hours, within 14 hours, within 15 hours, within 16 hours, within 17 hours, within 18 hours, within 19 hours, within 20 hours, within 21 hours, within 22 hours, within 23 hours, within 24 hours (within 1 day), within 2 days, within 3 days, within 4 days, within 5 days, within 6 days, within 7 days, within 8 days, within 9 days, within 10 days, within 11 days, within 12 days, within 13 days, within 14 days, within 15 days, within 16 days, within 17 days, within 18 days, within 19 days, within 20 days, within 21 days, within 22 days, within 23 days, within 24 days, within 25 days, within 26 days, within 27 days, within 28 days, within 29 days, within 30 days, within 31 days, within 1 month, within 2 months, within 3 months, within 4 months, within 5 months, within 6 months, within 7 months, within 8 months, within 9 months, within 10 months, within 11 months, and within 12 months of the subject receiving the CAR-T cell therapy;

[0035] Preferably, the therapeutically effective amount is 100 μg / kg to 10 mg / kg.

[0036] More preferably, the therapeutically effective amount is 1 mg / kg.

[0037] In some embodiments of the application, the CAR-T cell therapy comprises an ex vivo CAR-T cell therapy and an in vivo CAR-T cell therapy.

[0038] In some embodiments of the application, when the subject receives an ex vivo CAR-T cell therapy, the therapeutically effective amount of the TNFα antagonist depends on the amount of CAR-T cells administered to the subject.

[0039] In some embodiments of the application, when the subject receives an in vivo CAR-T cell therapy, the therapeutically effective amount of the TNFα antagonist depends on the amount of vector carrying a CAR gene administered to the subject.

[0040] In some embodiments of the application, the TNFα antagonist is a TNFα antibody, and the therapeutically effective amount is 100 μg / kg to 10 mg / kg; preferably, the therapeutically effective amount is 1 mg / kg.

[0041] In some embodiments of the present application, the CRS lethality comprises CRS lethality of grade ≥3.

[0042] In some embodiments of the present application, the CRS lethality comprises CRS-induced neurotoxicity lethality.

[0043] Preferably, the CRS-induced neurotoxicity lethality comprises immune effector cell-associated neurotoxicity syndrome (ICANS) lethality.

[0044] In some embodiments of the present application, the CRS lethality comprises CRS-induced ICANS lethality.

[0045] In some embodiments of the present application, the CRS lethality comprises CRS-induced ICANS lethality of grade ≥3.

[0046] The beneficial effects of the present application include:

[0047] The inventors of the present application first discovered that TNFα is a cytokine leading to CRS lethality induced by CAR-T cells, and that administration of a TNFα antagonist such as a TNFα antibody to a subject receiving CAR-T cell therapy can effectively prevent, treat, neutralize, alleviate or relieve CRS lethality induced by CAR-T cells; compared to the prior art using an IL-6 antibody and other non-TNFα antagonists to treat or relieve CRS, the present application provides a method for preventing and / or treating CRS lethality induced by CAR-T cells using a TNFα antagonist, which can significantly prevent, treat, alleviate, relieve or neutralize CRS lethality induced by CAR-T cells.

[0048] Definitions:

[0049] “TNFα antagonist”: In the present application, the TNFα antagonist is not particularly limited as long as it can bind to TNFα and inhibit or block the activity of TNFα, including but not limited to etanercept, infliximab, adalimumab, golimumab and certolizumab, etc.; the amount of TNFα antagonist is not particularly limited as long as it can effectively block the activity of TNFα, preferably, the therapeutically effective amount is 100 μg / kg-10 mg / kg (Kilogram, “kg”); more preferably, the therapeutically effective amount is 100 μg / kg, 200 μg / kg, 300 μg / kg, 400 μg / kg, 500 μg / kg, 600 μg / kg, 700 μg / kg, 800 μg / kg, 900 μg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg or 10 mg / kg; more preferably, the therapeutically effective amount is 1 mg / kg.

[0050] “CRS” stands for “Cytokine Release Syndrome”. CRS is a supraphysiological response that occurs after any immunotherapy that leads to the activation or involvement of endogenous or reinfused T cells and / or other immune effector cells. Symptoms may be progressive, and the initial presentation must include fever, which may be accompanied by hypotension, capillary leakage (leading to hypoxia), and end-organ dysfunction (Lee et al. Biol Blood Marrow Transplant, Dec 2018). It is graded from 0 to 4 according to the ASTCT (American Society for Transplantation and Cellular Therapy) consensus (2018-2019) (see Table 1 below).

[0051] Table 1

[0052] “ICANS” stands for “Immune Effector Cell-Associated Neurotoxicity Syndrome”. ICANS is a central nervous system toxicity reaction following immune effector cell therapy, manifested as cognitive, consciousness, language, motor abnormalities, seizures, and increased intracranial pressure (Lee et al. Biol Blood Marrow Transplant, Dec 2018). According to the ASTCT consensus, the ICE score is used in conjunction with five domains: consciousness, seizures, motor function, and intracranial pressure, and is graded into four levels, as detailed in Table 2 below.

[0053] Table 2

[0054] "Antibody" refers to a polypeptide or combination of polypeptides containing sufficient sequences from the variable regions of the immunoglobulin heavy chain and / or the variable regions of the immunoglobulin light chain, enabling it to specifically bind to an antigen. In this article, "antibody" encompasses various forms and structures, as long as they exhibit the desired antigen-binding activity.

[0055] By way of example, the antibodies of the present invention include, but are not limited to: immunoglobulins (full-length antibodies), haptens, Fab, Fab', F(ab')2, Fv fragments, single-chain variable region fragments (scFv), disulfide bond stable antibodies (dsFv), heavy chain variable regions (VH) or light chain variable regions (VL) of antibodies, Fd fragments composed of VH and CH1 domains, linear antibodies, heavy chain antibodies and nanobodies.

[0056] An "antibody" herein can be derived from any animal, including but not limited to humans and non-human animals, which can be selected from primates, mammals, rodents, and vertebrates, such as a camelid, a llama, an ostrich, an alpaca, a sheep, a rabbit, a mouse, a rat, or a chondrichthyan (e.g., a shark).

[0057] "Chimeric antigen receptor" or "CAR": refers to an artificial cell surface receptor engineered to be expressed on immune effector cells such as lymphocytes, which specifically binds to an antigen, comprising at least (1) an extracellular antigen binding domain, such as an antibody or antigen binding fragment thereof including a scFv or VHH, (2) a transmembrane domain anchoring the CAR molecule into the immune effector cell, and (3) an intracellular signaling domain; the extracellular structure of a CAR can further comprise a hinge region to improve the flexibility of the extracellular antigen binding domain, and the intracellular structure can further comprise one or more costimulatory molecules to form a costimulatory signaling domain to promote CAR-T cell proliferation and killing efficiency. The CAR molecule can redirect T cells and other immune effector cells to a selected target, such as a cancer cell, in a non-MHC restricted manner using the extracellular antigen binding domain.

[0058] "Nucleic acid": refers to any chemical and / or substance comprising a polymer of nucleotides, such as a polynucleotide. As used herein, "nucleic acid", "polynucleotide", and "gene" are used synonymously. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Typically, a nucleic acid molecule is described by the sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically represented as 5' to 3'. As used herein, the term "nucleic acid" encompasses deoxyribonucleic acids (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acids (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. A "nucleic acid" can be linear or circular. Furthermore, a "nucleic acid" includes both the sense (coding) strand and the antisense (template) strand, as well as single- and double-stranded forms. Moreover, a "nucleic acid" described herein can contain naturally-occurring or non-naturally-occurring nucleotides. Examples of non-naturally-occurring nucleotides include nucleotide bases modified with derivatized sugars, phosphate backbone linkages, or chemically modified residues.

[0059] “Viral envelope”: refers to the outermost layer of various viruses (HURLBERT, RONALD E., Fundamentals of Microbiology, 102. Chapter #11: Viruses.). The viral envelope protects the genetic material during its life cycle when the virus shuttles in host cells. Not all viruses have a viral envelope. Various human pathogenic viruses are enveloped in a lipid bilayer, which they use to infect target cells by fusing the viral envelope with the cell membrane. Viruses with a viral envelope include retroviruses, among others.

[0060] “Retrovirus”: i.e., Retroviruses. Retroviruses are double-stranded RNA enveloped viruses, whose main characteristic is the ability to “reverse transcribe” their genome from RNA to DNA. The virion measures 100-120 nm in diameter and contains a dimeric genome of identical positive RNA strands complexed with nucleocapsid proteins. The genome is encapsulated in a protein coat that also contains enzymatic proteins, i.e., reverse transcriptase, integrase, and protease, which are required for viral infection. A layer of matrix proteins is formed outside the core of the capsid, which interacts with the envelope of the lipid bilayer derived from the host cell membrane that surrounds the viral core particle. Anchored on this bilayer are the viral envelope glycoproteins responsible for recognizing specific receptors on host cells and initiating the infection process. The envelope (glyco)protein is formed by two subunits, anchoring the protein into the lipid membrane transmembrane (TM), and binding to cell receptors surface (SU).

[0061] Retroviruses contain reverse transcriptase and integrase. Upon entry into the target cell, retroviruses use their reverse transcriptase to transcribe their RNA molecule into a DNA molecule. Subsequently, the DNA molecule is integrated into the host cell genome using integrase. Upon integration into the host cell genome, the sequence from the retrovirus is referred to as a provirus (e.g., a sequence of a provirus or a proviral sequence).

[0062] Based on genomic organization, retroviruses are classified as simple retroviruses, such as MLV and murine leukemia virus; or complex retroviruses, such as HIV and EIAV. Retroviruses contain four genes: gag (group specific antigen), pro (protease), pol (polymerase), and env (envelope). The gag sequence encodes three major structural proteins: matrix protein, nucleocapsid protein, and capsid protein. The pro sequence encodes a protease responsible for cleavage of Gag and Gag-Pol during viral particle assembly, budding, and maturation. The pol sequence encodes a reverse transcriptase and an integrase, the former catalyzing the reverse transcription of viral genome from RNA to DNA during the infection process, and the latter responsible for the integration of proviral DNA into the host cell genome. The env sequence encodes the SU and TM subunits of the envelope glycoprotein. In addition, the retroviral genome presents non-coding cis-acting sequences, such as: two LTRs (long terminal repeat), which contain the elements required to drive gene expression, reverse transcription, and integration into the host cell chromosome; a sequence called the packaging signal (ψ) required for the specific packaging of viral RNA into newly formed virions; and a polypurine tract (PPT) as a site to prime plus-strand DNA synthesis during reverse transcription. In addition to gag, pro, pol, and env, complex retroviruses such as lentiviruses have accessory genes including vif, vpr, vpu, nef, tat, and rev, which modulate viral gene expression in infected cells, assembly of infectious viral particles, and regulation of viral replication.

[0063] “Lentivirus”: Lentiviruses are complex retroviruses that contain additional genes with regulatory or structural functions in addition to the common retroviral genes gag, pol, and env. The higher complexity allows the virus to modulate its life cycle, as it does during the process of latent infection. Lentiviruses belong to the Retroviridae family that can infect both dividing and non-dividing cells. Examples of lentiviruses include, but are not limited to, HIV (human immunodeficiency virus, including HIV type I and HIV type II), equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0064] “Pseudotyped”: As used herein, refers to an engineering approach to alter or expand the host cell tropism or stability of a viral vector by replacing or assembling the envelope glycoprotein of a viral vector of interest (such as an LVV) with a heterologous glycoprotein derived from another virus.

[0065] “Lentiviral Vector (LVV) and Retroviral Vector (RVV)”: As used herein, the term “Lentiviral Vector (LVV) or Retroviral Vector (RVV)” is intended to mean a self-inactivating viral particle that includes a viral envelope, has one or more features of a lentivirus or retrovirus, is capable of invading a target cell and delivering a gene of interest (GOI) of interest, and is not self-replicating.

[0066] LVVs and RVVs can stably integrate exogenous payload genes, such as CAR genes, into the chromosome of target cells, allowing the target cells to express the delivered transgene for a long period of time, providing a great advantage for gene therapy. In addition, they do not transfer viral genes, thus avoiding the problem of producing transduced cells that can be destroyed by cytotoxic T cells. And they have a relatively large clonal capacity, sufficient to meet most expected clinical applications.

[0067] “Packaging system”: As used herein, refers to a vector system comprising one or more nucleic acid vectors that contain nucleic acids necessary for the production, assembly, and / or packaging of lentiviral vectors or retroviral vectors in a packaging cell / packaging cell line when introduced into the packaging cell / packaging cell line for packaging of LVVs or RVVs.

[0068] In some embodiments of the present application, the packaging system comprises (a) nucleic acids encoding viral proteins necessary for the production, assembly, and / or packaging of LVVs or RVVs in a packaging cell line and (b) signals necessary to provide key functions such as viral replication, packaging, reverse transcription, and integration.

[0069] Commonly used packaging systems for lentiviral vectors include packaging systems for so-called third-generation LVVs. Packaging systems for third-generation LVVs include four plasmids, typically including a transfer plasmid and three packaging plasmids: a transfer plasmid / master plasmid containing a transgene / shuttle gene encoding a gene of interest / GOI, such as a CAR gene, a GagPol plasmid, a Rev plasmid, and an envelope plasmid containing a viral glycoprotein gene such as VSV-G or a variant thereof or Cocal-G or a variant thereof.

[0070] Generally, a “transfer vector” contains the lentiviral backbone genes, signals required for viral replication, packaging, reverse transcription, and integration, and other key functions. Transfer vectors typically have one or more transgenes flanked by long terminal repeat (LTR) sequences, which facilitate integration of the transgenes contained by the transfer vector, such as a CAR gene, into the host genome. The LTRs are responsible for the reverse transcription and integration processes of the viral genome. Through these sequences, lentiviruses can integrate transgenes into the genome of a host cell. For safety reasons, transfer vectors are often designed so that the resulting viral vectors are unable to self-replicate, e.g., the transfer vector lacks gene elements necessary for production of an infective LVV in a host cell. In addition, transfer vectors can be designed to have a 3’ LTR deleted, rendering the virus “self-inactivating.” The TAT gene is eliminated from third generation pseudotyped LVV packaging systems by adding a chimeric 5’ LTR fused to a heterologous promoter (e.g., CMV or RSV promoter) on the transfer vector, in contrast to traditional second generation pseudotyped LVV packaging systems, which are typically a single packaging plasmid containing nucleic acids encoding Gag, Pol, Rev, and Tat, and a separate envelope plasmid. Transfer vectors typically contain a Ψ sequence (Psi sequence, also known as the Ψ packaging signal) downstream of the 5’ LTR, which is responsible for packaging of the transgene RNA into the viral vector. The Ψ sequence ensures that only RNA containing the transgene is packaged into the viral vector. Transfer vectors can also optionally contain an Internal Ribosome Entry Site (“IRES”) to allow for the translation of two or more open reading frames (ORFs) on one mRNA, enabling multi-gene expression. Some transfer vectors, such as the master plasmid / transfer vector used in some embodiments of the present application, can also contain a selectable marker gene, such as an antibiotic resistance gene (e.g., PuroR, which encodes puromycin resistance) or a fluorescent protein gene (e.g., GFP), for selection or tracking of transduced cells.

[0071] Transfer vectors for packaging systems of LVVs are known in the art, see Naldini et al. (1996) Science 272:263-7; Zufferey et al. (1998) J. Virol. 72:9873-9880; Dull et al. (1998) J. Virol. 72:8463-8471; U.S. Patent No. 6,013,516; and U.S. Patent No. 5,994,136, each of which is incorporated by reference herein in its entirety. Generally, a transfer vector contains essential nucleic acid sequences configured to carry out the functions of selecting for cells containing the vector, incorporating foreign nucleic acids into lentiviral particles, and transferring nucleic acids to target cells.

[0072] Lentiviral backbone genes generally refer to those cis-acting elements that make up the most basic, essential portion of a transfer plasmid. These sequences do not encode viral proteins, but rather provide the signals necessary for viral replication, packaging, reverse transcription, and integration. Specifically, lentiviral backbone genes generally include: long terminal repeat sequences (LTRs): located at both ends of the genome, contain promoter, enhancer, and terminator functions, which regulate viral gene transcription and integration processes; packaging signal (Ψ): determines which RNA molecules are recognized and packaged into viral particles; central polypurine tract (cPPT) and central termination signal (CTS): help to increase reverse transcription efficiency and nuclear import; Rev response element (RRE): binds to the Rev protein, which regulates the transport of viral RNA from the nucleus to the cytoplasm.

[0073] In constructing transfer plasmids, to ensure safety and efficiency, only these necessary backbone sequences are generally retained in the transfer plasmid, while sequences encoding viral structural proteins and enzymes (e.g., gag, pol, env, etc.) are removed therefrom, to be provided by the packaging system in a helper plasmid. This design not only ensures the function of the vector, but also reduces the risk of generating replication-competent viruses.

[0074] In some embodiments of the present application, "backbone genes" are intended to include nucleic acids encoding lentiviral or retroviral cis nucleic acid sequences required for genome packaging. The backbone genes can also encode other cis nucleic acid sequences that are beneficial for gene delivery, including, for example, cis sequences required for reverse transcription, proviral integration, or genome transcription. Thus, the exact composition of the backbone genes will depend on the genetic material desired to be introduced into the target cell. Thus, the backbone genes can encode, for example, additional polypeptides or functions other than those required for packaging, reverse transcription, integration, or transcription. Such functions typically include encoding cis elements required for expression of the target nucleic acid / shuttle gene.

[0075] Transfer plasmids for lentiviral vectors and retroviral vectors are known in the art, see Naldini, et al., (1996) Science 272:263-7; Zufferey et al., (1998) J. Virol. 72:9873-9880; Dull et al., (1998) J. Virol. 72:8463-8471, U.S. Patent No. 6,013,516, and U.S. Patent No. 5,994,136, each of which is incorporated herein by reference in its entirety.

[0076] For a comparison and discussion of packaging systems for lentiviral vectors and packaging systems for retroviral vectors and their contained transfer plasmids see: Stripecke, R., Kasahara, N. (2007). Lentiviral and Retroviral Vector Systems. In: Hunt, K. K., Vorburger, S. A., Swisher, S. G. (eds) Gene Therapy for Cancer. Cancer Drug Discovery and Development. Humana Press.

[0077] Packaging systems for third generation lentiviral vectors typically also include three packaging plasmids: a GagPol plasmid, a Rev plasmid and an envelope plasmid. The envelope plasmid typically carries a viral envelope glycoprotein gene, for example wild-type VSV-G or Cocal-G is one of the commonly used viral glycoproteins; the viral glycoprotein gene is operably linked to a promoter, typically a CMV promoter, which initiates transcription of the viral glycoprotein gene. In some embodiments of the present application, the envelope plasmid comprises a nucleic acid sequence encoding any one of the aforementioned targeting molecules provided by the present application.

[0078] Packaging systems for third generation lentiviral vectors also include two packaging plasmids, one comprising genes encoding Gag and Pol proteins (GagPol packaging plasmid) and the other comprising a gene encoding Rev protein (Rev plasmid) as a further safety feature, which is an improvement over the single packaging plasmid of the so-called second generation packaging system. The Gag gene encodes a Gag polyprotein precursor comprising lentiviral structural proteins, including matrix, capsid and nucleocapsid; the Pol gene encodes a Pol polyprotein precursor providing lentiviral enzymatic functions necessary for replication, including protease, reverse transcriptase and integrase; the Rev gene encodes a Rev protein that binds to a Rev response element (RRE) to allow nuclear export of unspliced and singly spliced HIV RNAs during viral replication. The Gag and Pol polyprotein precursors are cleaved during viral vector production. The Rev protein binds to a Rev response element (RRE) sequence on the viral RNA, facilitating transport of incompletely spliced viral RNA from the nucleus to the cytoplasm by interacting with the nuclear export machinery of the host cell. These unspliced RNAs can be translated into viral structural proteins and enzymes in the cytoplasm or assembled into new viral vectors.

[0079] Exemplary, the packaging plasmids include, but are not limited to, pMD2.G, pRSV-rev, pMDLG-pRRE and pRRL-GOI.

[0080] In contrast to LVV packaging systems, RVV packaging systems generally do not include a Rev plasmid, as genomic RNA derived from retroviruses such as Moloney Murine Leukemia Virus ("MMLV") can naturally be transported from the nucleus to the cytoplasm for translation and assembly, thus obviating the need for a specific nuclear export mechanism, such as a Rev protein. RVV packaging systems generally include a transfer plasmid and two packaging plasmids: an envelope plasmid and a GagPol packaging plasmid. The transfer plasmid includes a transgene sequence flanked by long terminal repeat sequences (LTRs), which facilitate integration of the transfer plasmid sequence into the host genome. Generally, the sequence between and including the LTRs will be integrated into the host genome during viral transduction. The genome of MMLV or Murine Stem Cell Virus ("MSCV"), including their respective LTRs, are commonly used in the construction of transfer plasmids in RVV packaging systems. The GagPol packaging plasmid includes Gag and Pol genes; the envelope plasmid generally includes a polynucleotide encoding a viral glycoprotein, such as VSV-G or Cocal-G. In some embodiments of the present application, the envelope plasmid can also include a polynucleotide encoding any of the aforementioned targeting molecules.

[0081] In some embodiments, the production cells are transfected with a defined ratio of transfer plasmid, GagPol plasmid, envelope plasmid, and Rev plasmid. In some embodiments, the ratio of each plasmid is determined by mass, which is not particularly limited so long as a biologically active lentiviral vector or retroviral vector is packaged. In some embodiments, the mass of each of the transfer plasmid and the GagPol plasmid that package the lentiviral vector is higher than the mass of each of the envelope plasmid and the Rev plasmid. In some embodiments, the defined ratio of the transfer plasmid, the GagPol plasmid, the Rev plasmid, and the envelope plasmid is about 1 : 1 : 1 : 1 to about 10:5:4:4. In some embodiments, the defined ratio of the transfer plasmid, the GagPol plasmid, the Rev plasmid, and the envelope plasmid is about 9:4:2:2 to about 9:4:2:2. In some embodiments of the present application, the envelope plasmid can include a nucleic acid encoding the one or more targeting molecules.

[0082] In some embodiments, the enveloped plasmid comprises a polynucleotide encoding any of the preceding LVV or RVV surface-targeting molecules. In certain embodiments, the tandem expression cassette comprised in the enveloped plasmid comprises a polynucleotide encoding a first signal peptide, a polynucleotide encoding the targeting molecule, a polynucleotide encoding one of an internal ribosome entry site (IRES), a furin cleavage site, or a viral 2A peptide, a polynucleotide encoding a second signal peptide, and a polynucleotide encoding any of the preceding viral glycoproteins or variants thereof. In certain embodiments, the polynucleotide encoding any of the preceding viral glycoproteins or variants thereof is located 5' to the polynucleotide encoding the targeting molecule. In other embodiments, the polynucleotide encoding any of the preceding viral glycoproteins or variants thereof is located 3' to the polynucleotide encoding the targeting molecule. The polynucleotide encoding the targeting molecule and the polynucleotide encoding any of the preceding viral glycoproteins or variants thereof are separated in the tandem cassette by a polynucleotide encoding an IRES, a furin cleavage site, or a viral 2A peptide, which allows co-expression of the two proteins from a single mRNA. In certain embodiments, the viral 2A peptide is porcine teschovirus-1 (P2A), Thosea asigna virus (T2A), equine rhinovirus (E2A), foot-and-mouth disease virus (F2A), or variants thereof. In certain embodiments, the viral 2A peptide includes their derivatives, such as derivatives of the T2A peptide, T2A cleavage site with furin cleavage site and GSG linker, FT2A peptide. In certain embodiments, at least two different promoters independently drive the expression of the polynucleotide encoding any of the preceding viral glycoproteins or variants thereof and the polynucleotide encoding the targeting molecule, respectively.

[0083] “Packaging cell line”: The use of packaging systems for packaging lentivirus / retrovirus vectors relies on “packaging cells” and “packaging cell lines”. Generally, a packaging cell line is a cell line whose cells are capable of producing lentiviral or retroviral vectors that are not self-replicating, that can infect / transduce target cells upon introduction of a packaging system, e.g., comprising a transfer plasmid, one or more packaging plasmids, into the cells. An overview of available packaging lines is provided in JM Coffin, SM Hughes, et al. Cold Spring Harbour Laboratory Press, 1997, p. 447, incorporated herein by reference in its entirety.

[0084] Exemplarily, various plasmids can be introduced into the packaging cell line using transfection methods including chemical-mediated transfection methods, physical-mediated transfection methods, or biological-mediated transfection methods, etc. For example, the chemical-mediated transfection methods include transfection using chemical reagents such as calcium phosphate, DEAE-dextran, or PEI (Polyethylenimine, a transfection reagent), etc. The physical-mediated transfection methods include transfection using electroporation, etc.

[0085] Production / hosting / packaging cells that can be used to prepare the LVV or RVV provided herein include, but are not limited to, human embryonic kidney (HEK) 293 cells and their derivatives. The production cells can be adherent cell lines such as HEK-293T production cells, or suspension cell lines such as HEK-293T / 17SF production cells.

[0086] Exemplarily, the packaging cells / production cells are selected from CHO cells, BHK cells, MDCK cells, C3H-10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC-1 cells, BSC-40 cells, BMT-10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, HEK-293 cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, and 211 cells.

[0087] Preferably, the packaging cells / production cells are HEK-293T cells.

[0088] The packaging cells can be genetically engineered to otherwise improve the immunological properties of the LVV and RVV provided herein and / or to increase the efficiency of the LVV and RVV to transduce target cells; the otherwise including, but not limited to, adding genes, deleting genes, and introducing point mutations into genes, etc.

[0089] “Subject”: As used herein, “subject”, “patient”, and “individual” are used synonymously and include, but are not limited to, mammals, such as humans or non-human mammals, e.g., domestic animals, agricultural animals, or wild animals, as well as birds and aquatic animals. A “patient” is a subject afflicted with, at risk of developing, or otherwise in need of any one of the lentiviral or retroviral vectors, engineered immune cells, compositions, or therapeutic methods provided herein.

[0090] "Pharmaceutically acceptable excipient or carrier": Pharmaceutically acceptable excipients or carriers include, but are not limited to, diluents, solubilizers, emulsifiers, preservatives, and / or adjuvants. Preferably, the compositions disclosed herein also include an adjuvant that is nontoxic to recipients at the dosages contemplated. Such adjuvants include, but are not limited to, saline, buffers, glucose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, the compositions can contain substances that improve, maintain or preserve, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or permeation of the composition. The optimal composition can be determined depending on the intended route of administration, mode of delivery, and desired dosage.

[0091] "Therapeutically effective amount": The therapeutically effective amount of a TNFα antagonist or composition will depend, for example, on the degree and target of therapy. Those skilled in the art will know or be able to determine a proper dosage level for treatment, which will vary depending, inter alia, on the molecule being delivered, the indication, the route of administration, and the patient's condition (body weight, body surface or organ size) and / or status (age and general health).

[0092] The frequency of dosing will depend on the pharmacokinetic parameters of the TNFα antagonist or pharmaceutical composition in the formulation being used. The clinician typically will administer the pharmaceutical composition until a dosage is reached that achieves the desired effect. The pharmaceutical composition can therefore be administered as a single dose, or as two or more doses (which can or can not contain the same amounts of the desired molecule) over time, or by continuous infusion via an implantation device or catheter.

[0093] "Administration": Routes of administration of the pharmaceutical composition are routine in the art, e.g., oral, nasal, by injection intravenously, intraperitoneally, intracerebrally (intra-parenchymal), intracerebroventricularly, intramuscularly, intraocularly, intraarterially, portal, in situ, or intralesionally, and can also be by sustained release systems or by implantation devices.

[0094] "Treat": To subject a subject to a therapeutic method described herein to achieve at least one positive therapeutic effect (e.g., reduction of CRS and its associated neurotoxicity). The therapeutic method effective to treat a patient can vary depending on a variety of factors, such as the patient's disease state, age, body weight, and the ability of the therapy to elicit an anti-cancer response in the subject.

[0095] "Treat" as used herein includes any beneficial or desired effect associated with treatment. "Treatment" does not necessarily indicate complete eradication or cure of the disease or condition, or its associated symptoms.

[0096] “Prevention”: As used herein, “prevention” and similar terms, such as “avoidance”, indicate methods used to prevent, suppress, or reduce the likelihood of the occurrence or recurrence of a condition, for example, preventing CRS-related death in subjects receiving CAR-T cell therapy. As used herein, “prevention” and similar terms also include reducing the intensity, effectiveness, symptoms, and / or burden of a disease or condition before its onset or recurrence.

[0097] "And / or": should be understood as referring to one or two alternatives.

[0098] "Comprising": In this document, unless the context otherwise requires, the word "comprising" will be understood to mean including the specified steps, elements, or groups of steps or elements, but not excluding any other steps, elements, or groups of steps or elements. In some embodiments of the invention, the terms "comprising," "having," "containing," and "including" are used synonymously.

[0099] "Implementation Scheme": Throughout this specification, references to "some implementation schemes" and "some embodiments," or combinations thereof, mean that a particular feature, structure, or characteristic described in connection with an implementation scheme is included in at least one embodiment of the invention. Therefore, the appearance of the foregoing phrase in various places throughout this specification does not necessarily refer to the same implementation scheme. Furthermore, specific features, structures, or characteristics can be combined in any suitable manner in one or more implementation schemes.

[0100] “Transduction”: As used herein, the terms “transfection,” “transformation,” and “transduction” are used synonymously and refer to the process of transferring or introducing exogenous nucleic acids into host cells or packaging cells. “Transfected,” “transformed,” or “transduced” cells are cells that have been transfected, transformed, or transduced with exogenous nucleic acids. These cells include primary subject cells and their progeny.

[0101] Physical methods for introducing vectors or isolated polynucleotides into immune effector cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art (see Sambrook, J., Fritsch, E.F. and Maniatis, T. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.). In some embodiments of the present invention, the vector is introduced into cells via electroporation. In some embodiments of the present invention, the vector is introduced into cells via a PEI transfection reagent.

[0102] All publications, documents, and patent references cited herein are hereby incorporated by reference in their entirety as if each had been individually incorporated by reference herein, to the extent that they provide exemplary, procedural, or other appropriate guidance to the practice of the application. In case of conflict between the disclosure herein and any incorporated literature, the present application, including any definitions herein, will control. However, any reference, article, publication, patent, patent publication, and patent application cited herein is not intended by itself, nor in combination with anything else said herein, to be an admission that the information, material, or acts disclosed in the reference, article, publication, patent, patent publication, or patent application are prior art to the present application.

[0103] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. BRIEF DESCRIPTION OF DRAWINGS

[0104] Figure 1 is a graph showing the results of detecting whether different amounts of TNFα are lethal in mice in Example 1.

[0105] Figure 2 is a graph showing the results of detecting whether IL-6 or IFN-g is lethal in mice in Example 1.

[0106] Figure 3 is a plasmid map of the master plasmid 1 in Example 2.

[0107] Figure 4 is a graph showing the results of detecting whether a TNFα antagonist affects the efficiency of CAR-T cells in killing target cells in vitro in Example 2.

[0108] Figure 5 is a graph showing the growth curves of A20 modeling mice in each group in Example 3.

[0109] Figure 6 is a graph showing the survival results of A20 modeling mice in each group in Example 3.

[0110] Figure 7 is a plasmid map of the master plasmid 2 in Example 3. DETAILED DESCRIPTION

[0111] The concept of the present application and the technical effects produced thereby will be described below in conjunction with the embodiments so as to fully understand the technical solutions, the technical problems solved, and the beneficial effects of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present application.

[0112] The experimental methods not specified in the following examples are selected according to the conventional methods and conditions known in the art, or according to the instructions of the commercial product. The reagents and raw materials not specified in the present application are commercially available.

[0113] Example 1

[0114] 1. Detect whether TNFα is lethal

[0115] Day 0, inject different amounts of TNFα into each group of B6 mice (C57BL / 6 mice) according to the injection method shown in Table 3 below, and the survival results of each group of mice are shown in FIG. 1A-1E.

[0116] Table 3

[0117] As can be seen from FIG. 1, mice injected with more than 20 μg of TNFα all died on Day 1.

[0118] Therefore, excessive TNFα can cause mice to die, and TNFα is a lethal factor that causes CRS to be lethal.

[0119] TNFα: Brand: Yikai God, product name: Recombinant Mouse TNF-alpha Protein (ECD), item number: #50349-MNAE.

[0120] 2. Detect whether IL-6 or IFN-g is lethal

[0121] Day 0, inject IL-6 or IFN-g of the corresponding components into each group of B6 mice according to the injection method shown in Table 4 below, and the survival results of each group of mice are shown in FIG. 2A-2D.

[0122] Table 4

[0123] As can be seen from FIG. 2, unlike injecting excessive TNFα causing mice to die, injecting excessive IL-6 or IFN-g does not cause mice to die, and IL-6 and IFN-g are not lethal factors that cause CRS to be lethal.

[0124] IL-6 brand: Yikai God, product name: Recombinant Mouse IL-6 Protein, item number: #50136-MNAE;

[0125] IFN-g: Brand: Yikai God, product name: Recombinant Mouse IFN gamma Protein, item number: #50709-MNAH.

[0126] Example 2

[0127] Detect whether TNFα antibody affects the killing efficiency of CAR-T cells

[0128] 1. Package lentiviral vector-hCAR19 carrying CAR gene

[0129] A. Preparation of lentivirus vector packaging system:

[0130] The following four plasmids were prepared: pMD2.G envelope plasmid (wild type VSV-G), pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid and master plasmid 1; the master plasmid 1 comprises a polynucleotide encoding a chimeric antigen receptor targeting human CD19 (hCAR-19 molecule) and a lentivirus vector backbone gene; the plasmid map of the master plasmid 1 is shown in Figure 3.

[0131] The hCAR-19 molecule comprises, in order from N-terminus to C-terminus: an extracellular antigen binding region, a CD8a hinge region, a CD8a transmembrane region, a 4-1BB costimulatory signaling domain and a CD3 zeta intracellular signaling domain; the antigen binding region comprises a single chain variable fragment (scFv) that specifically binds to human CD19, FMC-63, the heavy chain variable region (VH) of the FMC-63 is connected to the light chain variable region (VL) of the scFv by a connecting peptide.

[0132] The polynucleotide encoding the hCAR-19 molecule is operably linked to a polynucleotide encoding a CD8a signal peptide, which is located at the N-terminus of the hCAR-19 molecule;

[0133] (1) the amino acid sequence of the CD8a signal peptide is shown in SEQ ID NO: 1;

[0134] (2) the amino acid sequence of the VH region of the FMC-63 is shown in SEQ ID NO: 2, and the amino acid sequence of the VL region of the FMC-63 is shown in SEQ ID NO: 3; the VH region is connected to the VL region by a connecting peptide, and the amino acid sequence of the connecting peptide is shown in SEQ ID NO: 4;

[0135] (3) the amino acid sequence of the hinge region of the CD8a is shown in SEQ ID NO: 5;

[0136] (4) the amino acid sequence of the transmembrane region of the CD8a is shown in SEQ ID NO: 6;

[0137] (5) the amino acid sequence of the 4-1BB costimulatory signaling domain is shown in SEQ ID NO: 7;

[0138] (6) the amino acid sequence of the CD3 zeta intracellular signaling domain is shown in SEQ ID NO: 8.

[0139] B. Packaging of lentivirus vector

[0140] The four plasmids were mixed and transfected into HEK-293T cells using PEI reagent to package a lentiviral vector-hCAR19 carrying a polynucleotide encoding an hCAR-19 molecule that targets human CD19.

[0141] The specific steps are as follows:

[0142] On Day 0, 9 μg of the main plasmid 1, 4 μg of pMDLg / pRRE packaging plasmid, 2 μg of pRSV-REV packaging plasmid, and 2 μg of pMD2.G envelope plasmid were added to 1 mL of Opti-MEM medium. After shaking well, 64 μL of PEI reagent was added, and the mixture was incubated for 10 minutes. Then, it was added to the culture medium of HEK-293T cells. The culture medium was replaced after 6 hours. 48 hours after transfection, the supernatant was collected, filtered through a 0.45 μm filter membrane, centrifuged at 50,000 g for 2.5 h, and the supernatant was discarded. The lentiviral vector-hCAR19 was resuspended in 200 μL of F12 medium and stored at -80 °C.

[0143] Opti-MEM alpha serum-depleted culture medium: Brand: GIBCO, Catalog No.: #SP0272;

[0144] HEK-293T cell culture medium: DMEM + 10% FBS; DMEM: Brand: GIBCO, Catalog No.: #C12430500BT; FBS: Brand: EXCELL, Catalog No.: #FSP500;

[0145] F12 medium: Brand: GIBCO, Product No.: #C11330500BT;

[0146] Needle filter: Brand: SORFA, Item No.: #622120.

[0147] 2. To determine whether the addition of TNFα antibody affects the killing efficiency of CAR-T cells.

[0148] A. Preparation of Mock-T cells

[0149] Day 0, take 1×10 7 Individual non-activated PBMCs (peripheral blood mononuclear cells, “PBMCs”) were resuspended in 10 mL of PBMCs culture medium consisting of 1640 medium + 10% FBS.

[0150] Add 1 pg / mL (final concentration) anti-CD3 antibody and 1 pg / mL (final concentration) anti-CD28 antibody to the PBMCs culture medium to activate the human non-activated PBMCs to prepare Mock-T cells.

[0151] B. Infection of human Mock-T cells to prepare human CAR-T cells

[0152] Day 1, use the lentiviral vector-hCAR19 to infect 5x10 6 Personal Mock-T cells.

[0153] Day 7, perform cell counting, and take 1x10 5 Personal Mock-T cells or human CAR-T cells, according to the effector-target ratio = 1:1, respectively mixed with Nalm-6 cells with luciferase reporter gene, a total of 8 groups, 3 replicates per group. When mixing, add 10 pg Adalimumab, 100 pg Adalimumab, 10 pg Etanercept, 100 pg Etanercept, 10 pg TNF alpha antibody, and 100 pg TNF alpha antibody to the 6 groups containing human CAR-T cells, respectively.

[0154] After 48 hours of culture, add the substrate, and perform chemiluminescence detection with an enzyme-labeled plate to calculate the killing efficiency. The specific calculation method is as follows: take the well containing only target cells Nalm-6 cells without human CAR-T cells as the reference well, and the fluorescence value of the original tumor cell number is the total fluorescence value. According to the tumor fluorescence value left after the 6 groups of human CAR-T cells containing various TNF alpha antagonists and 1 group of human CAR-T cells without any TNF alpha antagonist kill the target cells, the killing efficiency calculation formula is as follows: killing efficiency (%) = (total fluorescence value - residual fluorescence value) / total fluorescence value x 100%, and the results are shown in Figure 4.

[0155] As can be seen from Figure 4, the addition of various TNF alpha antagonists does not affect the killing efficiency of human CAR-T cells in killing tumor cells.

[0156] 1640 culture medium: brand: ELGBIO, item number: #EH80809;

[0157] Adalimumab: brand: MCE, item number: #HY-P9908;

[0158] Etanercept: brand: MCE, product name: Etanercept, item number: #HY-108847;

[0159] TNFα antibody: brand: Yiqi Shenzhou, commodity name: TNF-alpha / TNFA Neutralizing Antibody, product number: #10602-MM0N1.

[0160] Example 3

[0161] To detect whether the administration of TNFα antibody can reduce, neutralize or alleviate the damage caused by TNFα in mice.

[0162] 1. Preparation of retroviral vector-mCAR19

[0163] A. Construction of CAR-19 molecule containing mouse anti-CD19 antibody

[0164] Referring to the method for constructing hCAR-19 molecule containing human anti-CD19 antibody in Example 2, an anti-mouse CAR-19 molecule (mCAR-19 molecule) containing an antigen binding region of anti-mouse anti-CD19 antibody is constructed;

[0165] The mCAR-19 molecule comprises, in order from N-terminus to C-terminus, an extracellular antigen binding region, a CD8α hinge region, a CD8α transmembrane region, a 4-1BB costimulatory signaling domain and a CD3ζ intracellular signaling domain; the extracellular antigen binding region comprises an anti-mouse anti-CD19 antibody (scFv) 1D3, the VH region of the 1D3 is connected to the VL region of the 1D3 through the connecting peptide;

[0166] The polynucleotide encoding the mCAR-19 molecule is operably linked to a polynucleotide encoding a CD8α signal peptide, which is located at the N-terminus of the mCAR-19 molecule;

[0167] (1) the amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO: 1;

[0168] (2) the amino acid sequence of the VH region of the 1D3 is shown in SEQ ID NO: 9, and the amino acid sequence of the VL region of the 1D3 is shown in SEQ ID NO: 10; the VH region is connected to the VL region through a connecting peptide, and the amino acid sequence of the connecting peptide is shown in SEQ ID NO: 4;

[0169] (3) the amino acid sequence of the hinge region of the CD8α is shown in SEQ ID NO: 5;

[0170] (4) the amino acid sequence of the transmembrane region of the CD8α is shown in SEQ ID NO: 6;

[0171] (5) the amino acid sequence of the 4-1BB costimulatory signaling domain is shown in SEQ ID NO: 7;

[0172] (6) the intracellular signaling domain of the CD3 zeta is set forth in SEQ ID NO: 8.

[0173] B. Packaging retroviral vector-mCAR19 carrying polynucleotide encoding mCAR-19 molecule

[0174] (a) Preparation of mouse retroviral vector packaging system

[0175] Prepare the following 3 plasmids: pMD2.G envelope plasmid, psPAX2 packaging plasmid (gag / pol) and master plasmid 2, packaging retroviral vector-mCAR19 carrying polynucleotide encoding the mCAR-19 molecule; the master plasmid 2 is a transfer plasmid containing polynucleotide encoding the mCAR-19 molecule and derived from the MSGV (murine stem cell virus) genome, and the plasmid map of the master plasmid 2 is shown in Figure 7.

[0176] (b) Packaging retroviral vector-mCAR19

[0177] Using calcium phosphate transfection method, 4 μg of the master plasmid 2, 3 μg of psPAX2 packaging plasmid and 1 μg of pMD2.G envelope plasmid were transfected into HEK-293T cells, and the culture medium was replaced after 6 hours. The culture medium supernatant was collected 48-72 hours after transfection, filtered using a 0.45 μm filter, centrifuged at 50,000 g for 2.5 h, and the supernatant was aspirated. The retroviral vector-mCAR19 was resuspended using 200 μL of F12 medium and frozen at -80°C.

[0178] 2. Sorting mouse CD3 + T cells

[0179] Day-1, using Mouse CD3 + T Cell Isolation Kit (GIBCO, #CS101-01) to isolate CD3 + T cells from single cell suspension of mouse spleen, the specific method is:

[0180] (1) Preparation of single cell suspension: using the back of the needle to grind in a 70 μm cell screen, then rinsing the cell screen with pre-cooled sorting Buffer, collecting the suspension into a 50 mL centrifuge tube, centrifuging at 2,000 rpm (500 x g) for 5 min, and then discarding the supernatant; the sorting Buffer is PBS containing 1 mM EDTA and 2% fetal bovine serum (FBS), which is filtered with a 0.22 μm filter before use to remove bacteria.

[0181] (2) Add 3 mL of red blood cell lysing solution to the centrifuge tube, mix well by pipetting, and lyse at room temperature for 10 min. Then add 10 mL of PBS to terminate the reaction. Centrifuge at 2,000 rpm (500 x g) for 5 min.

[0182] (3) After centrifugation, discard the supernatant, resuspend the spleen cells in 1 mL of sorting buffer, filter them using a 70-μm cell strainer, and then count the cells. Centrifuge at 2,000 rpm (500 x g) for 5 min.

[0183] (4) After centrifugation, discard the supernatant, resuspend the cells in sorting buffer, and adjust the cell concentration to 1 x 10 8 cells / mL.

[0184] (5) Take 100 μL of the cell suspension (1 x 10 7 cells) and add it to the bottom of a flow tube, add 10 μL of CD3 + T Cell Isolation Cocktail, mix gently, and incubate on ice for 10 min.

[0185] (6) After incubation, add 20 μL of Streptavidin Beads, mix gently, and incubate at room temperature for 3 min. The beads should be mixed well by vortexing before use.

[0186] (7) After incubation, add 1 mL of sorting buffer, gently pipette 3-4 times, and then place the flow tube in a magnetic stand for 3 min.

[0187] (8) Slowly pour the cell suspension into a sterile centrifuge tube. This cell suspension is the purified CD3 + T cell suspension. The flow tube should not be removed from the magnetic stand during pouring.

[0188] (9) Wash the cells with PBS, centrifuge at 2,000 rpm (500 x g) for 5 min, discard the supernatant, and resuspend the isolated CD3 + T cells in mouse T cell culture medium, which includes RPMI 1640-GlutaMax (Thermo, #72400047), 10% FBS, NEAA (1x), 1 mM sodium pyruvate, 10 mM HEPES, and 50 μM 2-mercaptoethanol.

[0189] 3. Stimulate mouse CD3 + T cells to prepare mouse Mock-T cells

[0190] On Day-1, use the mouse T cell culture medium to stimulate the cells at a concentration of 1-1.5 x 10 6Mouse CD3 T cells were seeded at a density of 1 x 105cells / mL + T cells, and 50 IU / mL rh IL-2 and 10 ng / mL rm IL-7 were added to the mouse T cell medium. Anti-CD3 / CD28 antibody magnetic beads (trade name: DYNABEADS Mouse T Activator CD3 / CD28, brand: GIBCO, item number: #11456D) were added at a magnetic bead: cell ratio (bead: cell ratio) of 0.4: 1 to stimulate the mouse CD3 + T cells for 24 hours to prepare mouse Mock-T cells.

[0191] 4. Preparation of mouse CAR-T cells

[0192] On Day 0, 5 x 105Mock-T cells were infected with the retroviral vector-mCAR19 at an MOI of 1; on Day 7, cell counting was performed, and mouse CAR-T cells were collected. 6

[0193] 5. Detection of neutralization of TNFα antibody in mice

[0194] Four groups of mice, each with 4 mice, were prepared, and an A20 cell (mCD19 + mouse B cell lymphoma cell) model was established in each group of mice by the following method:

[0195] On Day 0, 1 x 106A20-Luci cells were injected into the tail vein of each mouse in each group, and live imaging showed that the modeling was successful; after the modeling was successful, 3 x 105mouse Mock-T cells (mouse Mock-T cell group), 3 x 105mouse CAR-T cells (mouse CAR-T cell group), 3 x 105mouse CAR-T cells + 100 μg TNFα antibody (anti-mouse) (CAR-T + TNFα antibody group), and 3 x 105mouse CAR-T cells + 100 μg Etanercept (CAR-T + Etanercept group) were injected into the tail vein of each mouse in each group, respectively, and live imaging was performed every four days. 6 6 6 6 6

[0196] The changes in A20-Luci cells in each group of mice were recorded by live imaging, and the growth curves of tumor cells A20 cells in each group of mice are shown in FIG. 5; the survival of mice is shown in FIG. 6;

[0197] ​​​​​​As shown in FIG. 5 and FIG. 6, all mice injected with mouse CAR-T cells only died at Day 4, while all mice injected with mouse CAR-T cells and TNFa antagonists, TNFa antibody and Etanercept survived.

[0198] Furthermore, the mouse CAR-T cells in the mice injected with mouse CAR-T cells and TNFa antagonists, TNFa antibody and Etanercept can continuously and effectively kill tumor cells A20 cells in vivo.

[0199] Therefore, TNFa antagonists, such as TNFa antibody and Etanercept, do not affect the killing of CAR-T cells in mice, and can effectively neutralize, alleviate, treat or prevent CRS induced by CAR-T cells.

[0200] TNFa antibody (anti-mouse): brand: Yikai Shenzhou, product name: TNF-alpha / TNFA / TNFSF2 Neutralizing Antibody, product number: 50349-RN023;

[0201] Substrate for in vivo imaging: brand: Yixing, product name: D-Luciferin, Sodium Salt: product number: #40901ES08.

Claims

1. Use of a cytokine antagonist for the manufacture of a medicament for the prevention and / or treatment of mortality from Cytokine Release Syndrome ("CRS"), characterized in that, The cytokine antagonist is a TNFα antagonist.

2. Use according to claim 1, characterized in that, The TNFα antagonist includes small molecule compounds, antibodies, nucleic acids, peptide fragments, polypeptides, fusion proteins, peptidomimetics, carbohydrates, glycomimetics, lipids, and aptamers that can antagonize TNFα; preferably, the TNFα antagonist includes anti-TNFα antibodies.

3. Use according to claim 1 or 2, characterized in that, The CRS occurs after the subject receives a cell therapy selected from at least one of a CAR-T cell therapy, a CAR-NK cell therapy, and a CAR-M (Macrophage) cell therapy; preferably, the cell therapy includes a CAR-T cell therapy.

4. Use according to claim 3, characterized in that, The subject is administered a therapeutically effective amount of the TNFα antagonist at least one time period selected from before, concurrently with, within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8 hours, within 9 hours, within 10 hours, within 11 hours, within 12 hours, within 13 hours, within 14 hours, within 15 hours, within 16 hours, within 17 hours, within 18 hours, within 19 hours, within 20 hours, within 21 hours, within 22 hours, within 23 hours, within 24 hours (within 1 day), within 2 days, within 3 days, within 4 days, within 5 days, within 6 days, within 7 days, within 8 days, within 9 days, within 10 days, within 11 days, within 12 days, within 13 days, within 14 days, within 15 days, within 16 days, within 17 days, within 18 days, within 19 days, within 20 days, within 21 days, within 22 days, within 23 days, within 24 days, within 25 days, within 26 days, within 27 days, within 28 days, within 29 days, within 30 days, within 31 days, within 1 month, within 2 months, within 3 months, within 4 months, within 5 months, within 6 months, within 7 months, within 8 months, within 9 months, within 10 months, within 11 months, and within 12 months of receiving the CAR-T cell therapy; Preferably, the therapeutically effective amount is 100 μg / kg to 10 mg / kg; More preferably, the therapeutically effective amount is 1 mg / kg.

5. Use according to any one of claims 1 to 4, characterized in that, The CRS lethality includes CRS-induced neurotoxicity lethality. Preferably, the CRS-induced neurotoxicity lethality includes immune effector cell-associated neurotoxicity syndrome (ICANS) lethality.

6. The use according to any one of claims 1 to 5, characterized in that, The CRS lethality includes ICANS lethality.

7. A composition characterized in that, The composition comprises the TNFα antagonist of any one of claims 1-6; Preferably, the composition can further comprise a pharmaceutically acceptable excipient or carrier.

8. A method of preventing and / or treating CRS lethal, which occurs after a subject receives a cell therapy, characterized in that, The method comprises administering to the subject a therapeutically effective amount of a TNFα antagonist.

9. The method of claim 8, wherein, The TNFα antagonist is selected from at least one of a small molecule compound, an antibody, a nucleic acid, a peptide segment, a polypeptide, a fusion protein, a peptidomimetic, a carbohydrate, a glycomimetic, a lipid, and an aptamer; preferably, the TNFα antagonist comprises an anti-TNFα antibody.

10. The method of claim 9, wherein, The cell therapy is selected from at least one of a CAR-T cell therapy, a CAR-NK cell therapy, and a CAR-M cell therapy; preferably, the cell therapy comprises a CAR-T cell therapy.

11. The method of claim 10, wherein, The TNFα antagonist is administered to the subject at least one time period prior to, concurrently with, within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8 hours, within 9 hours, within 10 hours, within 11 hours, within 12 hours, within 13 hours, within 14 hours, within 15 hours, within 16 hours, within 17 hours, within 18 hours, within 19 hours, within 20 hours, within 21 hours, within 22 hours, within 23 hours, within 24 hours (within 1 day), within 2 days, within 3 days, within 4 days, within 5 days, within 6 days, within 7 days, within 8 days, within 9 days, within 10 days, within 11 days, within 12 days, within 13 days, within 14 days, within 15 days, within 16 days, within 17 days, within 18 days, within 19 days, within 20 days, within 21 days, within 22 days, within 23 days, within 24 days, within 25 days, within 26 days, within 27 days, within 28 days, within 29 days, within 30 days, within 31 days, within 1 month, within 2 months, within 3 months, within 4 months, within 5 months, within 6 months, within 7 months, within 8 months, within 9 months, within 10 months, within 11 months, and within 12 months of the subject receiving the CAR-T cell therapy; Preferably, the therapeutically effective amount is 100 μg / kg to 10 mg / kg; More preferably, the therapeutically effective amount is 1 mg / kg.

12. The method according to any one of claims 8-11, characterized in that, The CRS lethality comprises CRS-induced neurotoxicity lethality; Preferably, the CRS-induced neurotoxicity lethality comprises immune effector cell-associated neurotoxicity syndrome (ICANS) lethality.

13. The method according to any one of claims 8-12, characterized in that, The CRS lethality comprises ICANS lethality.

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