Patent Description:
The theoretical basis of tumor immunotherapy is that the immune system can identify tumor-associated antigens and regulate the body to attack tumor cells (highly specific cytolysis).

In the <NUM>, Burnet and Thomas made the theory of "immunological surveillance" that holds that mutational tumor cells that often occur in the body can be identified and eliminated by the immune system, laying a theoretical foundation for tumor immunotherapy [Burnet FM. Immunological aspects of malignant disease. Then, a host of tumor immunotherapies, including cytokine therapy, monoclonal antibody therapy, adoptive immunotherapy and vaccine therapy, have been applied to clinical practice.

In <NUM>, CAR-T, a more advanced tumor immunotherapy, was successfully put to clinical use, and showed unprecedented clinical effects. CAR-T is short for Chimeric Antigen Receptor T-Cell Immunotherapy. Clinically, the most leading CAR-T is Novartis' CLT019. For patients with refractory-relapsed acute lymphoblastic leukemia and treated with CLT019, the six-month tumor progression-free survival rate can reach <NUM>%, and the longest response time can be more than two years. By cooperating with hospitals, Shanghai Unicar-Therapy Bio-Medicine Technology Co. , a Shanghai-based company, treated <NUM> patients with refractory-relapsed acute lymphoblastic leukemia, among whom <NUM> as a percentage of <NUM>% experienced complete remission. It's a subversive breakthrough in anti-cancer research. CAR-T may be one of the therapies that are the most likely to cure cancer, and was named the best in top <NUM> breakthroughs of science and technology <NUM> by the journal Science.

Although CAR-T is significantly effective, there will be a special clinical syndrome in the treatment, which is usually featured by fever, hypotension, chills and neurological symptoms related to markedly elevated levels of cytokines in serum, and is called Cytokine Release Syndrome (CRS). The mechanism of the CRS is that after the binding of antigen binding with T cell receptors, T cells are activated and release a series of cytokines including IL-<NUM>, resulting in a systemic inflammatory response (see <FIG> for the signal path of IL-<NUM>), which, if not treated timely, will lead to a series of symptoms including lung infection, abnormal blood coagulation index, abnormal liver function, damage to vital organs, and even cause death as a result of pulmonary edema.

At present, inflammatory response can be clinically inhibited by intravenously injecting anti-histamine drugs (such as chlorphenamine maleate) or corticosteroids (such as hydrocortisone), but correspondingly, the killing effect of CAR-T cells on tumors is also inhibited, making such patients have a higher rate of palindromia and affecting the efficacy of CAR-T.

A more viable option is to use commercialized tocilizumab (ACTEMRA®) to control the extent of CRS. Tocilizumab is a humanized IL-<NUM> receptor monoclonal antibody, and the specific binding of tocilizumab with IL-<NUM> receptor can block IL-<NUM> signal transduction, to reduce acute phase reactants, products of hepcidin, B cell activation, bone resorption and cartilage transformation, and inhibiting the differentiation of T lymphocytes into Th17 cells to effectively control inflammatory response. However, tombuzumab is very costly. The price of a piece of tocilizumab for <NUM> body weight of tombuzumab is about RMB <NUM>,<NUM>, and an adult patient usually needs <NUM> pieces at a time, which is hard for ordinary families to afford.

<CIT> discloses CD19-Redirected Chimeric Antigen Receptor T (CART19) Cells and IL-<NUM> Antagonist Tocilizumab for use in the treatment of Cytokine Release Syndrome associated with CART <NUM> therapy. <NPL>, discloses an approach to treat cytokine release syndrome (CRS) and neurotoxicity following CART cell therapy.

<NPL>, discloses that the cytokines IL-<NUM>, IL-<NUM>, and IFN-gamma are elevated in patients who develop CRS as a consequence of CAR T-cell treatment and that the anti-IL-<NUM> receptor Ab tocilizumab has been used to reverse CSR.

<NPL>, discloses cytokine release induced by anti-CD19 CAR T cells by blocking IL-<NUM> signaling using anti-IL-6R blocking antibody Tocilizumab, Siltuximaban IL-<NUM> antagonist, Sarilumab (anti-IL-6R) or Olokizumab (anti-IL-<NUM>).

<NPL>, discloses cytokine release induced by CD-<NUM>-specific CAR T cells (CAR19) using blocking antibodies Siltuximab o r Tocilizumab, respectively targeting IL-<NUM> or the IL-<NUM> receptor.

<CIT> discloses lentiviral vectors for the expression of chimeric antigen receptors in T cells, and specifies several elements of the lentiviral vector, including AmpR, pUC Ori, Sv40 Ori, IRES, eWEPRE, EF1α promoter, and lentiviral packaging cis-elements.

<CIT> and <CIT> D7 disclose constructs expressing CARS, some including two, three, or more of scFv against IL-<NUM>, a camelid VHH against IL-<NUM> receptor alpha, a bispecific camelid vHH targeting IL-<NUM> receptor alpha and albumin, Fx06 peptide, or combinations thereof. The CARs can be co-expressed with an scFV against IL6 and IL6R from a single vector without loss of activity. The vectors comprise IRES, packaging cis-elements, and a human EF1 alpha promoter.

<CIT> Al discloses lentiviral vectors for the co-expression of a CAR and an agent blocking IL-<NUM> by a siRNA to inhibit IL-<NUM>. The lentiviral vectors comprise AmpR, pUC Ori, SV40 Ori, IRES, and enhanced woodchuck hepatitis virus post-transcriptional regulatory element (eWPRE).

<CIT> discloses lentiviral and retroviral vectors for the expression of an anti-CD19 CAR and anti-IL-6Ra scFv.

<CIT> discloses lentiviral vectors to express interleukin-<NUM> acceptor (IL6R) single chain antibody (single-chain antibody) for alleviating cytokine release syndrome (Cytokine Release Syndrome, CRS); some of the vectors also comprise CAR constructs as well as AmpR, pUC ori, SV40 ori, eWPRE, and other elements.

Therefore, how to use low-cost methods to control or alleviate the occurrence of CRS without affecting the efficacy of CAR-T has become a technical problem in the field.

The technical problem to be solved by the invention is to provide an IL6R block CAR-T transgenic vector for alleviating CRS. First of all, it saves the cost and the expensive cost of purchasing antibody drugs. Secondly, it avoids the problem of low delivery efficiency of the scFv gene in vivo. Thirdly, the IL6R scFv gene transduced by lentivirus can effectively utilize the intracellular protein translation system and express a large number of corresponding IL6R scFv. Through fluid circulation, a good IL6R-blocking effect can be achieved, without affecting the curative effect of CAR-T.

The invention also relates to the use of the vector in alleviating CRS.

The above technical problem is solved by a CAR-T transgenic vector suitable for alleviating cytokine release syndrome (CRS) by blocking IL6R as defined in present claims <NUM> to <NUM>.

A second aspect of the invention relates to the use of the vector in alleviating CRS, see present claim <NUM>.

The inventive CAR-T transgenic vector suitable for alleviating cytokine release syndrome (CRS) by blocking IL6R can be prepared according to a method including the following steps:.

As the preferred technical scheme of the method, in step (<NUM>), the expression of the whole CAR gene is started by the human EFlalpha promoter; the CAR protein locates on the surface of the cell membrane, recognizes CD19 antigen, stimulates the T cell proliferation and cytokine secretion, and activates the expression of downstream signaling pathway; when scFv region binds to CD19 antigen, the signal is transmitted to cells through the chimeric receptor, which produces a series of biological effects, such as T cell proliferation, increased cytokine secretion, increased secretion of anti-apoptotic protein, delayed cell death, and lysis of target cells; the fusion protein of IL6RscFv and Fc is co-expressed by IRES ribosome binding sequence and secreted to extracellular space under the guidance of IL6 signal peptide. By binding with IL6R, the binding of IL-<NUM> and IL6R was blocked, thus the signal pathway of IL6 was blocked, thereby inhibiting CRS. As the preferred technical scheme of the method, in step (<NUM>), the filtration step is to control the volume of supernatant from <NUM> to <NUM>, the vacuum degree from - <NUM> MPa to <NUM> MPa to prevent the loss of vector caused by a blockage. The adsorption step is to control the pH value of the solution from <NUM> to <NUM> and prevent the vector from inactivating due to the change of pH, and the elution step is to control the ionic strength of eluent from <NUM> to <NUM> and prevent the change of ionic strength leading to incomplete elution or inactivation of the vector.

Compared with the existing technology, the invention has the following beneficial effects:
The invention has the human EF1α promoter start the whole CAR genetic expression by preparing and transferring into recombinant lentiviral vectors the IL6 signal peptide, the single chain antibody of human IL6R, the IRES ribosome binding sequence, the human antibody Fc fragment, the human EF1α promoter, the CD8 leader chimeric receptor signal peptide, the CD19 single chain antibody light chain VL, the Optimal Linker C, the CD19 single-chain antibody heavy chain VH, the CD8 Hinge chimeric receptor hinge, the CD8 Transmembrane chimeric receptor transmembrane region, the CD137 chimeric receptor co-stimulatory factor, and the TCR chimeric receptor T cell activation domain. The CAR protein locates on the surface of the cell membrane, recognizes the CD19 antigen, stimulates T cell proliferation and cytokine secretion, and activates the expression of the downstream signaling pathway; when the scFv region binds to the CD19 antigen, the signal is transmitted to cells through the chimeric receptor, which produces a series of biological effects, such as T cell proliferation, increased cytokine secretion, increased secretion of anti-apoptotic protein, delayed cell death, and lysis of target cells; the fusion protein of IL6RscFv and Fc is co-expressed by IRES ribosome binding sequence and secreted to extracellular space under the guidance of IL6 signal peptide. By binding with IL6R, the binding of IL-<NUM> and IL6R was blocked, thus the signal pathway of IL6 was blocked, thereby inhibiting CRS.

The blocking regimen adopted by the invention can be applied to the second or third generation of CAR design. Compared with the second design of the same, the third generation of CAR design adds CD28 chimeric receptor costimulatory factor (SEQ ID NO. <NUM>), which, according to a literature report, has stronger signal amplification [<NPL>].

The lentiviral vector column purification system (as shown in <FIG>) used in the invention is a lentivirus scale production process developed by the applicant (which has been disclosed in the patent for invention, <CIT>). Common ultracentrifugation or high-speed centrifugation methods use the principle of centrifugal sedimentation to separate lentivirus particles, with which many impurities with similar sedimentation coefficients will inevitably remain and subsequent experiments will be affected adversely. Moreover, complicated tube loading, cumbersome operation and multiple conversion of containers bring more chances for contamination, while the lentiviral vector column purification process developed by the company is operated by semi-automation, and the whole process is completed in the <NUM>-level experimental area, avoiding the cumbersomeness, errors and contamination probability of manual operation and making recovered lentiviral vector completely up to clinical standard in endotoxin, mycoplasma, host DNA residual and other indicators. The development of a fully automated purification instrument may be followed up subsequently.

The CAR design adopted by the invention can also be applied to the second generation of lentivirus vector structure (which has been disclosed in the patent for invention, <CIT>). The major difference in structure between the second and third generations of lentivirus vector (as shown in <FIG>) is that in the third generation of lentivirus vector, the U3 area in the second generation of <NUM>'LTR is replaced with the RSV promoter, so that the dependence of U3 transcription on Tat protein is eliminated, removing Tat sequences from lentiviral structural genes and improving the level and persistence of lentiviral genome transcription. Since the major difference between the second and third generations of lentivirus vector is in gene transcription, the CAR design employed by the invention can be applied to the two generations of lentivirus vector. The preferable option for the vector skeleton used in the invention is the third-generation lentivirus vector (as shown in <FIG>) (which has been disclosed in the patent for invention, <CIT>). In the <NUM>'SIN LTR, the U3 region is removed, eliminating the possibility of self-replication of the lentivirus vector, and greatly improving the security. The cPPT and WPRE elements were added to improve the transduction efficiency and the expression efficiency of the transgene. RSV promoter was used to ensure the continuous and efficient transcription of core RNA in the packaging of lentiviral vectors, and EF1α promoter was used to make the CAR gene continuously expressed in the human body for a long time.

The third generation of lentivirus skeleton plasmid developed the applicant (which has been disclosed in the patent for invention, <CIT>) employs enhanced WPRE components, which, compared with the WPRE components employed by Carl H. June and others at Pennsylvania State University (<NPL>. ) has the enhanced mutation of <NUM> nucleotides (g. <NUM>>A, g. <NUM>>A, g. 411A>T), and can enhance the polyadenylation of primary transcription products, increase the content of mRNA in cells, and improve transgenic expression efficiency.

The Lentivirus packaging system used in the invention is a four-plasmid packaging system without a helper virus (which has been disclosed in the patent for invention, <CIT>), which produces recombinant lentiviral vectors through the co-transfection of four plasmids into HEK293T/<NUM> cells. The recombined lentiviral vector is a single-use replication-defective vector, which can integrate the exogenous fragments into the host gene, and cannot be replicated and propagated, greatly improving safety.

The invention employs an scFv blocking technology for IL-6R. Composed of the heavy chain and light chain variable regions of antibody by linking <NUM>-<NUM> short peptides of amino acid, the single-chain antibody fragment (scFv) can better retain its affinity for antigen, and has the characteristics of small molecular weight, strong penetrability, weak antigenicity, etc. The human IL6R blocking single-chain antibody design employed by the invention can be overexpressed in T cells and secreted into extracellular space, and block the binding of IL-<NUM> with IL6R and the activation of IL6 signaling pathway, effectively. In the T-cell killing experiment, QPCR test shows that the design can effectively inhibit the transcription level of the mRNA of C-reactive protein in PBMC, and reduce the content of C-reactive protein in cell culture supernatant. The IL-6R blocking effect evaluation experiment proves that the vector of the invention can inhibit the IL-<NUM> signaling pathway in vivo, alleviating the symptoms of CRS.

The scFv fragment and the Fc fragment of antibody used in the invention are both humanized, can effectively reduce the production of human anti-mouse antibodies (HAMA) in vivo, and improve the half-life period and effects of scFv.

The Linker design of the scFv fragment used in the invention (which has been disclosed in the patent for invention, <CIT>) can markedly improve cytokine secretion as well as the killing effects and clinical treatment effects of CAR-T cells in vitro.

The invention adopts the action mode of IL6R scFv (as shown in <FIG>). First of all, it saves the cost and the expensive cost of purchasing antibody drugs. Secondly, it avoids the problem of low delivery efficiency of scFv gene in vivo. Thirdly, the IL6R scFv gene transduced by lentivirus can effectively utilize the intracellular protein translation system and express a large number of corresponding IL6R scFv. Through fluid circulation, good IL6R blocking effect can be achieved. The invention screens a series of bioinformatics information such as gene sequence and amino acid sequence of IL6R antibody, predicts the variable regions of heavy and light chains of IL6R scFv, analyses the secondary structure of IL6R scFv combination and its physicochemical properties, determines the affinity constants of IL6R scFv by soluble expression and indirect ELISA, from which selects three scFv for cell function level detection. Finally, IL6RscFv1 was determined as the best choice and may enter the clinical research stage in the future. The recombinant lentivirus vector skeleton of the invention can carry different therapeutic genes and be widely used in the field of adoptive cell therapy and can carry the IL6R scFv gene and be used to block IL6R. The recombinant lentivirus vector of the invention can express CD19 chimeric antigen receptor on human T lymphocytes, guide and activate the killing effect of T lymphocytes on CD19-positive cells, and is clinically used to treat B lymphocytic leukemia, B lymphoma and multiple myeloma. The scFv expressing IL6R in human T lymphocytes can effectively block IL6R and its signaling pathway and can be clinically used to alleviate CRS. It can be seen that the invention control or alleviate the occurrence of CRS with lost-cost methods and without prejudice to the effects of CAR-T, solving the technical problems in the field and achieving unexpected technical effects.

The IL6R single-chain antibody fragment expression frame and its gene expression products described by the invention can be used not only to eliminate or alleviate CRS in the treatment of ALL with CAR-T (CD19-CAR-T), but also to alleviate CRS caused in the treatment of all kinds of tumors such as pancreatic cancer, brain glioma and myeloma with CAR-T, and even to alleviate CRS caused by other kinds of treatment.

The invention is further described below in connection with specific implementation methods. It should be understood that the specific implementation methods described herein are expressed by way of examples and are not constrained by the invention. Without departing from the scope of the invention, the main features of the invention can be used in various implementation methods.

The preparation method of the recombinant lentiviral vector described in the invention is as follows:.

The human EF1α promoters, CD8 leader chimeric receptor signal peptide, CD19 single chain antibody light chain VL, Optimal Linker C, CD19 single chain antibody heavy chain VH, CD8 chimeric receptor hinge, CD8 transmembrane domain chimeric receptor, the chimeric receptor co-stimulation factor - CD137, TCR and T cell activation domain chimeric receptor fragments were cloned into the lentiviral cytoskeleton plasmid pLenti-<NUM> Basic2 to obtain recombinant lentiviral plasmid pCAR19-Basic2, and the siRNA fragments were connected into pCAR19-Basic2 respectively to obtain IL-<NUM> know-down recombinant lentiviral plasmids pCAR19-IL6RscFv1, pCAR19-IL6RscFv2, pCAR19-IL6RscFv3 and control pCAR19-scFv0.

(<NUM>) The lentiviral cytoskeleton plasmid pLenti-<NUM> Basic2 was double digested with Cla I and BamH I restriction enzymes. The product was electrophoresed on a <NUM>% agarose gel to confirm the 5854bp fragment V1 (see <FIG>), then such gel was recovered and placed in an Eppendorf tube. The corresponding fragments were recovered with Agarose Gel Recovery Kit of MN (see Table <NUM>), and the purity and concentration of the product were determined.

(<NUM>) Use the primers EF1α-F and EF1α-R with the synthesized SEQ ID NO. <NUM> as a template, and apply the system in Table <NUM>. PCR circulation condition was: <NUM> <NUM>, (<NUM> 10sec, <NUM> 15sec, <NUM> <NUM>)*35cycle, <NUM> <NUM>. The product was electrophoresed on a <NUM>% agarose gel to confirm the 1208bp fragment a, then such gel was recovered and placed in an Eppendorf tube. The corresponding fragments were recovered with Agarose Gel Recovery Kit of MN (see Table <NUM>), and the purity and concentration of the product were determined.

(<NUM>) Use the primers CD8 leader-F and CD8 leader-R with the synthesized SEQ ID NO. <NUM> as a template, and apply the system in Table <NUM>. PCR circulation condition was: <NUM> <NUM>, (<NUM> 10sec, <NUM> 15sec, <NUM> 30sec)*35cycle, <NUM> <NUM>. The product was electrophoresed on a <NUM>% agarose gel to confirm the 101bp fragment b, then such gel was recovered and placed in an Eppendorf tube. The corresponding fragments were recovered with Agarose Gel Recovery Kit of MN (see Table <NUM>), and the purity and concentration of the product were determined. (<NUM>) Use the primers VL-F and VL-R with the synthesized SEQ ID NO. <NUM> as a template, and apply the system in Table <NUM>. PCR circulation condition was: <NUM> <NUM>, (<NUM> 10sec, <NUM> 15sec, <NUM> 30sec)*35cycle, <NUM> <NUM>. The product was electrophoresed on a <NUM>% agarose gel to confirm the 336bp fragment c, then such gel was recovered and placed in an Eppendorf tube. The corresponding fragments were recovered with Agarose Gel Recovery Kit of MN (see Table <NUM>), and the purity and concentration of the product were determined. (<NUM>) Use the primers OLC-VH-F and VH-R with the synthesized SEQ ID NO. <NUM> as a template, and apply the system in Table <NUM>. PCR circulation condition was: <NUM> <NUM>, (<NUM> 10sec, <NUM> 15sec, <NUM> 30sec)*35cycle, <NUM> <NUM>. The product was electrophoresed on a <NUM>% agarose gel to confirm the 421bp fragment d, then such gel was recovered and placed in an Eppendorf tube. The corresponding fragments were recovered with Agarose Gel Recovery Kit of MN (see Table <NUM>), and the purity and concentration of the product were determined. (<NUM>) Use the primers CD8 Hinge-F and CD8 Hinge-R with the synthesized SEQ ID NO. <NUM> as a template, and apply the system in Table <NUM>. PCR circulation condition was: <NUM> <NUM>, (<NUM> 10sec, <NUM> 15sec, <NUM> 30sec)*35cycle, <NUM> <NUM>. The product was electrophoresed on a <NUM>% agarose gel to confirm the 147bp fragment e, then such gel was recovered and placed in an Eppendorf tube. The corresponding fragments were recovered with Agarose Gel Recovery Kit of MN (see Table <NUM>), and the purity and concentration of the product were determined. (<NUM>) Use the primers CD8 Transmembrane-F and CD8 Transmembrane-R with the synthesized SEQ ID NO. <NUM> as a template, and apply the system in Table <NUM>. PCR circulation condition was: <NUM> <NUM>, (<NUM> 10sec, <NUM> 15sec, <NUM> 30sec)*35cycle, <NUM> <NUM>. The product was electrophoresed on a <NUM>% agarose gel to confirm the 100bp fragment f, then such gel was recovered and placed in an Eppendorf tube. The corresponding fragments were recovered with Agarose Gel Recovery Kit of MN (see Table <NUM>), and the purity and concentration of the product were determined. (<NUM>) Use the primers CD137-F and CD137-R with the synthesized SEQ ID NO. <NUM> as a template, and apply the system in Table <NUM>. PCR circulation condition was: <NUM> <NUM>, (<NUM> 10sec, <NUM> 15sec, <NUM> 30sec)*35cycle, <NUM> <NUM>. The product was electrophoresed on a <NUM>% agarose gel to confirm the 142bp fragment g, then such gel was recovered and placed in an Eppendorf tube. The corresponding fragments were recovered with Agarose Gel Recovery Kit of MN (see Table <NUM>), and the purity and concentration of the product were determined. (<NUM>) Use the primers TCR-F and TCR-R with the synthesized SEQ ID NO. <NUM> as a template, and apply the system in Table <NUM>. PCR circulation condition was: <NUM> <NUM>, (<NUM> 10sec, <NUM> 15sec, <NUM> 30sec)*35cycle, <NUM> <NUM>. The product was electrophoresed on a <NUM>% agarose gel to confirm the 335bp fragment h, then such gel was recovered and placed in an Eppendorf tube. The corresponding fragments were recovered with Agarose Gel Recovery Kit of MN (see Table <NUM>), and the purity and concentration of the product were determined. (<NUM>) Applying the system in Table <NUM>, 1µl each of DNA fragments b, c and d were taken as templates to add to Eppendorf tubes except for primers. PCR circulation condition was: <NUM> <NUM>, (<NUM> 10sec, <NUM> 10sec, <NUM> 30sec)*6cycle. To add primer CD8 leader-F/VH-R with the conditions as (<NUM> 10sec, <NUM> 10sec, <NUM> 40sec)*24cycle, <NUM> <NUM>. The product was electrophoresed on a <NUM>% agarose gel to confirm the 814bp fragment i, then such gel was recovered and placed in an Eppendorf tube. The corresponding fragments were recovered with Agarose Gel Recovery Kit of MN (see Table <NUM>), and the purity and concentration of the product were determined.

(<NUM>) Applying the system in Table <NUM>, 1µl each of DNA fragments e, f, g and h were taken as templates to add to Eppendorf tubes except for primers. PCR circulation condition was: <NUM> <NUM>, (<NUM> 10sec, <NUM> 10sec, <NUM> 30sec)*6cycle. To add primer CD8 Hinge-F/TCR-R with the conditions as (<NUM> 10sec, <NUM> 10sec, <NUM> 30sec)*24cycle, <NUM> <NUM>. The product was electrophoresed on a <NUM>% agarose gel to confirm the 704bp fragment j, then such gel was recovered and placed in an Eppendorf tube. The corresponding fragments were recovered with Agarose Gel Recovery Kit of MN (see Table <NUM>), and the purity and concentration of the product were determined. (<NUM>) The DNA fragments V1, a, i, j were added to the Eppendorf tubes in a total volume of 5µl with a molar ratio of <NUM>:<NUM>:<NUM>:<NUM>. <NUM>µl of the homologous recombinase reaction solution was added to the tubes, and the mixtures were incubated at <NUM> for <NUM> minutes. Place them on ice for <NUM>-<NUM> minutes. Add the reaction solution to 50µl of TOP10, gently rotate to mix the contents, place them on ice for <NUM> minutes, then put the tubes in the thermostatic water bath pre-warmed to <NUM> for <NUM> seconds, and quickly transfer the tubes in an ice bath. The cells were allowed to cool for <NUM>-<NUM> minutes. Add <NUM>µl of LB medium to each tube, then put the tubes to a <NUM> shaker and incubate for <NUM> hour to resuscitate the bacteria. Take 100µl of transformant bacteria solution to apply to an Amp LB agar plate, invert the plate, and culture in a thermostatic incubator at <NUM> for <NUM> hours. The clones were picked for colony PCR identification, and the correct clones were identified as recombinant lentiviral plasmid pCAR19-Basic2. Enzyme digestion identification was performed for the correct clones (see <FIG>). (<NUM>) The recombinant lentiviral plasmid pCAR19-Basic2 was double digested with Sal I and Nhe I restriction enzymes. The product was electrophoresed on a <NUM>% agarose gel to confirm the 8491bp fragment V2, then such gel was recovered and placed in an Eppendorf tube. The corresponding fragments were recovered with Agarose Gel Recovery Kit of MN (see Table <NUM>), and the purity and concentration of the product were determined. (<NUM>) The primers IRES-F and IRES-R were used to synthesize SEQ ID NO. <NUM> as templates. Using the system in Table <NUM>, the conditions of PCR cycle were <NUM> <NUM>, (<NUM> 10sec, <NUM> 15sec, <NUM> <NUM>)*35cycle and <NUM> <NUM>. The product was by agarose gel electrophoresis of <NUM>%, and the fragment k of 605bp was confirmed. The tapping gel was recovered in Eppendorf tube, and the corresponding fragments were recovered by agarose gel recovery kit of MN company (see Table <NUM>), and the purity and concentration of the product were determined. (<NUM>) The primers IL6Rs1-F and IL6Rs1-R were used to synthesize SEQ ID NO. <NUM> as templates. Using the system in Table <NUM>, the conditions of PCR cycle were <NUM> <NUM>, (<NUM> 10sec, <NUM> 15sec, <NUM> <NUM>)*35cycle and <NUM> <NUM>. The product was by agarose gel electrophoresis of <NUM>%, and the fragment l of 754bp was confirmed. The tapping gel was recovered in Eppendorf tube, and the corresponding fragments were recovered by agarose gel recovery kit of MN company (see Table <NUM>), and the purity and concentration of the product were determined. (<NUM>) The primers IL6Rs2-F and IL6Rs2-R were used to synthesize SEQ ID NO. <NUM> as templates. Using the system in Table <NUM>, the conditions of PCR cycle were <NUM> <NUM>, (<NUM> 10sec, <NUM> 15sec, <NUM> <NUM>)*35cycle and <NUM> <NUM>. The product was by agarose gel electrophoresis of <NUM>%, and the fragment m of 777bp was confirmed. The tapping gel was recovered in Eppendorf tube, and the corresponding fragments were recovered by agarose gel recovery kit of MN company (see Table <NUM>), and the purity and concentration of the product were determined. (<NUM>) The primers IL6Rs3-F and IL6Rs3-R were used to synthesize SEQ ID NO. <NUM> as templates. Using the system in Table <NUM>, the conditions of the PCR cycle were <NUM> <NUM>, (<NUM> 10sec, <NUM> 15sec, <NUM> <NUM>)*35cycle and <NUM> <NUM>. The product was by agarose gel electrophoresis of <NUM>%, and the fragment n of 774bp was confirmed. The tapping gel was recovered in an Eppendorf tube, and the corresponding fragments were recovered by an agarose gel recovery kit of MN company (see Table <NUM>), and the purity and concentration of the product were determined. (<NUM>) The primers s0-F and s0-R were used to synthesize SEQ ID NO. <NUM> as templates. Using the system in Table <NUM>, the conditions of the PCR cycle were <NUM> <NUM>, (<NUM> 10sec, <NUM> 15sec, <NUM> <NUM>)*35cycle and <NUM> <NUM>. The product was by agarose gel electrophoresis of <NUM>%, and the fragment o of 729bp was confirmed. The tapping gel was recovered in an Eppendorf tube, and the corresponding fragments were recovered by an agarose gel recovery kit of MN company (see Table <NUM>), and the purity and concentration of the product were determined. (<NUM>) The primers Fc-F and Fc-R were used to synthesize SEQ ID NO. <NUM> as templates. Using the system in Table <NUM>, the conditions of the PCR cycle were <NUM> <NUM>, (<NUM> 10sec, <NUM> 15sec, <NUM> <NUM>)*35cycle and <NUM> <NUM>. The product was by agarose gel electrophoresis of <NUM>%, and the fragment p of 726bp was confirmed. The tapping gel was recovered in an Eppendorf tube, and the corresponding fragments were recovered by an agarose gel recovery kit of MN company (see Table <NUM>), and the purity and concentration of the product were determined. (<NUM>) DNA fragments (V2, k, l, p), (V2, k, m, p), (V2, k, n, p), (V2, k, o, p) were added into the Eppendorf tube with a total volume of 5µl and at a molar ratio of <NUM>:<NUM>:<NUM> respectively, and 15µl homologous recombinant enzyme reaction solution. After evenly mixed, they were incubated at <NUM> for <NUM> minutes and transferred to ice for <NUM>-<NUM> minutes. The reaction solution was added to 50µl TOP10 and rotated gently to evenly mix the content. Place the tube in ice for <NUM> minutes, and heatly shock the tube for <NUM> seconds in a constant temperature water bath pot preheated to <NUM>, quickly transfer the tube to the ice bath, cool the cells for <NUM>-<NUM> minutes, add <NUM>µl LB culture medium to each tube, then transfer the tube to a shaking bed at <NUM>, incubate for <NUM> hour to resuscitate the bacteria, take 100µl transformed bacteria solution and coat it on Amp LB agar plate, invert the flat dish, and put it in a constant temperature incubator at <NUM>, and culture for <NUM> hours. The correct clones were identified by colony PCR as recombinant lentivirus plasmids pCAR19-IL6RscFv1, pCAR19-IL6RscFv2, pCAR19-IL6RscFv3 and control pCAR19-scFv0. The correct clone would be identified with enzyme digestion (see <FIG>).

Purification of Recombinant Lentiviral Vectors by Ion Exchange Chromatography (see <FIG>);.

(<NUM>) 293T cells were inoculated with <NUM>-well plates. The number of cells in each well was <NUM>×<NUM><NUM>, and the volume of medium added was 500ul. As the growth rate of different types of cells was different, the rate of cell fusion during viral infection was <NUM>%-<NUM>%. (<NUM>) Three sterile EP tubes were prepared, and 90ul fresh complete medium (high glucose DMEM+<NUM>%FBS) was added into each tube to inoculate the cells. <NUM> hours later, the cells in the two pores were taken and counted with a hemocytometer to determine the actual number of cells at the time of infection, denoted as N. (<NUM>) 10ul of the virus stock to be determined was added to the first tube. After gently mixing, 10ul of the virus stock was added to the second tube, and then sequentially operated until the last tube; 410ul complete medium (high glucose DMEM+<NUM>%FBS) was added into each tube, and the final volume was 500ul. (<NUM>) <NUM> hours after the infection, the cultural supernatant was removed and replaced with 500µl complete medium (high glucose DMEM+<NUM>%FBS). The cells was continuously cultured for <NUM> hours in <NUM>% CO<NUM>. (<NUM>) After <NUM> hours, the fluorescence expression was observed. Under normal circumstances, the number of fluorescence cells decreased with the increase of dilution ratio. At the same time, photos were taken. (<NUM>) The cells were digested with <NUM> <NUM>% trypsin-EDTA solution, and then they were placed at <NUM> for <NUM> minute. The whole cellular surface were purged with medium, and the cells were collected by centrifugation. Genomic DNA was extracted according to the instructions of DNeasy kit. 200µl of eluent were added to each sample tube to remove DNA, and then they were quantified. (<NUM>) The DNA detection qPCRmix manifold I was prepared (QPCR primer sequences were SEQ ID NO. <NUM> --- SEQ ID NO.

n = number of reactions. For example, the total n were <NUM>. <NUM> of <NUM>× TaqMan Universal PCR Master Mix, 4µl of forward primer, 4µl of reverse primer, 4µl of probe and 788µl of H<NUM>O were mixed and Placed on ice after being shaken. (<NUM>) The reference DNA detection qPCRmix manifold II were prepared (QPCR primer sequences were SEQ ID NO. <NUM> --- SEQ ID NO.

n = number of reactions. For example, the total n were <NUM>. <NUM> of <NUM>× TaqMan Universal PCR Master Mix, 100µl pf <NUM>×RNaseP primer/probe mix and 700µl of H<NUM>O were mixed and placed on ice after being shaken. (<NUM>) The PCR system was established on a pre-cooled <NUM>-well PCR plate. Take <NUM>µl from each tube of manifold I to add to the wells of each row of A-D. Take <NUM>µl from each tube of manifold II to add to the wells of each row of E-G. (<NUM>) 5µl of the standard plasmid and the genomic DNA from the samples to be tested were taken respectively to add to the A-D row, and each sample was repeated once. <NUM> well was left to add 5µl of water as no-template control. (<NUM>) <NUM>µl of the genomic standards and the genomic DNA from the samples to be tested were taken respectively to add to the E-G row, and each sample was repeated once. <NUM> well was left to add <NUM>µl of water as no-template control. (<NUM>) The quantitative PCR instrument used was the ABI PRISM <NUM> quantitative system. The cyclic conditions were set to: <NUM> <NUM>, <NUM> <NUM>, (<NUM> 15sec, <NUM> <NUM>) × <NUM> cycle. Data analysis: the copy number of lentiviral vectors integrated in the measured DNA samples was calibrated with the number of genomes to obtain the copy number of viruses integrated in each genome. The calculation formula of integration units per ml (IU ml-<NUM>) was as follows: <MAT>.

(<NUM>) Titer results of recombinant lentiviral vectors lvCAR19-IL6RscFv1, lvCAR19-IL6RscFv2, lvCAR19-IL6RscFv3, lvCAR19-scFv0 (see <FIG>);
III. Endotoxin Determination.

(<NUM>) The working standard of endotoxin was 15EU per dose. (<NUM>) Sensitivity of Tachypiens Amebocyte Lysate (TAL) λ=<NUM>. 25EU/ml, <NUM>/tube. (<NUM>) Dilution of endotoxin standard: take one endotoxin standard, dilute it into 4λ and 2λ solution with BET water, seal with sealing film and vortex for <NUM>; During dilution, each dilution step should be mixed on the vertex mixer for <NUM>. (<NUM>) Adding: Several TAL were taken, each was dissolved in <NUM> of BET water, and then divided into several exdotoxin-free tubes (<NUM> each tube). Two of them were negative control which were added <NUM> of BET water to each of them.

Two tubes were positive control which were added <NUM> of endotoxin working standard solution with concentration of 2λ to each of them. Two tubes were positive control of sample which were added <NUM> sample solution contained 2λ endotoxin standard (<NUM> of 20x dilution of sample to be tested + <NUM> of solution contained 4λ endotoxin standard = <NUM> of 40x dilution of sample contained 2λ endotoxin standard). Two tubes were positive control of sample which were added <NUM> sample solution contained 2λ endotoxin standard (<NUM> of 20x dilution of sample to be tested + <NUM> of solution contained 4λ endotoxin standard = <NUM> of 40x dilution of sample contained 2λ endotoxin standard).

(<NUM>) The endotoxin detection results of the recombinant lentiviral vectors lvCAR19-IL6RscFv1, lvCAR19-IL6RscFv2, lvCAR19-IL6RscFv3, lvCAR19-scFv0 (as shown in Table <NUM>) showed that the endotoxin content was between <NUM>~<NUM> EU/ml, which met the requirements.

Measurement and Comparison of Mycoplasma.

Detection of Cellular Level Expression of CAR Gene:.

(<NUM>) After infection of PBMC cells by recombinant lentiviral vectors lvCAR19-IL6RscFv1, lvCAR19-IL6RscFv2, lvCAR19-IL6RscFv3 and lvCAR19-scFv0, and control virus Mock. RT-PCR was used to detect the mRNA transcriptional levels of CAR gene and scFv gene by collecting cells, and to verify the expression of CAR gene and scFv gene. If the mRNA transcriptional level of CAR gene and scFv gene increased, the expression of transcription level was successful. (<NUM>) After infection of PBMC cells by recombinant lentiviral vectors lvCAR19-IL6RscFv1, lvCAR19-IL6RscFv2, lvCAR19-IL6RscFv3 and lvCAR19-scFv0, and control virus Mock. western blot was used to detect the expression level of CAR protein by collecting cells, and to verify the expression of CAR gene. If the expression level of CAR protein increased, the translation level of CAR gene was successfully expressed. (<NUM>) After infection of PBMC cells by recombinant lentiviral vectors lvCAR19-IL6RscFv1, lvCAR19-IL6RscFv2, lvCAR19-IL6RscFv3 and lvCAR19-scFv0, and control virus Mock. The expression level of scFv protein was detected by ELISA in the supernatant of cultured cells to verify the expression of scFv gene. If the expression level of scFv protein increased, the translation level of scFv gene was successfully expressed. (<NUM>) Infect cells with lvCAR19-IL6RscFv1, lvCAR19-IL6RscFv2, lvCAR19-IL6RscFv3 and lvCAR19-scFv0 of MOI=<NUM> and control virus Mock, after <NUM> hours, extract the total RNA and total protein of cells from the <NUM> hole plate to carry out the quantitative fluorescence PCR and immunoblot experiment respectively. Specific steps: wrapping four holes of <NUM>-hole plate, adding corresponding PBS and RN to each hole, and staying overnight at <NUM>. After <NUM> hours, the virus was coated with MOI=<NUM> and placed in an incubator at <NUM> for <NUM> hours. The virus supernatant in the <NUM>-hole plate was discarded and washed twice with PBS. PBMC was coated with <NUM>*<NUM>/hole (separated from human blood with lymphocyte-separating fluid). Add 500ul culture medium (containing <NUM>% serum, 20U/ml IL-<NUM>, Polybrene 8ug/ml) and place static for <NUM> minutes, centrifuge for <NUM> minutes at <NUM> <NUM>, and culture for <NUM> hours at <NUM>. (<NUM>) Total RNA was extracted from PBMC cells in <NUM> hole plate by the Trizol method. Reverse transcription was used to amplify the cDNA. Quantitative fluorescence PCR experiment was performed with CAR gene QPCR primers (SEQ ID NO. <NUM>-SEQ ID NO. <NUM>) and scFv gene QPCR primers (SEQ ID NO. <NUM>-SEQ ID NO. The reaction system was shown in Table <NUM>. The internal reference Actin was used as the control group to verify the transcription of the mRNA.

(<NUM>) Western Blot was used to separate the total proteins extracted from PBMC in accordance with relative molecular mass by polyacrylamide gel electrophoresis. The protein was transferred to PVDF membrane by wet rotation (<NUM>, <NUM> mA, <NUM>). PVDF membranes were sealed at room temperature for <NUM> hour with a sealing solution (TBST solution containing <NUM>% skimmed milk), and Biotinylated protein L was diluted at <NUM>:<NUM> with the sealing solution. Then they were incubated with the sealed PVDF membranes at room temperature for overnight at <NUM>. TBST was washed three times, <NUM> minutes each time. The corresponding SA-HRP was diluted at <NUM>:<NUM>, and PVDF membrane was incubated at room temperature for <NUM> hours, and TBST was washed three times, <NUM> minutes each time. Amersham company's ECL + plus TM Western blotting system kit was used for color rendering. X-ray development produces film showing strips. (<NUM>) Enzyme Linked ImmunoSorbent Assay (ELISA) was used to coat the <NUM>:<NUM>, <NUM>:<NUM> and <NUM>:<NUM> diluted supernatant of cell culture into <NUM> hole plate. Negative control, positive control and blank hole were set up at the same time and overnight at <NUM>. Wash three times the next day, add fresh <NUM>:<NUM> diluted proteinG-HRP <NUM> to the reaction pore, incubate at <NUM> for <NUM>-<NUM> minutes, wash, and wash with pure water for the last time. The TMB substrate solution added into the reaction pore was <NUM> and incubated at <NUM> for <NUM>-<NUM> minutes. ELISA reaction termination solution added into each reaction pore was <NUM>. OD values of each pore were measured at <NUM> on the enzyme label. (<NUM>) RT-QPCR inspection showed that the transcription levels of CAR gene and scFv gene of recombinant lentivirus vector after infected with PBMC were significantly higher than those in empty cells (as shown in <FIG>), indicating that the transcription levels of CAR gene and scFv gene were successfully expressed. (<NUM>) Western Blot results showed that the expression level of CAR protein of recombinant lentivirus vector infected with PBMC was significantly higher than that of control virus MOCK and empty cells (as shown in <FIG>), indicating that the translation level of CAR gene was successfully expressed. (<NUM>) Enzyme linked immunosorbent assay (ELISA) results showed that the expression level of scFv protein of recombinant lentivirus vector infected with PBMC was significantly higher than that of control virus MOCK and empty cells (as shown in <FIG>), indicating that the translation level of scFv gene was successfully expressed. Evaluation of Cytotoxicity Test.

Claim 1:
A CAR-T transgenic vector suitable for alleviating cytokine release syndrome (CRS) by blocking IL6R, comprising:
an AmpR sequence comprising ampicillin resistance gene for amplifying target bacterial strains by a large number as shown in SEQ ID NO.<NUM>;
a prokaryotic replicon pUC Ori sequence for plasmid replication as shown in SEQ ID NO.<NUM>;
an SV40 Ori sequence of viral replicator for enhancing replication in eukaryotic cells as shown in SEQ ID NO.<NUM>;
an eWPRE enhanced posttranscriptional regulatory element of Groundhog hepatitis B virus for enhancing the expression efficiency of the transgene as shown in SEQ ID NO.<NUM>;
a human EFlalpha promoter for eukaryotic transcription of chimeric antigen receptor genes as shown in SEQ ID NO.<NUM>;
lentivirus packaging cis-elements for lentivirus packaging;
a sequence encoding a humanized single-chain antibody fragment specific for human IL6R (IL6RscFv), which is IL6RscFv1 as shown in SEQ ID NO.<NUM>, or IL6RscFv2 as shown in SEQ ID NO.<NUM>, or IL6RscFv3 as shown in SEQ ID NO.<NUM>;
an IRES ribosome binding sequence for co-transcription and expression of proteins as shown in SEQ ID NO.<NUM>;
a sequence encoding an IL6 signal peptide as shown in SEQ ID NO.<NUM>;
a sequence encoding a human antibody Fc segment as shown in SEQ ID NO.<NUM>; and
a sequence encoding a second-generation or third-generation chimeric antigen receptor (CAR);
wherein the expression is started by the human EF1alpha promoter; and
wherein (i) the CAR and, (ii) a fusion protein of IL6RscFv and Fc, are co-expressed by IRES ribosome binding sequence, wherein the CAR is expressed on the surface of the cell membrane and, wherein the fusion protein of IL6RscFv and Fc is secreted outside the cell under the guidance of IL6 signal peptide.