A car-t treatment vector for lymphoid leukemia based on octs technology and its construction method and application
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A technology of leukemia, vector, applied in the field of medical biology
Active Publication Date: 2019-05-17
SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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[0013] At present, there is no report on CAR-T therapy targeting CD19 and any one of the antigens selected from CD22, CD20, CD30, and CD123 to overcome the above shortcomings.
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[0060] Below in conjunction with specific embodiment further elaborates this invention. It should be understood that the specific embodiments described herein are presented by way of example and not as limitations of the invention. The principal characteristics of this invention can be employed in various embodiments without departing from the scope of the invention. Material
[0061] 1. Lentiviral backbone plasmid pLenti-3G basic, lentiviral packaging plasmids pPac-GP, pPac-R and membrane proteinplasmid pEnv-G, HEK293T / 17 cells, homologous recombinationenzyme, Oligo Annealing Buffer, mycoplasma detection kit, internal Toxin Detection Kit, CD19 + K562, CD20 + K562, CD22 + K562, CD30 + K562, CD123 + K562, CD19 + CD123 + K562, CD19 + CD20 + K562, CD19 + CD22 + K562, CD19 + CD30 + K562 and K562 cells were purchased from Shiao (Shanghai) Biomedical Technology Co., Ltd.; the specific preparation method of the lentiviral backbone plasmid pLenti-3G basic has been ...
Embodiment 1
[0103] Example 1 Construction of OCTS-CAR-T cells
[0104] 1. Construction, purification and detection methods of recombinant lentiviral vectors lvOCTS12319s, lvOCTS2219s, lvOCTS2019s, lvOCTS3019s, lvOCTS12319t, lvOCTS2219t, lvOCTS2019t, lvOCTS3019t.
[0105] see image 3 , the construction method of the recombinant lentiviral vector of the present invention is as follows:
[0106] 1. Human EF1α promoter (SEQ ID NO.14), OCTS structure [CD8leader chimeric receptor signalpeptide as shown in SEQ ID NO.15, two groups of single-chain antibodies: the first group is selected from the following four groups of single-chain antibodies Any group of chain antibodies: CD20 single-chain antibody light chain VL as shown in SEQ ID NO.18, CD20 single-chain antibody heavy chain VH as shown in SEQ ID NO.19; CD22 as shown in SEQ ID NO.20 Single-chain antibody light chain VL, CD22 single-chain antibody heavy chain VH as shown in SEQ ID NO.21; CD30 single-chain antibody light chain VL as shown i...
Embodiment 2
[0221] OCTS-CAR-T cellpathogen detection and expression detection.
[0222] 1. Endotoxin detection;
[0223] (1), endotoxin working standard is 15EU / branch;
[0225] (3) Dilution of endotoxin standard substance: Take one endotoxin standard substance, dilute it with BET water in proportion to dissolve into 4λ and 2λ respectively, seal with parafilm, shake and dissolve for 15min; each step of dilution should be mixed in the vortex Mix on the mixer for 30s;
[0226] (4) Adding samples: Take several LAL reagents, add 0.5 ml of BET water to each tube to dissolve, and distribute to several endotoxin-free test tubes, each tube has 0.1 ml. Two of them are negative control tubes, add 0.1ml of BET water;
[0228] 2 tubes are sample positive control tubes, add 0.1ml sample solution containing 2λ endotoxin ...
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Abstract
The invention discloses a CAR-T treatment vector for lymphoid leukemia based on OCTS technology, including lentiviral backbone plasmid, human EF1α promoter, OCTS chimeric receptor domain and IL6R single chainantibody; OCTS chimeric receptor domain Includes: CD8 leader chimeric receptorsignalpeptide, two groups of single chain antibodies: the first group is selected from any of the following four groups of single chain antibodies: CD20 single chain antibody light chain VL, CD20 single chain antibodyheavy chain VH; CD22 Single chain antibody light chain VL, CD22 single chain antibody heavy chain VH; CD30 single chain antibody light chain VL, CD30 single chain antibody heavy chain VH; CD123 single chain antibody light chain VL, CD123 single chain antibody heavy chain VH; Group 2 It is the CD19 single-chain antibody light chain VL and the CD19 single-chain antibody heavy chain VH; the intra-antibody hinge Inner‑Linker, the single-chain antibody inter-hinge Inter‑Linker, the CD8 Hinge chimeric receptor hinge, and the CD8 Transmembrane chimeric receptor transmembrane region. , TCR chimeric receptor T cell activation domain and chimeric receptor costimulator region. In addition, the present invention also discloses the construction method of the vector and its application in preparing drugs for treating lymphoid leukemia.
Description
technical field [0001] The invention belongs to the field of medical biology, in particular to a carrier, in particular to a CAR-T treatment carrier for lymphoid leukemia based on OCTS technology. In addition, the present invention also relates to the construction method and application of the carrier. Background technique [0002] The theoretical basis of tumor immunotherapy is that the immune system has the ability to recognize tumor-associated antigens and regulate the body to attacktumor cells (highly specific cytolysis). In the 1950s, Burnet and Thomas put forward the theory of "immune surveillance", thinking that the mutated tumor cells that often appear in the body can be recognized and eliminated by the immune system, which laid the theoretical foundation for tumor immunotherapy [Burnet FM. Immunological aspects of malignant disease. Lancet, 1967; 1:1171-4]. Subsequently, various tumor immunotherapies, including cytokine therapy, monoclonalantibody therapy, adopt...
Claims
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