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Transformed hinge and application thereof in construction of CAR skeleton

A hinge region, single-chain antibody technology, applied in the hinge region and its application in the construction of CAR skeleton, the hinge region, the application field in the construction of CAR skeleton, can solve the problem of CAR-T treatment with little effect, patient recurrence, CAR-T T cells affect the effectiveness of killing and other issues

Active Publication Date: 2018-07-03
CHONGQING PRECISION BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, even for acute lymphoblastic leukemia (ALL) with significant treatment effect, the median time of complete remission after treatment is generally about 8 months, and a large number of patients still relapse, which may be related to the short duration of CAR-T cells in patients. , Improving the duration of CAR-T in vivo is of great significance for the efficacy of CAR-T therapy
[0004] Although the use of CAR-T in the study of solid tumors has also been paid more and more attention, the effect of most CAR-T in the treatment of solid tumors is not satisfactory. Death soon affects the effectiveness of killing; on the other hand, most solid tumors will limit the control and killing of the immune system in many aspects due to their pluripotency in metabolism, immune escape, and tissue formation, resulting in CAR-T cells It is difficult to infiltrate into the tumor tissue of solid tumors, so CAR-T therapy for large solid tumors has little effect
Prolonging the persistence of CAR-T in vivo, promoting the infiltration of CAR-T cells in solid tumor tissues, can effectively kill tumors and inhibit the recurrence of tumor cells, but there is no effective way to prolong the persistence of CAR-T in vivo and promote the tumor infiltration of CAR-T cells Methods

Method used

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  • Transformed hinge and application thereof in construction of CAR skeleton
  • Transformed hinge and application thereof in construction of CAR skeleton
  • Transformed hinge and application thereof in construction of CAR skeleton

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0068] Example 2 Construction of chimeric antigen receptor viruses containing different hinge regions

[0069] In order to verify the influence of hinge regions with different structures on the continuity and tumor infiltration of CAR-T in vivo, experiments were carried out with CAR-T targeting PSCA as an example. designed as figure 1 CARs with 5 different hinge structures shown.

[0070] 1 Synthesize the chimeric antigen receptor gene sequence targeting PSCA containing the hinge sequence designed above

[0071] Synthesize single-chain antibodies containing leader peptide (also known as signal peptide) (abbreviated as LP), anti-human PSCA antigen, 6 different hinge regions, CD28 transmembrane region (abbreviated as TM), and intracellular signal domains of CD28, CD137 and CD3 The CAR structure.

[0072] 2 Construction of lentiviral vectors expressing chimeric antigen receptors

[0073] The following primers were designed and synthesized by a biotechnology company. The speci...

Embodiment 3

[0097] Example 3 Detection of the ability of CAR to transfect T lymphocytes

[0098] 1 Isolation of human peripheral blood mononuclear cells

[0099] Collect about 60ml of peripheral blood with a blood collection tube, divide it into a 50ml centrifuge tube, add 7.5ml of hydroxyethyl starch to dilute it; naturally settle at room temperature (18-25°C) for about 30min, collect the upper plasma, centrifuge the collected upper plasma and use physiological Resuspend the precipitate in saline, add it to the lymphocyte separation solution in a volume ratio of 1:1, and perform gradient centrifugation; after centrifugation, the centrifuge tube is divided into: the first layer: plasma layer; the second layer: annular milky white lymphocytes Cell layer; third layer: transparent separation liquid layer; fourth layer: red blood cell layer; take the second white lymphocyte layer, wash twice with normal saline, centrifuge for 5 min, resuspend cells in normal saline, add 10% FBS-containing Hu...

Embodiment 4

[0106] Example 4 Detection of long-term proliferation ability of CAR-T cells

[0107] The proliferation of infected PSCA-CAR-T cells under normal culture conditions was detected; the CAR-T cells obtained in Example 3 were cultured for 10 days using the culture method in Step 2 of Example 3. The 24-well plate was coated with PSCA antigen for four times overnight, and 1*10^6 / well of CAR-T cells were plated on the 24-well plate coated with PSCA antigen, and the survival time of CAR-T cells after antigen stimulation was observed. like Figure 4 As shown, the number of CAR-T cells was counted by a cell counter on the 3rd day, the 6th day, the 9th day and the 12th day after stimulation, and the CAR-T cell proliferation fold was calculated. According to the experience of in vivo experiments, antigen stimulation was performed every 7 days, Figure 4 As a result, the CAR-T cells no longer proliferated after the third antigen stimulation, so the experiment was terminated after the 15t...

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Abstract

The invention belongs to the field of immunological therapy and in particular relates to a transformed hinge region and application thereof in construction of a CAR skeleton. The hinge region providedby the invention can prolong in vivo existence of CAR-T cells and / or improve capability of the CAR-T cells for infiltrating tumors, and an amino acid sequence of the hinge region is shown in SEQ ID NO.1 and SEQ ID NO.2. The hinged region structure provided by the invention can prolong the in vivo existence of the CAR-T cells, a combined chimeric antigen receptor of the hinged region structure canbe more stably expressed in T lymphocytes, CAR-T containing the hinged region provided by the invention is longer in vivo existence, the capability of the cells for infiltrating the tumors is obviously enhanced, and killing effect is better.

Description

technical field [0001] The invention belongs to the field of immunotherapy, and relates to a modified hinge region and its application in constructing a CAR skeleton, in particular to a hinge region capable of prolonging the in vivo persistence of CAR-T cells and / or improving the ability of CAR-T cells to infiltrate tumors and its application in the construction of CAR skeletons. Background technique [0002] Chimeric antigen receptor (CAR) is an artificial receptor that mimics the function of TCR, including antigen recognition domain, hinge region, transmembrane region and intracellular signaling domain. The intracellular signaling domain is usually the CD3ζ chain or FcRγ, or is linked to one or more costimulatory molecules, such as 4-1BB (CD137), CD28, ICOS (CD278). When the antigen (receptor) on the surface of the tumor cell binds to the antibody (ligand) of the chimeric antigen receptor, the signal is transmitted into the cell through the hinge region and the transmembr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C07K19/00C12N15/62A61K35/17A61P35/00C12N15/867C12N15/66
CPCA61K35/17C12N15/66C12N15/86C07K14/7051C07K16/3069C07K2317/622C07K2319/03C12N2740/15043C12N5/0636A61P35/00C07K2317/53C07K2319/02C07K2319/33C12N2510/00Y02A50/30
Inventor 张巍赵文旭陈军黄霞赵永春单娟娟徐艳敏张茜真
Owner CHONGQING PRECISION BIOTECH CO LTD
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