Design of activated T cells of bispecific T cell activator and application thereof

A technology of cells and cell groups, applied in the direction of receptor/cell surface antigen/cell surface determinant, for targeting specific cell fusion, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can Solve the problems of neurotoxicity and CAR-T therapy not showing clinical efficacy

Pending Publication Date: 2021-03-12
普米斯生物技术(苏州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, there are still two major problems in the use of immunotherapy to treat solid tumors: on the one hand, the severe and even fatal clinical side effects associated with CAR-T therapy are still a huge risk in the clinical application of CAR-T therapy. These side effects mai

Method used

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  • Design of activated T cells of bispecific T cell activator and application thereof
  • Design of activated T cells of bispecific T cell activator and application thereof
  • Design of activated T cells of bispecific T cell activator and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0177] The design of embodiment 1CAB and contrast structure

[0178] 1.1 Control group CD3e-BBζ, 1 st -CAIX-BiTA, 1 st - Structure design of CAIX-CAB and CAIX-TRuC

[0179] In order to verify the anti-tumor activity of CAB-T, in a set of experiments, we first designed the first set of structures using nanobodies targeting CAIX: CD3e-BBζ (amino acid sequence is SEQ ID NO.: 7), CAIX-BiTA ( The amino acid sequence is SEQ ID NO.:8), CAIX-CAB (amino acid sequence is SEQ ID NO.:9) and CAIX-TRuC (amino acid sequence is SEQ ID NO.:10). In order to distinguish the second group of CAIX-CAB structures described later, we named the CAIX-BiTA and CAIX-CAB of this group as 1 st -CAIX-BiTA and 1 st -CAIX-CAB. Among them, 1 st -CAIX-BiTA is BiTA without label, 1 st -CAIX-CAB is the CAB structure of CD3e-BBζ and untagged BiTA, and CAIX-TRuC is the TCR-utilized 2 The comparative structure of the company's platform technology. The specific structure diagram of the above structure is as ...

Embodiment 2

[0184] Example 2 Packaging of CAB and its control structure lentiviral vector

[0185] In the present invention, we use lentivirus as a vector to prepare CAB-T cells. First, we prepared lentiviral vectors carrying genes encoding CAB and its control constructs. The specific process of lentivirus packaging is as follows:

[0186] 1) Spread 1×10 in a 10cm culture plate 7 For HEK 293T cells, add 10 mL of DMEM (Hyclone, SH30243.01) medium containing 10% FBS (Gibco, 10099-141C), mix the cells well, and culture overnight at 37 degrees;

[0187] 2) On the second day, when the HEK 293T (ATCC, CRL-3216) cell confluence reaches about 90%, replace with serum-free DMEM;

[0188] 3) Prepare the plasmid complex, the amount of each plasmid is: 8 μg plasmad DNA, 4 μg psPAX2 and 2 μg pMD2g, dissolve in 1 mL opti-MEM (Gibco, 31985-070) and add 42 μL PEI (Polysciences, 24765-2); vortex Spin and shake for 20s. After standing at room temperature for 15 minutes, gently add the mixture to the HE...

Embodiment 3

[0192] Example 3 Preparation of CAB and its control structurally engineered T cells

[0193] After the lentiviral vector carrying the CAB structure is prepared, the lentiviral vector can be used to infect immune cells to complete the preparation of CAB-T cells. The specific procedure for the preparation of CAB-T cells is as follows:

[0194] 1) Culture commercial PBMC (Sai ​​Li Bio, SLB-HP050B) cells with X-VIVO 15 (LONZA, 04-418Q) containing 5‰ human albumin (GRIFOLS, human albumin 20%), initially Cell density is 1 x 10 6 / mL;

[0195] 2) Add anti-CD3 / CD28beads (Miltenyi biotec, 130-091-441) according to the ratio of cells:Beads=3:1, and add 1000IU / mL IL-2 (Sihuan Biological, S10970016) to activate T cell expansion;

[0196] 3) After 48 hours of cell activation, an appropriate amount of virus and 12 μg / mL protamine (Sigma, P4005) were added to infect T cells;

[0197] 4) After 24 hours of lentivirus infection, the cell suspension was aspirated, and 1×10 6 Add complete f...

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PUM

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Abstract

The invention provides an immunotherapy scheme for inhibiting tumors, especially solid tumors, T cells expressing chimeric CD3e fusion protein are combined with BiTA, the chimeric CD3e fusion proteinand BiTA are combined with each other, and the functions of activating the T cells and targeting tumor cells are exerted. The invention also provides a CAB structure and a CAB editing T cell (CAB-T).BiTA secreted by the CAB-T cells can simultaneously realize activation of the CAB-T cells and activation of endogenous TCR of the non-edited T cells in tumor tissues, and the anti-tumor effect of theCAB-T and the anti-tumor effect of mobilizing the non-edited T cells are exerted. The chimeric CD3e expressed by CAB-T and BiTA cooperate with each other to exert an anti-tumor effect: activation of the chimeric CD3e depends on BiTA secreted by CAB-T, and secretion of the BiTA further stimulates the CAB-T to release more BiTA by activating the chimeric CD3e; a small amount of BiTA released by CAB-T in tumor tissues can stimulate the CAB-T to release more BiTA by activating chimeric CD3e, so that immune cell activation and anti-tumor effects of tumor tissue tendency are realized, and the safetyadvantage of CAB-T clinical application is ensured.

Description

technical field [0001] The invention belongs to the field of immunotherapy, in particular, the invention relates to the design and application of a bispecific T cell activator to activate T cells. Background technique [0002] In recent years, immune cell therapy has achieved unprecedented success in complete remission rates for hematological malignancies. In 2017, two CD19-targeting CAR-T products have been successfully launched, and were approved for the treatment of children and adolescents with acute leukemia and non-Hodgkin's lymphoma. [0003] However, there are still two major problems in the use of immunotherapy to treat solid tumors: on the one hand, the severe and even fatal clinical side effects associated with CAR-T therapy are still a huge risk in the clinical application of CAR-T therapy. These side effects mainly include cytokines Release syndrome (cytokine release syndrome, CRS), macrophage activation syndrome, hemophagocytic lymphohistiocytoma, neurotoxicit...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/29C12N5/10A61K39/00A61P35/00
CPCC07K14/7051C12N5/0636A61K39/001102A61P35/00C12N2510/00C07K2319/02C07K2319/03A61K35/17C07K16/2809C07K2319/33A61K2039/5156C07K2317/569C07K2317/31C07K16/30C07K16/32C07K2317/24A61K39/001154A61K39/001106A61K2039/5158C12N5/0638A61K38/00A61K31/506C07K2319/00A61K2300/00
Inventor 李志远伊刚曾竣玮刘晓林
Owner 普米斯生物技术(苏州)有限公司
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