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Therapeutic agent and application thereof comprising nucleic acid and car-modified immune cells

A therapeutic agent and tumor cell technology, applied in the field of medical bioengineering, can solve the problems of high risk of on-target/off-target toxicity, inability to exert the killing effect of cancer cells, poor T-cell tumor homing, etc. Effects of improved homing ability, reduced risk of on-target/off-target toxicity

Active Publication Date: 2022-05-27
HANGZHOU CONVERD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, CAR-T technology still has great challenges in the treatment of solid tumors. The main difficulties include: 1) Solid tumors express highly heterogeneous tumor antigens, that is, diversity, so that cancer cells can easily escape the surveillance of the immune system; This is different from blood cancers. For example, CD19 leukemia is basically CD19-positive; due to the diversity of solid tumor antigens, it is difficult to find a suitable target that can kill all cancer cells, and the remaining target-negative cancer cells will cause tumor recurrence; 2) Many tumor antigens of solid tumors are also expressed in normal tissues, so it is difficult to design tumor-specific CARs, the probability of off-target is high, and the risk of on-target / off-target toxicity is high; 3) T cell tumor homing is poor; Cells are surrounded by a dense matrix to form a tumor microenvironment; the matrix is ​​composed of recruited normal tissue and bone marrow-derived (stromal) cells, which prevent immune cells from penetrating this barrier; 4) solid tumors have a strong Immunosuppressive ability, many cells in the tumor microenvironment can inhibit the anti-cancer function of immune cells; therefore, CAR-T that infiltrates into solid tumors cannot exert efficient cancer cell killing effect

Method used

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  • Therapeutic agent and application thereof comprising nucleic acid and car-modified immune cells
  • Therapeutic agent and application thereof comprising nucleic acid and car-modified immune cells
  • Therapeutic agent and application thereof comprising nucleic acid and car-modified immune cells

Examples

Experimental program
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Effect test

preparation example 1

[0321] Preparation Example 1: Preparation of tumor cells labeled with TT1, TT2, TT3, C1&2a, C1&2b by electroporation

[0322] Will 5x10 6 Jurkat cells, HCT116-luc, SKOV3-luc or SK-HEP-1 with 5 μg of TT1 (SEQ ID NO:11), TT2 (SEQ ID NO:12) or TT3 (SEQ ID NO:13), C1 & 2a (SEQ ID NO:11), respectively SEQ ID NO: 14), C1 & 2b (SEQ ID NO: 15) mRNAs (obtained according to the method of Preparation Example 8) were mixed in electroporation solution P3 (product name "P3Primary Cell 4D-X Kit L", Lonza, article number V4XP-3012), placed in a 100 μl Nucleocuvette™ tube (product name “P3Primary Cell 4D-X Kit L”, Lonza, Cat. No. V4XP-3012), and subjected to an ice bath for 5 minutes. Then use the 4D-NucleofectorTM electroporator (Lonza) to select its own tumor cell electroporation program for electroporation. After electroporation, the cells were removed and placed in the corresponding tumor cell culture medium. Jurkat medium is RPMI (Gibco)+10%FBS (Hyclone), SKOV3-luc and HCT116-luc mediu...

preparation example 2

[0326] Preparation Example 2: Preparation of CAR-T cells targeting TT1, TT2 and TT3 respectively

[0327] Will 2 x 10 6 Human PBMC cells were resuspended in 1 ml of T cell culture medium (AIMV (Gibco) supplemented with 1% human AB serum (Valley Biomedical)) supplemented with OKT3 (eBioscience) at a final concentration of 100 ng / ml and hrIL-2 at 300 IU / ml (Peprotech), seeded into one well of a 24-well plate at 37°C, 5% CO 2 The humidification cell incubator (Thermo Fisher). Supplement hrIL-2 with a final concentration of 300IU / ml every 2-3 days. According to the growth of cells, add fresh T cell culture medium and adjust the number of cells to 1×10 6 cells / ml.

[0328] T cells (1 × 10 7 ) and 4 μg aTT3-CD8-41BB-CD3ζ mRNA (that is, the mRNA corresponding to the nucleotide sequence shown in SEQ ID NO: 55 (obtained according to the method of Preparation Example 8)) mixed in electroporation solution P3 (product name “P3Primary Cell4D-X”) Kit L", Lonza, Cat. No. V4XP-3012), pla...

Embodiment 1

[0330] Example 1: Tumoricidal ability of CAR-T cells targeting marker polypeptides to tumor cells labeled with marker polypeptides by electroporation

[0331] This example tests the killing ability of CAR-T cells directed against the labeled polypeptides to Jurkat cells after electrotransduction. The CAR-modified or unmodified T cells targeting TT1 or TT2 obtained by the method of Preparation Example 2 were co-cultured with Jurkat, a human T cell line labeled with TT1 or TT2 by electroporation obtained by the method of Preparation Example 1, respectively. In a U-shaped 96-well plate, the ratio of the number of CAR-T effector cells to target cells (E:T) ranged from 1.25:1 to 20:1. Each group of experiments was repeated 3 times. After 2 hours of co-culture, DELFIA EuTDA Cytotoxicity Kit (PerkinElmer, USA) was used to detect the ability of CAR-T cells to lyse tumor cells, and the killing effect was calculated by the following formula: % specific lysis = ((experimental group rele...

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Abstract

Therapeutic agent and application involving nucleic acid and CAR-modified immune cells. The therapeutic agent comprises a first composition and a second composition, the first composition comprises a nucleic acid having a coding sequence of a marked polypeptide for introduction into tumor cells and / or cancer cells; the marked polypeptide has an operable The linked extracellular epitope, spacer and transmembrane can be expressed and modified on the surface of the tumor cells and / or cancer cells; the amino acid sequence of the extracellular epitope contains one or more antigenic surface The amino acid sequence of the epitope polypeptide; wherein in the natural state, the amino acid sequence of the mammalian cell membrane protein or secreted protein does not contain the amino acid sequence of the antigen epitope polypeptide; the second composition comprises chimeric antigen receptor modified immune cells ; The chimeric antigen receptor-modified immune cells can specifically recognize and bind to the extracellular antigen-determining region of the labeled polypeptide. The invention realizes synergistic curative effect.

Description

technical field [0001] The present invention belongs to the field of medical bioengineering, and in particular, relates to a therapeutic agent comprising nucleic acid and CAR-modified immune cells, a labeled polypeptide, a chimeric antigen receptor, an encoding nucleic acid, an expression vector, a recombinant virus, a kit and applications thereof. Background technique [0002] Cancer immunotherapy is a treatment method that specifically removes minimal residual cancer foci or significantly inhibits cancer cell proliferation by activating the immune system in the body. This treatment method has the advantages of long duration of action and few side effects, and is known as the fourth mode of modern cancer treatment. In recent years, many advances have been made in cancer immunotherapy. The journal Science listed cancer immunotherapy as a major scientific breakthrough in 2013. Chimeric antigen receptor CAR-modified immune cells are now the most effective and promising tumor ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N5/10C12N7/01A61K35/761A61K35/763A61K35/765A61K35/766A61K35/768A61K35/17A61P35/00C07K19/00
CPCA61K35/17A61K35/761A61K35/763A61K35/765A61K35/766A61K35/768A61P35/00C07K14/70517C07K14/7051C07K14/70521C07K14/70514C07K14/70578C12N5/0636C12N5/0646C12N7/00C07K2319/00C07K2319/03C07K2319/33C07K2319/41C07K2319/42C07K2319/22C12N2510/00C12N2710/24021C12N2710/24032A61K39/4613A61K2239/38A61K39/0011A61K2239/59A61K39/4644A61K39/4631A61K2239/31A61K39/4611C12N5/0638A61K2239/50A61K2300/00Y02A50/30C07K2319/40C07K2319/20C12N2710/24121C12N2710/24143C12N15/86C12N2501/2302C12N2502/30A61K47/6891A61K38/1774A61K48/0058
Inventor 胡放陈璨肖琳傅瑾张蓉蔡金露
Owner HANGZHOU CONVERD CO LTD
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