CN-120795167-B - Chimeric antigen receptor CAR (CAR) targeting human EB (Epstein-Barr) virus, CAR-NK cell and application

Abstract

The invention discloses a chimeric antigen receptor CAR of targeting human EB virus, a CAR-NK cell and application thereof, wherein the chimeric antigen receptor CAR of targeting human EB virus comprises the following structures: a gH/gL single-chain antibody directed against EB virus, a transmembrane region, and an intracellular signaling region. The invention proposes that the target human EB virus CAR-NK cells can effectively identify and kill the nasopharyngeal carcinoma and B lymphomas infected by EBV, effectively kill tumor cells, can generate lasting immune response capability, does not generate serious toxic or side effect, and is helpful for preventing tumor recurrence.

Inventors

• LI GENGYU
• WANG KAI
• LI ZHISHENG

Assignees

• 佰芮慷(广州)细胞医药科技有限公司

Dates

Publication Date

20260508

Application Date

20250415

Claims (7)

1. 1. A chimeric antigen receptor CAR targeting human epstein barr virus, characterized in that said CAR is selected from any one of the following: (1) The chimeric antigen receptor consists of a signal peptide, gH/gL single-chain antibody aiming at EB virus, a hinge domain, a transmembrane region and an intracellular signal transduction region which are sequentially connected in series, wherein the gH/gL single-chain antibody aiming at EB virus consists of VH shown as SEQ ID NO. 2, connecting peptide and VL shown as SEQ ID NO. 3 which are sequentially connected in series; (2) The chimeric antigen receptor of (1) further comprises an IL-15/IL-15Rα cytokine, and the amino acid sequence of the IL-15/IL-15Rα cytokine is shown as SEQ ID NO. 1.
2. 2. The chimeric antigen receptor CAR targeting human epstein barr virus according to claim 1, characterized in that said transmembrane region is CD8 TM and said intracellular signal transduction region is the co-stimulatory signal domain CD137 of 4-1BB and the signal domain CD247 of the zeta chain of the human CD3 complex.
3. 3. The chimeric antigen receptor CAR for targeting human epstein barr virus according to claim 1, characterized in that the amino acid sequences of the hinge domain, the transmembrane region and the intracellular signal transduction region are shown in SEQ ID No. 4.
4. 4. A CAR-NK cell targeting human epstein barr virus, characterized in that it is prepared by transduction of NK cells with a chimeric antigen receptor CAR targeting human epstein barr virus according to any one of claims 1 to 3.
5. 5. The CAR-NK cell of claim 4, wherein the NK cell is derived from umbilical cord blood.
6. 6. The human EB virus targeting CAR-NK cell according to claim 5, wherein the preparation method comprises the steps of S1 constructing a retrovirus vector containing a chimeric antigen receptor CAR, S2 packaging the retrovirus, S3 separating and purifying NK cells from umbilical cord blood, activating the NK cells, culturing and activating the NK cells with factors CD16, OKE432 and IL15 by an enrichment method, carrying out CAR gene transduction virus infection on the 4 th day after activation, and S4 virus infection to collect the activated NK cells.
7. 7. The use of a CAR-NK cell targeted to human epstein barr virus according to any one of claims 4-5 for the preparation of a medicament for the treatment of a targeted human epstein barr virus infection.

Description

Chimeric antigen receptor CAR (CAR) targeting human EB (Epstein-Barr) virus, CAR-NK cell and application Technical Field The invention relates to the field of biotechnology engineering, in particular to a Chimeric Antigen Receptor (CAR) targeting human EB virus, a CAR-NK cell and application thereof. Background EB virus (Epstein Barr Virus, EBV) belongs to the herpesvirus family and is commonly susceptible to the population. EBV primary infection causes adenofever, but unlike other viruses, EBV is not cleared by the body's immune system after primary infection, but is carried throughout the life, and infected EBV can be activated under certain conditions to become oncogenic. EBV is a DNA tumorigenic virus of the lymphotropic genus, most common in a latent infection, more than 90% of people show a lifelong latent state, most prognosis is good, but EBV infection can be life threatening in immunocompromised persons. EBV can infect epithelial and B cells, although the proportion of cancers directly caused by EBV is low, 20 ten thousand new cancer cases and 14 ten thousand cancer deaths worldwide each year are associated with EBV infection due to the high proportion of infections in the population. Epithelial malignancy is the most common EBV-associated cancer including gastric and nasopharyngeal cancers, and EBV is also associated with Burkitt's malignant lymphoma and hodgkin's lymphoma. EBV is an enveloped virus that infects cells by a process of complete fusion of the membrane proteins exposed to itself with the host cell membrane, including proteins of fusion nature gB and accessory proteins of gp350, gp42, gHgL, etc. that interact directly or indirectly with host cell receptors. Entry of the EBV virus into B cells is initiated by binding of glycoprotein gp350 to complement receptor 2 (CR 2-Fc). The viral glycoprotein complex is composed of gH, gL and gp42, with gp42 binding to Human Leukocyte Antigen (HLA) class II molecules, gB being activated and promoting fusion of the viral membrane with the B cell membrane, both gH and gp42 being necessary for fusion of the virus with the B cell. Epithelial cell infection is caused by binding of EBV BMRF2 to integrin, binding of gH/gL to integrin and ephrin receptor A2, and then activating gB to promote virus-cell membrane fusion. The glycoproteins gH/gL and gB constitute the core fusion mechanism of the virus, which is conserved in all herpesviruses. Most of the serum neutralizing antibodies blocking B cell infection target gp350 at present, and almost all clinical trials for treating EBV infection use gp350 as the sole immunogen, as in chinese patent 202111646561.5, a neutralizing monoclonal antibody capable of recognizing gp350 protein of epstein barr virus with high affinity is provided, which is capable of blocking the binding of gp350 protein and its ligand CR 2. Although epithelial cell infection is of great importance in the EBV lifecycle and EBV-related epithelial malignancies are more common than B-cell cancers, no therapeutic effect of cell therapies directed against EBV glycoproteins (e.g. gH/gL) to target viral infection in B-cells and epithelial cells has been reported. Disclosure of Invention In view of the above, it is necessary to provide a chimeric antigen receptor CAR targeting human epstein barr virus, CAR-NK cells and uses thereof. In order to solve the technical problems, the first aspect of the invention provides a chimeric antigen receptor CAR targeting human EB virus, which comprises a gH/gL single-chain antibody aiming at the EB virus, a transmembrane region and an intracellular signal transduction region. Furthermore, the gH/gL single-chain antibody against EB virus contains the amino acid sequences of SEQ ID No.2 and SEQ ID No. 3. Furthermore, the chimeric antigen receptor CAR targeting human EB virus also comprises IL-15/IL-15Rα cytokines, wherein the IL-15/IL-15Rα cytokines contain the amino acid sequence of SEQ ID No. 1. Further, the transmembrane region is CD8 TM and the intracellular signaling region is the costimulatory signaling domain CD137 of 4-1BB and the signaling domain CD247 of the zeta chain of the human CD3 complex. Further, the chimeric antigen receptor CAR targeting human EB virus further comprises a hinge domain located between the single chain antibody and the transmembrane region. Furthermore, the chimeric antigen receptor CAR targeting the human EB virus is formed by sequentially connecting in series a signal peptide CD8 (SP), a gH/gL single-chain antibody aiming at the EB virus, a hinge domain, a transmembrane region, an intracellular signal transduction region and IL-15/IL-15 Ralpha. The second aspect of the present invention provides a CAR-NK cell targeting human epstein barr virus, which is prepared by transducing NK cells with any of the above chimeric antigen receptor CAR targeting human epstein barr virus. Further, the NK cells are derived from cord blood. The third aspect of the invention provides a preparation m