CN-122011198-A - NK immune cells with enhanced activity and application thereof in cancer treatment

Abstract

The invention discloses an NK immunocyte with enhanced activity and application thereof in cancer treatment. The NK cells stably express a conjugated bispecific antibody of anti-PD-L1/CD 16 through a lentiviral vector, and the antibody is formed by connecting an anti-PD-L1 single domain antibody and an anti-CD 16 single domain antibody in series through a flexible connecting peptide (G4S) 3. The NK immunocyte with enhanced activity has three functions of targeting tumor cells through the anti-PD-L1 sdAb and blocking a PD-1/PD-L1 immunosuppression channel, activating the self ADCC effect of the NK cells through the anti-CD 16 sdAb, and remarkably enhancing the penetration capacity to solid tumors by virtue of the small molecular weight characteristic of a single-domain antibody. In vitro experiments show that the killing rate of sdAb-NK cells on various PD-L1 positive tumor cells is improved more than that of unmodified NK cells, and the tumor penetrating capacity is improved by 3.7 times. In vivo animal experiments show that the tumor inhibition rate is up to 84.8%, and the tumor has no obvious toxicity, thus providing a new efficient and safe strategy for the immunotherapy of solid tumors.

Inventors

• SHANG PINGPING

Assignees

• 广东拓普灵生物医药科技有限公司

Dates

Publication Date

20260512

Application Date

20260304

Claims (10)

1. 1. The anti-PD-L1/CD 16 conjugated bispecific antibody is characterized in that the amino acid sequence of the anti-PD-L1/CD 16 conjugated bispecific antibody is shown as SEQ ID NO. 4.
2. 2. The anti-PD-L1/CD 16 coupled bispecific antibody coding gene is characterized in that the nucleotide sequence of the anti-PD-L1/CD 16 coupled bispecific antibody coding gene is shown as SEQ ID NO. 3.
3. 3. An expression vector, wherein the expression vector is a pCHO-sdAb-BsAb comprising the coding gene of claim 2.
4. 4. A CHO cell, characterized in that, the CHO cell comprising the expression vector of claim 3.
5. 5. An enhanced activity NK immune cell, wherein said enhanced activity NK immune cell stably expresses the anti-PD-L1/CD 16 conjugated bispecific antibody of claim 1 mediated by a lentiviral vector.
6. 6. The NK immune cell of claim 5, wherein the lentiviral vector has a backbone of pLVX-EF1 a-IRES-ZsGreen 1, comprising an EF 1a promoter, an expression cassette of the anti-PD-L1/CD 16 conjugated bispecific antibody coding gene of claim 2 and an IRES-ZsGreen1 reporter.
7. 7. A pharmaceutical composition comprising the NK immune cells of claim 5 that enhance activity and a pharmaceutically acceptable adjuvant.
8. 8. Use of an anti-PD-L1/CD 16 conjugated bispecific antibody according to claim 1 for the preparation of an NK immune cell with enhanced activity.
9. 9. Use of an NK immune cell of claim 5 that enhances activity in the preparation of a medicament for the treatment of cancer, wherein the cancer is a PD-L1 positive cancer.
10. 10. The use according to claim 9, wherein the PD-L1 positive cancer comprises melanoma, non-small cell lung cancer, liver cancer, colorectal cancer.

Description

NK immune cells with enhanced activity and application thereof in cancer treatment Technical Field The invention belongs to the technical field of immune cell treatment, and particularly relates to an NK immune cell with enhanced activity and application thereof in cancer treatment. Background Natural Killer (NK) cells are core effector cells of the innate immune system, have the ability to rapidly recognize and clear malignant cells, and do not cause graft versus host disease. Its function depends on the activation/inhibition of receptor signal balance and on CD 16-mediated antibody-dependent cellular cytotoxicity (ADCC). These properties make NK cells exhibit unique advantages in adoptive cell therapy of hematological tumors (e.g., leukemia, lymphoma) and partial solid tumors. However, traditional NK cell therapies face a double bottleneck in the treatment of solid tumors. On one hand, tumor microenvironment inhibits NK cell function through multiple mechanisms, namely, after high-expression immune checkpoint ligands such as PD-L1 and the like are combined with NK cell surface receptors (such as PD-1), inhibitory signal channels such as SHP-1/2 and the like are activated, release of cytotoxic particles and production of effector are weakened, and meanwhile, inhibiting factors such as TGF-beta, adenosine and the like further exacerbate NK cell function exhaustion. On the other hand, solid tumors escape natural recognition of NK cells by down-regulating or proteolytically shedding NKG2D ligands (e.g., MICA/B), whereas unmodified NK cells lack tumor-specific targeting ability, lack infiltration at focal sites, and may accidentally injure normal tissues. The prior art scheme has the defects that CAR-NK cells targeting blood tumor antigens such as CD19 and the like have limited effect on solid tumors and do not integrate immune checkpoint blocking functions, NK cells secreting PD-1 antibodies can block partial inhibition signals but cannot enhance tumor targeting, systemic antibody secretion can disturb immune homeostasis, and CAR-NK cells aiming at solid tumor antigens (such as EGFR) are easy to inactivate in an immune inhibition microenvironment. These single function modification strategies have difficulty breaking through the complex defense system of solid tumors. Therefore, developing an integrated strategy capable of synchronously solving three challenges of targeted recognition, immunosuppression and in vivo persistence becomes a key for improving the curative effect of NK cell solid tumors. The ideal scheme is to block the key immune check point and provide endogenous cytokine support while enhancing tumor specific targeting through multiple gene modification, thereby realizing synergy and breaking through the bottleneck of the existing therapy. Disclosure of Invention In order to solve the technical problems, the invention provides NK immunocytes with enhanced activity and application thereof in cancer treatment. Therefore, in one aspect, the invention discloses an anti-PD-L1/CD 16 conjugated bispecific antibody, wherein the amino acid sequence of the anti-PD-L1/CD 16 conjugated bispecific antibody is shown as SEQ ID NO. 4. The nucleotide sequence of the encoding gene of the anti-PD-L1/CD 16 coupled bispecific antibody is shown as SEQ ID NO. 3. The invention also discloses an expression vector which is pCHO-sdAb-BsAb, wherein the pCHO-sdAb-BsAb comprises the coding gene. The invention also discloses a CHO cell, which comprises the expression vector. In one aspect, the invention also discloses an NK immunocyte with enhanced activity, which is mediated by a lentiviral vector and stably expresses the anti-PD-L1/CD 16 conjugated bispecific antibody. The framework of the lentiviral vector is pLVX-EF1 alpha-IRES-ZsGreen 1, and the lentiviral vector comprises an EF1 alpha promoter, an anti-PD-L1/CD 16 coupled bispecific antibody coding gene and an IRES-ZsGreen1 reporter gene. In one aspect, the invention also discloses a pharmaceutical composition comprising the NK immunocytes with enhanced activity and pharmaceutically acceptable auxiliary materials. In one aspect, the invention also discloses an application of the anti-PD-L1/CD 16 coupled bispecific antibody in preparing NK immune cells with enhanced activity. The invention also discloses application of the NK immunocytes with enhanced activity in preparing medicines for treating cancers, wherein the cancers are PD-L1 positive cancers, and the PD-L1 positive cancers comprise melanoma, non-small cell lung cancer, liver cancer and colorectal cancer. The invention is designed by bispecific antibody, The slow virus vector optimizes and prepares the synergistic innovation with natural killer immune cells, effectively overcomes the defects of the existing natural killer cell treatment technology, has the core advantages that a bispecific antibody adopts a serial structure of a single domain antibody and (G4S) 3 connecting peptide, has the molecular weight of