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A tumor suppressive antibody targeting PD-1 and its application

A PD-1 and antibody technology, applied in the medical field, can solve problems such as inability to effectively stimulate T cell immunity

Active Publication Date: 2022-05-13
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the lack of expression of B7 molecules in tumor cells may be an important factor for the body's inability to effectively stimulate T cell immunity

Method used

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  • A tumor suppressive antibody targeting PD-1 and its application
  • A tumor suppressive antibody targeting PD-1 and its application
  • A tumor suppressive antibody targeting PD-1 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1. Preparation of PD-1.5 Antibody

[0053] 1. Construction of PD-1 recombinant expression plasmid

[0054] Using the cDNA of PD-1 (NM_005018.2) as a reference, the DNA sequence of PD-1 (amino acid 1-170) was synthesized, and restriction sites EcoRI and BglII were respectively introduced, and six His amino acid tags were introduced at the C-terminal, where The EcoRI restriction site is located at the 5' end of the sequence, and the 6-His amino acid tag and the restriction site BglII are located at the 3' end of the sequence in turn. The DNA sequence of the synthesized PD-1 was cloned into the expression vector pCAGGS (Addgene) by using the restriction sites EcoRI and BglII to establish a recombinant eukaryotic expression plasmid for the full-length PD-1 protein.

[0055] 2. Expression and purification of PD-1 recombinant protein

[0056] 1) Transfection of HEK293T cells: HEK293T cells (ATCC) were transferred to culture dishes at a ratio of 1:3; 7.5mL of DMEM (w...

Embodiment 2

[0074] Example 2. PD-1 blocking antibody screening and affinity analysis

[0075] Mice were immunized with the PD-1 protein expressed in vitro by 293T cells, and the obtained monoclonal antibodies were screened for antibodies that could specifically block the interaction between PD-1 and PD-L1 by blocking experiments on PD-1 and PD-L1.

[0076] 1. Preparation of 293T cells expressing full-length PD-L1

[0077] In this example, 293T cells (ATCC) expressing PD-L1 were obtained by transfecting the PD-L1-GFP-p plasmid (Clontech) containing the full length of PD-L1 (pEGFP-N1 vector-GFP tag plasmid) into 293T cells (ATCC). Full-length 293T cells. 1 day before transfection according to 0.5~2×10 5 The cells were inoculated into each well of a 24-well culture plate, and 500 μl of complete DMEM medium (GIBCO Company) without antibiotics was added to ensure that the cells reached 70-80% confluence during transfection. 1 μg of PD-L1-GFP-p plasmid was diluted in 50 μL medium without ser...

Embodiment 3

[0080] Example 3. In vitro activation ability of PD-1 blocking antibody on tumor-specific T cells in non-small cell lung cancer cases

[0081] One of the important applications of PD-1 blocking antibody is its anti-tumor effect. In this example, the peripheral blood of 15 cases of non-small cell lung cancer was collected, and tumor-specific polypeptides were used for in vitro culture, and then the effect of PD-1.5 antibody on tumor polypeptides was evaluated. The activation ability of library-specific T cells is detected to evaluate the anti-tumor potential of the PD-1 blocking antibody screened by the present invention at the cellular level in vitro.

[0082] 1. Case enrollment screening

[0083] The cases included in this example were non-small cell lung cancer cases with positive tumor cells in needle biopsy.

[0084] 2. PBMCs sorting

[0085] The lymphocytes used in the present invention are obtained from the venous peripheral blood of an individual. After the selected ...

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Abstract

The present invention provides an anti-PD-1 antibody or an antibody fragment thereof, which can specifically bind to a PD-1 molecule, and after binding, can block the binding of PD-1 to PD-L1, PD-L2 or a combination thereof, and It can produce biological effects such as T cell activation and anti-tumor.

Description

technical field [0001] The invention belongs to the field of medicine, and in particular relates to an antibody or an antigen-binding fragment thereof, which specifically recognizes programmed cell death 1 (PD-1), and can be used as an immune activator to stimulate the body's immunity Reaction, thereby producing the effect of anti-tumor and other diseases. Background technique [0002] In 2011, cancer surpassed heart disease as the leading cause of death worldwide. The WHO announced in December 2013 that the number of new cancer patients worldwide has exceeded 14 million each year, which is a substantial increase compared with the 2008 statistics of 12.7 million. During the same period, the number of deaths of cancer patients also increased, from 7.6 million in the past to 8.2 million. In 2013, the immune anti-cancer therapy was rated as the first among the top 10 scientific and technological breakthroughs of the year by Science magazine. Since then, the research of tumor ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00
CPCA61P35/00C07K16/2818A61K2039/505C07K2317/76C07K2317/92
Inventor 严景华陈丹青谭曙光仝舟高福
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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