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Anti-EGFR single-chain antibody, anti-PD1 single-chain antibody and fusion protein

A single-chain antibody and antibody technology, applied in the fields of immunology and molecular biology, can solve the problems of affecting the anti-tumor effect of antibody-conjugated complexes, difficulty in ensuring product uniformity, and increasing preparation costs, etc., to achieve enhanced anti-tumor effects, Broaden the anti-tumor mode, the effect of small molecular weight

Active Publication Date: 2019-06-14
北京科立思维生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the antibody-conjugated complex has good tumor recognition ability and anti-tumor effect, it needs to be coupled with another chemical group during the preparation process, which not only increases the preparation cost, but also makes it difficult to ensure the uniformity of the product. The chemical group and the antigen recognition region of the antibody are often prone to steric hindrance, and sometimes have toxic side effects on the body. These factors affect the actual anti-tumor effect of the antibody-conjugated complex

Method used

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  • Anti-EGFR single-chain antibody, anti-PD1 single-chain antibody and fusion protein
  • Anti-EGFR single-chain antibody, anti-PD1 single-chain antibody and fusion protein
  • Anti-EGFR single-chain antibody, anti-PD1 single-chain antibody and fusion protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Preparation of anti-EGFR and anti-PD1 bispecific antibodies

[0032] 1.1 The establishment of a high-capacity natural antibody library.

[0033] Isolation of human peripheral blood mononuclear lymphocytes: 100 healthy adults were randomly selected, and 10 ml of peripheral blood was drawn from each person. Dilute 1:1 with RPMI-1640 culture medium containing 10% heparin, and add it to a centrifuge tube containing lymphocyte separation liquid (the volume ratio of diluted venous blood to lymphocyte separation liquid is 2:1), 2,000×g, Centrifuge for 17 minutes. Aspirate the milky white mononuclear cell layer at the interface of the lymphocyte separation solution and wash twice with PBS buffer.

[0034] 1.2 Extraction of total cell RNA

[0035] Every 5×10 6 Trizol reagent is added at the ratio of cells / ml, and the cells are lysed by pipetting. Incubate at room temperature for 5 minutes, transfer to a DEPC-treated EP tube, add 1 / 5 volume of chloroform, shake vigorously fo...

Embodiment 2

[0075] Example 2. Purification of bispecific antibodies

[0076] Regenerate the two ScFv sequences to connect the light chain variable region and heavy chain of the anti-CD3E single chain antibody according to the heavy chain variable region and light chain variable region of the anti-epidermal growth factor receptor single chain antibody The sequence of variable regions is reconstituted into a new bispecific antibody. The amino acid sequences of the CDR1, CDR2 and CDR3 regions of the light chain of the anti-epidermal growth factor receptor single-chain antibody are shown in SEQ ID NOs. 1, 2 and 3, respectively, and the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the heavy chain are shown in SEQID NO. 5, 6 and 7 are shown. The amino acid sequences of the light chain variable region and the heavy chain variable region are shown in SEQ ID NO. 4 and 8, respectively. The amino acid sequence of the linking polypeptide is shown in SEQ ID NO.9.

[0077] The amino acid se...

Embodiment 3

[0090] Example 3. Anti-EGFR-PD1 bispecific antibody on the proliferation of T cells

[0091] CCK8 detects T cell proliferation

[0092] Collect the suspended T cells, centrifuge at 1200 rpm for 3 min, and discard the supernatant;

[0093] Add 3ml saline, centrifuge at 1200rpm for 3min, discard the supernatant, add 4ml AIM-V culture solution,

[0094] count;

[0095] Take out 4 96-well plates and add 4×10 4 / Well PIK-PD1 T cells and blank T cells, the total volume is 100ul, set up 3 multiple wells;

[0096] Put it at 37℃, 5% C0 2 Cultured in the incubator, add 10ul of CCK8 reagent at 0h, 24h, 48h, 72h and culture for 6h in 37℃ incubator (the culture time varies from cell to cell, but the culture time for the same experiment should be the same), the culture solution becomes After brown-red, measure the OD value at 450nm on the microplate reader;

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Abstract

The invention discloses an anti-epidermal growth factor receptor single-chain antibody, an anti-PD1 single-chain antibody and a fusion protein, and an application thereof. The double-specific single-chain antibody has highly specific antigen recognition ability, not only has the specific recognition function of the antibody, but also has excellent anti-tumor effect; moreover, the antibody fusion protein has small molecular weight, can be expressed in a prokaryotic cell expression system, greatly reduces the production cost of antibody drugs, and has good clinical application prospects.

Description

Technical field [0001] The invention discloses an anti-EGFR single-chain antibody and an anti-PD1 single-chain antibody bispecific antibody, belonging to the fields of immunology and molecular biology. Background technique [0002] Tumor is the biggest disease threatening human survival. According to a study by the World Health Organization, the number of cancer cases in China in 2012 was 3.065 million, accounting for about one-fifth of the global incidence; the number of cancer deaths was 2.205 million, accounting for about a quarter of the global cancer deaths. [0003] The formation and development of tumors have very complex regulatory mechanisms, and many biological effect molecules are involved. Clarifying the mechanism of action of these biological effect molecules is of great significance for the prevention and treatment of tumor diseases. [0004] Epidermal growth factor receptor is a transmembrane receptor with tyrosine kinase activity. It is commonly expressed in human e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K19/00
Inventor 李峥郝中禾
Owner 北京科立思维生物科技有限公司
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