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Single-chain bispecific antibody resistant to VEGF and EGFR and application thereof

A bispecific, antibody technology, applied in specific peptides, chemical instruments and methods, preparations for in vivo tests, etc., can solve the problems of low antibody specificity, increased detection cost, single target, etc., and achieve improved sensitivity. Effect

Active Publication Date: 2018-05-04
北京格根生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the antibodies in the prior art also have poor tumor binding effect due to their low specificity, single target so that there is a risk of missed detection, single coupling agent labeling leads to increased detection costs, and the coupling of antibodies and indicators is adapted The further application of this diagnostic method is limited by problems such as low precision and limited imaging effects.

Method used

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  • Single-chain bispecific antibody resistant to VEGF and EGFR and application thereof
  • Single-chain bispecific antibody resistant to VEGF and EGFR and application thereof
  • Single-chain bispecific antibody resistant to VEGF and EGFR and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0031] The preparation of embodiment 1 anti-VEGF and EGFR single-chain antibody

[0032] 1.1 Establishment of high-capacity natural antibody library.

[0033] Isolation of human peripheral blood mononuclear lymphocytes: 100 healthy adults were randomly selected, and 10ml of peripheral blood was extracted from each person. Dilute 1:1 with RPMI-1640 culture medium containing 10% heparin, add to the centrifuge tube containing lymphocyte separation medium (the volume ratio of diluted venous blood to lymphocyte separation medium 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 cellular RNA

[0035] Press every 5×10 6 Add Trizol reagent at the ratio of cells / ml, and lyse the cells by pipetting. Incubate at room temperature for 5 minutes, transfer to a DEPC-treated EP tube, add 1 / 5 volume of chloroform, shake vigorousl...

Embodiment 2

[0090] Example 2. FITC-labeled VEGF VL, EGFR VH and VEGF-EGFR single-chain bispecific ScFv to the immunofluorescence results of pancreatic cancer tumor cell panc-1 (confocal)

[0091] The pancreatic cancer cell line panc-1 was used to verify the binding of VEGF VL, EGFR VH and VEGF-EGFR single-chain bispecific ScFv to tumor cells.

[0092] First, FITC (fluorescein isothiocyanate) labeled VEGF VL, EGFR VH and VEGF-EGFR single-chain bispecific ScFv, the steps are as follows:

[0093] (1) Dialyze the antibody to be cross-linked (concentration ≥ 1 mg / ml) against the cross-linking reaction solution three times (4° C.) until pH = 9.0. Preparation method of cross-linking reaction solution: 7.56g NaHCO 3 , 1.06 g Na 2 CO 3 , 7.36g NaCl, add water to make up to 1L.

[0094] (2) FITC was dissolved in DMSO at a concentration of 1 mg / ml. The FITC used for each cross-linking should be freshly prepared and protected from light.

[0095] (3) Slowly add FITC to the antibody solution acc...

Embodiment 3

[0117] Example 3. Fluorescence and nuclear magnetic imaging of near-infrared fluorescence (irdye800) and DOTA-gd-labeled VEGF-EGFR ScFv on mouse colorectal cancer animal model

[0118] (1) Stability analysis of irdye800 and DOTA-gd labeled VEGF-EGFR ScFv probes

[0119] In recent years, optical imaging has been widely used in the field of tumor research due to its non-invasive, real-time, and high-resolution advantages, which can be used for early diagnosis of tumors and reflect the anatomical structure and metabolism of tumors. Near-infrared fluorescence imaging is currently a research hotspot in the field of optical molecular imaging. Its spectral range is 700-1000nm. In this range, the autofluorescence interference of the monitored organisms and tissues is small, and the penetration distance of tissues can be as high as several centimeters. Improving the accuracy and sensitivity of imaging.

[0120] DOTA-gd (Chinese name is gadolinium-macrocyclic ligand) is an MRI contrast...

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Abstract

The invention discloses a single-chain bispecific antibody resistant to VEGF and EGFR. The single-chain antibody is prepared by fusing a VEGF-resistant antibody light chain and an EGFR-resistant antibody heavy chain through a connecting peptide, has two groups of unique CDR region sequences capable of respectively recognizing the VEGF and EGFR, has highly specific recognition capability on the VEGF and EGFR and an excellent tumor binding effect, and has specific binding properties on different types of tumor cell lines. The invention further discloses application of the antibody conjugated with an indicator in preparation of an in-vitro tumor diagnosis reagent and an in-vivo bio-imaging agent. When the imaging agent is applied onto a tumor animal model, the single-chain antibody labeled with near infrared fluorescence and magnetic resonance imaging agents can clearly show the size and position of tumors in mice, and has extremely high in-vivo imaging capability.

Description

technical field [0001] The present invention relates to an antibody, more particularly, the present invention relates to a single chain antibody. Background technique [0002] Tumor is the biggest disease that threatens human survival. The World Health Organization research shows that in 2012, the number of cancer cases in China 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 one-quarter of the global cancer deaths. [0003] The clinical diagnosis technology of tumor is also improving gradually. From the initial detection of tumor markers, ultrasound, fluoroscopy, etc., to the detection of more protein and molecular levels of tumor-related factors, CT, nuclear magnetic resonance, PET, SPET, and minimally invasive biopsy of tumors, etc. have been widely used clinically. [0004] Tumor diagnosis is divided into in vitro and in vivo diagnosis. In vitro, immunohistochemical stainin...

Claims

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Application Information

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IPC IPC(8): C07K16/46A61K49/00A61K49/16
CPCA61K49/0058A61K49/16C07K16/2863C07K2317/31C07K2317/565C07K2317/622C07K2317/92
Inventor 谢珞琨满来李峥许春萍
Owner 北京格根生物科技有限公司
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