Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Anti-CD38 nano-antibody, encoding gene and application

A nanobody and gene-encoding technology, applied in the field of biomedicine or biopharmaceuticals, can solve problems such as insufficient activity, difficulty in high-affinity antibodies, and high production costs of monoclonal antibodies

Active Publication Date: 2019-01-18
PEKING UNIV SHENZHEN GRADUATE SCHOOL
View PDF3 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Compared with single-domain antibodies, the production cost of monoclonal antibodies is very high, because it is generally produced by mammalian cells; in addition, it is relatively difficult to obtain high-affinity antibodies with cytotoxic activity; and its efficacy is also related to the immune system of the body. State-related, often due to insufficient activity and need to be combined with other drugs and other shortcomings

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Anti-CD38 nano-antibody, encoding gene and application
  • Anti-CD38 nano-antibody, encoding gene and application
  • Anti-CD38 nano-antibody, encoding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Construction and Screening of Anti-CD38 Nanobody Phage Display Library

[0039] 1.1 Immunization of dromedary camels: select a healthy adult dromedary, mix the ectodomain of CD38 expressed by yeast at a ratio of 10 μg / Kg and Freund’s adjuvant at a ratio of 1:1, and inject it subcutaneously at multiple points on the back. Vaccines were immunized six times with an interval of 4 weeks. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the remaining several immunizations. After the immunization, 100 mL of peripheral blood from the neck of the dromedary camel was collected for the construction of a phage display library.

[0040] 1.2 Separation of camel-derived lymphocytes: analyze lymphocytes from the collected camel-derived anticoagulated whole blood according to the routine procedures in this technical field, every 3×10 7 Add 1mL Trizol RNA separation solution to each cell, take 2mL for RNA extraction...

Embodiment 2

[0068] Example 2 Expression and Purification of Nanobodies in Escherichia coli

[0069] Select clones with positive phage ELSIA results, extract plasmids and transform into competent cells of strain BL21 DE3, induce nanobody protein expression with 100mM IPTG, collect and break up the bacteria, use Ni-NTA (GE Healthcare) resin for purification, use different concentrations Imidazole was eluted and collected, and the collected samples were subjected to reduced protein electrophoresis analysis. For the results, see Figure 4 , M is the molecular weight marker; 1-5 are nanobody cell disruption solution, penetration solution, low-concentration imidazole elution of impurity protein and elution of target protein, respectively.

[0070] The recovered target protein was dialyzed into a low-salt solution and subjected to ion exchange chromatography. For the results, see Figure 5 , M is the molecular weight marker; 1-9 are nanobody loading solution, penetration solution, and different...

Embodiment 3

[0072] Example 3 Determination of Nanobody and CD38 Affinity

[0073] Through biofilm interferometry (Octet RED96, PALL Fortebio), we coated the nanobody on the biosensor and measured its affinity with the target antigen CD38, Figure 6 And Table 1 is the measurement result.

[0074] In the present invention, through camel immunization, cell separation, phage library construction, and nanobody screening, a total of one anti-CD38 nanobody was screened out. And analyze the antibody light chain and heavy chain genes to determine the framework regions (framework regions, FR) and complementarity determining regions (complementarity determining regions, CDR) of the variable region, as follows:

[0075] FR1:Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly

[0076] FR2: Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Val Val Ala

[0077] FR3: Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn AlaGlu Asn Thr Val Tyr ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
affinityaaaaaaaaaa
Login to View More

Abstract

The invention relates to an anti-CD38 nano-antibody, an encoding gene and an application. The anti-CD38 nano-antibody is following protein a) or b): a), protein consisting of an amino acid sequence shown as a sequence 2 in a sequence table; b), protein which is obtained by the amino acid sequence shown as the sequence 2 in the sequence table through substitution and / or deletion and / or addition ofone or more amino acids, related with specifically recognized CD38 and derived from a). The anti-CD38 nano-antibody can be efficiently and specifically bound with CD38, affinity is as high as 4 nM, large-scale production with low cost can be realized in an Escherichia coli expression system, and the anti-CD38 nano-antibody has significant application prospect in the fields of detection and pharmaceuticals.

Description

technical field [0001] The invention belongs to the technical field of biomedicine or biopharmaceuticals, and relates to an anti-CD38 nanobody, a coding gene and an application. Background technique [0002] In the 1980s, CD38 was discovered on the surface of T lymphocytes and used as a marker of cell differentiation. In 2004, it was found that CD38 is generally highly expressed in multiple myeloma cells, while it is in a low expression state on normal lymphocytes, bone marrow cells and some non-hematopoietic cells, which makes CD38 an ideal target for the treatment of myeloma. At present, several anti-CD38 monoclonal antibodies are in clinical use or trial stage, including daratumumab, SAR650984 and MOR202, etc. [0003] In 1993, Hamers-Casterman et al. found heavy chain antibodies consisting only of heavy chains in the blood of camelids. The variable region of the heavy chain antibody is the smallest antibody unit capable of binding to an antigen, and has similar antigen...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13G01N33/53
CPCC07K16/2896C07K2317/569C07K2317/92
Inventor 赵永娟李汉璋侯运楠
Owner PEKING UNIV SHENZHEN GRADUATE SCHOOL
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com