Anti-β-ngf nanobody and its application

A nanobody and antibody technology, applied in the field of biomedicine or biopharmaceuticals, can solve the problems such as the inability to meet the diverse treatment needs of the majority of patients and the single form, and achieve the effect of significant pain relief/inhibition, good effect and low immunogenicity.

Active Publication Date: 2022-06-28
SHANDONG BIOANTY BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, the NGF-targeting antibodies currently under development are all IgG antibodies, which ...

Method used

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  • Anti-β-ngf nanobody and its application
  • Anti-β-ngf nanobody and its application
  • Anti-β-ngf nanobody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1. Generation of Anti-Human Nerve Growth Factor (β-NGF) Nanobodies

[0067] 1.1 Immunization

[0068] Alpacas were immunized with recombinant β-NGF protein (Yiqiao Shenzhou, 11050-HNAC) together with adjuvant, complete Freund's adjuvant was used for the first immunization, and incomplete Freund's adjuvant was used from the second immunization. This time, 3 alpacas were immunized for 3-5 times. After the second immunization, blood was collected to measure the titer, and the alpacas with higher serum titers were selected for neck blood collection, and lymphocytes were separated for subsequent experiments. Elisa method to detect blood titer such as Figure 1A -B shown.

[0069] 1.2 Establishment of phage library

[0070] Take cryopreserved lymphocytes, add Trizol (Thermo Scientific, 15596-026), add 1 / 5 volume of chloroform after fully lysed, mix well, place at room temperature for 20 minutes, centrifuge at 4°C for 20 minutes at 12,000 rpm, take the upper aqueous...

Embodiment 2

[0094] Example 2. Characterization of Candidate Antibodies

[0095] 2.1 Elisa detects the binding activity of candidate antibodies to β-NGF protein

[0096] The purified candidate antibodies were tested for binding activity to determine the binding sensitivity of the candidate antibodies to β-NGF protein. This test is mainly for candidate antibodies NGF-8TTT-BA22\BA81\BA85\BA91\BA94 (hereinafter referred to as BA22\BA81\BA85\BA91\BA94), NGF-2389-CA27\CA56\BA15\BA20\BA42\BA176\ BA177 (hereinafter referred to as CA27\CA56\BA15\BA20\BA42\BA176\BA177), of which 8TTT / 2389 represent the libraries established by alpaca number 8TTT and 2389 respectively, CA represents plate screening, and BA represents magnetic bead screening.

[0097] Refer to Example 1.3 for the method of Elisa to detect the binding activity of candidate antibodies to β-NGF protein. The results are shown in Figure 2. The 12 antibodies have similar binding activities.

[0098] 2.2 Candidate antibodies block the bi...

Embodiment 3

[0108] Example 3. Humanization of BA22, BA81, BA176, CA66, CA30 sequences

[0109]Comprehensive analysis of antibody sequences, blocking data, cell activity data, etc., we selected CA66, CA30, BA22, BA81, BA176 for humanization, and continued to optimize the non-germline amino acids at both ends of the antibody sequence to reduce the risk of immunogenicity.

[0110] Table 3. Humanized Amino Acid Sequences

[0111]

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Abstract

Provided is a nanobody or a multivalent nanobody that binds to a β-NGF antigen, which can be used for treating, preventing, detecting or diagnosing diseases associated with β-NGF, such as pain or inflammation, especially postoperative pain, rheumatoid arthritis Pain and Osteoarthritis Pain.

Description

technical field [0001] The present invention relates to the technical field of biomedicine or biopharmaceuticals, and more particularly relates to a nanobody of β-NGF, its coding sequence and its use in the treatment and / or prevention of diseases related to β-NGF. Background technique [0002] There is an antibody in the blood of camel that naturally lacks the light chain and only contains the heavy chain, which is called a heavy chain antibody. The variable region of the cloned heavy chain antibody can be obtained by only one heavy chain variable region with a size of about 12-15KDa, which is only one-tenth of the traditional IgG antibody fragment, which is called a nanobody (Nanobody). Nanobodies have the advantages of strong tissue penetration, stable physical and chemical properties, easy sources, high yields, and the ability to be expanded by microorganisms. [0003] Nerve growth factor (NGF) is a key survival and maintenance factor in the development of peripheral sym...

Claims

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Application Information

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IPC IPC(8): C07K16/22
CPCA61P19/02C07K16/22A61K39/395A61P25/00
Inventor 刘红宋德勇邢平平
Owner SHANDONG BIOANTY BIOLOGICAL TECH CO LTD
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