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Sustained-release anti-FcRn antibody or antigen binding fragment and application thereof

A technology for combining fragments and antibodies, which is applied in the field of slow-release anti-FcRn antibodies or antigen-binding fragments, treatment, prevention and/or diagnosis of FcRn-related diseases, and can solve the problem of short half-life of FcRn antibodies or Fc fragments, fast metabolism of antibody drugs, etc. question

Active Publication Date: 2022-01-11
BEIJING KOHNOOR SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims to solve the problem of fast metabolism of some antibody drugs in the prior art, specifically, to solve the technical problem of short half-life of FcRn antibody or Fc fragment

Method used

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  • Sustained-release anti-FcRn antibody or antigen binding fragment and application thereof
  • Sustained-release anti-FcRn antibody or antigen binding fragment and application thereof
  • Sustained-release anti-FcRn antibody or antigen binding fragment and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Nanobody construction

[0044] Camels were immunized with antigens, peripheral blood mononuclear cells (PBMC) were isolated and total RNA was extracted for reverse transcription, and the variable domain of the heavy-chain of heavy chain antibody was amplified using the reverse transcription product as a template , VHH) and connected to the phage display vector, electroporated into Escherichia coli TG1 competent cells, and camel immune library was constructed.

[0045] Specifically, camels are immunized once every two weeks, a total of 4 times. Each injection of 0.8 mg HSA extracellular region recombinant protein (purchased from Hualan Bioengineering Co., Ltd., product batch number: 201405030), accompanied by Freund's complete / incomplete adjuvant (Sigma, F5881, F5506), was taken subcutaneously at multiple points The way of injection. Two weeks after each immunization, 1 mL of blood was collected to separate the serum, and the immunogen was used as the assay ...

Embodiment 2

[0047] Example 2: Construction of FcRn sustained release antibody

[0048] In this example, the HSA nanobody HzH4-3 and Hz2MG6m19 sequences obtained in Example 1 were connected to the C-terminus of the FcRn antibody Efgartigimod (Ef for short, the sequence is shown in SEQ ID NO: 9) to construct H4-3-Ef and m19 -Ef eukaryotic expression vector, and then transfected into eukaryotic cells for expression and purification to obtain slow-release antibodies against hFcRn H4-3-Ef and m19-Ef.

Embodiment 3

[0049] Example 3: ELISA method to detect the metabolism of H4-3-Ef and m19-Ef in HSA / FcRn double-transformed humanized mice

[0050] The experiment was conducted with hFcRn female 6-week-old transgenic mice, divided into 3 groups, 4 mice in each group, and the first two groups were intravenously injected with H4-3-Ef and m19-Ef, both at 300 μg / mouse. Then blood was collected by docking at 2h, 4h, 8h, 24h, 48h, 79h, 120h, 151h and 192h. Another group was injected with Ef at a rate of 300 μg / bird, and then blood was collected at 0.5h, 1h, 3h, 4h, 5h, 6h, 8h, 24h, 30h, 48h, 79h and 96h, and the serum was collected and stored at -20°C. save.

[0051] After all the blood collection is completed, the collected serum is tested by ELISA, and the steps are as follows:

[0052] 1) Coating anti-Fc Ab (Sigma, I2136) onto 96-well ELISA plate, 0.5 μg / mL, 100 μL / well, overnight at 4°C;

[0053] 2) After washing the plate 3 times with PBS, add 0.5% casein-PBS, block at 37°C for 60 min, and...

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Abstract

The invention discloses a sustained-release anti-FcRn antibody or antigen binding fragment and application thereof. The sustained-release anti-FcRn antibody or antigen binding fragment comprises a heavy chain variable region and a heavy chain constant region. The heavy chain variable region is VHH of a heavy chain antibody of camelidae or a polypeptide consisting of the VHH of the heavy chain antibody of the camelidae; the VHH comprises CDRs 1, 2 and 3; the VHH CDR1 region, the VHH CDR2 region and the VHH CDR3 region respectively comprise amino acid sequences shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 or amino acid sequences with at least 80% identity with amino acid sequences shown as SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; and the heavy chain constant region has an amino acid sequence which is at least 80% identical with an amino acid sequence as shown in SEQ ID NO: 9. By conjugating an HSA antibody sequence to an FcRn antibody, the half-life period of an original antibody is prolonged, and the number of times of drug administration is reduced.

Description

technical field [0001] The invention relates to the field of biological medicine. Specifically, the present invention relates to a novel artificially designed antibody with extended half-life, especially a slow-release anti-FcRn antibody or antigen-binding fragment. The present invention also relates to the therapeutic and diagnostic uses of these FcRn-binding antibodies, especially in the treatment, prevention and / or diagnosis of FcRn-related diseases, such as autoimmune-related diseases. Background technique [0002] For the problem of fast metabolism of some antibodies, several methods have been developed in the past to prolong the half-life of antibody fragments and prepare sustained-release drugs. These methods include reducing the sensitivity of the fragment to serum proteases using specific slow-release formulations, or reducing the intrinsic clearance of antibody fragments by amino acid substitutions that reduce receptor binding affinity in the endosomal compartment...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13A61K39/395A61K47/68A61P37/02
CPCC07K16/2833A61K47/6803A61K47/6849A61P37/02C07K2317/22C07K2317/52C07K2317/565C07K2317/569A61K2039/505
Inventor 王荣娟张畅曾大地焦莎莎王双张锦超
Owner BEIJING KOHNOOR SCI & TECH CO LTD
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