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Anti-IL5 nano antibody and application thereof

A Nanobody, Antibody Technology

Active Publication Date: 2021-01-15
SHANGHAI NOVAMAB BIOPHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Up to now, there is no nanobody drug targeting IL5 released on the market, and the nanobody (nanobody, Nb), that is, the heavy chain nanobody VHH (variable domain of heavy chain of heavy-chainantibody) - there is a natural deficiency in camels The heavy-chain antibody of the light chain (heavy-chain antibody, HCAb), the Nanobody composed of only one heavy-chain variable region obtained by cloning its variable region, is a stable antigen-binding antigen with complete functions currently available The smallest unit of

Method used

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  • Anti-IL5 nano antibody and application thereof
  • Anti-IL5 nano antibody and application thereof
  • Anti-IL5 nano antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0225] Example 1: Screening of IL5-specific Nanobodies by Phage Display Technology

[0226] In the early stage, high-purity human IL5 protein was mixed with immune adjuvant, and 2 Xinjiang Bactrian camels were immunized. After 7 times of immunization, peripheral blood was collected, and then lymphocytes were extracted from it to isolate RNA. After reverse transcription of the RNA, the VHH gene was isolated by nested PCR amplification, and then the VHH fragment was cloned into the pMECS vector, and then electrotransformed into TG1 competent cells to establish an anti-IL5 nanobody phage display library. It was determined that the storage capacities of the two libraries were 6.4×10 8 CFU and 1.3×10 8 The insertion rates of CFU and VHH of the target fragment of the library were 91.7% and 100%, respectively. Then, phage display technology was used to screen IL5-specific Nanobodies. For the specific screening method, refer to the scheme in Example 3 of patent CN110144011B. After ...

Embodiment 2

[0227] Example 2: Blocking IL5 Nanobody Screening

[0228] Inoculate IL5 nanobody clones with different sequences in 1 mL of TB medium containing appropriate concentration of ampicillin, and culture on a constant temperature shaker at 37°C until logarithmic growth phase, add IPTG inducer, induce at 28°C for 16h; after 16h, use osmotic Shred the bacteria by pressure shock method to obtain the crude nanobody extract; take 1E6 HEK293F / IL5Ra cells from each sample and resuspend in 0.5% BSA-PBS buffer, add 200 μL of the above IL5 nanobody crude extract, and set a negative control at the same time (lysate), add appropriate concentration of IL5-Biotin to all samples, incubate at 4°C for 20 minutes; wash twice with 1×PBS, add SA-PE, incubate at 4°C in the dark for 20 minutes, wash cells twice with PBS and use Flow cytometry (BD Caliber) detection, the results are as follows figure 2 Shown: 11 strains of IL5 nanobodies with good blocking effect were preliminarily screened, and the lo...

Embodiment 3

[0232] Example 3: Species-specific detection of IL5 Nanobodies

[0233] ELISA was used to detect whether the 11 strains of Nanobodies obtained in Example 2 could cross-react with IL5 from other species. Add 1 μg / mL human IL5, mouse IL5, cynomolgus IL5 and IgG1 proteins to the microtiter plate and coat overnight, 4°C, 100uL / well; after washing 5 times with PBST, add 300uL 1% BSA to each well at room temperature Block for 2 hours; after washing 5 times with PBST, add 100uL 2μg / mL prokaryotic expressed Nanobodies (Nb6, Nb13, Nb20, Nb21, Nb25, Nb26, Nb50, Nb66, Nb71, Nb85, Nb92) and incubate at 37°C 1 hour; wash 5 times with PBST, add 100uL diluted mouse anti-HA antibody (diluted 1:2000) and incubate at 37°C for 1 hour; wash 5 times with PBST, add 100uL diluted alkaline phosphatase modified anti-mouse Antibody (diluted at 1:2000) was incubated at 37°C for 1 hour; then washed 5 times with PBST, added with chromogenic solution, and the absorbance was measured with a microplate read...

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Abstract

The invention provides an IL5 nanobody and application thereof. Specifically, the invention provides the IL5 nanobody. The invention also provides a coding sequence for coding the nanobody, a corresponding expression vector, a host cell capable of expressing the nanobody, and a production method of the nanobody. The nanobody can specifically recognize IL5 of human beings and cynomolgus monkeys, does not recognize IL5 of mice, and has good binding activity; the nanobody disclosed by the invention has good IL5 / IL5R blocking activity, and the blocking activity is obviously superior to that of a control antibody Nucala; the nanobody can effectively inhibit proliferation of IL5-induced TF-1 cells, and the inhibition activity of the nanobody is superior to that of the control antibody Nucala; and the expression yield of the nanobody in pichia pastoris can reach 13 g / L, and the purity of target protein expression is high.

Description

technical field [0001] The invention relates to the technical field of biomedicine or biopharmaceuticals, and more specifically relates to an anti-IL5 nanobody and its application. Background technique [0002] Eosinophils play an important role in protecting the body from infection. But in some individuals, elevated levels of eosinophils may contribute to inflammation and play a role in the development of certain inflammatory diseases. IL5 is the most selective for eosinophils among the known cytokines, and can regulate the growth, differentiation, recruitment, activation and survival of eosinophils. And its receptor IL5Rα is highly expressed on eosinophils, which plays a key role in clearing allergens in blood and tissues by eosinophils. IL5 is currently considered to be one of the key drivers of the Th2 pathway, and the binding of IL5 to its receptor will further activate the downstream JAK-STAT signaling pathway. [0003] In recent years, as an important target in the...

Claims

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Application Information

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IPC IPC(8): C07K16/24C07K16/46C12N15/13C12N15/81C12N1/19A61K39/395A61K51/10A61K47/68A61K49/00A61P7/00A61P9/14A61P11/00A61P11/02A61P11/06A61P17/00A61P37/08C12R1/84
CPCC07K16/244C12N15/815C07K2317/569C07K2317/565C07K2317/567C07K2317/52C07K2317/64C07K2317/22C07K2317/76C07K2317/33C07K2317/92C07K2317/24C07K2317/35C07K2317/73C07K2317/31G01N33/563A61K2039/505G01N33/6869G01N2333/5409
Inventor 万亚坤朱敏盖军伟李光辉沈晓宁
Owner SHANGHAI NOVAMAB BIOPHARM CO LTD
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