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Humanized anti-IL-4R alpha single-domain antibody and application thereof

A single-domain antibody, humanized technology, applied in the direction of antibody, application, anti-animal/human immunoglobulin, etc.

Pending Publication Date: 2022-06-10
REGENECORE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently a lack of humanized antibodies targeting IL-4Rα camel-derived single domain antibodies

Method used

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  • Humanized anti-IL-4R alpha single-domain antibody and application thereof
  • Humanized anti-IL-4R alpha single-domain antibody and application thereof
  • Humanized anti-IL-4R alpha single-domain antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Humanization

[0055] Humanized transformation was carried out on the basis of the anti-IL-4Rα single domain antibody of SEQ ID NO.22 (named 4E9-V0).

[0056] The sequences of the transformed humanized single domain antibodies (respectively named 4E9 V1-V14) are shown in Table 1, and the alignment results of the partially humanized antibody sequences are shown in figure 2 shown.

[0057] Table 1 Sequence identifiers (SEQ ID NO.) corresponding to each region of each humanized single domain antibody

[0058] FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 4E9-V0 1 3 4 12 13 18 20 4E9-V1 2 3 5 12 14 18 21 4E9-V2 2 3 6 12 14 18 21 4E9-V3 2 3 6 12 15 18 21 4E9-V4 1 3 4 12 13 19 20 4E9-V5 2 3 5 12 14 19 21 4E9-V6 2 3 7 12 15 18 21 4E9-V7 2 3 8 12 16 18 21 4E9-V8 2 3 9 12 16 18 21 4E9-V9 2 3 10 12 15 18 21 4E9-V10 2 3 11 12 15 18 21 ...

Embodiment 2

[0060] Detection of TF1 cell proliferation induced by humanized single domain antibody neutralizing IL-4 or IL-13

[0061] (1) Place 10,000 TF-1 cells passaged 3-4 times after recovery into 96-well plates;

[0062] (2) Different Tabs and humanized single domain antibodies in Table 1 were prepared as a 10 μg / mL solution, and diluted 5-fold;

[0063] (3) The Tab antibody (the reference application number is CN202010576200.7, the invention name is anti-IL-4Rα single domain antibody and the method obtained by the Chinese invention patent of application and medicine), humanized single domain antibody and EC80 concentration of IL-4 or IL13 (reference application number is CN202010576200.7, the invention name is anti-IL-4Rα single domain antibody and the method obtained by the Chinese invention patent of application and medicine) is mixed at 1:1 to make a mixed solution ;

[0064] (4) Add the mixed solution in the previous step to the cell culture well by an equal volume of the cel...

Embodiment 3

[0073] Detection of thermal stability of humanized single domain antibody by differential scanning method

[0074] experimental method:

[0075] (1) Add 45 μL of 0.1 mg / mL humanized single domain antibody solution above to 8-tube or 96-well PCR plate, then add 5 μL of 100×Sypro orange dye, and the final concentration of the dye is 5×; Each sample was repeated 3 times, and 1×PBS was used as a blank.

[0076] (2) Experiment type Melt curve, reporter group: ROX, quencher group: None; heating program: 25°C for 5 minutes, scanning range 25-95°C, heating rate 1% (about 1°C / min).

[0077] (3) Take the temperature corresponding to the maximum value of the first derivative of the melting curve as the corresponding denaturation temperature (Tm value) of the protein.

[0078] The experimental results are shown in Table 4.

[0079] Table 4

[0080]

[0081]

[0082] The results show that among all humanized single domain antibodies, 4E9-V13 has the highest thermal stability, 4E9...

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Abstract

The invention discloses a humanized anti-IL-4R alpha single-domain antibody and application thereof, and relates to the technical field of antibodies. The anti-IL-4R alpha single-domain antibody disclosed by the invention has a complementary determining region and a humanized modified skeleton region. The anti-IL-4R alpha single-domain antibody provided by the invention is subjected to humanized transformation, so that the affinity and tumor cell proliferation blocking effect of the anti-IL-4R alpha single-domain antibody are maintained, and immune side reactions are effectively reduced.

Description

technical field [0001] The present invention relates to the technical field of antibodies, in particular to a humanized anti-IL-4Rα single domain antibody and its application. Background technique [0002] The type 2 inflammatory response of the human body is caused by type 2 helper T cells (T helper 2, TH2). These responses have unified characteristics, so they are called allergic diseases, such as asthma and a series of other inflammatory diseases. Traditional anti-inflammatory methods generally use broad-spectrum immunosuppressants to suppress disease pain, or use targeted drugs that specifically target the downstream products of TH2 cells, such as IgE, for disease treatment. Compared with traditional treatment methods, targeting type 2 cytokines secreted by TH2 cells—IL-4, IL-5, IL-13—has shown great promise in the treatment of various immune diseases, and patients A better therapeutic effect was obtained. Following several disappointing clinical results of therapies t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K16/46C12N15/13A61K39/395A61P5/16A61P7/06A61P9/14A61P11/00A61P11/06A61P13/12A61P17/00A61P17/02A61P19/02A61P27/02A61P31/06A61P31/22A61P37/02A61P37/08
CPCC07K16/2866C07K16/244A61P11/06A61P37/08A61P17/00A61P19/02A61P31/22A61P17/02A61P11/00A61P37/02A61P9/14A61P7/06A61P5/16A61P27/02A61P31/06A61P13/12C07K2317/24C07K2317/569C07K2317/565C07K2317/567C07K2317/76C07K2317/74C07K2317/94C07K2317/92C07K2317/31A61K2039/505C07K2319/00C07K2317/73C12N15/63
Inventor 苏志鹏张云孟巾果王乐飞姚尧
Owner REGENECORE BIOTECH CO LTD
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