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Modified Fc fragment, antibody comprising same, and use thereof

A fragment and antibody technology, applied in applications, antibodies, antibody mimics/scaffolds, etc., can solve problems such as the inability to completely eliminate the non-specific activation of anti-CD3 antibodies, the deterioration of antibody stability, and the increase of immunogenicity.

Active Publication Date: 2020-03-24
WUHAN YZY BIOPHARMA CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, when the above-mentioned technology for Fc modification is applied to the multifunctional antibody structure, it cannot completely eliminate the non-specific activation of T cells by anti-CD3 antibodies (such as in human IgG1, L234F / L235E / P331S mutation, L234A / L235A / P329G mutation and L234A / L235A mutation), and the modification of certain sites will lead to poor stability of the antibody (such as the 297th asparagine mutation of human IgG1 to remove glycosylation), and in the literature "J Immunol 2003 ; 170:3134-3138; Human IgG2 can form covalent dimers.” It is mentioned that human IgG2 is prone to dimerization to form a tetravalent complex with a molecular weight of 300kD, and amino acid mutations of up to 7 positions are carried out on human IgG2 (such as V234A / G237A / P238S / H268A / V309L / A330S / P331S), risk of increased immunogenicity

Method used

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  • Modified Fc fragment, antibody comprising same, and use thereof
  • Modified Fc fragment, antibody comprising same, and use thereof
  • Modified Fc fragment, antibody comprising same, and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0361] Example 1. Antibody Preparation

[0362] A. Construction of antibody expression plasmids

[0363] According to the sequences in Table 28-30, the coding sequence DNAs were respectively synthesized by Wuhan Jinkairui and cloned into the vector pcDNA3.1 (purchased from Invitrogen). Then transform Trans10 competent cells (purchased from Beijing Quanshijin Biological Company) respectively. After sequencing and identification, the expression plasmid was obtained.

[0364] Specifically involved in the construction of expression plasmids as follows:

[0365] 1) The multifunctional antibody structure 1 in Figure 1 involves the construction of three plasmids. The three plasmids are: light chain expression plasmid (pL), heavy chain expression plasmid (pH), and fusion peptide 1 expression plasmid (pF1).

[0366] 2) The multifunctional antibody structure 2 in Figure 2 involves the construction of three plasmids. The three plasmids are: light chain expression plasmid (pL), heavy...

Embodiment 2

[0433] Example 2: Detection of biological activity of antibodies

[0434] 1. Cell affinity

[0435] 1) Cell preparation: T cells isolated from CD3-positive human whole blood are used for the detection of the affinity of the CD3 end of the multifunctional antibody molecule, and the detection of the affinity of the tumor antigen is carried out with the positive tumor cells corresponding to the antigen: for example, CD38-positive MM is used for the detection of the CD38 antigen. 1S cells (purchased from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) or RPMI 8226 cells (purchased from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences), PD-L1 antigen detection with PD-L1 positive H358 cells (purchased From the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences), etc. Resuspend the cells with 1% FBS-PBS and adjust the density to 4×10 6 / ...

Embodiment 3

[0532] Embodiment 3: Stability detection of antibody

[0533] Experimental operation:

[0534] A. Thermally accelerated stability test at 40°C, the specific operation steps are:

[0535] 1) Replace the sample into the buffer, the composition of the buffer is 20mM citric acid, pH5.5, and adjust the sample concentration to 1mg / mL;

[0536] 2) Divide each sample into 500 μL per tube (6 tubes in total) and seal it and place it in a water bath at 40°C. Samples were taken on day 0, day 3, day 5, day 7, day 10 and day 14 for HPLC- For SEC detection, the water bath time was 14 days in total.

[0537] B. Acid resistance test, also known as low pH stability, is to investigate whether antibody molecules can maintain their original state after being neutralized to physiological conditions after being treated in an acidic environment for a period of time. The specific operation steps are:

[0538] When antibody molecules are subjected to protein A affinity chromatography, in the acid e...

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Abstract

The present invention relates to a modified Fc fragment, an antibody comprising the same, and uses thereof. The Fc fragment is a human IgG1 source, and the constant region CH2 domain of the Fc fragment comprises a plurality of substitutions. The substitution can significantly reduce the binding capacity of the Fc fragment and an Fc gamma receptor (Fc gamma R), and reduce the non-specific activation of the antibody (such as an anti-CD3 antibody) on T cells.

Description

technical field [0001] The present invention relates to the field of antibodies. In particular, it relates to engineered Fc fragments, antibodies comprising the same. Background technique [0002] Human natural antibodies, such as IgG1, IgG2, IgG3 and IgG4, all have the ability to bind to FcγR. Human FcγR is divided into three types: FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16), each of which corresponds to It is expressed on the surface of different monocytes, and each type of receptor is divided into a, b, c and other subtypes. Natural antibodies produce the following immune effector functions through the combination of their own Fc and FcγR: antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated cellular phagocytosis (ADCP), and complement-mediated cytotoxicity (CDC )Wait. In the process of antibody drug research, for some types of antibodies, it is necessary to reduce the ability to bind to FcγR to reduce the generation of ADCC, ADCP ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/13C12N5/10A61K47/68
CPCA61K47/68C07K2317/31C07K2317/622C07K16/2896C07K16/2809C07K2317/24C07K2317/56C07K2317/732C07K2317/71C07K2317/52C07K2317/524C07K16/2827C07K2317/92C07K2317/33C07K16/2878C07K16/3007C07K2317/73A61P35/00C07K2317/94C07K2317/60C07K16/2803C07K16/30C07K16/283C07K16/2851C07K16/468A61K2039/505C07K16/28C07K2317/522C07K2317/526C07K2317/53C07K2317/565C07K2319/30
Inventor 张敬方丽娟严永祥曾亮周鹏飞
Owner WUHAN YZY BIOPHARMA CO LTD
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