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Tetravalent symmetric bispecific antibody

A bispecific antibody and light chain technology, applied in the direction of antibodies, antibody mimics/scaffolds, specific peptides, etc., can solve the problems of unstable molecules, low expression levels, long half-life, etc.

Pending Publication Date: 2022-01-25
WUHAN YZY BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This type of bispecific antibody has pharmacokinetics close to that of natural antibodies, but has the disadvantage that impurities are difficult to identify and remove
[0006] The second type: additional IgG, with natural monoclonal antibody IgG as the backbone, fused with an antigen-binding domain at the N-terminal or C-terminal of the heavy or light chain of the antibody, such as variable region fragment (Fv), single-chain variable region fragment ( ScFv), antigen-binding fragment (Fab) or polypeptide, etc., the molecular weight of this type of bispecific antibody is greater than 150kDa, it is mainly a symmetrical structure, its pharmacokinetics is similar to that of natural antibodies, and even its half-life is longer. Disadvantages such as low quantity and insufficient molecular stability
This type of antibody has a complicated process and a low final yield, so it is basically not used in the world at present
[0010] The above-mentioned types of bispecific antibodies have some disadvantages, which lead to potential risks in clinical use of the products

Method used

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  • Tetravalent symmetric bispecific antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0305] Example 1. Preparation of bispecific antibody

[0306] 1. Plasmid construction method

[0307] The vector used was pcDNA3.1 (purchased from invitrogen) or pCHO1.0 (purchased from Gibco).

[0308] 1.1 Polymerase chain reaction (PCR) amplifies the target fragment DNA

[0309]

[0310] * Template DNA was synthesized by Wuhan Jinkairui. The DNA sequence is obtained by reverse translation according to the amino acid sequence of SEQ ID NO: 1-145.

[0311]

[0312] The resulting PCR product is the target fragment DNA.

[0313] 1.2 Restriction endonuclease digestion vector plasmid

[0314] Vector plasmid DNA* 10μg buffer 10μl Restriction enzymes (such as NotI, NruI or BamHI-HF, etc.) 5μl h 2 o

Add to 100μl total capacity 100μl

[0315] The optimum temperature for enzyme digestion was 4 hours.

[0316] The resulting enzyme-digested product is the enzyme-cleaved carrier DNA.

[0317] * There are two types of vectors: he...

Embodiment 2

[0407] Example 2. Detection of biological activity of bispecific antibodies

[0408] 1. Antigen affinity

[0409] 1) Antigen preparation: construct expression plasmids with His tags for antigen proteins VEGFA, TGF-β1, IL-10, PD-1, CTLA-4, TIGIT, SLAMF7, LAG-3 and CD16a (respectively see Table 26) pcDNA3.1 (purchased from Invitrogen), in which five proteins, PD-1, CTLA-4, TIGIT, SLAMF7, and CD16a, were selected to construct extracellular domains, and then transiently transfected into 293E cells for expression and purification. The purification method is two steps of nickel column purification and molecular sieve purification, and finally the purity of the antigenic protein SDS-PAGE is not less than 95%; the concentration of each antigenic protein is adjusted to 2 μg / ml, 100 μl / well is coated with a microtiter plate, and 4 overnight; Discard the supernatant, and add 250 μl of blocking solution (3% BSA in PBS) to each well;

[0410] 2) Antibody addition: According to the experi...

Embodiment 3

[0429] Example 3. Stability detection of antibodies

[0430] Experimental operation:

[0431] 1. Thermally accelerated stability test at 40°C, the specific operation steps are:

[0432] 1) Replace the sample into a specific buffer solution, the composition of the buffer solution is 20mM citric acid, pH5.5, and adjust the sample concentration to 1mg / mL;

[0433] 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.

[0434] 2. 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:

[0435] When antibody molecules are subjected to protein A affinity chrom...

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Abstract

The invention relates to a bispecific antibody, which is a tetravalent symmetric bispecific antibody with a structure of F(ab)2-(Fv)2-Fc, and comprises two identical fusion heavy chains and two identical fusion light chains.

Description

[0001] This application has the international application number PCT / CN2019 / 095603, the international application date is July 11, 2019, the application number for entering the Chinese national phase is 201980050120.3, the date for entering the Chinese national phase is January 27, 2021, and the title of the invention is " Divisional application for tetravalent symmetric bispecific antibody". technical field [0002] The present invention relates to the field of antibodies. In particular, it relates to bispecific antibodies, pharmaceutical compositions comprising them and uses thereof. Background technique [0003] Bispecific antibody (Bispecific antibody, BsAb) is an antibody or antibody-like molecule with two different binding specificities. Bispecific antibodies are widely used in biomedicine, especially in immunotherapy against tumors. [0004] Bispecific antibodies can be prepared by methods such as chemical engineering, cell engineering and genetic engineering. The ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46C07K19/00C12N15/13G01N33/68G01N33/574A61K39/395A61P35/00
CPCC07K16/30C07K16/18G01N33/6854G01N33/574A61P35/00C07K2317/31C07K2317/52C07K2317/56C07K2317/51C07K2317/515C07K2317/53C07K2319/00C07K2319/31A61K2039/505C07K16/22C07K16/244C07K16/2803C07K16/2818C07K16/283C07K2317/35C07K2317/54C07K16/32C07K2317/90C07K2317/92C07K2317/94C07K2317/64C07K16/2827C07K16/2863C07K2317/55C07K16/468C07K2317/24C07K2317/73
Inventor 张敬方丽娟严永祥曾亮周鹏飞
Owner WUHAN YZY BIOPHARMA CO LTD
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