Method for obtaining high-purity heterologous antibody

A high-purity, bispecific antibody technology, applied in the field of obtaining high-purity heterologous antibodies, can solve the problem of high immunogenicity of the heavy chain constant region, achieve broad commercial application prospects, simplify purification steps, and reduce production costs

Active Publication Date: 2021-07-16
周易
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the limitation of this method is that the heavy chain constant regions of mice and rats are highly immunogenic (document 7), and the half-life of the antibody Catumaxomab (Catumaxomab) prepared by this method in the human body is about 2.1 days, which is very short compared with the usual human IgG half-life of 2-3 weeks (Reference 8)

Method used

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  • Method for obtaining high-purity heterologous antibody
  • Method for obtaining high-purity heterologous antibody
  • Method for obtaining high-purity heterologous antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1. Design of Amino Acid Modifications in the Heavy Chain Constant Region

[0059] The binding site of antibody Fc fragment to protein A and FcRn is as follows: figure 1 shown. figure 2 Shows the complex crystal structure of Fc and protein A (PDB code: 4WWI), M252, I253, S254, L309, H310, Q311, L314, N315, K317, H433, N434, H435, Y436 on the Fc segment of the antibody (EU number) on the Fc and protein A interaction interface. image 3 Shows the complex crystal structure of Fc and FcRn (PDB code: 4NOU), M252, I253, S254, T256, L309, H310, Q311, L314, N315, K317, H433, N434, H435, Y436 (EU numbering) are located in Fc and FcRn interaction interface. The present invention creatively proposes that mutating I253 into a positively charged amino acid, such as Lys, Arg, or mutating I253 into a polar amino acid such as Asn, Gln, may make the interaction between Fc and protein A polar- Water repulsion, thereby changing the affinity of Fc for protein A.

Embodiment 2

[0060] Example 2. Construction and expression of antibody expression vectors

[0061] It can be known from Example 1 that mutating the I253 of the heavy chain of the antibody into a positively charged amino acid, such as Lys, Arg, or a polar amino acid such as Asn, Gln, may weaken the interaction between the antibody and protein A, while minimizing Effect on antibody binding to FcRn. Therefore, in this example, a series of PD1×Her2 bispecific antibody molecules with I253 mutation were constructed. Such as Figure 4 As shown, one heavy chain of the bispecific antibody carries the I253 mutation; the other heavy chain does not carry the I253 mutation, and a single-chain antibody fragment is concatenated at the N-terminal of the heavy chain. This allows the molecular weight of the bispecific antibody to be differentiated from the two cognate antibodies. The heterologous antibodies or heterodimers described in this example refer to bispecific antibodies, and the homologous antib...

Embodiment 3

[0070] Example 3. Research on the Elution Conditions of Protein A Affinity Chromatography

[0071] The antibody gene was transfected into 293E cells. After the cells were cultured for 7 days, the culture medium was subjected to high-speed centrifugation and vacuum filtration through a microporous membrane, and then loaded onto a HiTrap MabSelectSuRe column (purchased from GE). The staged use is shown in Table 3 Washing 1, Elution 1-5 The antibody proteins in Table 2 were purified, and the pH was neutralized with Tris buffer at pH 9.0 after elution. The eluted fractions were collected and concentrated, then added to the reduced protein electrophoresis loading buffer and non-reduced protein electrophoresis loading buffer respectively, boiled and then detected by SDS-PAGE.

[0072] table 3

[0073] balance PBS cleaning 1 PBS Elution 1 100mM citric acid, pH5.5 Elution 2 100mM citric acid, pH5.0 Elution 3 100mM citric acid, pH4.5 Elutio...

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Abstract

The invention belongs to the field of antibody engineering, and provides a method for obtaining a high-purity heterologous antibody. The heterologous antibody contains two heavy chains with different amino acid sequences, wherein a mutation is introduced into the I253 of the constant region of one heavy chain, and through protein A affinity chromatography purification, a high-purity bispecific antibody can be obtained, wherein the position of the above amino acid is determined according to an EU index numbered by KABAT. By use of the method disclosed by the invention, one mutation is introduced into the heavy chain, Ile of which the EU number is on the 253rd site in the constant region of the heavy chain is mutated into Asn, i.e., the purity of the heterologous antibody can be improved to be 99% or above only through one-step protein A affinity chromatography, purification steps are greatly simplified, preparation cost is lowered, and the method has a wide commercial application prospect.

Description

technical field [0001] The invention belongs to the field of antibody engineering, and in particular relates to a method for obtaining a high-purity heterologous antibody containing two heavy chains with different amino acid sequences. Background technique [0002] Bispecific antibodies can be constructed in a variety of ways, among which IgG bispecific antibodies have similar structures, physicochemical properties and Fc segment functions as ordinary antibodies. Generally, IgG bispecific antibodies consist of two heavy chains with different amino acid sequences (i.e., a heavy chain against antigen A and a heavy chain against antigen B) and two light chains with different amino acid sequences (i.e., a light chain against antigen A and a heavy chain against antigen B). light chain against antigen B). When 4 polypeptide chains are combined, 8 different combinations will be produced, only one of which is the desired target antibody molecule. However, the efficiency of separat...

Claims

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Application Information

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IPC IPC(8): C07K16/00C07K1/22C07K16/46
CPCC07K16/00C07K1/22C07K16/2809C07K16/32C07K2317/31C07K16/46C12N15/00A61K39/395
Inventor 周易
Owner 周易
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