Bispecific antibodies and uses thereof

A bispecific antibody and antibody technology, applied in the field of protein engineering, can solve the problems of affecting affinity, producing immunogenicity, and insufficient production of bispecific antibodies effectively, so as to achieve improved binding ability, strong activation ability, and good binding active effect

Pending Publication Date: 2022-03-01
EXCELMAB INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method of transplanting the TCRαβ constant region interface may also affect affinity or produce immunogenicity
However, the method of replacing the CH1 and CL domains of the constant region is not enough to effectively produce the target bispecific antibody

Method used

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  • Bispecific antibodies and uses thereof
  • Bispecific antibodies and uses thereof
  • Bispecific antibodies and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0335] Example 1 The CH1 / CL binding interface can be used for the evaluation of modified amino acid sites

[0336] The amino acids at the CH1 / CL binding interface of the Fab constant region of the antibody maintain the interaction between the side chains through hydrophobic interactions, charge interactions, hydrogen bonds, etc. Inside. In this example, by analyzing the crystal structure of the CH1 / CL binding interface protein, the potential amino acid sites in the CH1 and CL domains of the bispecific antibody that can be used for engineering are obtained. The protein crystal structure was analyzed by PyMOL software (PDB ID: 3EO9), and the binding interface of E-strand in the CH1-CL domain can be found in figure 1 . Among them, the amino acid positions with strong interactions have been summarized in Table 1 (Summary of Modified Sites).

[0337] In the present invention, unless otherwise specified, amino acid numbers are described according to Kabat numbering. In this exa...

Embodiment 2

[0340] Example 2 Screening of amino acid site mutations at the CH1 / CL binding interface

[0341] In order to screen different CH1 / CL interfaces that can produce specific binding, the following methods are used to screen for mutation combinations with better CH1 / CL interface pairing effects:

[0342] Referring to the method of Hust et al. (Methods in Molecular Biology-Phage Display Volume 1701||Construction of Synthetic Antibody Phage-Display Libraries[J].2018.), the CH1-kCL binding interface in Table 1 can be used for the modified amino acid sites , design primers for random mutation of amino acids, and construct a Fab display library for random mutation of amino acid sites on the phage CH1 / CL interface.

[0343] After releasing, packaging, and precipitating the phage library, after three rounds of screening with the corresponding biotinylated antigen, the clones obtained from the screening were further detected by enzyme-linked immunosorbent assay, and the absorbance value (O...

Embodiment 3 4

[0352] Example 3 Design of four-chain bispecific antibody and construction of expression vector

[0353] (1) Design of four-chain bispecific antibody

[0354] The exemplary bispecific antibody designed and constructed in this example is a "Y"-shaped IgG antibody (see figure 2 ), which includes 2 complete heterologous heavy chains and 2 complete heterologous light chains, which together form the Fab and Fc domains of the antibody.

[0355] The target combination selected in this embodiment is GPC3 and CD3, but not limited to this target combination. The first Fab region of the bispecific antibody in this example binds to human CD3, and the second Fab region binds to human GPC3. The part specifically binding to human CD3 has a first heavy chain and a first light chain, and the part specifically binding to human GPC3 has a second heavy chain and a second light chain, and its sequence is derived from the GPC3 sequence of our company. The Fc part of the antibody is modified acc...

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Abstract

The invention provides a bispecific antibody and application thereof, the bispecific antibody contains an antibody or an antibody fragment, and the antibody or the antibody fragment is the bispecific antibody or the antibody fragment with a first heavy chain and a first light chain as well as a second heavy chain and a second light chain; the first heavy chain and the first light chain form a first Fab region; the second heavy chain and the second light chain form a second Fab region; a plurality of mutations are introduced into the first Fab region and the second Fab region. According to the present invention, amino acid size and charge mutation is performed on the interface amino acids of the heavy chain CH1 and the light chain CL of the antibody or the antibody fragment, such that the heavy/light chain correct pairing aiming at different antigens is enhanced, the binding capacity of the specific antigen is maintained, the mismatching is reduced, the binding specificity is improved, and the yield of the target product is improved.

Description

technical field [0001] The invention belongs to the technical field of protein engineering and relates to a modification method within or between protein molecules. Specifically, the present invention relates to a method for performing amino acid size and charge mutations on the interface amino acids of the heavy chain CH1 and light chain CL of bispecific antibodies comprising two different light chains to prevent mismatches and improve their binding specificity, especially Involves bispecific antibody and its application. Background technique [0002] Bispecific antibodies can simultaneously bind two different epitopes, have special biological functions, and can produce additive or synergistic effects superior to single antibodies. It is a research and development hotspot in the field of antibody engineering and tumor immunotherapy. Some previous studies have made many attempts to engineer antibodies for new bispecific formats (such as DVD-Ig, CrossMab, and BiTE, etc.) (Sp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46C12N15/13C12N15/85C12N5/10A61K39/395A61K47/68A61P35/00
CPCC07K16/2809C07K16/303C12N15/85C12N5/0686A61K47/68A61P35/00C12N2510/00C12N2800/107C07K2317/31C07K2317/522C07K2317/52C07K2317/76C07K2317/55A61K2039/505
Inventor 张文军李峰刘敏潘志豪覃陆英曹广灿
Owner EXCELMAB INC
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