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Tetravalent bispecific antibody as well as preparation method and application thereof

A bispecific antibody and specific technology, applied in the field of antibodies, can solve the problems affecting the safety and effectiveness of bispecific antibodies, affecting the yield, purity and physical and chemical stability of bispecific antibodies.

Pending Publication Date: 2021-10-29
SUNSHINE GUOJIAN PHARMA (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although a large number of new bispecific antibody constructs have been reported recently, however, the main technical difficulty in producing bispecific antibodies lies in obtaining the correct pairing of molecules
The forms of the above-mentioned bispecific antibodies all have the problem of mismatching, so by-products or aggregates caused by one or more mismatches will be produced, thereby affecting the yield, purity and physical and chemical stability of the target bispecific antibody, and further Affect the safety and efficacy of bispecific antibodies in vivo

Method used

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  • Tetravalent bispecific antibody as well as preparation method and application thereof
  • Tetravalent bispecific antibody as well as preparation method and application thereof
  • Tetravalent bispecific antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0134] Example 1 Construction of bispecific antibodies against PD-1 and VEGF

[0135] Example 1.1 Sequence

[0136] From the published literature (Magdelaine-Beuzelin C, Kaas Q, Wehbi V, et al.Structure–function relationships of the variable domains of monoclonal antibodies approved for cancer treatment[J].Critical reviews in oncology / hematology,2007,64(3) :210-225.) Obtain the heavy chain variable region and light chain variable region sequences (SEQ ID NO: 1 and 2) of Bevacizumab (replaced with 601 hereinafter) antibody. The DNA encoding the above variable regions was synthesized by Shanghai Sangon Bioengineering Co., Ltd. The heavy chain variable region (601-VH) and the light chain variable region (601-VL) of 601 are respectively associated with the human IgG1 heavy chain constant region (SEQ ID NO: 147) and the human Kappa light chain constant region (SEQ ID NO: 149 ) were connected to construct the heavy chain and light chain genes of the full-length 601 antibody, named...

Embodiment 13

[0152] Example 1.3 Construction of bispecific antibody

[0153] The heavy chain variable region of 20-Hu was connected to the CH1 domain of human IgG4, and then the heavy chain variable region of 601 was connected through an artificial linker (the linker used here was three GGGGS in series), and finally The heavy chain constant region of human IgG4 (CH1+CH2+CH3, the hinge region contains the S228P mutation), and the long heavy chain gene constructed by this procedure containing two heavy chain variable regions and two CH1 domains is named 20 - Fab-601-IgG4 (SEQ ID NO: 16 and 17). Similarly, the heavy chain variable region of 601 was connected to the CH1 domain of human IgG4, and then the heavy chain variable region of 20-Hu was connected through an artificial linker (the linker used here was three GGGGS in series) , and finally connect the heavy chain constant region of human IgG4 (CH1+CH2+CH3, the hinge region contains the S228P mutation), the long heavy chain gene containin...

Embodiment 14E

[0155] Embodiment 1.4ELISA measures relative affinity

[0156] Such as Figure 3A As shown, 20-Hu-HC+601-LC-V94L, 20-Fab-601-IgG4-V94L and 601-Fab-20-IgG4-V94L can effectively bind to PD1-His, with EC50 of 0.3314nM, 0.4768nM and 1.772nM. Such as Figure 3B As shown, 601-HC+601-LC-V94L, 20-Fab-601-IgG4-V94L and 601-Fab-20-IgG4-V94L can effectively bind VEGF165-His, with EC50 of 0.01872nM, 0.05859nM and 0.03886nM. 20-Fab-601-IgG4-V94L and 601-Fab-20-IgG4-V94L can bind both PD-1 and VEGF, indicating that they are bispecific antibodies.

[0157] Embodiment 1.5Biacore measures affinity

[0158] Here, the affinity between the above antibodies and PD-1 or VEGF was detected by Biacore 8K (GE healthcare). On Biacore 8K, use the chip coupled with Protein A / G to capture various antibodies, and then inject the recombinant protein PD1-His or VEGF165-His to obtain the binding-dissociation curve, wash with 6M guanidine hydrochloride regeneration buffer After detachment, the next cycle ...

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Abstract

The invention discloses a common light chain-based tetravalent bispecific antibody with a symmetrical structure and a construction method of the tetravalent bispecific antibody. The tetravalent bispecific antibody has biological activity and physicochemical properties similar to or even better than those of a monoclonal antibody, and can be used for treating various inflammatory diseases, cancers and other diseases.

Description

technical field [0001] The invention relates to the field of antibodies, and more specifically, the invention discloses a class of tetravalent bispecific antibodies, its preparation method and use. Background technique [0002] Bispecific antibodies refer to antibody molecules that can specifically bind two antigens or two epitopes at the same time. Based on symmetry, bispecific antibodies can be divided into structurally symmetric and asymmetric molecules. According to the number of binding sites, bispecific antibodies can be divided into bivalent, trivalent, tetravalent and multivalent molecules. [0003] ScFv consists of a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH and VL are linked together by a flexible peptide linker. In ScFv, the order of domains can be VH-linker-VL or VL-linker-VH. Various linkers have been reported for linking VH and VL, such as short alanine linkers, glycine-serine-rich linkers, linkers adopting a helical...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46C12N15/13A61K39/395A61P35/00A61P35/02A61P29/00A61P37/02A61P27/02A61P11/06A61P19/02A61P17/06A61P17/00
CPCC07K16/2818C07K16/22C07K16/32C07K16/2803C07K16/2878C07K16/2863C07K16/468A61P35/00A61P35/02A61P29/00A61P37/02A61P27/02A61P11/06A61P19/02A61P17/06A61P17/00C07K2317/31C07K2317/35C07K2317/51C07K2317/515C07K2317/92C07K2317/76C07K2317/24C07K16/46C07K2317/64C07K2317/55C07K2317/94A61K2039/505
Inventor 赵杰黄浩旻朱祯平朱云霞
Owner SUNSHINE GUOJIAN PHARMA (SHANGHAI) CO LTD