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Bivalent antibody having single-domain antigen-binding fragment fused to conventional Fab fragment

A bivalent antibody and binding fragment technology, applied in the direction of antibodies, anti-tumor drugs, drug combinations, etc.

Inactive Publication Date: 2016-12-07
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Despite these advances, major challenges for bispecific antibodies remain, such as improving manufacturing efficiency, retaining immunogenicity, and maintaining half-life

Method used

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  • Bivalent antibody having single-domain antigen-binding fragment fused to conventional Fab fragment
  • Bivalent antibody having single-domain antigen-binding fragment fused to conventional Fab fragment
  • Bivalent antibody having single-domain antigen-binding fragment fused to conventional Fab fragment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0150] Example 1. Bispecific antibodies induce T-cell-mediated cytotoxicity

[0151] This example provides a novel bispecific antibody format by linking a single-domain anti-CEA VHH targeting CEA-positive tumor cells to an anti-CD3 Fab that binds T cells. Such an antibody (S-Fab) can recruit T cells to CEA-positive tumor cells. By involving T cells, this S-Fab antibody effectively kills CEA-positive tumor cells in vitro. In xenograft models, the S-Fab antibody exhibited potent tumor suppressive effects, suggesting that such a strategy could be used in many immunotherapies. Furthermore, the S-Fab antibody can be efficiently and stably expressed and purified from E.coli.

[0152] This example utilizes the combination of single domain antibodies (VHH) and Fab fragments to produce heterodimeric antibodies. In this format, an anti-CEA VHH is linked to the C-terminus of a partial heavy chain of an anti-CD3 antibody (VH-CH1), and the resulting VH-CH1-VHH chain is combined with an ...

Embodiment 2

[0191] Example 2. Bispecific antibodies induce NK-cell-mediated cytotoxicity

[0192] This example provides a new bispecific antibody form Her2-S-Fab, by linking single domain anti-CD16 VHH to trastuzumab (Trastuzumab, ) constructed from the C-terminus of the Fab. Her2-S-Fab can be expressed and purified by bacterial cells. In vitro cell experiments, Her2-S-Fab can specifically kill Her2 overexpressed cancer cells by recruiting NK cells. Enhanced tumor cell killing was observed compared with trastuzumab. In vivo studies have shown that Her-2-S-Fab can inhibit tumor progression.

[0193] Methods and Materials

[0194] Fab design and protein purification

[0195] The constructs of Her2-S-Fab and control Her2-Fab are shown in Fig. 6a. By standard DNA cloning techniques, first, trastuzumab anti-Her2 VL-CL and VH-CH1 (see Table 5) were chemically synthesized and cloned into pET21a vector and pET26b vector. Subsequently, VHH-CD16 was cloned into the C-terminus of trastuzumab...

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Abstract

Provided is a bivalent antibody, comprising: (a) an antigen-binding fragment (Fab) comprising a light chain and a portion of a heavy chain of the antibody, and (b) a single-domain antigen-binding fragment (VHH) fused to a C terminal of the light chain or the portion of the heavy chain, wherein the portion of the heavy chain does not comprise a CH2 or CH3 domain. The bivalent antibody can kill cancer cells in vivo, inhibits tumor growth, and can be expressed with soluble protein form production through E.coli expression in vitro.

Description

technical field [0001] The present invention relates to a bivalent antibody, in particular a bivalent antibody having a single domain antigen-binding fragment fused to a conventional Fab fragment. Background technique [0002] Bispecific antibodies (BsMAb, BsAb) are man-made proteins composed of fragments of two different monoclonal antibodies and thus can bind to two different types of antigens. For example, in cancer immunotherapy, engineered BsMAbs bind to both cytotoxic cells and targets to be killed (such as tumor cells). [0003] At least three types of bispecific antibodies have been proposed or tested, including trifunctional antibodies, chemically linked Fabs, and bispecific T cell engagers. To overcome manufacturing difficulties, first generation BsMAbs known as trifunctional antibodies have been developed. It consists of two heavy chains and two light chains, each from a different antibody. These two Fab regions are directed against two antigens. The Fc region...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46C12N1/21C12N1/19A61K39/395A61P35/00
CPCC07K16/468A61K2039/505C07K2317/569
Inventor 王忠
Owner SUN YAT SEN UNIV
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