Novel bispecific antibody and purpose thereof

A bispecific and antibody technology, applied in the field of new bispecific antibodies and their medical and biological applications, can solve problems such as small molecular weight, inability to mediate biological functions, and short half-life

Pending Publication Date: 2017-06-13
BEIJING WISDOMAB BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The advantage of this type of structural antibody is that it has a small molecular weight, can be expressed in prokaryotic cells, and does not need to consider the problem of correct assembly; the disadvantage is that because there is no antibody Fc segment, it cannot mediate the corresponding biological functions, and the half-life is short, so clinical applications are restricted. certain limit

Method used

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  • Novel bispecific antibody and purpose thereof
  • Novel bispecific antibody and purpose thereof
  • Novel bispecific antibody and purpose thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0132] Example 1 Preparation and Confirmation of Monoclonal Antibody

[0133] As an example demonstrating the bispecific human IgG1 antibody of the present application, a bispecific human IgG1 antibody targeting human CD3E and HER2 was prepared.

[0134] As the basis for preparing the bispecific human IgG1 antibody, the inventors of the present application first prepared two monoclonal antibodies against human CD3E and HER2. This process required the use of a variety of different recombinant proteins, including the extracellular region (CD3E, SEQ ID NO: 32), human CD3D-extracellular domain (CD3D, SEQ ID NO: 33), monkey CD3E extracellular domain (mfCD3E, SEQ ID NO: 34), monkey CD3D extracellular domain (mfCD3D, SEQ ID NO: 35), mouse CD3E extracellular region (mCD3E, SEQ ID NO: 36), mouse CD3D extracellular region (mCD3D, SEQ ID NO: 37), recombinant HER2 extracellular region D1D2D3 part (HER2, SEQ ID NO :38). In the natural environment, CD3E and CD3D form a heterodimer, so in ...

Embodiment 2

[0138] Example 2 Design and preparation of bispecific antibodies with different structures

[0139] Based on the two monoclonal antibodies prepared and verified in Example 1, a series of bispecific antibodies against human CD3E and HER2 were designed.

[0140] Since each arm of a bispecific antibody can contain Fab fragments or scFv, four structural bispecific antibodies were designed according to different combinations (see Figure 1a-Figure 1d ).

[0141] In addition, in order to promote the formation of heterodimers, Fc fragment mutants (FcK, SEQ ID NO: 41 or FcH, SEQ ID NO: 42) of human IgG1 antibodies based on KIH (Knob-Into-Hole) technology were used, That is, the antigen-binding region for human CD3E (derived from H10B7+L1A7) was fused to the N-terminus of Fc (FcK) containing Knob mutation, and the antigen-binding region for HER2 (derived from C6G9+L1A7) was fused to the N-terminus of Fc (FcK) containing mutation of Hole N-terminus of Fc(FcH).

[0142] According to th...

Embodiment 3

[0147] Example 3 Affinity Analysis of Various Bispecific Antibodies

[0148] Antibody affinity determination was performed with GE's Biacore X100plus. Amine coupling kit (Amine coupling kit), human antibody capture kit (human antibody capture kit), His capture kit (his capture kit), CM5 chip and pH7.4 10×HBS-EP and other related reagents and consumables were purchased from GE Healthcare.

[0149] The affinities of different bispecific antibodies were determined using a capture method. When measuring the affinity of the monoclonal antibody or bispecific antibody prepared in Example 2 to CD3E, the anti-His antibody was coupled to the surface of the CM5 chip, and the CD3E antigen with His tag (CD3E-FcK-His / CD3D -FcH) was captured on the surface of the CM5 chip as a stationary phase, and a series of concentration gradients of various anti-CD3E antibody proteins were set to flow through the surface of the stationary phase by a single cycle method, and the affinity of each antibod...

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Abstract

The invention provides a novel bispecific human IgG1 antibody, which comprises two kinds of Fc fragments with the same hinge regions, wherein the amino acid sequence of the hinge region is SEQ ID NO:1 or SEQ ID NO:28; the 216th to 230th site sequences in a natural human IgG1 antibody constant region are replaced. The amino acid position of the antibody constant region is determined according to EU numbering. In addition, the invention also provides a purpose of the bispecific antibody.

Description

technical field [0001] The present application generally relates to the field of antibody medicine, and in particular, the present application provides novel bispecific antibodies and their medical and biological uses. Background technique [0002] Bispecific antibodies (BsAbs) are a class of artificial antibodies that contain two different antigen-binding sites. Bispecific antibodies are widely used in the field of biomedicine, especially in tumor immunotherapy. [0003] Bispecific antibodies can be divided into dual signal blocking type and cell function-mediated type from the mechanism of action. Generally, the bispecific antibody that mediates cell function refers to the anti-CD3 bispecific antibody that mediates T cell killing. In 1985, the concept of using T cells to kill tumor cells was proposed (Stearz at al. Nature 1985, 314:628-631). It is generally believed that effective activation of T cells requires dual signals. The first signal comes from the combination o...

Claims

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Application Information

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IPC IPC(8): C07K16/46A61K39/395A61P35/00
CPCC07K16/2809C07K16/30A61K2039/505C07K2317/524C07K2317/522C07K2317/622C07K2317/55C07K16/32C07K16/2863C07K16/40A61P35/00A61P35/02C07K2317/31C07K2317/73C07K2317/92C07K16/468
Inventor 刘志刚刘玉兰郝小勃郭晶晶
Owner BEIJING WISDOMAB BIOTECHNOLOGY CO LTD
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