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Trivalent bispecific antibody and its preparation process and use

A bispecific antibody, antibody technology, applied in the direction of antibodies, chemical instruments and methods, specific peptides, etc., can solve the problems of low target molecule affinity and poor guiding effect, and achieve enhanced effector function, reduced concentration, and alleviated side effects. Effect

Inactive Publication Date: 2005-04-06
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, bivalent bispecific or trivalent trispecific antibodies are constructed using the first constant region (CH1) of human IgG1 heavy chain and the constant region (CL) of kappa chain as the heterodimerization domain. The antigen has only one binding domain, which has a low affinity for the target molecule and poor guiding effect

Method used

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  • Trivalent bispecific antibody and its preparation process and use
  • Trivalent bispecific antibody and its preparation process and use
  • Trivalent bispecific antibody and its preparation process and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Preparation of anti-Her2 / neu×anti-CD16 trivalent BsAb

[0050] 1. Materials

[0051] Mouse anti-human CD16 monoclonal antibody light chain, heavy chain variable region genes (VL, VH) were cloned from hybridoma cell B88-9, commercially available (refer to "Molecular Cloning" (Science Press, second edition) for experimental methods); mouse anti-P185 erbB2 The VL and VH genes of the monoclonal antibody were cloned from hybridoma cells Her2 (commercially purchased), and the scFv was synthesized according to conventional methods. editor-in-chief); human IgG1 heavy chain first constant region CH1 and κ chain constant region CL genes were provided by Dr. Tong Yigang, Academy of Military Medical Sciences (GenBank AF027159, X95747); Escherichia coli strains JM109, BL21(DE3), prokaryotic expression vector pET22b(+) It is a standard strain, commercially available; highly expressed P185 erbB2 The human breast cancer cell line SK-BR-3 is the standard strain (ATCC Number...

Embodiment 2

[0067] Example 2 FACS analysis and detection of anti-Her2 / neu×anti-CD16

[0068] Activity detection of trivalent BsAb

[0069] 1. Materials

[0070] Same as embodiment one.

[0071] 2. Methods and results

[0072] 1. FACS analysis of the binding activity of BsAb to SK-BR-3 cells

[0073] SK-BR-3 cells were digested with 0.02% EDTA and washed with cold PBA (PBS, 2% BSA, 0.1% NaN 3) and washed twice by centrifugation; incubated with BsAb (0.1mg / L) at 0°C for 30min; washed twice by cold PBA, incubated with FITC-GAH by shaken at 0°C for 30min; washed by centrifuge twice with cold PBA, and resuspended cells in PBS , analyzed with a Becton-Dickenson FACsort instrument. The results show that BsAb can specifically combine with intact SK-BR-3 cells, and the relative average fluorescence intensity is 92.08, which is 25.4 times that of the negative control, see Figure 12 .

[0074] 2. FACS analysis of the binding activity of BsAb to CD16 surface ...

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Abstract

This invention discloses a preparation of tervalence double specificity antibody and its service application. The antibody includes person IgG1 heavy chain first constant region CH1, k chain constant region CL structure field, heavy chain heavy chain variable region VH and light chain variable region VL antibody A, and single chain variable region scFv of antibody B. VH is connected to N terminal of CH1, scFv connected to C terminal of CH1, VL connected to N terminal of CL, scFv to C terminal of CL. The preparation procedures include building of express carrier, express and detect of double specificity antibody, purification of double specificity antibody, combination analysis of double specificity antibody etc. there are little side effect, good guidance quality etc. advantages of the tervalence double specificity antibody of this invention, it can be used to prepare curing medicine of cancer.

Description

technical field [0001] The present invention relates to a bispecific antibody, in particular to a trivalent bispecific antibody, as well as its preparation method and application. Background technique [0002] Bispecific antibody (bispecific antibody, BsAb) is an artificial antibody containing two specific antigen-binding sites, which can build a bridge between target cells and functional molecules (cells) to produce oriented effector functions. BsAb has broad application prospects in biomedicine, especially in tumor immunotherapy. Killing tumor cells through BsAb-mediated cytotoxicity is a hot topic in current immunotherapy application research. Its main feature is that BsAb can simultaneously bind to tumor-associated antigens and target molecules on effector cells, directly triggering the specific killing of tumor cells by effector cells . Currently, BsAbs can be prepared by methods such as chemical engineering, cell engineering, and genetic engineering. The biggest adv...

Claims

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Application Information

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IPC IPC(8): A61K39/395A61P35/00C07K16/44C12N15/63C12P21/00
Inventor 解志刚郭宁沈倍奋
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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