Recombinant single-chain antibody g5-4scfv of anti-human γδtcr monoclonal antibody and its encoding gene and application
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A kind of technology of monoclonal antibody and single chain antibody
Active Publication Date: 2017-04-12
北京佳德和细胞治疗技术有限公司
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Antibody solid-phase in vitro expansion is an important means to obtain γδT cells, but because the current anti-γδTCR monoclonal antibody is of mouse origin, it will produce human anti-mouse antibody reaction (HAMA), so it is greatly limited in clinical treatment
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Embodiment 1
[0059] Example 1. Obtaining the recombinant single-chain antibody G5-4ScFv of anti-human γδTCR monoclonal antibody and its encoding gene
[0060] (1) Acquisition of anti-human γδTCR monoclonal antibody
[0061] The anti-human γδTCR monoclonal antibody of the present invention can be prepared according to conventional methods known in the art (as prepared by referring to the methods recorded in the following documents: Kohler and Milrtein, Nature 256: 495-96, 1975; Harlow and Lane, Aatibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988); the corresponding humanized anti-human γδTCR monoclonal antibody can also be prepared according to the method described in US Patent No. 5,585,089. The following description only provides a specific operation for the purpose of disclosure, and is not intended to limit the present invention.
[0062] 1. Animal immunity
[0063] 1. Primary immunization
[0064] 100 μg recombinant γ9δ2(OT3)-Fc ("Preliminary Identification of Expre...
Embodiment 2
[0140] Example 2. Expression and purification of recombinant single-chain antibody G5-4ScFv
[0141] 1. The expression vector of recombinant single-chain antibody G5-4ScFv and the construction of engineering bacteria
[0142] Take the recombinant plasmid inserted into the recombinant gene G5-4ScFv fragment constructed in Example 1 and have the correct sequence, carry out double digestion with BamH I / EcoR I or NdeI / EcoR I respectively, reclaim the double digestion product of 750bp, and separate the recovered fragments with The pET22b(+) vectors digested by BamH I / EcoR I or NdeI / EcoR I were ligated to obtain the expression vectors of recombinant single-chain antibody G5-4ScFv, which were named G5-4ScFv-PET22b(+)-sp and G5- 4ScFv-PET22b(+), double enzyme digestion results are as follows Figure 6 Shown (1, Marker; 2, before BamH I / EcoRI double digestion; 3, after BamH I / EcoR I double digestion; 4, after Nde I / EcoR I double digestion; 5, Nde I / EcoRI double Before enzyme digestio...
Embodiment 3
[0166] Example 3, Identification of recombinant single-chain antibody G5-4ScFv
[0167] The recombinant single-chain antibody G5-4ScFv obtained in Example 2 was identified.
[0168] 1. Physical and chemical properties
[0169] Mass spectrometry analysis of the molecular weight of the recombinant single-chain antibody G5-4ScFv, the mass spectrogram is as follows Figure 9 As shown, the molecular weight of the recombinant single-chain antibody G5-4ScFv is 29840.9kD.
[0170] 2. Identification by SDS-PAGE and Western blot
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Abstract
The invention discloses a recombinant single-chain antibody G5-4ScFv of an anti-human gamma delta T cell receptor (TCR) monoclonal antibody, and an encoding gene and the application of the recombinant single-chain antibody G5-4ScFv. A heavy chain variable region of the recombinant single-chain antibody of the anti-human gamma delta TCR monoclonal antibody has an amino acid residue sequence in a sequence table SEQ ID No.1, and a light chain variable region of the recombinant single-chain antibody of the anti-human gamma delta TCR monoclonal antibody has an amino acid residue sequence in a sequence table SEQ ID No.2. Experimental results show that the recombinant single-chain antibody G5-4ScFv can specifically bond with a gamma delta TCR; when the recombinant single-chain antibody G5-4ScFv and the gamma delta TCR are in a solid phase, the proliferation of gamma delta T cells in peripheral blood mononuclear cells (PBMC) of a human body can be stimulated, and the purity of the gamma delta T cells can be up to 90% in two weeks; and the gamma delta T cells obtained through the proliferation can effectively secrete interferon-gamma (IFN-gamma) and transforming growth factor-alpha (TGF-alpha) when being stimulated, and can effectively kill daudi cells which are in a tumor cell line, and therefore the recombinant single-chain antibody G5-4ScFv can be described to have a good cytotoxicity effect, and can be used in clinical adoptive immunotherapy to a tumor. Besides, the recombinant single-chain antibody G5-4ScFv has the following advantages in application: immunogenicity is lowered, and the safety of the clinical application is improved; the molecular weight is small, the penetration ability is excellent, and the recombinant single-chain antibody G5-4ScFv can easily enter the microenvironment around the solid tumor; and the recombinant single-chain antibody G5-4ScFv can be produced in a bulk, so that the output is increased, and the cost is lowered. The recombinant single-chain antibody G5-4ScFv plays an important role in the medical field, especially in the gamma delta T cell proliferation and tumor treatment, and has broad application prospects.
Description
technical field [0001] The present invention belongs to the monoclonal antibody and its encoding gene and application, in particular to the recombinant single-chain antibody G5-4ScFv (also referred to as "G5-4ScFv" herein) of the anti-human γδTCR monoclonal antibody and its encoding gene and application. Background technique [0002] T cells can be divided into two types according to the differential expression of surface receptors (TCR): TCRαβ cells and TCRγδ cells. γδT cells are a small subset of T lymphocytes, accounting for about 1%-5% of the total number of normal peripheral blood T lymphocytes, but they are relatively enriched in epithelial tissues such as epidermis and mucosa. In tumor immunity, the recognition of γδT cells to tumor antigens is not MHC-restricted and does not require specific antigen presentation, and has cytotoxic activity against a variety of tumor cells, so it has the potential to be used in tumor adoptive immunotherapy. Antibody solid-phase in vi...
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