Preparation method and application of PD-1/CTLA-4 (programmed death-1/cytotoxic T lymphocyte antigen-4) bispecific antibody

A CTLA-4, bispecific antibody technology, applied in the field of life sciences, can solve the problem that there is no efficient preparation method for bispecific antibodies, etc.

Inactive Publication Date: 2016-07-13
杨晶
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If T cells are genetically modified to directly express specific antibodies against PD-1 and CTLA-4, it can block the immunosuppressive signals derived from the tumor and tumor microenvironment, thereby rescuing exhausted T cells and enhancing the ability of CTL to kill cancer cells. function; but there is currently no efficient preparation method for PD-1 / CTLA-4 bispecific antibody

Method used

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  • Preparation method and application of PD-1/CTLA-4 (programmed death-1/cytotoxic T lymphocyte antigen-4) bispecific antibody
  • Preparation method and application of PD-1/CTLA-4 (programmed death-1/cytotoxic T lymphocyte antigen-4) bispecific antibody
  • Preparation method and application of PD-1/CTLA-4 (programmed death-1/cytotoxic T lymphocyte antigen-4) bispecific antibody

Examples

Experimental program
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preparation example Construction

[0043] The invention provides a method for preparing a PD-1 / CTLA-4 bispecific antibody gene fragment, comprising the following steps:

[0044] Step 1, using the anti-PD-1dsFv gene as a template to amplify the gene fragment PD-1V H -GGGGS, the sequences of upstream and downstream amplification primers are shown in SEQNO.1 and SEQNO.2;

[0045] Step 2, using the anti-CTLA-4dsFv gene as a template to amplify the gene fragment GGGGS-CTLA-4V L , the sequences of upstream and downstream amplification primers are shown in SEQNO.3 and SEQNO.4;

[0046] Step 3, OverlapPCR splicing step 1 and step 2 gene fragment PD-1V H -GGGGS and GGGGS-CTLA-4V L , construct the gene fragment PD-1V H -GGGGS-CTLA-4V L , the amplification primer sequence is shown in SEQNO.1 and SEQNO.5;

[0047] Step 4, using the anti-CTLA-4dsFv gene as a template to amplify the gene fragment CTLA-4V H -GGGGS, the sequences of upstream and downstream amplification primers are shown in SEQNO.6 and SEQNO.2;

[0048...

Embodiment 1

[0052] Example 1 Preparation of PD-1 / CTLA-4 bispecific antibody gene fragment

[0053] Using anti-PD-1 and CTLA-4DsFv genes as templates, construct PD-1V by PCR and OverlapPCR respectively H -GGGGS-CTLA-4V L , CTLA-4V H -GGGGS-PD-1V L Gene fragments, and then construct PD-1V through furin-GSGS-2A linker peptide H -GGGGS-CTLA-4V L -furin-GSGS-2A-CTLA-4V H -GGGGS-PD-1V L (if attached figure 1 ).

[0054] Table 1 PCR primer sequence

[0055] Tab.1 Primersequences

[0056]

[0057]

[0058] Step 1, gene fragment PD-1V H -Amplification of GGGGS

[0059] Using the DsFv gene as a template, use primer 1 and primer 2 to amplify the gene fragment PD-1V H -GGGGS, the amplification system is as follows:

[0060] Table 2 PCR reaction system

[0061] Table.2 PCR reaction system

[0062]

[0063] The PCR amplification program was: pre-denaturation at 98°C for 30s, denaturation at 98°C for 5s, extension at 72°C for 30s, and after 30 cycles, extension at 72°C for 5min. ...

Embodiment example 2

[0109] Example 2 Construction of PD-1 / CTLA-4 bispecific antibody lentiviral expression vector and packaging of recombinant lentivirus

[0110] Use EcoRI and NotI respectively to obtain PD-1V in embodiment one H -GGGGS-CTLA-4V L -furin-GSGS-2A-CTLA-4V H -GGGGS-PD-1V L And the lentiviral expression vector was subjected to double digestion (Table 11, 12), digested at 37° C. for 8 hours, and the digested products were separated by 1% agarose gel electrophoresis and then recovered from the gel.

[0111] Table 11 enzyme digestion system

[0112] Table.11digestionsystem

[0113]

[0114] Table 12 enzyme digestion system

[0115] Table.12 digestion system

[0116]

[0117] Ligate the recovered gene fragments with the digested vector (Table 13), ligate at 16°C for 8 hours, take 5 μl of the ligated product and transform into 50 μL Escherichia coli DH5α, plate, pick a single colony and extract the plasmid, and finally use EcoRI and NotI to The plasmid was subjected to double...

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Abstract

The invention discloses a preparation method and application of a PD-1 / CTLA-4 (programmed death-1 / cytotoxic T lymphocyte antigen-4) bispecific antibody. The preparation method comprises the following steps of using a single-chain antibody gene as a template and introducing disulfide bond into a variable region of the antibody by a particular high-efficiency amplification primer through PCR (polymerase chain reaction) and Overlap PCR through a gene engineering technique, so as to prepare a PD-1 / CTLA-4 bispecific antibody fragment; cloning the gene fragment to a chronic virus expression carrier, and transfecting 293T cells to package the virus; transducing a CIK (cytokine induced killer) cell, separating PBMC (peripheral blood mononuclear cell) from peripheral blood of a health person, using CD3 / CD28 magnetic beads to stimulate the growth of the T cell, and co-culturing the chronic virus and the T cell, so as to obtain a novel anti-tumor T cell which can block an immunosuppression signal of the tumor and tumor micro environment source, and enhance the function of killing cancer cells.

Description

technical field [0001] The present invention relates to the field of life sciences, in particular to a method for preparing a PD-1 / CTLA-4 bispecific antibody and related applications thereof Background technique [0002] Tumor immunotherapy (mainly referring to CTL cells, DC cells, CIK cells, NK cells, TIL cells, etc.) aims to control and kill tumor cells by stimulating or mobilizing the body's immune function and enhancing the anti-tumor immunity of the tumor microenvironment. With the obvious advantages of good curative effect, low or no side effects, and no drug resistance, it has become the most advanced tumor treatment field after traditional therapy (surgery, chemotherapy and radiotherapy) and targeted therapy (small molecule targeted drugs and monoclonal antibodies). One of the promising research directions. [0003] Tumor immunotherapy is one of the most promising research directions in the treatment of tumors. By stimulating or mobilizing the immune function of th...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N15/13C12N5/10C12N15/867C12N15/66C07K16/46A61K39/395A61P35/00
CPCA61K39/395C07K16/46C12N5/10C12N15/10C12N15/66C12N15/867C07K16/468C12N15/86C12N2740/15043
Inventor 杨世成
Owner 杨晶
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