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

[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...

[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. ...

[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...