WO2017128534A1

WO2017128534A1 - Method for preparing pd-1/ctla-4 bispecific antibody and use thereof

Info

Publication number: WO2017128534A1
Application number: PCT/CN2016/080419
Authority: WO
Inventors: 杨世成
Original assignee: 杨世成
Priority date: 2016-01-29
Filing date: 2016-04-27
Publication date: 2017-08-03
Also published as: CN105754990A

Abstract

Provided is a preparation of a PD-1/CTLA-4 bispecific antibody fragment produced by using a single chain antibody gene as a template and specific high-efficiency amplification primers, performing PCR and overlap PCR, and introducing a disulfide bond into a variable region of the antibody. The gene fragment is cloned into a lentiviral expression vector, transfects a 293T cell to achieve virus packaging, and then a CIK cell comprising a PBMC separated from healthy human peripheral blood is transduced, stimulating T-cell growth using CD3/CD28 magnetic beads and co-culturing the lentivirus with the T cell, so as to obtain a novel anti-tumor T cell capable of blocking immunosuppression signals of tumors and tumor microenvironments, and enhancing anti-tumor cytotoxicity.

Description

Preparation method of PD-1/CTLA-4 bispecific antibody and application thereof

Technical field

The invention relates to the field of life sciences, in particular to a preparation method of PD-1/CTLA-4 bispecific antibody and related application thereof

Background technique

Tumor immunotherapy (mainly refers to CTL cells, DC cells, CIK cells, NK cells, TIL cells, etc.) enhances the anti-tumor immunity of the tumor microenvironment by stimulating or modulating the immune function of the motor, thereby controlling and killing the tumor cells. It has the advantages of good curative effect, low or no toxic side effects, no drug resistance, and has become the most in cancer treatment field after traditional therapy (surgery, chemotherapy and radiotherapy), targeted therapy (small molecule targeted drugs and monoclonal antibodies). One of the research directions of the future.

Tumor immunotherapy is one of the most promising research directions for the treatment of tumors. By stimulating or modulating the immune function of the motivational body, the tumor microenvironment is enhanced against the tumor immunity, thereby controlling and killing the tumor cells. Tumor immunotherapy mainly includes three categories: immunoassay monoclonal antibody therapy, adoptive cellular immunotherapy (ACT) and tumor vaccine. At present, the major international tumor immunotherapy treatments are concentrated in the field of immunoassay monoclonal antibodies, especially inhibitors of negative co-stimulatory factors - CTLA4, PD1 and other monoclonal antibodies, which are the focus of global research and development competition.

Cellular immunotherapy is a hotspot in the world of cancer treatment research. Biological treatment with traditional small molecule and monoclonal antibody drugs is a brand new tumor treatment mode and a living drug. Cytotoxic T lymphocyte (CTL) plays an important role in the individualized treatment of tumors because of its characteristics of targeting tumor cells. Currently used tumor-specific CTLs include TIL, DC-induced CTL, and genetically modified T cells (TCR-T and CAR-T), which specifically kill tumor cells expressing the antigen, and because of their excellent efficacy, CTL immunity Therapy has become an ideal solution for individualized treatment of tumors, but its efficacy is also limited, such as immunological checkpoint molecules PD-1, CTLA-4. Because tumor cells have the magical ability to evade immune responses, immunologists are also trying to activate the anti-tumor activity of T cells and maintain their ability to detect and attack cancer cells. The generation of tumor immune response requires the body's immune system to effectively identify and present tumor antigens, thereby activating specific T cells; costimulatory molecules and their regulatory networks Played an extremely important role in this process. In recent years, with the deepening of research on the mechanism of tumor immunosuppression, especially the discovery of immunological checkpoints PD-1 and CTLA-4, it provides a new method for improving the efficacy of CTL. If genetically modified T cells directly express specific antibodies against PD-1 and CTLA-4, they can block the immunosuppressive signals from tumor and tumor microenvironment, thereby saving depleted T cells and enhancing CTL killing of cancer cells. Functionality; however, there is currently no efficient method for the preparation of PD-1/CTLA-4 bispecific antibodies.

Summary of the invention

The present invention is directed to the defects of the prior art, and provides a method for preparing a PD-1/CTLA-4 bispecific antibody gene fragment, comprising the following steps:

Step 1. The anti-PD-1 dsFv gene is used as a template to amplify the gene fragment PD-1 V _H -GGGGS, and the upstream and downstream amplification primer sequences are shown in SEQ NO. 1 and SEQ NO. 2;

Step two, using the anti-CTLA-4dsFv gene as a template, the gene fragment GGGGS-CTLA-4 V _L is amplified, and the upstream and downstream amplification primer sequences are shown in SEQ NO. 3 and SEQ NO.

Step 3, Overlap PCR splicing the gene fragments PD-1 V _H -GGGGS and GGGGS-CTLA-4 V _L obtained in

steps

1 and 2 to construct the gene fragment PD-1 V _H -GGGGS-CTLA-4 V _L , amplification primer The sequences are shown in SEQ NO. 1 and SEQ NO. 5;

Step four, using the anti-CTLA-4 dsFv gene as a template, the gene fragment CTLA-4 V _H -GGGGS is amplified, and the upstream and downstream amplification primer sequences are shown as SEQ NO. 6 and SEQ NO. 2;

In step 5, the gene fragment GGGGS-PD-1 V _L is amplified by using the anti-PD-1 dsFv gene as a template, and the upstream and downstream amplification primer sequences are shown in SEQ NO. 3 and SEQ NO.

Step 6. Overlap PCR splicing the gene fragments CTLA-4 V _H -GGGGS and GGGGS-PD-1 V _L obtained in

steps

4 and 5 to construct the gene fragment CTLA-4 V _H -GGGGS-PD-1 V _L , amplification primers The sequences are shown in SEQ NO. 7 and SEQ NO. 8;

Step seven, six Overlap PCR fragment obtained gene splicing step III and step _{PD-1 V H -GGGGS-CTLA} -4 V L and _{CTLA-4 V H -GGGGS-PD} -1 V L, constructing a gene fragment of PD-1 V _H -GGGGS-CTLA-4 V _L -furin-GSGS-2A-CTLA-4 V _H -GGGGS-PD-1 V _L , which is a PD-1/CTLA-4 bispecific antibody gene fragment. The 2A peptide may be 2A having a self-shearing function such as F2A (foot-and-mouth disease virus), E2A (equine Rhinitis A virus), P2A (porcine teschovirus-1), and T2A (Thosea asigna virus).

Preferably,

Furin interval sequence:

The 2A sequence is preferably:

Accordingly, the present invention also provides a PD-1/CTLA-4 bispecific antibody gene fragment prepared by the above preparation method.

Further, the present invention provides a T cell modified with the PD-1/CTLA-4 bispecific antibody gene fragment described above.

Further, the present invention provides a specific antibody expressed by a PD-1/CTLA-4 bispecific antibody gene fragment.

Further, the present invention also provides the use of a PD-1/CTLA-4 bispecific antibody gene fragment for the preparation of an antitumor drug or kit.

In another aspect, the present invention provides a method for constructing and packaging a PD-1/CTLA-4 bispecific antibody lentiviral expression vector, comprising the steps of:

In the first step, the PD-1/CTLA-4 bispecific antibody gene fragment and the lentiviral expression vector of claim 2 are digested with EcoR I and Not I, respectively, and the digested product is separated by agarose gel electrophoresis. After the rubber recovery;

In the second step, the recovered gene fragment is ligated with the digested lentiviral vector, and the ligation product is transformed into E. coli DH5α, plated, single colony is picked and the plasmid is extracted, and finally the plasmid is double-enzymed with EcoR I and Not I. Cut to obtain a PD-1/CTLA-4 bispecific antibody lentiviral expression vector;

In step 3, the PD-1/CTLA-4 bispecific antibody lentiviral expression vector and the packaging plasmid obtained in the second step are co-transfected into 293T cells and cultured, the culture supernatant is collected, the virus is isolated, and the virus titer is determined. .

With the optimization of the above construction and packaging methods, the technical measures adopted by the present invention also include:

Preferably, the PD-1/CTLA-4 bispecific antibody gene fragment and lentivirus are described in the above step 1. The expression vector was subjected to double digestion conditions and digested at 37 ° C for 8 h.

Preferably, the above step 2 is specifically carried out: the gene fragment recovered in the first step is ligated with the digested vector at 16 ° C for 8 h, 5 μl of the ligation product is transformed into 50 μL of Escherichia coli DH5α, plated, single colonies are picked, and the plasmid is extracted. Finally, the plasmid was digested with EcoR I and Not I for 8 h at 37 °C, and the digested product was identified by 1% agarose gel electrophoresis to obtain a PD-1/CTLA-4 bispecific antibody expression vector.

Preferably, the above step 3 is specifically carried out: passage of 293T cells one day prior to transfection, so that the degree of fusion on the day of transfection is 60%-80%, the medium is changed 4 h before transfection, and the cell culture dish is from 5% CO _{2 .} The medium containing the double antibody was replaced with the pre-warmed DMEM+10% FBS medium without the double antibody, and the recombinant lentiviral plasmid and the packaging plasmid were co-transfected into 293T cells with Lipofectamine 2000; On the next day, the medium was changed to a normal volume of DMEM + 10% FBS + 1% double-antibody medium, and the culture supernatant was collected for 30-48 hours, centrifuged at 3000 rpm for 10 minutes, and then filtered with a 0.45um needle filter to high-speed sterilizing. Centrifuge in a centrifuge tube at 2 ° C for 2 h at 4 ° C, gently discard the supernatant after centrifugation, and fold the tube onto a clean paper towel until no clear supernatant remains, then add pre-cooled 1 ml PBS per centrifuge tube. After dispersing the virus cluster, 100 ul of virus solution per tube was placed in a clean sterile 1.5 ml centrifuge tube, and the virus titer was detected and stored at -80 ° C until use.

In a final aspect, the present invention also provides a method for preparing a T cell modified with a PD-1/CTLA-4 bispecific antibody gene fragment, comprising the following steps:

Step one, separating PBMC from healthy human peripheral blood, and stimulating T cell growth with CD3/CD28 magnetic beads for 3 days;

In the second step, the lentivirus prepared by the packaging method according to any one of claims 7 to 9 is co-cultured with the T cell culture in the first step to obtain a T cell modified with the PD-1/CTLA-4 bispecific antibody gene fragment.

The invention adopts the above technical solution, and has the following technical effects compared with the prior art:

The present invention prepares PD-1/CTLA-4 bispecific by genetic engineering technology using a single-chain antibody gene as a template, specific high-efficiency amplification primers, PCR and Overlap PCR, and introduction of disulfide bonds into the variable region of the antibody. The antibody fragment was cloned into a lentiviral expression vector and transfected into 293T cells for viral packaging; then transduced with CIK cells, PBMCs were isolated from healthy human peripheral blood, and T cells were stimulated with CD3/CD28 magnetic beads. Lentivirus is co-cultured with T cells to obtain novel anti-tumor T cells capable of blocking the immunosuppressive signals of tumor and tumor microenvironment sources while enhancing the function of killing cancer cells.

DRAWINGS

Figure 1 shows PD-1 V _H -GGGGS-CTLA-4 V _L -furin-GSGS-2A-CTLA-4 V _H -GGGGS-PD-1 V _L V _H -GGGGS-PD-1 prepared by the method of the present invention Schematic diagram of the structure of the V _L expression vector.

2 is a 1.5% agarose gel electrophoresis identification result of PD-1 V _H -GGGGS-CTLA-4 V _L and CTLA-4 V _H -GGGGS-PD-1 V _{L in the} first embodiment. The reference numerals are M:marker; 1:PD-1 V _H -GGGGS-CTLA-4 V _L ; 2: CTLA-4 V _H -GGGGS-PD-1 V _L .

Figure 3 is a 1.5% agarose gel electrophoresis identification of PD-1 V _H -GGGGS-CTLA-4 V _L -furin-GSGS-2A-CTLA-4 V _H -GGGGS-PD-1 V _L after splicing in Example 1. result. The reference numeral is M:marker; 1:PD-1 V _H -GGGGS-CTLA-4 V _L -furin-GSGS-2A-CTLA-4 V _H -GGGGS-PD-1 V _L .

Figure 4 is a graph showing the results of detection of the expression of intracellular PD-1/CTLA-4 bispecific antibody by intracellular staining in Example 3.

Figure 5 is the result of ELISA detection (coated with PD-1 protein) of PD-1/CTLA-4 bispecific antibody in Example 3.

Figure 6 is the result of ELISA detection (coated with CTLA-4 protein) of PD-1/CTLA-4 bispecific antibody in Example 3.

Figure 7 is a graph showing the results of detection of IFN-? release in Example 4.

Figure 8 is a graph showing the results of detection of target cell killing activity by genetically engineered T cells modified with PD-1/CTLA-4 bispecific antibody in Example 4.

detailed description

The invention provides a preparation method of a PD-1/CTLA-4 bispecific antibody gene fragment, comprising the following steps:

Step two, using the anti-CTLA-4 dsFv gene as a template, the gene fragment GGGGS-CTLA-4 V _L is amplified, and the upstream and downstream amplification primer sequences are shown in SEQ NO. 3 and SEQ NO.

steps

Step 4, using the anti-CTLA-4dsFv gene as a template, the gene fragment CTLA-4 V _H -GGGGS is amplified, and the upstream and downstream amplification primer sequences are shown in SEQ NO. 6 and SEQ NO. 2;

Step 5, using the anti-PD-1dsFv gene as a template, the gene fragment GGGGS-PD-1 V _L is amplified, and the upstream and downstream amplification primer sequences are shown in SEQ NO. 3 and SEQ NO.

Step 6. Overlap PCR splicing the gene fragments CTLA-4 V _H -GGGGS and PD-1 V _L -GGGGS obtained in

steps

Step 7. Overlap PCR splicing step III and step 6 gene fragments PD-1 V _H -GGGGS-CTLA-4 V _L and CTLA-4 V _H -GGGGS-PD-1 V _L to construct gene fragment PD-1 V _H -GGGGS-CTLA-4 V _L -furin-GSGS-2A-CTLA-4 V _H -GGGGS-PD-1 V _L , which is a PD-1/CTLA-4 bispecific antibody gene fragment.

Meanwhile, the present invention also provides a T-1 cell modified with a PD-1/CTLA-4 bispecific antibody gene fragment and a correspondingly expressed specific antibody, and the use of the above aspect in tumor immunotherapy. The present invention will be described in detail and by the following detailed description of the preferred embodiments of the invention

Example 1 Preparation of PD-1/CTLA-4 Bispecific Antibody Gene Fragment

The PD-1 V _H -GGGGS-CTLA-4 V _L and CTLA-4 V _H -GGGGS-PD-1 V _L genes were constructed by PCR and Overlap PCR using anti-PD-1 and CTLA-4DsFv genes as templates. Fragment, then construct the PD-1 V _H -GGGGS-CTLA-4 V _L -furin-GSGS-2A-CTLA-4 V _H -GGGGS-PD-1 V _L by furin-GSGS-2A ligation peptide (as shown in the attached figure 1).

Table 1 PCR primer sequences

Tab.1 Primer sequences

Step one, amplification of the gene fragment PD-1 V _H -GGGGS

Using the DsFv gene as a template, primer 1 and primer 2 were used to amplify the gene fragment PD-1 V _H -GGGGS. The amplification system was as follows:

Table 2 PCR reaction system

Table.2 PCR reaction system

The PCR amplification procedure was: pre-denaturation at 98 ° C for 30 s, denaturation at 98 ° C for 5 s, extension at 72 ° C for 30 s, 30 cycles, and extension at 72 ° C for 5 min. The reaction product was identified by 1.5% agarose gel electrophoresis. After the identification was correct, the PCR product was recovered by gel.

Step 2, amplification of the gene fragment GGGGS-CTLA-4 V _L

Using the DsFv gene as a template, the gene fragment GGGGS-CTLA-4 V _{L was} amplified using primer 3 and primer 4, and the amplification system was as follows:

Table 3 PCR reaction system

Table.3 PCR reaction system

Step 3, amplification of the gene fragment PD-1 V _H -GGGGS-CTLA-4 V _L

Overlap PCR splicing gene fragments PD-1 V _H -GGGGS and GGGGS-CTLA-4 V _L , construct gene fragment PD-1 V _H -GGGGS-CTLA-4 V _L , the reaction system is as follows:

Table 4 Overlap PCR reaction system

Table.4 Overlap PCR reaction system

The PCR amplification procedure was: pre-denaturation at 98 ° C for 30 s, denaturation at 98 ° C for 5 s, extension at 72 ° C for 30 s, 7 cycles, and extension at 72 ° C for 5 min. Then, an equal volume of the amplification reaction solution was added to the above spliced product to amplify PD-1 V _H -GGGGS-CTLA-4 V _L , and the amplification system was as follows:

Table 5 Overlap PCR reaction system

Table.5 Overlap PCR reaction system

The PCR amplification procedure was: pre-denaturation at 98 ° C for 30 s, denaturation at 98 ° C for 5 s, extension at 72 ° C for 30 s, 30 cycles, and extension at 72 ° C for 5 min. The reaction product was identified by 1.5% agarose gel electrophoresis (Fig. 2). After the identification was correct, the PCR product was recovered by gel.

Step 4, amplification of the gene fragment CTLA-4 V _H -GGGGS-PD-1 V _L

Amplification of the gene fragment CTLA-4 V _H -GGGGS

Using the DsFv gene as a template, the gene fragment CTLA-4 V _H -GGGGS was amplified using primer 6 and primer 2, and the amplification system was as follows:

Table 6 PCR reaction system

Table.6 PCR reaction system

Step 5, amplification of the gene fragment GGGGS-PD-1 V _L

Using the DsFv gene as a template, the gene fragment GGGGS-PD-1 V _{L was} amplified using primer 7 and primer 8, and the amplification system was as follows:

Table 7 PCR reaction system

Table.7 PCR reaction system

Step 6. Amplification of the gene fragment CTLA-4 V _H -GGGGS-PD-1 V _L

Overlap PCR splicing gene fragment CTLA-4 V _H -GGGGS and GGGGS-PD-1 V _L , construct gene fragment CTLA-4 V _H -GGGGS-PD-1 V _L , the reaction system is as follows:

Table 8 Overlap PCR reaction system

Table.8 Overlap PCR reaction system

The PCR amplification procedure was: pre-denaturation at 98 ° C for 30 s, denaturation at 98 ° C for 5 s, extension at 72 ° C for 30 s, 7 cycles, and extension at 72 ° C for 5 min. Was then added to the splicing reaction product solution volume amplification and the like, amplified _{CTLA-4 V H -GGGGS-PD} -1 V L, amplification system as follows:

Table 9 Overlap PCR reaction system

Table.9 Overlap PCR reaction system

Step 7. Amplification of the gene fragment PD-1 V _H -GGGGS-CTLA-4 V _L -furin-GSGS-2A-CTLA-4 V _H -GGGGS-PD-1 V _L

Overlap PCR splicing gene fragment PD-1 V _H -GGGGS-CTLA-4 V _L and CTLA-4 V _H -GGGGS-PD-1 V _L , construct gene fragment PD-1 V _H -GGGGS-CTLA-4 V _L - furin-GSGS-2A-CTLA-4 V _H -GGGGS-PD-1 V _L , the reaction system is as follows:

Table 10 Overlap PCR reaction system

Table.10 Overlap PCR reaction system

The PCR amplification procedure was: pre-denaturation at 98 ° C for 30 s, denaturation at 98 ° C for 5 s, extension at 72 ° C for 30 s, 30 cycles, and extension at 72 ° C for 10 min. The reaction product was identified by 1.5% agarose gel electrophoresis. After the identification was correct, the PCR product was recovered by gel (Fig. 3).

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

PD-1 V _H -GGGGS-CTLA-4 V _L -furin-GSGS-2A-CTLA-4 V _H -GGGGS-PD-1 V _L and lentiviral expression were obtained in Example 1 using EcoR I and Not I, respectively. The vector was double-digested (Tables 11, 12), and digested at 37 °C for 8 h. The digested product was separated by 1% agarose gel electrophoresis and then recovered by gel.

Table 11 Enzyme digestion system

Table.11 digestion system

Table 12 Enzyme digestion system

Table.12 digestion system

The recovered gene fragment was ligated with the digested vector (Table 13), ligated at 16 ° C for 8 h, and 5 μl of the ligated product was transformed into 50 μL of Escherichia coli DH5α, plated, single colonies were picked and plasmids were extracted, and finally EcoR I and Not were used. The plasmid was double-digested (Table 14), digested at 37 °C for 8 h, and the digested product was identified by 1% agarose gel electrophoresis.

Table 13 Connection system

Table.13 Ligase reaction system

Table 14 Enzyme digestion system

Table.14 digestion system

The 293T cells were passaged one day before transfection, so that the degree of fusion on the day of transfection reached 60%-80%. The medium was changed 4 hours before transfection, and the cell culture dish was taken out from the 5% CO ₂ 37 °C incubator. The medium containing the double antibody was replaced with the pre-warmed DMEM+10% FBS medium without the double antibody, and the recombinant lentiviral plasmid and the packaging plasmid were co-transfected into 293T cells with Lipofectamine2000. On the second day of transfection, the medium was changed to a normal volume of DMEM + 10% FBS + 1% double-antibody culture medium, and the culture supernatant was collected for 30-48 hours, centrifuged at 3000 rpm for 10 minutes, and then filtered with a 0.45um needle filter to a high speed. Centrifuge the tube at 2 ° C for 2 h at 4 ° C, gently discard the supernatant after centrifugation, and fold the tube down on a clean paper towel until no clear supernatant remains, then add pre-cooled 1 ml PBS per centrifuge tube After dispersing the virus cluster, 100 ul of virus solution per tube was placed in a clean sterile 1.5 ml centrifuge tube and stored at -80 ° C for infection of T cells. The resulting virus titer was detected using a Lenti-X p24 Rapid Titer Kit to be 5 x 10 ^{7 -} 5 x 10 ⁸ IFU.

Example 3 Preparation of PD-1/CTLA-4 bispecific antibody gene-modified T cells and detection of antibody expression

PBMC were isolated from healthy human peripheral blood, and T cells were stimulated with CD3/CD28 magnetic beads for 3 days to detect CD8+/CD4+ ratio, and CD8+ cells could reach 80% or more. The lentivirus packaged in Example 2 was cultured for 3 days with T cells. , PD-1/CTLA-4 bispecific antibody gene-modified T cells were obtained.

Collect cells, add Brefeldin A at 1:1000, mix and incubate for 4-6 hours at 37 ° C in 5% CO 2 incubator; harvest cells, add 100 μL MEDIUM A, incubate for 15 min at room temperature; wash 3 times with PBS (3% FBS) Add 100 μL of MEDIUM B and 1 μL of protein-L-biotein; incubate for 30 min at 4 °C; wash 3 times with PBS (3% FBS); add 5 μL of SA-PE, incubate for 30 min at 4 °C; flow detection (test results are shown in Figure 4) Shown).

The expression of PD-1/CTLA-4 bispecific antibody in T cells was detected by ELISA, and the PD-1 protein was coated at 100 ng/well. The supernatant of 100 μl PD-1/CTLA-4 bispecific antibody was added and detected by ELISA. Binding activity with PD-1 protein (detection results are shown in Figure 5); coating CTCL-4 protein 100ng/well, adding 100μl PD-1/CTLA-4 bispecific antibody expression supernatant, ELISA detection The binding activity of CTLA-4 protein (detection results are shown in Figure 6).

Example 4 Detection of killing activity of target cells by PD-1/CTLA-4 bispecific antibody gene-modified T cells

In vitro detection of melanoma cell killing experiments: chromium isotope labeled tumor cells, 4 hours gamma technician to determine chromium isotope release, while measuring IFN-γ release, the results are shown in Figures 7, 8.

Effector cell grouping: PBMC control group; CIK control group; PD-1/CTLA-4 bispecific antibody gene transfection group;

Effective target ratio grouping: effective target ratio is 3:1; 6:1; 12:1; 25:1; 50:1;

The effector cells were mixed with the target cells in a pre-experimental design ratio, and then co-cultured at 37 ° C for 4 h;

The supernatant was discarded, and an appropriate amount of 10% SDS was added to collect cells, and the isotope activity was measured by an isotope analyzer, and the tumor killing activity of each group of T cells was calculated.

The PD-1/CTLA-4 bispecific antibody-modified genetically engineered T cells were co-cultured with 51Cr-labeled tumor cells for four hours, and the ratio of effector cells to target cells was as shown. The tumor cell killing activity of each group was calculated according to the following formula: tumor cell kill percentage = (experimental group - lymphocyte) / (maximum lysis group - target cell control group) × 100%.

The present invention prepares PD-1/CTLA-4 bispecific by genetic engineering technology using a single-chain antibody gene as a template, specific high-efficiency amplification primers, PCR and Overlap PCR, and introduction of disulfide bonds into the variable region of the antibody. Antibody fragment, and the gene fragment was cloned into a lentiviral expression vector by transfecting 293T cells The virus is packaged; then, the CIK cells are transduced, PBMC is isolated from healthy human peripheral blood, T cells are stimulated by CD3/CD28 magnetic beads, and lentivirus and T cells are co-cultured to obtain a tumor and tumor microenvironment source. Immunosuppressive signals, while enhancing novel anti-tumor T cells that kill the function of cancer cells.

The specific embodiments of the present invention have been described in detail above, but are merely exemplary, and the invention is not limited to the specific embodiments described above. Any equivalent modifications and substitutions to the invention are also within the scope of the invention. Accordingly, equivalents and modifications may be made without departing from the spirit and scope of the invention.

Claims

A method for preparing a PD-1/CTLA-4 bispecific antibody gene fragment, comprising the steps of:

Step 1. The anti-PD-1 dsFv gene is used as a template to amplify the gene fragment PD-1 V H -GGGGS, and the upstream and downstream amplification primer sequences are shown in SEQ NO. 1 and SEQ NO. 2;

Step two, anti-CTLA-4 dsFv gene as a template, amplification of gene fragments GGGGS-CTLA-4 V L, upstream and downstream amplification primer sequence as shown in SEQ NO.3 and SEQ NO.4;

Step 3, Overlap PCR splicing the gene fragments PD-1 V H -GGGGS and GGGGS-CTLA-4 V L obtained in steps 1 and 2 to construct the gene fragment PD-1 V H -GGGGS-CTLA-4 V L , amplification primer The sequences are shown in SEQ NO. 1 and SEQ NO. 5;

Step four, using the anti-CTLA-4 dsFv gene as a template, the gene fragment CTLA-4 V H -GGGGS is amplified, and the upstream and downstream amplification primer sequences are shown as SEQ NO. 6 and SEQ NO. 2;

In step 5, the gene fragment GGGGS-PD-1 V L is amplified by using the anti-PD-1 dsFv gene as a template, and the upstream and downstream amplification primer sequences are shown in SEQ NO. 3 and SEQ NO.

Step 6. Overlap PCR splicing the gene fragments CTLA-4 V H -GGGGS and PD-1 V L -GGGGS obtained in steps 4 and 5 to construct the gene fragment CTLA-4 V H -GGGGS-PD-1 V L , amplification primers The sequences are shown in SEQ NO. 7 and SEQ NO. 8;

Step 7. Overlap PCR splicing step III and step 6 gene fragments PD-1 V H -GGGGS-CTLA-4 V L and CTLA-4 V H -GGGGS-PD-1 V L to construct gene fragment PD-1 V H -GGGGS-CTLA-4 V L -furin-GSGS-2A-CTLA-4 V H -GGGGS-PD-1 V L , that is, a PD-1/CTLA-4 bispecific antibody gene fragment is obtained, wherein 2A is selected from F2A, E2A or T2A with self-shearing function.
The PD-1/CTLA-4 bispecific antibody gene fragment produced by the production method according to claim 1.
The PD-1/CTLA-4 bispecific antibody gene fragment-modified T cell according to claim 2.
The specific antibody expressed by the PD-1/CTLA-4 bispecific antibody gene fragment of claim 2.
The use of the PD-1/CTLA-4 bispecific antibody gene fragment of claim 2 for the preparation of an antitumor drug or kit.
Construction and packaging method of PD-1/CTLA-4 bispecific antibody lentiviral expression vector, characterized in Therefore, the following steps are included:

In the first step, the PD-1/CTLA-4 bispecific antibody gene fragment and the lentiviral expression vector of claim 2 are digested with EcoR I and Not I, respectively, and the digested product is separated by agarose gel electrophoresis. After the rubber recovery;

In the second step, the recovered gene fragment is ligated with the digested lentiviral vector, and the ligation product is transformed into E. coli DH5α, plated, single colony is picked and the plasmid is extracted, and finally the plasmid is double-enzymed with EcoR I and Not I. Cut to obtain a PD-1/CTLA-4 bispecific antibody lentiviral expression vector;

In step 3, the PD-1/CTLA-4 bispecific antibody lentiviral expression vector and the packaging plasmid obtained in the second step are co-transfected into 293T cells and cultured, the culture supernatant is collected, the virus is isolated, and the virus titer is determined. .
The construction packaging method according to claim 6, wherein in step 1, the PD-1/CTLA-4 bispecific antibody gene fragment and the lentiviral expression vector are subjected to restriction enzyme digestion at 37 ° C for 8 h.
The method of constructing a package according to claim 6, wherein the step 2 is specifically carried out by: linking the gene fragment recovered in the step 1 to the vector after digestion at 8 ° C for 8 hours, and connecting 5 μl of the ligation product to transform 50 μL of Escherichia coli DH5α. , plating, picking a single colony and extracting the plasmid, and finally digesting the plasmid with EcoR I and Not I for 8 h at 37 °C, and digesting the product with 1% agarose gel electrophoresis to obtain PD-1/CTLA-4 bispecific. Sexual antibody expression vector.
The method for constructing packaging according to claim 6, wherein the specific operation in step 3 is: passage of 293T cells one day before transfection, so that the degree of fusion on the day of transfection is 60%-80%, and the medium is changed 4 hours before transfection. The cell culture dish was taken out from the 5% CO 2 37 ° C incubator, and the medium containing the double antibody was replaced with the preheated DMEM+10% FBS medium without the double antibody, and the recombinant lentiviral plasmid and the recombinant lentiviral plasmid were The packaging plasmid was co-transfected into 293T cells with Lipofectamine 2000; the medium was transfected into the normal volume of DMEM + 10% FBS + 1% double antibody medium for the next day, and the culture supernatant was collected for 30-48 hours, and centrifuged at 3000 rpm for 10 min. Then, filter it into a high-speed sterile centrifuge tube with a 0.45um needle filter, centrifuge at 25000 rpm for 2 hours at 4 ° C, gently discard the supernatant after centrifugation, and fold the centrifuge tube onto a clean paper towel until no clear supernatant remains. After adding the pre-cooled 1 ml PBS per centrifuge tube, the virus cluster was blown off, and then 100 ul of virus solution per tube was placed in a clean sterile 1.5 ml centrifuge tube, and the virus titer was detected, and stored at -80 ° C for use.
Preparation method of PD-1/CTLA-4 bispecific antibody gene fragment modified T cell The question is to include the following steps:

Step one, separating PBMC from healthy human peripheral blood, and stimulating T cell growth with CD3/CD28 magnetic beads for 3 days;

In the second step, the lentivirus prepared by the packaging method according to any one of claims 7 to 9 is co-cultured with the T cell culture in the first step to obtain a T cell modified with the PD-1/CTLA-4 bispecific antibody gene fragment.