WO2017128534A1 - Procédé de préparation d'un anticorps bispécifique dirigé contre pd-1 et ctla-4 et son utilisation - Google Patents

Procédé de préparation d'un anticorps bispécifique dirigé contre pd-1 et ctla-4 et son utilisation Download PDF

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WO2017128534A1
WO2017128534A1 PCT/CN2016/080419 CN2016080419W WO2017128534A1 WO 2017128534 A1 WO2017128534 A1 WO 2017128534A1 CN 2016080419 W CN2016080419 W CN 2016080419W WO 2017128534 A1 WO2017128534 A1 WO 2017128534A1
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ctla
ggggs
gene fragment
bispecific antibody
seq
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杨世成
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杨世成
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • 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
  • 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.
  • ACT adoptive cellular immunotherapy
  • tumor vaccine 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.
  • CTL Cytotoxic T lymphocyte
  • 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.
  • costimulatory molecules and their regulatory networks Played an extremely important role in this process.
  • it provides a new method for improving the efficacy of CTL.
  • 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.
  • 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;
  • 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).
  • F2A foot-and-mouth disease virus
  • E2A equine Rhinitis A virus
  • P2A porcine teschovirus-1
  • T2A Thosea asigna virus
  • Furin interval sequence
  • the 2A sequence is preferably:
  • the present invention also provides a PD-1/CTLA-4 bispecific antibody gene fragment prepared by the above preparation method.
  • the present invention provides a T cell modified with the PD-1/CTLA-4 bispecific antibody gene fragment described above.
  • the present invention provides a specific antibody expressed by a PD-1/CTLA-4 bispecific antibody gene fragment.
  • 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.
  • the present invention provides a method for constructing and packaging a PD-1/CTLA-4 bispecific antibody lentiviral expression vector, comprising the steps of:
  • 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;
  • the recovered gene fragment is ligated with the digested lentiviral vector, and the ligation product is transformed into E. coli DH5 ⁇ , 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;
  • 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 technical measures adopted by the present invention also include:
  • 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.
  • 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 ⁇ l of the ligation product is transformed into 50 ⁇ L of Escherichia coli DH5 ⁇ , 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.
  • 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.
  • 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;
  • 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.
  • 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.
  • the invention provides a preparation method of 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-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.
  • 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 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 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 , which is a PD-1/CTLA-4 bispecific antibody gene fragment.
  • 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
  • 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).
  • Step one amplification of the gene fragment PD-1 V H -GGGGS
  • primer 1 and primer 2 were used to amplify the gene fragment PD-1 V H -GGGGS.
  • the amplification system was as follows:
  • 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
  • the gene fragment GGGGS-CTLA-4 V L was amplified using primer 3 and primer 4, and the amplification system was as follows:
  • 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 3 amplification of the gene fragment PD-1 V H -GGGGS-CTLA-4 V L
  • 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:
  • 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
  • the gene fragment CTLA-4 V H -GGGGS was amplified using primer 6 and primer 2, and the amplification system was as follows:
  • 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 5 amplification of the gene fragment GGGGS-PD-1 V L
  • the gene fragment GGGGS-PD-1 V L was amplified using primer 7 and primer 8, and the amplification system was as follows:
  • 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 6 Amplification of the gene fragment CTLA-4 V H -GGGGS-PD-1 V L
  • 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:
  • 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 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
  • 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).
  • 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.
  • the recovered gene fragment was ligated with the digested vector (Table 13), ligated at 16 ° C for 8 h, and 5 ⁇ l of the ligated product was transformed into 50 ⁇ L of Escherichia coli DH5 ⁇ , 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.
  • 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.
  • 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.
  • PBMC peripheral blood cells 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 ⁇ L MEDIUM A, incubate for 15 min at room temperature; wash 3 times with PBS (3% FBS) Add 100 ⁇ L of MEDIUM B and 1 ⁇ L of protein-L-biotein; incubate for 30 min at 4 °C; wash 3 times with PBS (3% FBS); add 5 ⁇ L 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 ⁇ l 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 100 ⁇ l PD-1/CTLA-4 bispecific antibody expression supernatant, ELISA detection The binding activity of CTLA-4 protein (detection results are shown in Figure 6).
  • 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 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.

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Abstract

L'invention concerne une préparation d'un fragment d'anticorps bispécifique dirigé contre PD-1 et CTLA-4, qui est produit par les étapes consistant à utiliser un gène d'anticorps simple chaîne en tant que matrice et des amorces d'amplification spécifiques à haute efficacité, effectuer une PCR et une PCR de chevauchement et introduire une liaison bisulfure dans une région variable de l'anticorps. Le fragment de gène est cloné dans un vecteur d'expression lentiviral, il transfecte une cellule 293T pour effectuer une encapsidation virale, puis une cellule CIK comprenant un PBMC séparé à partir de sang périphérique humain sain est transduite, en stimulant la croissance des lymphocytes T à l'aide de billes magnétiques CD3/CD28 et en co-cultivant les lymphocytes T avec le lentivirus, de manière à obtenir un nouveau lymphocyte T antitumoral capable de bloquer les signaux d'immunosuppression des tumeurs et des micro-environnements tumoraux, et d'accroître la cytotoxicité antitumorale.
PCT/CN2016/080419 2016-01-29 2016-04-27 Procédé de préparation d'un anticorps bispécifique dirigé contre pd-1 et ctla-4 et son utilisation WO2017128534A1 (fr)

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CN114195900A (zh) * 2020-09-17 2022-03-18 普米斯生物技术(珠海)有限公司 一种抗4-1bb/pd-l1双特异性抗体及其用途
US11479608B2 (en) 2016-08-23 2022-10-25 Akeso Biopharma, Inc. Anti-CTLA4 antibodies
US11578128B2 (en) 2016-08-23 2023-02-14 Akeso Pharmaceuticals, Inc. Anti-CTLA4 and anti-PD-1 bifunctional antibody, pharmaceutical composition thereof and use thereof
CN117402248A (zh) * 2023-12-14 2024-01-16 成都美杰赛尔生物科技有限公司 抗体组合物的应用、检测免疫细胞抗肿瘤活性的方法及试剂盒

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WO2018014260A1 (fr) 2016-07-20 2018-01-25 Nanjing Legend Biotech Co., Ltd. Protéines de liaison antigènes multi-spécifiques et leurs procédés d'utilisation
WO2018068201A1 (fr) 2016-10-11 2018-04-19 Nanjing Legend Biotech Co., Ltd. Anticorps à domaine unique et ses variants contre ctla-4
CA3046082A1 (fr) 2016-12-07 2018-06-14 Agenus Inc. Anticorps et procedes d'utilisation de ceux-ci
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WO2019179421A1 (fr) * 2018-03-19 2019-09-26 Wuxi Biologics (Shanghai) Co., Ltd. Nouvelles molécules d'anticorps pd-1/ctla-4 bispécifiques
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CN110563851B (zh) * 2019-08-20 2020-06-12 启辰生生物科技(珠海)有限公司 具有免疫调节功能的融合蛋白、药物组合物、细胞及应用
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US20220072159A1 (en) * 2015-11-11 2022-03-10 Precigen, Inc. Compositions and methods for expression of multiple biologically active polypeptides from a single vector for treatment of cardiac conditions and other pathologies
US11479608B2 (en) 2016-08-23 2022-10-25 Akeso Biopharma, Inc. Anti-CTLA4 antibodies
US11578128B2 (en) 2016-08-23 2023-02-14 Akeso Pharmaceuticals, Inc. Anti-CTLA4 and anti-PD-1 bifunctional antibody, pharmaceutical composition thereof and use thereof
CN114195900A (zh) * 2020-09-17 2022-03-18 普米斯生物技术(珠海)有限公司 一种抗4-1bb/pd-l1双特异性抗体及其用途
CN114195900B (zh) * 2020-09-17 2024-02-23 普米斯生物技术(珠海)有限公司 一种抗4-1bb/pd-l1双特异性抗体及其用途
CN117402248A (zh) * 2023-12-14 2024-01-16 成都美杰赛尔生物科技有限公司 抗体组合物的应用、检测免疫细胞抗肿瘤活性的方法及试剂盒
CN117402248B (zh) * 2023-12-14 2024-02-13 成都美杰赛尔生物科技有限公司 抗体组合物的应用、检测免疫细胞抗肿瘤活性的方法及试剂盒

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