United use of LGR4/RSPO blocker and anti-immune check point inhibitor to immunotherapy of tumors
A technology of immune checkpoints and blockers, applied in the direction of anti-tumor drugs, antibody medical components, antibodies, etc., can solve the problems of limiting tumor broad-spectrum action and low response
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[0142] Antibody preparation
[0143] Any method suitable for producing monoclonal antibodies can be used to produce anti-immune checkpoints (such as PD-1, PD-L1, CTLA-4, TIM3, TIGIT\ICOS, LAG3, OX40, CD47, VISTA, 4- 1BB) antibodies. For example, linked or naturally occurring immune checkpoints (such as PD-1, PD-L1, CTLA-4, TIM3, TIGIT\ICOS, LAG3, OX40, CD47, VISTA, 4-1BB) homodimers or Fragments thereof are used to immunize animals. Suitable immunization methods may be used, including adjuvants, immunostimulants, repeated booster immunizations, one or more routes may be used.
[0144] Any suitable form of immune checkpoint (such as PD-1, PD-L1, CTLA-4, TIM3, TIGIT\ICOS, LAG3, OX40, CD47, VISTA, 4-1BB) can be used as an immunogen (antigen) for Generate non-human antibodies specific to immune checkpoints (such as PD-1, PD-L1, CTLA-4, TIM3, TIGIT\ICOS, LAG3, OX40, CD47, VISTA, 4-1BB), and screen the biology of the antibodies active. The eliciting immunogen can be a full-leng...
Embodiment 1
[0227] Example 1 Construction of Lgr4 prokaryotic expression vector and expression and purification of Lgr4 extracellular segment protein
[0228] The Lgr4 prokaryotic expression vector was constructed by the applicant in the early stage. The expression vector skeleton used was PET28a(+). The polynucleotide sequence of the LGR4 gene was connected into its multiple cloning site through genetic engineering operation technology. After expression, the extracellular segment of the target protein Lgr4 Protein N-terminal fusion expresses 6×his-tagged protein. Transform BL21 Escherichia coli with the recombinant plasmid, and coat the LB agarose culture plate with kanamycin resistance. After culturing overnight, pick a single positive colony and place it in the LB liquid medium containing kanamycin resistance for shaking culture. Overnight, transfer all the overnight bacterial liquid into 1L of fresh LB liquid medium containing kanamycin resistance, shake the bacteria at 37°C and 220rp...
Embodiment 2
[0246] Example 2 Anti-tumor effect of LGR4 / Rspondin blocker synergistically with PD-1 antibody
[0247] LGR4-ECD can block the interaction between LGR4 and Rspondin1, Rspondin2, Rspondin3, Rspondin4, and Rspondin1 antibody can block the interaction between LGR4 and Rspondin1.
[0248] The present invention uses C57BL / 6 mice to establish a subcutaneous melanoma model of B16F10, and the tumor-forming mice with uniform tumor volume are given LGR4-ECD, anti-Rspondin1 monoclonal antibody, and anti-PD-1 monoclonal antibody respectively. Single drug and combined drug treatment to detect and evaluate the synergistic effect of blockade of Rspondin / lgr4 signaling on the therapeutic effect of anti-PD-1 monoclonal antibody.
[0249] The result is as figure 2 shown. The results show that the LGR4 / Rspondin blocker LGR4-ECD can significantly enhance the anti-tumor effect of the immune checkpoint PD-1 antibody and prolong the survival of tumor-bearing mice; the LGR4 / Rspondin blocker RSPO1 ...
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