Tumor vaccine, dendritic cell tumor vaccine and preparation method thereof
A technology for tumor vaccines and tumor cells, which is applied in the fields of tumor vaccines, dendritic cell tumor vaccines and their preparation, can solve the problems of low therapeutic effect of tumor vaccines and importing dendritic cells, etc., and achieve the goal of avoiding immunosuppression and satisfying commercial production Effect
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Embodiment 1
[0070] Example 1 Preparation of stable phagocytosis receptor ligand-avidin-biotin-antigen conjugate
[0071] 1. Metabolic markers of tumor cells
[0072] 1.1 Selection of metabolic markers
[0073] The purpose of metabolic labeling of tumor cells is to incorporate azide group-containing compounds into tumor cell surface antigens. Alternative compounds include unnatural sugars and unnatural amino acids. For example, non-natural sugars or their precursors that can be incorporated into sugar chains modified by tumor antigens through the cellular sugar metabolism pathway after being co-cultured with cells, including sialic acid containing azido groups, glucose, galactose, One of salhalose, mannose or their precursors.
[0074] 1.2 We co-cultured tumor cells with Ac4ManNAz.
[0075] Co-cultivation system: according to 2×10 5 cells / mL 4T1 cells were seeded in a 96-well plate, 100 μl per well, and cultured for 24 hours. After aspirating the supernatant, add such as figure 1 Ac4M...
Embodiment 2
[0087] Example 2: Crosslinking of Biotin to Azidosialic Acid Expressed by Tumor Cells
[0088] As described in Example 1, there are two types of strategies for modifying the ligands of phagocytosis receptors. The first type requires the corresponding metabolic markers of tumor cells to achieve azidation of tumor cell surface antigens; the second type requires the corresponding A requirement for metabolic labeling of tumor cells is to achieve biotinylation of tumor cell surface antigens.
[0089] In this embodiment, we use the mIgG1Fc-NeutrAvidin hybrid protein prepared in Example 1 to couple to tumor cell surface antigens.
[0090] First we follow the 2×10 5 cells / mL 4T1 cells were inoculated into 96-well plates, 100 μl per well, and the culture medium (the medium was the same as in Example 1) containing 3 mM Ac4Man-NAz or Sialic acid was cultured for 24 hours. Cells were washed 4 times with PBS containing 1% FBS. Add 50 μM Click-iT DIBO-biotin (Thermo Fisher, C10412), and ...
Embodiment 3
[0093] Example 3 Conjugation of Modified Phagocytosis Receptor Ligands to Tumor Cell Surface Antigens
[0094]As described in Example 1, strategies for modifying ligands for phagocytosis receptors fall into two categories. The first type is to modify the ligands of phagocytosis receptors with compounds containing orthogonal reactive groups (alkynes), so that the modified ligands can be directly conjugated with azidated tumor cell surface antigens through a bioorthogonal reaction. The second type is the use of avidin (or streptavidin) to modify the ligand of the phagocytic receptor to form a hybrid protein or a recombinant fusion protein, so that the modified ligand cannot directly interact with the azidated tumor cell surface antigen However, the azide tumor cell surface antigen needs to be further modified with biotin, and then the high-affinity properties of biotin-avidin (streptavidin) are used to realize the binding between the modified ligand and the tumor antigen. conju...
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