Composition for culturing t cells and method for culturing t cells using same
A composition and cell technology, applied in the field of T cell culture, to achieve the effect of increasing proliferation and activity, and safe preparation
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preparation Embodiment 1
[0160] Preparation Example 1, hCD80-Fc-IL-2 variant (2M): preparation of GI101
[0161] To generate a fusion protein containing a human CD80 fragment, an Fc domain and an IL-2 variant, a polynucleotide comprising the nucleotide sequence (SEQ ID NO: 8) was synthesized by ThermoFisher Scientific's Invitrogen GeneArt Gene Synthesis service from the N-terminal Encoding sequence containing signal peptide (SEQ ID NO:1), CD80 fragment (SEQ ID NO:2), linker-bound Ig hinge (SEQ ID NO:3), Fc domain (SEQ ID NO:4), linker ( SEQ ID NO: 5) and a fusion protein of IL-2 variant (2M) (SEQ ID NO: 6) with two amino acid substitutions (R38A, F42A) and cloned into the pcDNA3_4 vector. In addition, this vector was introduced into CHO cells (Expi-CHO TM ), used to express the fusion protein with SEQ ID NO:9. After introduction of the vector, the CHO cell culture solution was incubated at 37°C, 125RPM and 8% CO 2 cultured for 7 days, and then harvested to purify the fusion protein. The purified f...
preparation Embodiment 2
[0164]Preparation Example 2, Fc-IL-2 variant (2M) dimer: Preparation of Fc-IL-2v2
[0165] To generate a fusion protein containing the Fc domain and the IL-2 variant, a polynucleotide comprising the nucleotide sequence (SEQ ID NO: 45), in order from the N-terminus, was synthesized by ThermoFisher Scientific's Invitrogen GeneArt Gene Synthesis service The encoding contains a signal peptide (SEQ ID NO: 1), an Ig hinge (SEQ ID NO: 38), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5) and has two amino acid substitutions (R38A, F42A) fusion protein of IL-2 variant (2M) (SEQ ID NO: 6) and cloned into pcDNA3_4 vector. In addition, this vector was introduced into CHO cells (Expi-CHO TM ), used to express the fusion protein with SEQ ID NO:44. After introduction of the vector, the CHO cells were incubated at 37°C, 125 RPM and 8% CO 2 cultured for 7 days, and cells were harvested to purify fusion protein dimers. The purified fusion protein dimer was named "Fc-IL2v2".
[0166] P...
preparation Embodiment 3
[0167] Preparation Example 3, Fc-IL-2 dimer: Preparation of Fc-IL-2wt
[0168] In order to generate a fusion protein containing the Fc domain and wild-type IL-2, a polynucleotide comprising the nucleotide sequence (SEQ ID NO: 43) was synthesized by ThermoFisher Scientific's Invitrogen GeneArt Gene Synthesis service, which sequentially encodes from the N-terminal Signal peptide (SEQ ID NO:1), Ig hinge (SEQ ID NO:38), Fc domain (SEQ ID NO:4), linker (SEQ ID NO:5) and wild-type IL-2 (SEQ ID NO:38) 10) and cloned into the pcDNA3_4 vector. In addition, this vector was introduced into CHO cells (Expi-CHO TM ), used to express the fusion protein with SEQ ID NO:42. After introduction of the vector, the CHO cells were incubated at 37°C, 125 RPM and 8% CO 2 cultured for 7 days, and then harvested to purify the fusion protein dimer. The purified fusion protein dimer was named "Fc-IL2wt".
[0169] Purification and collection of the fusion protein were performed using the same method ...
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