Co-expression and purification method of conditionally activated binding proteins
An expression vector and protease cleavage technology, applied in the field of co-expression and purification of conditionally activated binding proteins, can solve the problems of reduced tumor accumulation, reduced specificity, and lack of affinity
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0290] A. Example 1: Method for generating half-COBRA pairs
[0291] Half-COBRA cannot act as a single molecule to induce T cell killing even after protease activation, however, two activated complementary half-COBRA molecules can induce potent T cell-mediated cytotoxicity against target-expressing cells (Figures 1 and 1). figure 2 ). This creates the problem of producing these molecules as drugs, since two protein molecules need to be produced. One solution is to form two expressing cell lines, each expressing a different half-COBRA, these two half-COBRAs can then be expressed and purified separately, and mixed to produce a prodrug mixture. Unfortunately, this is a time-consuming and expensive process to produce large doses of drug mixtures.
[0292] The solution to this problem is to co-express two half-COBRAs in the same cell and then purify them separately or together from the same conditioned medium.
[0293] image 3 Half-COBRA is shown to be co-expressed from transie...
Embodiment 2
[0320] B. Example 2: Generation of stable cell lines co-expressing half-COBRA pairs
[0321] This example illustrates an exemplary method for generating cell lines that co-express complementary half-COBRA pairs.
[0322] Materials and methods
[0323] All cell culture media, transfections were from Life Technologies. Transfection reagents include Expi293 Transfection Kit. The growth and expression medium used in the study was Expi293 expression medium. Selection medium included DMEM + 10% FBS + 0.5 mg / ml G418. All biosensors used for Octet analysis were from ForteBio, including SAX (high precision streptavidin) and His1K (Anti-Penta-His).
[0324] result
[0325] Figure 41 A schematic diagram of the two half-COBRA pairs used to generate co-expressing stable cell lines is shown in . Half-COBRA pairs are labeled differently to facilitate detection and purification. Each test pair included a first polypeptide with the following structure (N-terminal to C-terminal): s...
PUM
| Property | Measurement | Unit |
|---|---|---|
| molecular weight | aaaaa | aaaaa |
| affinity | aaaaa | aaaaa |
| volume | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More