Antigen-binding molecule containing two antigen-binding domains that are linked to each other
A technology of antigen-binding molecules and binding domains, applied in the field of increasing the resistance of antigen-binding molecules to protease cleavage, and the field of pharmaceutical compositions containing such antigen-binding molecules, which can solve problems such as difficulty in obtaining agonist antibodies
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0634] [Example 1] Concept of Fab cross-linked antibody
[0635]Compared with natural ligands and their fusion proteins, agonist antibodies have superior properties such as stability, pharmacokinetics, and production methods, and their drug development is ongoing. In general, however, agonist antibodies with strong activity are more difficult to obtain than binding or neutralizing antibodies alone. Therefore, there is a need for a solution to this problem.
[0636] The desired properties of an agonist antibody may depend on the type of ligand. For agonist antibodies against the TNF receptor superfamily represented by death receptors (DR), OX40, 4-1BB, CD40, etc., it has been reported that multimerization of antibodies or ligands contributes to activation. As techniques for enhancing this effect, the following techniques have been reported to enhance agonist activity: using natural ligands, by cross-linking of anti-Fc antibodies, by cross-linking of FcγRs, by multimerization ...
Embodiment 2
[0639] [Example 2] Preparation of expression vector of modified antibody, and expression and purification of modified antibody
[0640] Using PCR, In-Fusion Advantage PCR cloning kit (TAKARA), etc., by methods known to those skilled in the art, the amino acid residue sequence of the antibody gene inserted into the animal cell expression vector is replaced to construct the expression vector of the modified antibody. The nucleotide sequence of the resulting expression vector is determined by methods known to those skilled in the art. Transient introduction of the resulting expression vector or cells (Invitrogen) and allowed to express the modified antibody in the culture supernatant. Using rProtein A Fast Flow (GE Healthcare), the modified antibody was purified from the obtained culture supernatant by methods known to those skilled in the art. Absorbance at 280 nm was measured using a spectrophotometer. Absorption coefficients were calculated from the measured values u...
Embodiment 3
[0650] [Example 3] Preparation of bispecific antibody
[0651] The purified antibody was dialyzed into TBS (WAKO) buffer and its concentration was adjusted to 1 mg / mL. Prepare 10x reaction buffer 250 mM 2-MEA (SIGMA). The two different homodimeric antibodies prepared in Example 2 were mixed in equal amounts. To this mixture, 1 / 10 volume of 10x reaction buffer was added and mixed. The mixture was allowed to stand at 37°C for 90 minutes. After the reaction, the mixture was dialyzed into TBS to obtain a solution of bispecific antibodies in which the above two different antibodies were heterodimerized. The antibody concentration was measured by the method described above, and the antibody was subjected to subsequent experiments.
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More