Targeted compounds for the site-specific coupling of chemical moieties comprising a peptide linker
A compound, specific technology, used in medical applications to solve problems such as structural instability
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[0057] The present invention will now be further described in more detail. Each embodiment defined below may be combined with any other embodiment unless explicitly stated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature indicated as being preferred or advantageous.
[0058] The present invention relates to targeting compounds for conjugation of chemical moieties comprising at least one targeting domain selected from the group capable of binding with a binding constant K of 500 nM or below D a non-immunoglobulin protein or antibody fragment that binds a target, and at least one linking moiety comprising up to about 80 amino acids, wherein the linking moiety consists essentially of or consists of alanine, proline and serine, and at least one coupling site at the C-terminus or N-terminus of the linking moiety, wherein the coupling site consists of Cys(C), CXC, CXXC or CXXXC, wherein X is selected fro...
Embodiment 1
[0120] Example 1. Generation of expression constructs
[0121] figure 1 Schematic representations of preferred compounds of the invention are provided. The fusion protein has the following structural features: targeting domain-(linker-coupling site) n -cap. Specific fusion proteins were generated comprising (i) as targeting domain Her2-specific binding protein or EGFR-specific binding protein; (ii) as linking moiety selected from Ala, Pro, Ser (in Table 2 22 to 74 amino acids called "APS") with 2, 3 or 4 linking moieties in the fusion protein; (iii) amino acid Cys or peptide motif Cys-Pro-Ala-Cys as coupling site, It is regularly distributed between linking moieties with a minimum distance of 22 amino acids and a maximum distance of 74 amino acids between each coupling; (iv) 10 to 30 amino acids at the C-terminus as a cap (selected from Ala, Pro, Ser) to avoid the end position of the conjugation site.
[0122] Table 2. Examples of fusion proteins
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[0124] A...
Embodiment 2
[0126] Example 2. Expression of fusion proteins
[0127] HMS174(DE3) competent cells were transformed with the expression plasmid. Cells were plated onto selective agar plates (kanamycin) and incubated overnight at 37°C. Inoculate 100 ml of super-rich medium (modified H15 medium, 2% glucose, 5% yeast extract, 1% casamino acids, 0.76% glycerol, 0.7% lactose, 1% cyclin) with a preculture from a single colony. Yeast RNA, 250mM MOPS, 202mM TRIS, 10mg / L RNase A, pH 7.4, antifoam SE15) and at 37°C, in a regular orbital shaker at 160rpm, added with 150μg / ml kanamycin but Incubate for 16 hours in a 1 L baffled Erlenmeyer flask without lactose and antifoam. The main culture from the above overnight culture was grown at an adjusted initial-OD of 0.5 600 Inoculate into 400 ml of ultra-rich medium in a 1 L thick-walled Erlenmeyer flask supplemented with glycerol, glucose, lactose, antifoam, and 150 μg / ml kanamycin. The culture was transferred to a resonant acoustic mixer (RAMbio) and ...
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