Improved stable tethering structures with defined composition for multifunctionality or binding specificity
A stable tethering, covalently bound technology, applied in the field of improved stable tethering structures with well-defined composition with multifunctionality or binding specificity, which can solve problems such as unstable aggregation, reduction, low expression level, etc.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
preparation example Construction
[0294] Methods for making and screening aptamers capable of binding a particular target of interest are well known, eg, US Patent No. 5,475,096 and US Patent No. 5,270,163, each of which is incorporated herein by reference. Techniques typically involve selection from a mixture of candidate aptamers and stepwise iterative binding, separation of bound from unbound aptamers, and amplification. Since in a mixture only a small number of sequences (possibly only one molecule of aptamer) are present corresponding to the highest affinity aptamer, it is usually best to set the partitioning criteria so that during the separation, an effective amount of aptamer in the mixture (approx. 5-50%). Each round enriches aptamers with high affinity for the target. 3-6 rounds of selection and amplification cycles can be repeated to generate aptamers that bind the target with high affinity and specificity.
[0295] Avimers
[0296] In certain embodiments, the precursors, components and / or comple...
Embodiment 1
[0353] Example 1. General strategy for generating modular Fab subunits
[0354] Fab modules were generated as fusion proteins containing DDD or AD sequences. Independent transgenic cell lines were developed for each Fab fusion protein. Once the modules are produced, the modules can be purified if necessary, or retained in the cell culture supernatant. Any (Fab-DDD) 2 After the module is produced, it can be combined with any Fab-AD module to generate a bispecific trivalent Fab (bsTF).
[0355] The plasmid vector pdHL2 has been used to generate various antibodies and antibody-based constructs. See Gillies et al., J Immunol Methods (1989), 125:191-202; Losman et al., Cancer (Phila) (1997), 80:2660-6. A bicistronic mammalian expression vector directs the synthesis of IgG heavy and light chains. For many different IgG-pdHL2 constructs, the vector sequence is mostly identical, only in the variable region (V H and V L ) differences in the sequence. These IgG expression vector...
Embodiment 2
[0399] Embodiment 2: expression vector
[0400] Construction of h679-Fd-AD1-pdHL2
[0401] h679-Fd-AD1-pdHL2 is an expression vector for producing h679 Fab, in which AD1 is linked to the carboxy-terminal of FdCH1 region through a flexible Gly / Ser peptide spacer composed of 14 amino acid residues. The pdHL2-based vector containing the variable region of h679 was transformed into h679-Fd-AD1-pdHL2 by replacing the SacII / EagI fragment with the CH1-AD1 fragment that was shuttled from CH1-AD1-SV3 with SacII and EagI cut from the carrier.
[0402] Construction of C-DDD1-Fd-hMN-14-pdHL2
[0403] C-DDD1-Fd-hMN-14-pdHL2 is an expression vector that produces a stable dimer, which contains two copies of the fusion protein C-DDD1-Fab-hMN-14, in which DDD1 is at the carboxy-terminal of CH1 through a flexible The peptide spacer was linked to hMN-14Fab. The plasmid vector hMN14(I)-pdHL2, which had been used to produce hMN-14 IgG, was converted to C-DDD1-Fd by digestion with SacII and Eag...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More