Albumin binding antibodies and use thereof
An albumin-binding, anti-albumin technology, applied in the field of albumin-binding antibodies and their uses, can solve the problems of increased medical system costs and decreased patient compliance.
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Embodiment 1
[0500] Example 1. Production of anti-HSA single domain antibodies
[0501] Human serum albumin (HSA) expressed in mice (Sigma, #A9731) was used to immunize llamas to produce anti-HSA single domain antibodies (V H H antibody). After immunization, peripheral mononuclear cells (PBMCs) were isolated for RNA extraction. Use mRNA / cDNA encoding antibody genes to construct V H H Antibody Phage Display Library. The constructed phage display library was screened by multiple rounds of affinity binding to the antigen. pass Affinity measurement ( RED96) to identify positive clones. Sequence the antibody genes of positive clones and clone these genes into Vector (EMD Millipore, #CS221284) for expression in Chinese hamster ovary (CHO) cells.
[0502] Table 4 lists exemplary anti-HSA single domains (e.g. V H H) Antibodies (eg antibodies A1 to A18). These exemplary V H The CDR sequences of the H antibody are listed in Table 5. Among these antibodies, antibody A8 interacts with h...
Embodiment 2
[0509] Example 2. Production of Humanized Anti-HSA Single Domain Antibodies
[0510] Each llama sequence of the anti-HSA single domain antibody (sdAb) was directed against Databases were compared to identify the best matching human IGHV3 germline. When multiple germlines have similar homology, use The similarity of the amino acid at position 52 of FR2 in the numbering system was used to determine which germline to use as the humanized scaffold. FR1 and FR3 from the selected germline were then used as "humanized" FR1 and FR3. For FR2, most of the human sequences were adopted except for positions 42 and 52, where the respective llama sequences were retained. Table 6 shows exemplary human FR region sequences for each of the anti-HSA antibodies. For human FR4, the humanized antibodies A1-A9, A19 and A20 each independently have the amino acid sequence of WGQGTQVTVSS (SEQ ID NO: 145), WGQGTLVTVSS (SEQ ID NO: 146) or SGQGTQVTVSS (SEQ ID NO: 147). In one embodiment, humanized a...
Embodiment 3
[0514] Example 3. Binding-dissociation constants (K for anti-albumin antibodies D )
[0515] Evaluation V H The ability of H anti-albumin antibody to interact with human, monkey and mouse serum albumin at different pH values (eg, pH 7.4 or pH 5.5) to mimic the pH of acidifying endosomes. In short, put V H H anti-albumin antibody fused to human IgG1-Fc. The expressed protein was then loaded onto the Protein A biosensor ( RED96). The biosensor is then immersed in a solution containing human, monkey or mouse serum at three or more different concentrations, for example 50 nM, 100 nM or 200 nM, or 100 nM, 200 nM or 400 nM. The main experimental data were analyzed with an overall fit to determine the binding-dissociation constant (K D ). The results are summarized in Table 7 (pH 5.5) and Table 8 (pH 7.4), where A represents a value not greater than 10 nM, B represents a value greater than 10 nM but not greater than 100 nM, and C represents a value greater than 100 nM but n...
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