Noncompetitive domain antibody formats that bind interleukin 1 receptor type 1
A technology of interleukin and domain antibody, applied in the direction of antibody, non-central analgesic, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve the problems that cannot meet the requirements
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[0199] Preparation of immunoglobulin-based ligands
[0200]Ligands according to the present invention (such as dual-specific ligands, dAb monomers) can be prepared according to previously established techniques in the field of antibody engineering, and can be used for the preparation of scFv, "phage" antibodies and other genetically engineered antibody molecules. Techniques for preparing such antibodies are described in the following reviews and citations therein: Winter & Milstein, (1991) Nature 349:293-299; Pluckthun (1992) Immunological Reviews 130:151-188; Wright et al., (1992) Crti. Rev. Immunol. 12: 125-168; Holliger, P. & Winter, G. (1993) Curr. Op. Biotechn. 4, 446-449; Carter, et al. (1995) J. Hematother .4, 463-470; Chester, K.A. & Hawkins, R.E. (1995) Trends Biotechn. 13, 294-300; Hoogenboom, H.R. (1997) Nature Biotechnol. 15, 125-126; Fearon, D. (1997) Nature Biotechnol. 15 , 618-619; Plückthun, A. & Pack, P. (1997) Immunotechnology 3, 83-105; Carter, P. & Merchan...
Embodiment 1
[0290] method
[0291] selection and screening
[0292] For primary screening, the Vκ domain antibody monomer 4G-K2 library was panned with IL-1R1-Fc fusion protein (Axxora, Nottingham, UK). Domain antibodies obtained from the initial screening were subjected to three further rounds of screening. The 1st round was screened with protein G-coated magnetic beads (Dynal, Norway) and 100 nM IL-1R1-Fc; the 2nd round was with anti-human IgG magnetic beads (Novagen, Merck Biosciences, Nottingham, UK) and 10 nM IL-1R1 - Fc selection; round 3 selection with protein G magnetic beads and 1 nM IL-1R1-Fc. (Henderikx et al., Selection of antibodies against biotinylated antigens. Antibody Phage Display: Methods and protocols, Ed. O'Brien and Atkin, Humana Press (2002).). Elution at each stage was performed with 1 mg / ml trypsin-phosphate buffered saline (PBS). For the affinity maturation screen, the method described above was used, but with the following modifications: Two rounds of select...
Embodiment 2
[0317] Embodiment 2 protease stability
[0318] protease stability
[0319] dAbs and ligands containing dAbs can be used to treat a variety of diseases, such as inflammatory diseases. In addition, the half-life of dAbs as well as ligands can be altered, such as by pegylation, as described herein. Thus dAbs and ligands can be used eg for systemic administration (eg pegylated dAbs for arthritis) or topical administration (eg dAb monomers for COPD).
[0320] The stability of two dAbs binding IL-1R1 to the action of elastase or trypsin was investigated. These two proteases are found to be low in the lung in the natural state, but in disease states such as COPD, the levels of proteases such as elastase are elevated. The dAB monomer DOM4-130-54, and a DOM4-130-54 variant containing a point mutation providing a cysteine residue specifically attached to PEG was used in this study.
[0321] A 1 mg / ml DOM4-130-54 solution in PBS was incubated with 0.04% trypsin or elastase (human ...
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