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Knobs-in-holes (KIH) BsAb Generation Service

Based on years of experience in antibody discovery and manufacture, Creative Biolabs has established a second to none bispecific antibodies (BsAbs) generation platform. With our comprehensive platform, we are proud to offer knobs-in-holes (KIH) BsAb generation service in a timely and cost-effective manner.

Knobs-in-holes (KIH) Technology

In recent years, BsAbs have rising as a potential field of research for therapies in oncology, infectious and inflammable diseases. Their ability of dual target recognition enables for new therapeutic hypothesis to be tested, where typical mono-specific antibodies would lack the required mode of target engagement. Among diverse approaches of BsAbs production, KIH technology has been extensively used. This technology refers to produce either a “knob” or a “hole” in each heavy chain to promote heterodimerization. In this method, the two heavy chains have been engineered, thus, knob half-antibody preferentially partners with the hole half antibody, and application of disulfide reduction/reoxidation covalently binds the two to generate a BsAb with two different arms. The KIH technology has also been applied to develop the one-armed antibody onartuzumab (MetMAb), which is comprised of a single antigen-binding arm and a human Fc region.

Expression of human bispecific antibodies. Heterodimerization of antibody heavy chains specific for different antigens enable to be enforced through knobs-into-holes mutations in CH3 domains.

Figure 1. Expression of human bispecific antibodies. Heterodimerization of antibody heavy chains specific for different antigens enable to be enforced through knobs-into-holes mutations in CH3 domains. (Rouet, R., 2014)

Amino Acid Changes in KIH

KIH amino acid (AA) change is a useful design strategy in antibody engineering to produce BsAbs. H chains of human immunoglobulin IgG directly interact at the level of their CH3 domains, however, as to CH2 domains, they interact through the carbohydrate bound to the asparagine (Asn) N84.4 in the DE turn. For purpose of making a knob on the CH3 of the H chains of mAb1 and a hole on the CH3 of the H chains of mAb2, AA changes are engineered. The knob is showed by a tyrosine (Y) which is part of the 'very large' IMGT volume class of AA. Nevertheless, the hole is showed by a threonine (T) that is part of the 'small' IMGT volume class. mAb1 and mAb2 stand for two antibodies with different specificities. As showed in figure 2, the light chains (L) of the two antibodies had the same VL domain. Then, coexpression of these engineered H chains in HEK-293S cells allowed a mixture of products with the bispecific heterodimer representing the major fraction (92%).

Strategy for creating bispecific mAb using the Knobs-into-holes AA changes. (Xu, Y.,2015)

Figure 2. Strategy for creating bispecific mAb using the Knobs-into-holes AA changes. (Xu, Y.,2015)

Expression Systems

Generation of KIH BsAbs to high purity has been challenging, on account of the inherent complexity of the molecule. We offer mammalian cell expression systems for KIH BsAbs production. We can also provide glycosylated, functional and competent heterodimeric antibodies after the expression step in mammalian host.

With the well-established KIH technology platform, the experienced scientists here at Creative Biolabs are dedicated to help you develop therapeutic BsAbs. We also provide other various services regarding BsAbs development. Please feel free to contact us for more information and a detailed quote.

References

1. Rouet, R.; Christ D. Bispecific antibodies with native chain structure. Nature biotechnology. 2014, 32(2): 136.
2. Xu, Y.; et al. Production of bispecific antibodies in “knobs-into-holes” using a cell-free expression system. Mabs. 2015, 7(1): 231-242.

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