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ScFv Fragments BsAb Generation Service

Creative Biolabs is one of the world’s most trusted experts for bispecific antibody (BsAbs) development and production. Based on the unchallenged experience in recombinant antibody synthesis, we are dedicated to developing scFv fragments with promised high affinity for both academic and clinical purposes.

BsAbs are a class of biological therapeutics developed to bind to two distinct epitopes that express on either the same target molecule or two totally dissimilar molecules. They hold particular interest on account of their capacity to simultaneously recognize and engage two distinct targets. Heterodimerizing two distinct single chains of IgG or its derivatives is a common strategy for obtaining a BsAb. However, the hybrid products always contain homodimers contamination. To overcome this chain-pairing problem, diverse modifications are introduced into the constant regions of antibodies, the parts of which are not in charge of binding targets. In the past years, a range of innovative BsAb formats has been designed with single chain Fv (scFv) fragments being widely used as fundamental building blocks. ScFv fragments are single polypeptide antigen binding domains containing a VH domain linked to a VL domain via a synthetic peptide linker, generally a 15 amino acids (Gly4/Ser)3 peptide.

Schematic diagram of scFv fragments BsAbs.

Figure 1. Schematic diagram of scFv fragments BsAbs.

scFv BsAb Fragments

This class of BsAbs mainly include three constructs utilizing scFv as units combined with connecting portions: scFv-CH3, scFv-Fc and scFv-CH-CL-scFv. In the formats of scFv-CH3 and scFv-Fc, the engineering of knobs-in-holes is introduced into the CH3 to force the formation of heterodimers. While the format of scFv-CH-CL-scFv utilizes the natural heterodimerization of CH1 and CL, similarly to triple body. The first scFv is either linked to a CH or a CL to construct the first scFv-Fc chain; the second scFv is linked to the CL from the same origin when the first scFv is linked to the CH, or vice versa. Consequently, specific recognizing and disulfide bond between CH and CL can lead to the proper heterodimerization.

Knobs Into Holes

"Knobs into Holes" (KIH) is the most famous one among these proven techniques. Creative Biolabs can significantly increase the efficient formation of heterodimers with a rate around 95% in both scFv-Fc and scFv-CH3 BsAb construction based on KIH method. Recently, other heterodimeric Fc variants have also been developed by using novel CH3 variant-pair strategies, including DD–KK variants, HA–TF variants and IgG/IgA strand-exchange engineered domains. All of these strategies can be applied in Creative Biolabs depending on customer’s requirements.

With our well-established scFv fragments BsAb generation platform, the experienced scientists here at Creative Biolabs are dedicated to helping you develop unique BsAbs. We also provide other various services regarding BsAbs development. Please feel free to contact us for more information and a detailed quote.


1. Miller, B. R.; et al. Stability engineering of scFvs for the development of bispecific and multivalent antibodies. Protein Engineering, Design & Selection. 2010, 23(7): 549-557.
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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