== In vitro T cell activation induced by each anti-HER2/CD3 BsAb with FAST-Ig mutations, with or without target cells

== In vitro T cell activation induced by each anti-HER2/CD3 BsAb with FAST-Ig mutations, with or without target cells. reporter cell assay demonstrated the combined effects of epitope, affinity, and subclasses. Our findings highlight the potential of FAST-Ig technology for efficiently generating mouse BsAbs for preclinical studies. Keywords:bispecific antibody, FAST-Ig, surrogate mouse bispecific antibody, orthogonal Fab, single-cell production == 1. Introduction == Bispecific antibodies (BsAbs) are capable of binding to two distinct antigens or epitopes, enabling them to exhibit mechanisms of action that cannot be achieved by monospecific antibodies. This unique property has shown promise in treating various diseases, including cancer, hemophilia, and eye diseases [1]. Although several molecular formats for BsAbs have been reported [2], IgG-type BsAbs are preferred formats for therapeutic antibodies due to their stability and lower immunogenicity. However, when IgG-type BsAbs Xanthotoxol are expressed in a single cell, the random assembly of two heavy chains (HCs) and two light chains (LCs) can result in nine mispairs, except for the correctly assembled BsAb. To promote the correct assembly of human BsAbs, strategies have been developed separately for the HC heterodimerization and for HC/LC pairings. For the Rabbit polyclonal to ADD1.ADD2 a cytoskeletal protein that promotes the assembly of the spectrin-actin network.Adducin is a heterodimeric protein that consists of related subunits. interface of HCs, knobs-into-holes (KiH) [3,4] and charge pairs designs [5,6] have been described, while for the HC/LC interfaces, CrossMab [7], Orthogonal Fab (fragment antigen-binding) [8], and DuetMab [9] have been described in several reviews [1,2]. In antibody drug discovery, animal immunization, particularly immunization of mice, is still the primary method for generating new paratopes [10]. Additionally, surrogate mouse antibodies are often required to demonstrate a drug discovery concept for animal studies using mice [11]. Therefore, efficient methods for generating mouse BsAbs are crucial for preclinical studies. However, as mentioned in the following paragraph, there have been a limited number of published reports on the creation of mouse BsAbs. There are two primary methods reported for generating mouse BsAbs. One example is the controlled Fab arm exchange (cFAE), which efficiently generates mouse BsAbs in mouse IgG1 (mIgG1), mIgG2a, and mIgG2b [12]. However, cFAE requires additional steps beyond affinity purification to obtain Xanthotoxol BsAbs. These steps involve separately preparing the two parental antibodies, mixing them together, and then placing them under reducing conditions. Following this, the reducing agent is removed, allowing for the formation of BsAbs. The other example is a single-cell expression system that necessitates technologies to facilitate not only the correct assembly of HC/HC but also HC/LC. Strategies such as KiH and charge modification have been reported to promote correct HC heterodimerization in mouse BsAbs [13,14,15]. However, there are few reports on the orthogonal Fab design between HC/LC for mouse antibodies. One example involves applying a charge modification to mIgG2a, which is located at the core of the interface between the constant HC domain 1 (CH1) and the light chain constant region (CL) [16]. It should be noted that this report has limited applications, and that the variable region is derived from a human antibody. Additionally, the application of the DuetMab to mouse C and CrossMab has been reported [13,15]. However, these reports have not compared the correct HC/LC pairing efficiency with and without the introduction of modifications, leaving the extent of the effects of these modifications unclear. Recently, a novel technology called FAST-Ig (Four-chain Assembly by electrostatic Steering TechnologyImmunoglobulin) was developed to promote the correct assembly of HC/HC and HC/LC within a single cell [17]. To promote correct HC and LC assembly, FAST-Ig uses charge-based orthogonal Fab design for human IgGs. However, there are no reports on the application of FAST-Ig for mouse IgGs. This study aimed to investigate whether the orthogonal Fab design of FAST-Ig can be applied to mIgG1 and mIgG2a, the two major types of antibodies often used in mouse studies. To promote HC heterodimerization, KiH mutations, which have already been reported [14,15], Xanthotoxol were utilized. To confirm the applicability for preclinical screening, new mouse BsAbs comprising known anti-cluster of differentiation 3 (CD3) and anti-human epidermal growth factor receptor 2 (HER2) antibodies were generated using FAST-Ig mutations. The activities of these BsAbs were then compared using the Jurkat NFAT luciferase reporter cell line. FAST-Ig demonstrated high BsAb production efficiency after simple affinity purification, even for mouse antibodies, without the need for downstream processing. Therefore, FAST-Ig is a useful technology for preclinical BsAb screening and preparing mouse surrogate BsAbs. == 2. Materials and Methods == == 2.1. Preparation of FAST-Ig Antibodies == Site-directed mutagenesis for FAST-Ig mutations and sub-cloning into mammalian expression vectors was performed using.