Overview of Fab-scFv-Fc

Bispecific antibodies (BsAbs) are a class of antibody molecules that can simultaneously recognize two different antigens or epitopes, and have a variety of novel therapeutic mechanisms, such as immune cell redirection, multiple antigen or epitope targeting, immune checkpoint modulation, and antibody-drug conjugate carrier enhancement. Currently, nearly a hundred BsAbs have entered clinical trial stages, mainly for the treatment of cancer and other inflammatory diseases. BsAb formats are diverse and can be classified into Fc-deficient and Fc-rich types based on whether they contain an Fc region. Fab-scFv-Fc is an asymmetric BsAb format that consists of an scFv and a Fab connected to a heterodimeric Fc (Figure 1). This format uses scFv and Fab, two common building blocks for generating bispecific fragments. Bispecific fragments composed of both Fab and scFv include Bibody (Fab-scFv), Fab-scFv-Fc, F(ab')2-scFv2, etc. Fab-scFv-Fc has the following advantages: (1) it avoids the possibility of light chain mispairing, because only one light chain is present; (2) it improves heterodimerization efficiency, because forced heterodimerization technology (such as KIH technology) is used; (3) it retains Fc-mediated effector functions and serum half-life extension; (4) it achieves monovalent binding to two targets, avoiding cross-bridging toxicity or dose limitation. Fab-scFv-Fc also has some drawbacks: (1) expression imbalance, because there is a difference between scFv and Fab chains; (2) scFv-induced aggregation, because scFv itself tends to form higher-order structures; (3) structural complexity, because four polypeptide chains need to be expressed and correctly assembled.

Structural Features of Fab-scFv-Fc

The structure of Fab-scFv-Fc consists of an scFv and a Fab connected to the N-terminus of a heterodimeric Fc, respectively. scFv is the smallest antibody binding fragment consisting of variable heavy chain (VH) and variable light chain (VL) connected by a single polypeptide chain, with high affinity and specificity. Fab is an antibody fragment formed by heterodimerization of light chain and heavy chain fragment, containing a complete antibody binding site. Fc is an antibody fragment formed by homodimerization of the constant region (CH2 and CH3) of the heavy chain, with the ability to mediate effector functions and extend serum half-life. In order to ensure the correct heterodimerization of Fc, forced heterodimerization technology (such as KIH technology) is usually required, that is, mutations are introduced in the CH3 domain of the heavy chain, so that two different heavy chains form complementary interfaces between them, thereby increasing the thermodynamic stability of heterodimerization relative to homodimerization.

Fig.1 Schematic diagram of Fab-scFv-Fc (Creative Biolabs)

The structural variations of Fab-scFv-Fc mainly involve aspects such as the position, number and arrangement of scFv and Fab. For example, scFv can be connected to the C-terminus of light chain or heavy chain, or both; Fab can be connected to the N-terminus of heavy chain or light chain, or both; scFv and Fab can be arranged in different orders on Fc, such as scFv-Fab-Fc or Fab-scFv-Fc; scFv and Fab can exist in different proportions on Fc, such as 1:1, 2:1, 1:2, etc. These structural variations will affect the properties of Fab-scFv-Fc such as affinity, valency, stability, expression level, etc., so they need to be optimized and selected according to different application goals.

There is a close relationship between the structure and function of Fab-scFv-Fc. On the one hand, scFv and Fab respectively endow Fab-scFv-Fc with the recognition ability for two different antigens or epitopes, thus achieving bispecific binding. On the other hand, Fc endows Fab-scFv-Fc with the ability to interact with molecules such as Fc receptors or FcRn, thus achieving effects such as effector functions or serum half-life extension. Therefore, scFv and Fab need to maintain an appropriate spatial distance and angular relationship between them and between scFv, Fab and Fc, to avoid interference or hindrance. In addition, scFv and Fab themselves also need to have sufficiently high affinity, specificity, stability and other properties to ensure that Fab-scFv-Fc has good activity, safety and tolerability.

Clinical Data of Fab-scFv-Fc

Fab-scFv-Fc, as a novel BsAb format, has shown its potential and advantages in clinical trials and marketing. According to different targets and disease areas, Fab-scFv-Fc can achieve various therapeutic mechanisms, such as immune cell redirection, multiple antigen or epitope targeting, immune checkpoint modulation, antibody-drug conjugate carrier enhancement, etc. Currently, several Fab-scFv-Fc products have entered clinical trial stages, mainly for the treatment of cancer and other inflammatory diseases. In addition, one Fab-scFv-Fc product has been approved for marketing, for the treatment of psychostimulant effects caused by methamphetamine (METH) abuse. This section will introduce the Fab-scFv-Fc products that have been approved for marketing and those that are in clinical trial stages, respectively, and list the relevant information and references.

Currently, only one Fab-scFv-Fc product has been approved by the US Food and Drug Administration (FDA) for the treatment of psychostimulant effects caused by METH abuse. The product is composed of an scFv and Fc of an anti-METH monoclonal antibody, which is expressed in mice by gene therapy and can effectively reduce the concentration of METH in the central nervous system. In addition, several Fab-scFv-Fc candidates are in different stages of clinical trials, mainly for the treatment of cancer and other inflammatory diseases. These candidates involve various targets, such as CD3, Her2, PSMA, etc., and various mechanisms of action, such as T-cell redirection, multiple antigen or epitope targeting, immune checkpoint modulation, etc.

References

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