Overview of Tandem scFv-Fc

Tandem scFv-Fc is a common bispecific antibody (bsAb) format, consisting of two single-chain variable fragments (scFvs) connected by a linker peptide, each scFv consisting of a heavy chain variable region (VH) and a light chain variable region (VL) connected by another linker peptide. Tandem scFv-Fc has an IgG-like structure and can utilize the functions of the Fc region, such as extending the half-life, mediating effector cell functions, regulating immune complex formation, etc. Tandem scFv-Fc has advantages over traditional full-length IgG antibodies, such as low molecular weight, high affinity, good stability, high expression level, etc. Tandem scFv-Fc exerts its effects in tumor immunotherapy mainly by redirecting T cells or NK cells to kill tumor cells, blocking the signaling pathways required for tumor growth, or enhancing tumor antigen presentation. Tandem scFv-Fc exerts its effects in autoimmune disease treatment mainly by blocking pro-inflammatory factors or enhancing inhibitory factors.

Structure and Generation Methods of Tandem scFv-Fc

Tandem scFv-Fc is an IgG-like BsAbs, consisting of two scFvs connected by a linker peptide, each scFv consisting of a VH and a VL connected by another linker peptide. Tandem scFv-Fc has the functions of the Fc region, such as extending the half-life, mediating effector cell functions, regulating immune complex formation, etc. The structural features of tandem scFv-Fc can be adjusted according to its affinity, valency and stability. For example, the interaction between scFvs can be influenced by changing the length and sequence of the linker peptide, resulting in monovalent, bivalent or multivalent tandem scFv-Fc. Bivalent or multivalent tandem scFv-Fc can improve the affinity and specificity for antigens, but may also increase its molecular weight and immunogenicity. In addition, the "Knobs into Holes" mechanism can be introduced to enhance the formation of heavy chain heterodimers, thereby improving the stability and expression level of tandem scFv-Fc. This mechanism is achieved by replacing an amino acid in the CH3 region of each heavy chain, resulting in a protrusion (knob) on one heavy chain and a recess (hole) on the other heavy chain, thus promoting the complementary pairing of the two heavy chains.

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

There are mainly two methods for generating tandem scFv-Fc. Based on recombinant DNA technology, using genetic engineering methods to connect two scFv genes together and then transfect them into host cells for expression. This method can achieve high efficiency, high flexibility, and low-cost production, but it also has problems such as low expression levels, purification difficulties, and poor stability. Based on antibody engineering technology, using different platform technologies to connect two scFv molecules together, such as the XmAb platform and CrossMab platform. This method can produce high quality, high stability and high affinity products, but also has problems such as high technical complexity, patent protection difficulty and high cost. To improve the expression level and stability of tandem scFv-Fc, some optimization strategies can be adopted, such as changing the length and sequence of the linker peptide, introducing the "Knobs into Holes" mechanism to enhance the formation of heavy chain heterodimers, selecting suitable host cells and expression systems, etc.

Clinical Data of Tandem scFv-Fc

Tandem scFv-Fc is mainly used for tumor immunotherapy and autoimmune disease treatment in clinical settings. So far, one tandem scFv-Fc has been approved for marketing, and several tandem scFv-Fc are in clinical trials.

The approved tandem scFv-Fc is Blinatumomab (Blincyto), developed by Amgen, which is a tandem scFv-Fc targeting CD19 and CD3. Blinatumomab received accelerated approval in the United States in 2014 for the treatment of relapsed/refractory acute lymphoblastic leukemia (r/r ALL), and later expanded its indications, including high-risk r/r B-cell precursor ALL, MRD-positive B-cell precursor ALL, etc. Blinatumomab's mechanism of action is to redirect T cells to kill CD19-positive tumor cells, thereby inducing tumor remission and prolonging survival. Blinatumomab's clinical efficacy has been confirmed in several randomized controlled trials, such as significantly improving the complete remission rate and overall survival of r/r ALL patients. Blinatumomab's safety has also been evaluated in several trials, finding that its main adverse reactions include neurotoxicity, cytokine release syndrome, infection, etc.

The tandem scFv-Fc products in clinical trials are various, mainly for tumor immunotherapy and autoimmune disease treatment.

In summary, tandem scFv-Fc, as a BsAbs format with high affinity, high stability, high expression level and other advantages, shows a wide range of application prospects in clinical settings, but also needs further optimization of its structure design, reduction of its adverse reactions, enhancement of its efficacy and so on.

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