22.5.1 Single-Chain Variable Fragment (scFv)

Antibody Fv fragment is the smallest unit responsible for antigen-binding activity,

and single-chain variable fragment (scFv) format consists of variable region of heavy

(VH) and light (VL) chains, which are joined together by a flexible peptide linker.

The commonly used peptide linkers comprise streches of glycine and serine residues

which provides scFv flexibility. Inclusion of glutamic acid and lysine residues in the

linker enhances the solubility of scFv. The scFv format of therapeutic antibodies is a

good delivery vehicle for radionucleotides as these can rapidly penetrate tissues as

compared to whole antibodies and thus used as reagents for radio-imaging and

radioimmunotherapy (Hudson and Souriau 2009). These can also be used to deliver

a range of toxins or drugs to the specific cells. The physiological disadvantage of

scFv format is rapid elimination from the body. Intrabodies which can penetrate the

cells to target various viruses or cancers by neutralizing a range of oncogene or

signaling molecules have been developed as variant of scFv (Strube and Chen 2002).

22.5.2 Bispecific and Tri-Specific MAbs

Bispecific antibodies with ability to engage two different antigens have been pro-

duced with clinical applications. Bispecific antibodies are made in two formats:

(1) IgG-like bispecific antibodies which carry Fc region and (2) non-IgG format.

IgG-like bispecific antibodies have Fc-mediated effector functions such as antibody-

dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phago-

cytosis (ADCP), and complement-dependent cytotoxicity, whereas non-IgG format

primarily mediates its action by binding to two different antigens. The first bispecific

antibody catumaxomab (Removab^®) approved by the European Union has been

used for malignant ascites. It is based on IgG format with Fc region. It recognizes

CD3 antigen on cytotoxic T cells and epithelial cell adhesion molecule (EpCAM)

which is a type 1 transmembrane glycoprotein associated with malignant ascites

(Seimetz 2011). Another bispecific antibody, blinatumomab, approved by the US

FDA in December 2014 comprised two scFv connected by peptide linker (Newman

and Benani 2016). Bilnatumomab recognized simultaneously CD19 antigen

expressed on all stages of B cell lineage and CD3 T cell receptor complex and

approved for treatment of relapsed or refractory Ph-negative acute lymphoblastic

leukemia in adults. The first full-length bispecific MAb engineered on the structure

of a humanized IgG4 (emicizumab, Hemlibra^®) was approved in 2017 by the US

FDA. It has been used to reduce the frequency of bleeding episodes in hemophilia A

patients. In addition to bispecific, a tri-specific antibody recognizing a tumor antigen,

CD16 on NK cells, and IL15 has also been made. Such a tri-specific antibody

directly triggers NK cell activation through CD16, amplifying NK cell cytolytic

activity and cytokine production against various tumor cell antigen targets (Tay et al.

2016). Even a tetra-specific antibody simultaneously directed against HER1, HER3,

C-MET (hepatocyte growth factor receptor), and insulin-like growth factor

1 (IGFIR) with enhanced antitumor effect has also been made (Castoldi et al. 2016).

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