Antibody variable region engineering for improving cancer immunotherapy

Abstract

The efficacy and specificity of conventional monoclonal antibody (mAb) drugs in the clinic require further improvement. Currently, the development and application of novel antibody formats for improving cancer immunotherapy have attracted much attention. Variable region-retaining antibody fragments, such as antigen-binding fragment (Fab), single-chain variable fragment (scFv), bispecific antibody, and bi/trispecific cell engagers, are engineered with humanization, multivalent antibody construction, affinity optimization and antibody masking for targeting tumor cells and killer cells to improve antibody-based therapy potency, efficacy and specificity. In this review, we summarize the application of antibody variable region engineering and discuss the future direction of antibody engineering for improving cancer therapies.

Keywords: antibody engineering; bi/trispecific killer engager; cancer immunotherapy; chimeric antigen receptor-T (CAR-T) cell; nanobody; natural killer (NK) cell; scFv.

FIGURE 1

Graphic summary of antibody format engineering, including variable region humanization, peptide masking, CDR region mutagenesis, replacement of antibody variable region, and Fc inactivation by mutagenesis.Abbreviations: CDR, complementarity‐determining region; Fc, fragment crystallizable region; VH, heavy variable region; VL, light variable region; CH1, 2, 3, heavy constant region 1, 2, 3; VHH, single variable domain on a heavy chain

FIGURE 2

Illustration of four classes of antibody formats. I: mono‐specific antibody, which includes full IgG, nanobody, scFv and VH‐scFv; II: bispecific antibodies, which includes bi‐specific VHH, bi‐specific scFv, diabody, DART, minibody and scFv‐Fab; III: multi‐specific antibodies, which includes trivalent VHHs, triabody, tetrabody, tandem diabody, (scFv)₂‐Fab; IV: antibody format applied on CAR, which includes nanobody‐based CAR, bi‐specific nanobody‐based CAR, scFv CAR, and bi‐specific scFv‐based CAR.Abbreviations: IgG, immunoglobulin G; VH, heavy variable region; VL, light variable region; VHH: single variable domain on a heavy chain; CH1, 2, 3, heavy constant region 1, 2, 3; scFv: single‐chain variable fragment; DART, Dual‐Affinity Re‐Targeting; Fab, fragment antigen‐binding

FIGURE 3

Neural network‐aided high‐throughput bispecific antibody screening. The multivalent antibody fragment library is screened for their binding to target cancer cells in a high‐throughput manner. The sequences of target cancer cell binders are fed into the neural network as training dataset. The sequences of the larger multivalent antibody fragment library are fed into the neural network as testing dataset. The neural network can then predict the multivalent antibodies that bind to target cancer cells, which can be validated by high‐throughput screening.Abbreviations: H, heavy chain sequences; L, light chain sequences