Higher affinity human D MoAb prepared by light-chain shuffling and selected by phage display

Area of Science:

• Immunology
• Biotechnology
• Hematology

Background:

• Monoclonal antibodies (MoAbs) are vital for blood group phenotyping.
• Current anti-D MoAbs often fail to detect weak D variants on red blood cells (RBCs).
• Higher affinity MoAbs are needed to improve detection of weak D variants.

Purpose of the Study:

• To enhance the affinity of the anti-D monoclonal antibody 43F10.
• To improve the detection of weak D-expressing RBCs.
• To explore light-chain shuffling and phage display for antibody engineering.

Main Methods:

• Constructed a kappa light-chain repertoire from polyimmunized individuals.
• Recombined light chains with the 43F10 heavy chain using a phagemid vector system.
• Selected engineered anti-D phages on intact RBCs and characterized variants using ELISA and agglutination assays.

Main Results:

• Selected a 43F10 MoAb variant (p3.17) with enhanced reactivity to weak D RBCs.
• Agglutination assays demonstrated improved performance of the p3.17 variant.
• Sequence analysis revealed significant differences in light-chain variable (VL) and joining (J) segments between p3.17 and wild-type 43F10.

Conclusions:

• The engineered p3.17 variant shows potential for increasing the potency of anti-D reagents.
• This variant could improve the detection of weak D variants in blood banks.
• The study highlights the significant role of the heavy chain in anti-D specificity, even after light chain shuffling.