More Than Meets the Kappa for Antibody Superantigen Protein L (PpL)

Abstract

Immunoglobulin superantigens play an important role in affinity purification of antibodies and the microbiota-immune axis at mucosal areas. Based on current understanding, Staphylococcal Protein A (SpA), Streptococcal Protein G (SpG) and Finegoldia Protein L (PpL) are thought to only bind specific regions of human antibodies, allowing for selective purification of antibody isotypes and chains. Clinically, these superantigens are often classified as toxins and increase the virulence of the producing pathogen through unspecific interactions with immune proteins. To perform an in-depth interaction study of these three superantigens with antibodies, bio-layer interferometry (BLI) measurements of their interactions with a permutation panel of 63 IgG1 variants of Pertuzumab and Trastuzumab CDRs grafted to the six human Vκ and seven human VH region families were tested. Through this holistic and systemic analysis of IgG1 variants with various antibody regions modified, comparisons revealed novel PpL–antibody interactions influenced by other non-canonical antibody known light-chain framework regions, whereas SpA and SpG showed relatively consistent interactions. These findings have implications on PpL-based affinity antibody purification and design that can guide the engineering and understanding of PpL-based microbiota-immune effects.

Variable-heavy (VH) families influencing IgA1&2 engagement to the antigen, FcαRI and superantigen proteins G, A, and L

“… 65 , 66 and S. aureus 67 , respectively, and that both are part of the normal human flora at mucosal areas and skin surfaces 68 , this finding expands the potential interaction to isotypes such as IgE 50 present on mast cells 62 to trigger allergic reactions. Apart from unraveling the possible mechanisms underlying microbiome-antibody interactions at the mucosal area, there is a clear need to exploit or mitigate the IgA-superantigen properties beyond therapeutics to that of diagnostics, given the interactions with IgGs 69 , 70 .…”

“…All Trastuzumab and Pertuzumab VH and Vκ sequences used were described previously 49 . The genes were transformed into competent E. coli (DH5α) strains 79 followed by plasmid extraction (Biobasic Pte Ltd) and sub-cloning into pTT5 vector (Youbio, China) using restriction enzyme sites, as previously performed 48 – 50 , 57 , 70 , 76 .…”

“…For Her2 binding, IgA variants were immobilized using PpL biosensor (Sartorius, Cat: 18–5185) or biotinylated anti-IgA antibody (Thermo Scientific, Cat: 7,102,882,500) bound on streptavidin biosensor (Sartorius, Cat: 18–5119) and subjected to free floating Her2 in solution to obtain the rate of association (ka), dissociation (kd), and equilibrium dissociation constant (KD). The program and steps used were as previously described 48 – 51 , 57 , 58 , 70 , 76 .…”

“…His-tagged FcαRI (Sino Biological, Cat: 10,414-H08H) were immobilized via Ni–NTA biosensor (Sartorius, Cat: 18–5101) and subject to free floating IgA variants to obtain the ka, kd, and KD. The program and steps used were as previously described 48 – 51 , 57 , 58 , 70 , 76 .…”

“…PpL (Sartorius, Cat: 18–5185), SpA (Sartorius, Cat: 18–5012), and SpG (Sartorius, Cat: 18–18-5083) biosensors were subject to free floating IgA variants to obtain the ka, kd and KD. The program and steps used were as previously described 48 – 51 , 57 , 58 , 70 , 76 .…”