Engineering of pH-dependent antigen binding properties for toxin-targeting IgG1 antibodies using light-chain shuffling

Summary Immunoglobulin G (IgG) antibodies that bind their cognate antigen in a pH-dependent manner (acid-switched antibodies) can release their bound antigen for degradation in the acidic environment of endosomes, while the IgGs are rescued by the neonatal Fc receptor (FcRn). Thus, such IgGs can neutralize multiple antigens over time and therefore be used at lower doses than their non-pH-responsive counterparts. Here, we show that light-chain shuffling combined with phage display technology can be used to discover IgG1 antibodies with increased pH-dependent antigen binding properties, using the snake venom toxins, myotoxin II and α-cobratoxin, as examples. We reveal differences in how the selected IgG1s engage their antigens and human FcRn and show how these differences translate into distinct cellular handling properties related to their pH-dependent antigen binding phenotypes and Fc-engineering for improved FcRn binding. Our study showcases the complexity of engineering pH-dependent antigen binding IgG1s and demonstrates the effects on cellular antibody-antigen recycling.


Target
(LCS = light-chain shuffled clone, NC = negative control, PC = positive control).Positive control means that the IgG showed a drastically increased off-rate at pH 5.5 compared to pH 7.4.Negative control implies that the off-rates at pH 5.5 and pH 7.4 were similar.

Figure S1 .
Figure S1.Primary screening of monoclonal scFvs obtained from phage display selection campaigns, related to Figure 1.Binding signal of 184 monoclonal scFvs from the library (a) B04, (b) B12, and (c) C08 against their target antigen.Clones showing a binding signal above the set threshold of 10,000 units (shown by dotted lines) were selected for further analysis.

Figure S2 .
Figure S2.Light chain sequence alignments of parental and light-chain shuffled antibodies, related to Figure 2. (a) Sequence alignment of light chains of the anti-M-II parent antibody B04 and the light-chain shuffled antibody A05.(b) Sequence alignment of light chains of the anti-αcbtx parent antibody C08 and the light-chain shuffled antibody B01.Amino acid residues that are identical in the sequence alignments are marked with a blue background, while distinct amino acids are marked with a white background.The light-chain CDRs are shown in red boxes and the gained histidine residues in the light-chain shuffled antibodies are shown in yellow boxes.The sequence alignments were made using Geneious Prime.(LCS = Light-chain shuffled, LC = Light chain, CDRL= light chain complementarity-determining region).

Figure S3 :
Figure S3: Structural characterization of the influence of changes in protonation at pH 5.5 and pH 7.4 on antigen binding, related to STAR methods.(a) Electrostatic interaction energies at pH 7.5, pH 5.5, and pH 5.0 for the antibodies C08 and B01 binding to -cbtx and B04 and A05 binding to M-II.(b) The differences in the light chain between the variants are depicted in light purple for B01/A05 and in light blue for B04/A05 on the surface of the structures.For each antibody, the protonated residues at pH 5.5 are depicted and highlighted as sticks and color-coded based on their location in the heavy and light chains respectively.Additionally, the predicted antigen-binding sites (epitope fingerprints) are colorcoded in shades of blue for -cbtx and in shades of purple for M-II.

Figure S4 . 3 .
Figure S4.Differential scanning fluorimetry of parental (C08 and B04) and light-chain shuffled (B01 and A05) antibodies, related to STAR methods.(a) Fluorescence emission at 350 nM plotted against temperature.(b) The slope of the fluorescence emission at 350 nM plotted against temperature.(a-b) Dashed vertical lines indicate the detected melting temperatures (TM).

Figure S7 .
Figure S7.Net charge of the variable regions of the anti--cbtx and anti-M-II antibodies, related to Figure 3.The charge of (a) whole variable regions (Fvs), (b) complementarydetermining regions (CDRs), and (c) frameworks across a pH gradient was calculated by using EMBOSS iep software.

Figure S8 :
Figure S8: Mass photometry of antibody-antigen complexes, related to Figure 3. (a) Histograms of detected masses of antibodies at 33 nM, with (blue) and without (red) the presence of 667 nM of either M-II or α-cbtx.(b) Bar charts of the observed difference in mass of the dominant species upon addition of antigen.Black horizontal lines indicate the mass of two antigens.(c) Log-scaled histograms of detected masses of three anti-M-II antibodies at 33 nM with (blue) and without (red) 667 nM M-II.
(kon), off-rates (koff), and affinities (KD) of the Fabs at pH 7.4 as determined by BLI.(LCS = light-chain shuffled clone, NC = negative control, PC = positive control).Positive control means that the Fab showed a drastically increased off-rate at pH 5.5 compared to pH 7.4.Negative control implies that the off-rates at pH 5.5 and pH 7.4 were similar.

Table S3 :
Similarity between parental and light-chain shuffled (LCS) antibody variable regions and germline antibody variable regions of the light chain, related to Figure2.

Table S5 .
Amount of IgG1 immobilized onto biosensor chip by amine coupling, related to STAR methods.