Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2022 Nov 28;10(11):e004850. doi: 10.1136/jitc-2022-004850

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.

PMC Copyright notice

Siglec-6-targeting antibody clones selected from a post-alloHSCT library exhibit specificity and shared epitope. (A) Comparison of post-alloHSCT antibody clones including V_H and V_L amino acid sequence identities, IMGT-defined CDR3 alignments, and affinity data from SPR analysis of Fab binding to Siglec-6–Fc fusion protein. (B) Heatmap of ELISA data (triplicate) from directly coating human or rhesus (85% amino acid sequence identity within the ectodomain) Siglec–Fc fusion proteins, incubating with Fabs, and detecting with antihuman Fab or antihuman Fc (positive control). The post-alloHSCT Fabs are all specific for human Siglec-6. (C) Flow cytometry histograms (left) and quantification (right) demonstrating that JML-1 Fab can block the binding of biotinylated RC-1 (RC-1-bio) Fab (40 nM) by competing for Siglec-6 on the surface of U937 cells. Statistics (n=3) were calculated using Student’s t-test. ****p<0.0001. (D) Homology model of Siglec-6 (V and C2i domains, 27–236; see online supplemental material), colored according to the differential deuterium uptake in the presence of RC-1 Fab as determined by HDX-MS. Negative differential D₂O values represent decreased solvent exposure in the presence of Fab. Black regions were not observed, and gray regions indicate no significant change in deuterium uptake. (E) Homology model of Siglec-6 illustrating with shades of yellow and orange the peptides that were individually substituted with corresponding CD33 sequences, and green indicating the C2i domain that was replaced entirely for epitope mapping. (F) Epitope mapping ELISA with Siglec-6×CD33 chimeric Fc-fusion proteins indicating the dependence of the mutated regions for binding with human Fabs but not for the commercial mouse mAb or sheep pAb to human Siglec-6 (hS6). alloHSCT, allogeneic hematopoietic stem cell transplantation; C2i, C2-type I; CDR3, complementarity determining region 3; HDX-MS, hydrogen–deuterium exchange mass spectrometry; mAb, monoclonal antibody; MFI, mean fluorescence intensity; pAb, polyclonal antibody; SPR, surface plasmon resonance; V_H, variable heavy; V_L, light chain domain.