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Blood, Vol. 108, Issue 9, 3103-3111, November 1, 2006
Synthetic anti-BR3 antibodies that mimic BAFF binding and target both human and murine B cells
Blood Lee et al.
108: 3103
Supplemental materials for: Lee et al
Files in this Data Supplement:
- Table S1. Shotgun alanine-and homolog-scanning of CB3s-Fab for binding mBR3 or hBR3 (PDF, 16.3 KB) -
For each of the listed residues, the effects of mutation to alanine (m1), or additional mutations (m2, m3; due to limitations of shotgun-alanine codons), or to a homologous amino acid (m4) were calculated as occurrence ratios of wild type/mutant (wt/mut) among the clones binding to mBR3 or hBR3 (in parentheses). When only the wild type residue was selected, the ratio is shown as > the wild type count. Display selection was performed independently by selection against antibody to gD tag. Most of the wt/m ratios from the gD-selection are not significantly different from one, indicating the proper folding and display of these Fab mutants. As only the Fab heavy chain is fused to phage coat, the phage display of gD tag, which is fused to the light chain, requires proper folding and association of light chain and heavy chain. The functional ratio (Fwt/mut) for each residue was derived by dividing the antigen selection wt/mut ratio by the display selection ratio, and represents an estimate for the energetic cost of each substitution on antigen binding. The substituted amino acid type is indicated by a superscript on the F value except for the alanine mutation (m1). When the wild type residue is alanine, glycine was substituted as m1. F values are grouped in three categories as the color-coding groups in Figure 5: >3, >10, >30. F >30 in m1 and m4 are in bold print. F values that belong to a higher category for binding mBR3 than for hBR3 or vice versa are underlined. These residues appear to contribute differently in binding the two orthologs.
- Figure S1. Affinity measurement of CB1 Fab variants (PDF, 35.6 KB) -
(A) Kinetic parameters based on SPR determination of CB1 variants to immobilized hBR3 or mBR3 are shown. (B) SPR sensorgrams generated by injecting 100 nM CB1- or CB2-Fab over flow sorted (CHECK WIT AUTHORS) cells immobilized with mBR3-ECD (left) or hBR3-ECD (right).
- Figure S2. BR3 binding specificity of CB2-IgG (PDF, 212 KB) -
(A) Binding to other BAFF receptors was not observed when incubating CB2-IgG with TACI or BCMA coated directly on ELISA wells, or (B) in solution, where soluble receptor Fc-fusion proteins were tested for binding and inhibiting CB2-IgG binding to BR3coated wells.
- Figure S3. Immunohistochemical staining of B cells with CB antibody (PDF, 233 KB) -
Organ sections from normal 8-12 week old BALB/c mice (from OCT blocks) were stained with biotinylated CB2 followed by avidin peroxidase (ABC-HRP Elite; Vector) and developed with metal-enhanced diaminobenzidine (Pierce Chemical, St. Louis, MO). B cell areas appear in brown in the spleen (A) and in the small mucosal lymphoid follicles of the colon (B-f). The specificity of CB2 specificity is shown by absent staining in tissues lacking substantial numbers of B cells (B): brain (a), liver (b), lung (c), duodenum and pancrease (d), and colon (e). B cell areas were confirmed with serial sections stained with other B cell markers and with dual immunofluorescent stains (not shown).
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