Figure 1.
Two steps of engineering accelerate clonal diversification.
Schematic diagram of the rearranged and expressed IgH locus, showing the variable (VDJ) region, the constant (Cμ) region, and the upstream ψVH array. IgH was first modified by insertion of PolyLacO within the ψVH array in DT40 PolyLacO-λR cells, which carry PolyLacO targeted to the rearranged and expressed Igλ locus [13], [14], [15]. Next, this locus was further modified by substitution of the endogenous VH (VDJ) region with VH regions from a naive chick.
Figure 2.
Clonal diversification rate accelerated in DTLacO cells.
(A) sIgM loss assay of three representative clonal DTLacO LacI-HP1 transfectants. Fraction of sIgM− cells in each culture indicated at lower right in each panel. (B) Summary of sIgM loss assays. Each open circle represents the percentage of sIgM− cells in one clonal transfectant, analyzed 3 weeks post-transfection. Cells analyzed were: DT40 PolyLacO-λR GFP-LacI control transfectants (n = 27); DT40 PolyLacO-λR LacI-HP1 transfectants (n = 16) and DTLacO LacI-HP1 transfectants (n = 20). (C) Median sIgM loss of DT40 PolyLacO-λR LacI-HP1 and DTLacO LacI-HP1 transfectants relative to GFP-LacI control transfectants.
Figure 3.
Rapid evolution of anti-SAv antibodies in DTLacO cells.
(A) SAv binding profile of successive selected cell populations of DTLacO (left) or DTLacO E47-LacI (right) cells. Selection was carried out on average weekly. Cell numbers plotted relative to SAv-PE fluorescent signal. Populations at successive rounds of selection designated above peaks (S0–S7). Pre, populations prior to any sorting (gray fill). (B) Saturation binding kinetics of DTLacO E47-LacI S7 population. (C) Sequences of high affinity selected anti-SAv mAb compared to the germline [16], [17]. CDRs are identified by background shading. The D6 sequence was chosen as the germline D element for comparison [17]. Note the 18-residue insertion/duplication in CDR1 of Vλ of the anti-SAv mAb, recapitulating an insertion in light-chain CDR1 reported by others selecting anti-SAv mAbs from DT40 cells that had not undergone any genetic engineering [8].
Figure 4.
High affinity mAbs selected from DTLacO cells.
(A) Above, binding profiles of successive DTLacO LacI-HP1 populations selected for recognition of cell surface receptors, VEGFR2, TIE2 and TROP2. Rounds of selection designated above peaks (S0–S8). Below, saturation binding kinetics, indicating apparent kD. (B) Specificity of selected DTLacO populations. FACS analysis of binding of cell populations selected for high affinity recognition of VEGFR2, TIE2 or TROP2 to recombinant VEGFR2, TIE2, TROP2, SAv or ovalbumin (OVA). Solid peaks represent the negative reference control (secondary antibody alone), and green lines represent staining with antigen. (C) Schematic alignment of VH and Vλ regions of mAbs selected for binding to VEGFR2, TIE2 and TROP2. Thin horizontal blue lines represent chicken framework regions, thicker horizontal lavender lines against background shading identify CDRs, vertical bars indicate single residue differences relative to the most common DTLacO sequence, and triangle indicates insertion.
Figure 5.
Selection and humanization of anti-FN14 and anti-FZD10 mAbs.
(A) Schematic of time course of selection of anti-FN14 and anti-FZD10 mAbs, with selection steps indicated by S, and apparent affinities (kD) of recombinant chimeric mAbs shown below. (B) Schematic alignment of VH and Vλ regions of mAbs selected for binding to FN14 and FZD10. Thin horizontal lines represent chicken framework regions, thicker horizontal lines against background shading identify CDRs, vertical bars indicate single residue differences relative to the most common DTLacO sequence, and triangle indicates insertion. (C) Antibody humanization. VH and Vλ regions of humanized mAbs hFS24 and hFZ2 schematically aligned to the human VH-III or Vλ-III consensus (top lines). Thin horizontal lines represent human framework regions; asterisks denote the two residues eliminated from the N-terminal of the light chain; vertical lines outside background shading identify Vernier zone residues preserved in humanized mAbs; other notations as in Panel B. (D) Apparent affinities (kD) of humanized and progenitor mAbs.