Antibody VH and VL recombination using phage and ribosome display technologies reveals distinct structural routes to affinity improvements with VH-VL interface residues providing important structural diversity

Abstract

In vitro selection technologies are an important means of affinity maturing antibodies to generate the optimal therapeutic profile for a particular disease target. Here, we describe the isolation of a parent antibody, KENB061 using phage display and solution phase selections with soluble biotinylated human IL-1R1. KENB061 was affinity matured using phage display and targeted mutagenesis of VH and VL CDR3 using NNS randomization. Affinity matured VHCDR3 and VLCDR3 library blocks were recombined and selected using phage and ribosome display protocol. A direct comparison of the phage and ribosome display antibodies generated was made to determine their functional characteristics.In our analyses, we observed distinct differences in the pattern of beneficial mutations in antibodies derived from phage and ribosome display selections, and discovered the lead antibody Jedi067 had a ~3700-fold improvement in KD over the parent KENB061. We constructed a homology model of the Fv region of Jedi067 to map the specific positions where mutations occurred in the CDR3 loops. For VL CDR3, positions 94 to 97 carry greater diversity in the ribosome display variants compared with the phage display. The positions 95a, 95b and 96 of VLCDR3 form part of the interface with VH in this model. The model shows that positions 96, 98, 100e, 100f, 100 g, 100h, 100i and 101 of the VHCDR3 include residues at the VH and VL interface. Importantly, Leu96 and Tyr98 are conserved at the interface positions in both phage and ribosome display indicating their importance in maintaining the VH-VL interface. For antibodies derived from ribosome display, there is significant diversity at residues 100a to 100f of the VH CDR3 compared with phage display. A unique deletion of isoleucine at position 102 of the lead candidate, Jedi067, also occurs in the VHCDR3.As anticipated, recombining the mutations via ribosome display led to a greater structural diversity, particularly in the heavy chain CDR3, which in turn led to antibodies with improved potencies. For this particular analysis, we also found that VH-VL interface positions provided a source of structural diversity for those derived from the ribosome display selections. This greater diversity is a likely consequence of the presence of a larger pool of recombinants in the ribosome display system, or the evolutionary capacity of ribosome display, but may also reflect differential selection of antibodies in the two systems.

Figure 1. The lead generation strategy. (A) Lead isolation phage display. (B) Affinity maturation phage display. (C) Affinity maturation phage and ribosome display.

Figure 2. Selection strategy used for phage and ribosome display to generate potent anti-IL-1RI Antibodies. (A) Gel photograph of cDNA outputs from Round 2 of Ribosome Display Selections with antigen concentrations at 1 nM, 500 pM and 50 pM. (B) Selection pathway (high-lighted in light blue) followed to generate high affinity IL-1RI variants using phage and ribosome display.

Figure 3. Inhibition of IL-1 receptor ligand binding to IL-1 Receptor. Anti-IL-1 receptor lead panel antibodies from ribosome display completely inhibit both IL-1Ra* binding and IL-1β binding to IL-1R1Fc in biochemical binding HTRF assay. Data representative of n = 2 independent experiments. KENB061 inhibited IL-1β binding to IL-1RI with an IC₅₀ of 473 nM (n = 3, Table 1). *Anakinra is a drug used to treat rheumatoid arthritis. It is an interleukin-1 (IL-1) receptor antagonist. Interleukin-1 receptor antagonist IL-1Ra binds to IL-1RI, but cannot initiate signaling and plays an important role limiting the extent of IL-1 pathway activation in vivo

Figure 4. (A) Amino acid usage within the VH CDR3 loops of the improved potency anti-IL-1R1 variants from phage and ribosome display. (B) Amino acid usage within the VL CDR3 loops of the improved potency anti-IL-1R1 variants from phage and ribosome display

Figure 5. A structural model of Jedi067 Fv. A view of the structural model of Jedi067 Fv looking down the CDRs is shown. The VH-VL interface residues discussed in the text are shown as sticks and labeled. Color code: cyan-VH; pink-VL; blue-VHCDRs; magenta-VLCDRs; yellow dotted lines-hydrogen bonds. The conserved aspartate at position 101 in VH domain is substituted by phenylalanine.