Combining Cellular Immunization and Phage Display Screening Results in Novel, FcγRI-Specific Antibodies

Abstract

Antibodies that specifically bind to individual human fragment crystallizable γ receptors (FcγRs) are of interest as research tools in studying immune cell functions, as well as components in bispecific antibodies for immune cell engagement in cancer therapy. Monoclonal antibodies for human low-affinity FcγRs have been successfully generated by hybridoma technology and are widely used in pre-clinical research. However, the generation of monoclonal antibodies by hybridoma technology that specifically bind to the high-affinity receptor FcγRI is challenging. Monomeric mouse IgG2a, IgG2b, and IgG3 bind human FcγRI with high affinity via the Fc part, leading to an Fc-mediated rather than a fragment for antigen binding (Fab)-mediated selection of monoclonal antibodies. Blocking the Fc-binding site of FcγRI with an excess of human IgG or Fc during screening decreases the risk of Fc-mediated interactions but can also block the potential epitopes of new antibody candidates. Therefore, we replaced hybridoma technology with phage display of a single-chain fragment variable (scFv) antibody library that was generated from mice immunized with FcγRI-positive cells and screened it with a cellular panning approach assisted by next-generation sequencing (NGS). Seven new FcγRI-specific antibody sequences were selected with this methodology, which were produced as Fc-silent antibodies showing FcγRI-restricted specificity.

Keywords: ELISA; Fc receptor; FcγRI; NGS; cellular panning; phage display.

Figure 1

Antibodies from hybridoma clones selected for FcγRI specificity lack or show reduced binding to FcγRI after elimination of the Fc part. (a) F(ab’)₂ were prepared from antibodies of hybridoma clones (in-house-produced 1E2 and 1E3, mIgG2c) and 197 (mIgG2a) and show missing or strongly reduced binding to Ba/F3-FcγRI after elimination of the Fc part. (b) For comparison, commercially available FcγRI antibodies 197 (mIgG2a) and 10.1 (mIgG1) also show a reduced signal but remain able to bind to Ba/F3-FcγRI after elimination of the Fc part. The isotype of the in-house-produced hybridoma (mIgG2c) mediates Fc-restricted binding to human FcγRI. Data represent mean values of two to five experiments +/− SEM. Cells were stained with 10 µg/mL antibodies and APC-labeled goat F(ab’)₂ anti-mouse IgG (H + L). MFI: mean fluorescence intensity.

Figure 2

Binding of serum antibodies to FcγRI-transduced mouse cells after cellular immunization using a flow cytometry-based screening method. Ba/F3 and Ba/F3-FcγRI were first blocked with hIgG Fc, to reduce the Fc-mediated binding of serum antibodies. Serum was stepwise diluted with the dilution factor indicated. Sera from day 0 (before immunization) and day 91 (final bleed) were tested. Antibody binding was determined with a goat F(ab’)₂ anti-mouse IgG-APC antibody. Screening data of the mouse with the highest antibody titers for FcγRI-positive cells are shown. MFI: mean fluorescence intensity.

Figure 3

FcγRI-specific scFvs were enriched and selected from an antibody library by phage display. (a) Schematic overview of “Fc-free” detection of bound phages in flow cytometric analyses using anti-myc Fab and polyclonal anti-kappa light chain FITC-conjugated F(ab’)₂. (b) Flow cytometric analysis of phage antibodies of the initial library, after the 1st panning and after the 2nd panning. Ba/F3-FcγRI (green) and Ba/F3 (blue) cells were incubated with polyclonal phages and fluorescence was measured in comparison to a control phage (white). (c) Schematic overview of initial whole-cell ELISA experiments using anti-M13 HRP-conjugated mouse IgG1 for the detection of cell-bound phages. (d) A total of 1 × 10⁶ Ba/F3-FcγRI (green) or Ba/F3 (blue) cells were incubated with monoclonal phage antibodies from 25 single bacterial colonies (C01–C25), and bound phages were detected with anti-M13 HRP-conjugated mouse IgG1. For comparison, polyclonal phage antibodies of the initial library were added to the analysis. Candidates binding with higher intensity to FcγRI-transfected cells than to non-transfected Ba/F3 cells can be grouped according to their intensities in group A (OD 0.5–1) and group B (OD 1–2).

Figure 4

Enrichment of FcγRI-specific antibodies was monitored by NGS. (a) VH and (b) VL amino acid sequences were aligned and are shown as dendrograms. Eight candidates that show the same VH are combined with three different VLs and can be assigned to group A (green). Seven candidates that show the same VH are combined with four different VLs and can be assigned to group B (orange). The VH of C15 differs in one amino acid from the candidates of group B. (c) VHs of the initial immune library the library after the 1st panning and the library after the 2nd panning were sequenced by NGS, the clonotypes were determined by ARResT/Interrogate, and the frequencies (% of VH sequences) in each sample were calculated. Clonotypes with a low frequency (<0.5%, light gray) are summed up. After the 2nd panning, 54% and 25% of all VH sequences share the same clonotypes of groups A (green) and B (orange), respectively.

Figure 5

Selected scFvs show Fab-mediated and specific binding to FcγRI after conversion in Fc-silent IgG1. Antibody candidates, produced as chimeric IgG1 with LALAPG mutations, show Fab-mediated binding (green) to (a) Ba/F3-FcγRI cells and (b) IFN-γ-stimulated PMNs in cytometric analysis. As proven by control antibodies, inserting LALAPG mutations into human IgG1 (IgG1-LALAPG, dotted lines) silences Fc-mediated binding to human FcγRI compared to human wildtype IgG1 (grey). The FcγRI-specific antibody 10.1 (mIgG1) was used as a positive control (black). To exclude cross-reactivity with other human FcγRs, binding to (c,d) FcγRIIa/b-transduced IIa1.6-cells and (e,f) FcγRIIIa/b-transduced Ba/F3 cells was measured by flow cytometry, showing FcγRI-specific binding of the generated antibodies (green) in comparison to FcγRIIa/b- and FcγRIIIa/b-specific antibodies (grey). Data represent mean values of two to three experiments +/− SEM. Cells were stained with 10 µg/mL of FITC-labeled antibodies. MFI: mean fluorescence intensity.