In situ antibody phage display yields optimal inhibitors of integrin α11/β1

Abstract

Integrins are transmembrane multi-conformation receptors that mediate interactions with the extracellular matrix. In cancer, integrins influence metastasis, proliferation, and survival. Collagen-binding integrin-α11/β1, a marker of aggressive tumors that is involved in stroma-tumor crosstalk, may be an attractive target for anti-cancer therapeutic antibodies. We performed selections with phage-displayed synthetic antibody libraries for binding to either purified integrin-α11/β1 or in situ on live cells. The in-situ strategy yielded many diverse antibodies, and strikingly, most of these antibodies did not recognize purified integrin-α11/β1. Conversely, none of the antibodies selected for binding to purified integrin-α11/β1 were able to efficiently recognize native cell-surface antigen. Most importantly, only the in-situ selection yielded functional antibodies that were able to compete with collagen-I for binding to cell-surface integrin-α11/β1, and thus inhibited cell adhesion. In-depth characterization of a subset of in situ-derived clones as full-length immunoglobulins revealed high affinity cellular binding and inhibitory activities in the single-digit nanomolar range. Moreover, the antibodies showed high selectivity for integrin-α11/β1 with minimal cross-reactivity for close homologs. Taken together, our findings highlight the advantages of in-situ selections for generation of anti-integrin antibodies optimized for recognition and inhibition of native cell-surface proteins, and our work establishes general methods that could be extended to many other membrane proteins.

Keywords: Enhanced diversity; ITGA11; antibody selections; cancer therapeutics; cellular selections; integrin-α11/β1 receptor.

Figure 1.

Sequences of integrin-α11/β1 Abs. Abs were isolated by screening a phage-displayed Fab library for binding to (a) purified integrin-α11/β1 or (b) integrin-α11/β1 displayed on live cells. Sequences are shown for positions that were diversifed in the library and are numbered according to the IMGT nomenclature.³⁹ Dashes indicate gaps in the alignment. Underlined bold text indicates Abs that inhibited integrin-α11/β1 binding to collagen-1, and asterisks (*) indicate Abs that were also characterized as full-length immunoglobulins.

Figure 2.

Binding and function of anti-integrin-α11/β1 Fabs. (a) Binding of Fabs (x-axis) to purified integrin-α11/β1 (white bars, y-axis, left) or C2C12-α11/β1 cells (black bars, y-axis, right). Binding to purified or cell-surface integrin-α11/β1 was assessed by ELISA or flow cytometry, respectively. Data are shown for single-point measurements, and error bars indicate the standard deviation (SD) of two independent experiments. (b) Effects of Fabs (x-axis) on adhesion of C2C12-α11/β1 cells to collagen-I (y-axis). Asterisks (*) indicate Fabs that inhibited cell adhesion to collagen-I, and double asterisks (**) indicate Abs that were also characterized as full-length immunoglobulins. See Materials and Methods for details. Data are shown for single-point measurements, and error bars indicate SD of two independent experiments.

Figure 3.

NGS analysis of Fab-phage clones selected for binding to cells displaying integrin-α11/β1. (a) The abundance of each sequence in Fab-phage pools selected for binding to integrin-α11/β1 overexpressing cells (x-axis) is plotted versus the ratio of the abundance in pools selected for binding to cells overexpressing integrin-α11/β1 over pools selected for binding to cells not expressing integrin-α11/β1 (positive/negative, y-axis). Selections were performed with C2C12 mouse myoblast cells or human CAF cells in the presence of the indicated cation. Each circle represents a unique paratope (i.e., a unique combination of CDRs L3, H1, H2 and H3). The dashed lines define an upper-right quadrant that contains putative integrin-α11/β1-binding clones, defined arbitrarily as those occurring more than 100 times in the positive pool and being greater than two-fold enriched relative to the negative pool. Clones that were validated as specific integrin-α11/β1-binding Abs by ELISA screening (Figure 1) are shown as filled circles, colored as indicated. Each red circle represents the highest abundance and enrichment clone of a family cluster based on unique CDR L3 and H3 sequences (see Materials and Methods) that was not validated by ELISA, but is predicted to bind to integrin-α11/β1. The number in parentheses in the lower right quadrant is the number of unique potential integrin-α11/β1-binding clones in each upper-right quadrant, which is the sum of the validated (filled circles) and putative binders (red circles). (b) Venn diagrams for potential integrin-α11/β1-binding clones selected with the two different cell lines in the presence of Mg⁺², Ca⁺², or Mn⁺². The total unique clones are indicated at the bottom. (c) Venn diagram for total unique potential integrin-α11/β1-binding clones selected under the indicated cation conditions.

Figure 4.

Characterization of full-length anti-integrin-α11/β1 immunoglobulins. (a) Binding of anti-integrin-α11/β1 IgGs and a negative control IgG (x-axis) to C2C12 cells engineered to express the indicated integrins, assessed by flow cytometry fluorescence (y-axis). Data are shown for single-point measurements, and error bars indicate SD of two independent experiments. (b) Dose response curves for anti-integrin-α11/β1 IgGs and a negative control IgG (x-axis) binding to CAF094-α11/β1 or C2C12-α11/β1 cells, assessed by flow cytometry fluorescence (y-axis). Mean fluorescence intensity signals were normalized to the highest concentration value for each sample, and error bars indicate SD of two independent experiments. (c) Peptide counts for IP-MS analysis of CAF094-α11/β1 cell lysates immunoprecipitated with anti-integrin-α11/β1 IgGs or a negative control IgG. (d) Blocking of anti-integrin-α11/β1 IgGs (x-axis) binding to CAF094-α11/β1 cells by indicated Fabs, assessed by flow cytometry fluorescence (y-axis). Data are shown for single-point measurements, and error bars indicate SD of two independent experiments.

Figure 5.

Effects of immunoglobulins on integrin-α11/β1 function in cells. (a) Dose response curves for the effects of anti-integrin-α11/β1 IgGs and a negative control IgG (x-axis) on adhesion of C2C12-α11/β1 cells to collagen-I (y-axis). Assays were performed in DPBS containing 10 mM CaCl₂ and 5 mM MgCl₂ (b) Dose response curves for the effects of anti-integrin-α11/β1 IgGs and a negative control IgG (x-axis) on collagen-I gel contraction with C2C12-α11/β1cells. Assays were performed in serum-free DMEM. The means of at least three independent experiments are plotted and bars denote the standard deviation.