Specificity tuning of antibody fragments to neutralize two human chemokines with a single agent

Abstract

Chemokines are important mediators of the immune response that are responsible for the trafficking of immune cells between lymphoid organs and migration towards sites of inflammation.Using phage display selection and a functional screening approach, we have isolated a panel of single-chain fragment variable (scFv) capable of neutralizing the activity of the human chemokine CXCL10 (hCXCL10). One of the isolated scFv was weakly cross-reactive against another human chemokine CXCL9,but was unable to block its biological activity. We diversified the complementarity determining region 3 (CDR3) of the light chain variable domain (VL) of this scFv and combined phage display with high throughput antibody array screening to identify variants capable of neutralizing both chemokines. Using this approach it is therefore possible to engineer pan-specific antibodies that could prove very useful to antagonize redundant signaling pathways such as the chemokine signaling network.

Figure 1

The scFv, E7, binds to hCXCL9 and hCXCL10. Capacity of scFv E7 to bind to a panel of human chemokines was tested in ELISA. NusA fusion chemokine was coated at 20 µg/ml and 5 µg/ml E7 scFv in 1% milk-PBS buffer was incubated for 1 hour at room temperature. Coating was controlled using specific mAb for each chemokine and NusA protein was also added to the assay as negative control (data not shown). Results are expressed as mean ± S.D. of duplicates of two representative experiments.

Figure 2

Strategy for library construction and VL CDR3 randomization of the scFv E7. Schematic representation of scFv E7 with complementarity determining regions (CDR) shown in black. The VL CDR3 amino sequence is magnified in order to show the overlapping stretches of four amino acids targeted by the libraries L3.1 to L3.4. The glycine-rich linker between the heavy and light chains as well as the c-terminal hexa-histine and c-myc tags are indicated.

Figure 3

High-density array screening of scFv variants. (A,B) Bacterial clones grown in 384 well plates were double spotted on membranes and probed for binding to NusA-hCXCL9 (A) or to the control protein, NusA (B). Clones from each plate were double spotted in a defined pattern to facilitate identification and retrieval of the hits. (C) Schematic representation of a double spotted pattern corresponding to plate 8. Signals corresponding to clones C1, F13, J5, J9 and P8 are indicated. As control, the parental clone E7 was grown in all the wells in column 24 of plate 8. No spot in orientation 8 could be detected in column 24 thus indicating that under the conditions used E7 did not generate a positive signal, thus facilitating the identification of potentially improved variants for binding to hCXCL9.

Figure 4

Dose-response of the variants for hCXCL9 and hCXCL10 binding. ELISA were performed using different concentrations of purified scFv against hCXCL10 (A) or hCXCL9 (B). Chemokine target was coated either at 2 µg/ml for NusA-hCXCL10 or 5 µg/ml for NusA-hCXCL9 fusion protein. Serial 3-fold dilutions of scFv were performed in 1% milk-PBS buffer and scFv was incubated for one hour at room temperature. Results are expressed as mean ± S.D. of duplicates.

Figure 5

Specificity of improved scFv variants. Binding of the selected scFv candidates was tested in an ELISA against a panel of human chemokines. NusA fusion chemokine was coated at 20 µg/ml and 5 µg/ml variant scFv in 1% milk-PBS buffer was incubated for 1 hour at room temperature. Coating was controlled using specific mAb for each chemokine and NusA protein was also added to the assay as negative control (data not shown). Results are expressed as mean ± S.D. of duplicates of two representative experiments.

Figure 6

Dose dependant chemotaxis inhibition of improved scFv variants. (A) The assay involves L1.2 cells expressing hCXCR3 which migrate in response to an established chemokine gradient which was visualized and quantified using FMAT. (B and C) Dose response data for the chemokines, CXCL9 (B) or CXCL10 (C) for the scFv E7, P8 and C1. Different concentrations of purified scFv were incubated with either 1 nM of hCXCL10 or 10 nM of hCXCL9. Results are expressed as mean ± S.D. of duplicates of three representative experiments.