Development and in vitro characterization of canine CD40-Ig

Abstract

We recently reported that blockade of the CD40-CD154 ligand interaction with the cross-reacting mouse anti-human CD154 antibody, 5c8, together with donor-specific transfusion led to enhanced but not completely successful engraftment in a canine model of DLA-identical marrow transplantation after 100cGy total body irradiation (TBI). In order to improve the transplantation outcomes, we sought to develop a canine-specific reagent. To that end, we fused the extracellular domain of the canine CD40 with a mouse IgG2a Fc tail and tested the immunosuppressive effectiveness of the fusion protein in mixed leukocyte reactions. The extracellular domain of canine CD40 was fused with the Fc portion of mouse IgG2a in a pcDNA3.1+vector. Dhfr-deficient CHO cells were co-transfected with the CD40-Ig vector and a dhfr-containing vector. Stable, high producing clones were selected under increasing methotrexate concentrations. The fusion protein was purified, tested in mixed leukocyte reactions, and its immunosuppressive effect compared to that of the anti-CD154 antibody 5c8. The transfected cell line produced a CD40-Ig dimer whose identity was confirmed by mass spectroscopy. The purified canine CD40-Ig blocked mixed leukocyte reactions at a concentration of 1nM, which was 10 times more effective than the anti-CD154 antibody. Canine CD40-Ig is more immunosuppressive than the anti-human CD154 antibody 5c8 in canine mixed leukocyte reactions and may be more effective in vivo in a model of marrow transplantation.

Figure 1

A 68% identity of amino acid sequences of the canine CD40 (GenBank Accession No. NM001002982) compared to human CD40 (Stamenkovic et al., 1989) The derived sequence of the extracellular domain of the canine CD40 is underscored with the first 20 amino acid representing the leader sequence. Matching amino acids are shown in black, non-matching in grey.

Figure 2

Schematic diagram of CD40-Ig expression vector containing the leader and extracellular domain of canine CD40 fused to a Gly4Ser linker and the hinge through CH3 regions of murine IgG2a.

Figure 3

SDS-PAGE and Immunoblot of rcCD40-Ig. rc CD40-Ig run under reducing conditions (A), and non-reducing conditions (B), and immunoblot of nonreduced rcCD40-Ig (C). Molecular weight standard “SeeBlue2” (Invitrogen) was used in all three experiments (right of each). The molecular weight marker is enlarged for technical reasons in Figure 3C. The monomer band between marker 49kDa and 62 kDa in (A) and the dimer band at marker 98kDa in (B) were proven to be canine CD40-Ig by tandem mass spectroscopy.

Figure 4

Canine CD40-Ig in canine allogenic MLR: Purified rcCD40-Ig was added on day 0 to the MLR at a dose of 10µg/mL. The irrelevant mouse antibody 31A was used as negative control. RhCTLA4-Ig and mouse anti-human CD154 antibody 5c8 served as positive controls (dose 10µg/mL). Four independent experiments were performed. All data points in each individual experiment were done in triplicate. The ³H-thymidine uptake with medium alone was set at 100%. The asterisks mark statistically significant reduction compared to medium alone (P< 0.05).

Figure 5

Dose responses of rcCD40-Ig and mouse anti-human CD154 antibody 5c8 in canine MLR. Seven individual MLR were performed. All data points in each individual experiment were done in triplicate. The ³H-thymidine cellular uptake with medium alone was set at 100%. The asterisks mark statistically significant reductions compared to medium alone (P < 0.05).