Characterization of the biological anti-staphylococcal functionality of hUK-66 IgG1, a humanized monoclonal antibody as substantial component for an immunotherapeutic approach

Abstract

Multi-antigen immunotherapy approaches against Staphylococcus aureus are expected to have the best chance of clinical success when used in combinatorial therapy, potentially incorporating opsonic killing of bacteria and toxin neutralization. We recently reported the development of a murine monoclonal antibody specific for the immunodominant staphylococcal antigen A (IsaA), which showed highly efficient staphylococcal killing in experimental infection models of S. aureus. If IsaA-specific antibodies are to be used as a component of combination therapy in humans, the binding specificity and biological activity of the humanized variant must be preserved. Here, we describe the functional characterization of a humanized monoclonal IgG1 variant designated, hUK-66. The humanized antibody showed comparable binding kinetics to those of its murine parent, and recognized the target antigen IsaA on the surface of clinically relevant S. aureus lineages. Furthermore, hUK-66 enhances the killing of S. aureus in whole blood (a physiological environment) samples from healthy subjects and patients prone to staphylococcal infections such as diabetes and dialysis patients, and patients with generalized artery occlusive disease indicating no interference with already present natural antibodies. Taken together, these data indicate that hUK-66 mediates bacterial killing even in high risk patients and thus, could play a role for immunotherapy strategies to combat severe S. aureus infections.

Keywords: MRSA; Staphylococcus aureus; bacterial killing; fc-receptors; humanization; immunotherapeutics; immunotherapy; monoclonal antibodies; opsonophagocytosis; vaccinology.

Figure 1. Binding affinities of scFv hUK-66 fragments after humanization analyzed by comparative ELISA studies. (A) Affinity of scFv hUK-66 fragments against rIsaA were analyzed by ELISA studies. The scFv fragment against Gal12 served as control. Binding of scFv fragments were detected by HRP-conjugated anti-Myc antibody. (B) Specificity of scFv UK-66 fragments against rIsaA was determined by competitive ELISA studies using HRP-conjugated anti-Myc antibody. Presented data illustrate one representative experiment done in triplicate.

Figure 2. Comparative binding studies of complete humanized UK-66 IgG isotypes against rIsaA. (A) ELISA plates were coated with 10 µg/ ml rIsaA and incubated with the indicated full humanized UK-66 IgG isotypes. Binding of isotypes were determined by isotype specific, HRP-conjugated anti human IgG antibodies. (B) Specificity of hUK-66 IgGs was analyzed by competitive ELISA. Binding of UK-66 IgGs to coated rIsaA was analyzed by isotype specific, HRP-conjugated anti-human IgG antibodies. Presented data illustrate one representative experiment done in triplicate.

Figure 3. Impact of immunoglobulins isotype structure on hUK-66-dependent staphylococcal killing. Heparinized blood samples from healthy donors were infected with S. aureus Newman and either humanized UK-66 IgG1, IgG2, or IgG4 for 60 min at 37°. The monoclonal antibody Trastuzumab served as matched IgG1 isotype control (IC) and the intravenous immunoglobulin mixture Gammunex^® (IVIg) as a second positive control. Viable bacteria were recovered after whole eukaryotic cell lysis and enumerated by plate counting. The values were expressed as mean percentage with standard deviation (SD) in parenthesis relative to the level of untreated blood/bacterial samples (set at 100%). Data represent three independent experiments from three different donors. The dashed line in the graphs demonstrates the 100% reference value of control bacteria samples. *P > 0.05, t test.

Figure 4. Quantitative analysis of the interaction of hUK-66 IgG1 with rIsaA performed using surface plasmon resonance (SPR). Various concentrations of rIsaA (0.39 to 400 nM) were flushed over the antibody hUK-66 IgG1, immobilized on the sensor chip surface. Sensorgrams were recorded at a flow rate of 30 µl/ min at 25 °C. From these Sensorgrams, an equilibrium dissociation constant (K_D) of 4.8 nM was determined. Rate constants for association (k_a) and dissociation (k_d) were determined to be 3.7 × 10⁵ M⁻¹s⁻¹ and 1.8x10⁻³ s⁻¹, respectively.

Figure 5. IsaA conservation and expression by different clinically relevant S. aureus strains. (A) Alignment of IsaA sequence of S. aureus strain N315, MW2, and MU 50 (B) Immunoblot analysis of IsaA in cell pellets of different clinically relevant strains. (C) Specific binding of IsaA by hUK-66 IgG1 was performed with S. aureus MA12 spa^- and its isogenic isa^- mutant by indirect immunofluorescence studies.

Figure 6. hUK-66 IgG1 augments the respiratory burst in neutrophils from clinical relevant patient groups. Heparinized human blood samples (n = 4–7) from healthy donors, patients undergoing dialysis, diabetes, and artery occlusive disease (AOD) were infected with S. aureus Newman in the presence of two different concentrations of hUK-66 as indicated for 20 min at 37°. The monoclonal antibody Trastuzumab served as matched IgG1 isotype control (IC). The generation of reactive oxygen species (ROS) by neutrophils was analyzed by flow cytometry and the values were plotted relative to the burst of unstimulated samples (not shown). (A) ROS-positive neutrophils in blood from healthy donors (B–D) ROS-positive neutrophils in blood from patients with dialysis (B), diabetes, (C) and artery occlusive disease (D). Individual data points of one blood sample (triplicate) were superimposed with bar. Mean values ​​of each group are shown as percentage.

Figure 7. hUK-66 IgG1 binds to FcγRIa and FcγRIIa expressing HEK293 transiently transfected by CaPO₄. The expression of the FcγRs was analyzed by α-CD64 and α-CD32-PE antibodies, respectively. Binding of different concentrations of hUK-66 IgG between 6.61 pM (1.024 ng/ ml) and 20.65 µM (3.2 mg/ ml) on FcγRs was detected via a α-human Fab IgG1-PE.

Figure 8. Killing capacity of S. aureus by whole blood from patients at risk for staphylococcal infections is enhanced by the humanized UK-66 IgG1. Heparinized human blood samples (n = 7) were infected with S. aureus Newman in the presence of two different concentrations of hUK-66 as indicated for 60 min at 37°. The monoclonal antibody Trastuzumab served as matched IgG1 isotype control (IC) and the intravenous human immunoglobulin preparation Gammunex^® (IVIg) as a second positive control. Total viable bacteria were recovered after eukaryotic cell lysis and enumerated by plate counting. The values were plotted relative to the level of control bacteria samples (set at 100%). Results were expressed in mean percentage (SD) of viable S. aureus recovered in the blood from (A) healthy donors, (B) dialysis patients, (C) diabetes patients, and (D) patients with artery occlusive disease (AOD). Data are illustrated as box-and-whisker plot. The dashed line in the graphs demonstrates the 100% reference value of control bacteria samples. *P < 0.05, t test.