Development of a Bispecific Antibody Targeting Clinical Isolates of Acinetobacter baumannii

Abstract

Background: We previously reported developing 2 anticapsular monoclonal antibodies (mAbs) as a novel therapy for Acinetobacter baumannii infections. We sought to determine whether a bispecific mAb (bsAb) could improve avidity and efficacy while maximizing strain coverage in one molecule.

Methods: Humanized mAb 65 was cloned into a single-chain variable fragment and attached to humanized mAb C8, combining their paratopes into a single bsAb (C73). We tested bsAb C73's strain coverage, binding affinity, ex vivo opsonic activity, and in vivo efficacy compared to each mAb alone and combined.

Results: The bsAb demonstrated strain coverage, binding affinity, opsonization, and in vivo efficacy superior to either original mAb alone or combined.

Conclusions: A humanized bsAb targeting distinct A. baumannii capsule moieties enabled potent and effective coverage of disparate A. baumannii clinical isolates. The bsAb enhances feasibility of development by minimizing the number of components of a promising novel therapeutic for these difficult-to-treat infections.

Keywords: Acinetobacter baumannii; bispecific monoclonal antibody; carbapenem resistant; extreme drug resistance; immunotherapy.

Figure 1.

Confocal microscopy shows binding of bispecific monoclonal antibody (BsAb C73) to the surface of Acinetobacter baumannii. A. baumannii strains HUMC1, VA-Ab41, HUMC9, and LAC-4 were grown to log phase and incubated with primary antibody (isotype control or bsAb C73), followed by secondary antibody (Alexa Fluor 647-conjugated anti-human IgG heavy and light chain) to assess antibody binding (green) by confocal microscopy. Bacteria were counter-stained with propidium iodide (red).

Figure 2.

Bispecific monoclonal antibody (BsAb) C73 opsonizes Acinetobacter baumannii for macrophage uptake. A, By flow cytometry, monoclonal antibody (mAb) C8 and bsAb C73 were both able to bind HUMC1; mAb 65 and bsAb C73 were both able to bind VA-Ab41; mAb C8, mAb 65, and bsAb C73 were all able to bind HUMC9; and the antibodies were unable to bind LAC-4. B, Primary human macrophage cells were unable to appreciably uptake extremely drug-resistant hypervirulent A. baumannii strains in the absence of mAb. Addition of mAb C8, mAb 65, mAbs C8 and 65, or bsAb C73 helped opsonize bacteria and facilitate macrophage uptake, whereas the isotype control did not. A. baumannii strains opsonized by mAb C8, mAb 65, or both were also opsonized by bsAb C73. *P ≤ .05 versus isotype control group; **P ≤ .05 versus isotype control group, mAb C8 group, and Combo C8 + 65 group by the Wilcoxon rank-sum test for unpaired comparisons.

Figure 3.

Bispecific monoclonal antibody (bsAb) C73 rescues C3HeB/Fe mice with Acinetobacter baumannii bacteremia. Mice were infected intravenously (IV) via the tail vein with hypervirulent extremely drug-resistant A. baumannii and treated with antibody (isotype control, monoclonal antibody [mAb] C8, mAb 65, Combo C8 + 65, or bsAb C73) at equimolar amounts. A, Mice were infected with HUMC1 and treated with 1.5 μg bsAb C73 or molar equivalent of another antibody. All survived when treated with bsAb C73 or mAb C8, most survived when treated with Combo C8 + 65, few survived when treated with mAb 65, and none survived when treated with isotype control. B, Mice were infected with VA-Ab41 and treated with 5 μg bsAb C73 or molar equivalent of another antibody. All survived when treated with bsAb C73, most survived when treated with Combo C8 + 65 or mAb 65, few survived when treated with isotype control, and none survived when treated with mAb C8. C, Mice were infected with HUMC9 and treated with 7 μg bsAb C73 or molar equivalent of another antibody. Half survived when treated with bsAb C73, one-third survived when treated with Combo C8 + 65 or mAb C8, and none survived when treated with mAb 65 or isotype control. D, Mice were infected with LAC-4 and treated with 7 μg bsAb C73 or molar equivalent of another antibody. No mice survived, regardless of what treatment they received. n = 4–10 mice/group/experiment over 2–3 experiments; *P ≤ .05 versus isotype control group by the nonparametric log-rank test.

Figure 4.

Bispecific monoclonal antibody (bsAb) C73 works synergistically with colistin, in the setting of delayed treatment, and against pneumonia. A, Combined with colistin (0.0045 mg/kg), a low dose of bsAb C73 (1.5 μg) was able to rescue all C3HeB/Fe mice from lethal bacteremia that both monotherapies failed to treat. B, Delayed intraperitoneal (IP) treatment with bsAb C73 (100 μg) was also capable of rescuing all mice from lethal bacteremia. C, bsAb C73 (300 μg) rescued most mice in an otherwise lethal aspiration pneumonia infection model. n = 5 mice/group/experiment over 2 experiments; *P ≤ .05 versus isotype control group by nonparametric log-rank test.