Increased macrophage phagocytic activity with TLR9 agonist conjugation of an anti- Borrelia burgdorferi monoclonal antibody

Abstract

Borrelia burgdorferi (Bb) infection causes Lyme disease, for which there is need for more effective therapies. Here, we sequenced the antibody repertoire of plasmablasts in Bb-infected humans. We expressed recombinant monoclonal antibodies (mAbs) representing the identified plasmablast clonal families, and identified their binding specificities. Our recombinant anti-Bb mAbs exhibit a range of activity in mediating macrophage phagocytosis of Bb. To determine if we could increase the macrophage phagocytosis-promoting activity of our anti-Bb mAbs, we generated a TLR9-agonist CpG-oligo-conjugated anti-BmpA mAb. We demonstrated that our CpG-conjugated anti-BmpA mAb exhibited increased peak Bb phagocytosis at 12–24 h, and sustained macrophage phagocytosis over 60+ hrs. Further, our CpG-conjugated anti-BmpA mAb induced macrophages to exhibit a sustained activation morphology. Our findings demonstrate the potential for TLR9-agonist CpG-oligo conjugates to enhance mAb-mediated clearance of Bb, and this approach might also enhance the activity of other anti-microbial mAbs.

Introduction

Lyme disease results from B. burgdorferi (Bb) infection of humans [1], and without adequate antibiotic treatment Bb infections can lead to long lasting and severe symptoms [2]. Bb has evolved multiple mechanisms to evade immune clearance and establish persistent infection. The bacterium has a robust repertoire of outer surface lipoproteins that have anti-complement activity [3]. Some of these outer surface proteins, such as OspC, interfere with phagocytosis of the Lyme disease spirochete by macrophages [4]. There is great need for next-generation approaches to treat Bb infections.

The innate immune system uses a variety of signaling pathways that can be activated by microbial infectious agents, like Bb [5]. Toll-like receptors (TLRs) are pattern-recognition receptors (PRRs), which recognize foreign infectious agents and their pathogen-associated molecular patterns (PAMPs) to initiate inflammatory reactions [6,7]. TLRs are expressed in macrophages and dendritic cells [8]. In mammals, different TLRs can detect diverse range of bacterial ligands including lipopolysaccharide (LPS), nucleic acids, and certain proteins found in gram-positive and gram-negative bacteria [6,9]. TLRs are translated in the ER and then transferred to their designated cellular compartments, which includes the plasma membrane and endosomes. TLR9 is an endosomal TLR that recognizes unmethylated bacterial CpG DNA [10]. Among the TLRs expressed by macrophages, TLR9 represents a compelling therapeutic target, due to its ability to bind bacterial ligands, like CpG DNA, in the endosome and further activate macrophages [[11], [12], [13]]. Further, studies have shown that TLR9 agonists can be used to enhance the efficacy of vaccines and cancer therapies [14,15].

Macrophages are innate immune cells that play a key role in preventing and clearing microbial infections by using a variety of mechanisms for recognition, signal transduction and phagocytosis [9]. Although macrophages have an immense ability to destroy cells by antibody-dependent phagocytosis, the literature does not specifically show the effect of opsonins on clearing of Bb. Macrophages utilize TLR expression and activation to generate robust immune responses against Bb infection [7,16]. Specifically, TLR7 and TLR9 stimulation increased phagocytosis and macrophage longevity while inducing polarization [17]. Further, recent studies showed that antibody drug conjugates can be used as an effective treatment for cancer immunotherapy by a mechanism of augmented macrophage phagocytosis [18,19]. To our knowledge, induction of macrophage phagocytic activity by patient derived, anti-Bb mAbs conjugated with a TLR9-agonist have not been developed as a therapeutic for Lyme disease.

Here, we informatically analyzed plasmablast antibody repertoires from Bb-infected humans to identify antibodies representative of the clonal families. Through recombinant expression of the encoded monoclonal antibodies (mAbs), we identified patient-derived mAbs that bind a spectrum of Bb antigens. Several of these mAbs promoted macrophage phagocytosis of Bb. To develop a more potent anti-Bb mAb, we conjugated a CpG oligo TLR9 agonist to a Bb specific clone (anti-BmpA). We found that this CpG-conjugated anti-Bb BmpA mAb was able to significantly increase Bb phagocytosis activity across multiple donors. This work provides a potential path to the development of next-generation therapeutics for Bb and other microbial infections.