Redirecting the Host Immune Response to Bacterial Infection with Antibody-Recruiting Molecules (ARMs)

Abstract

The increasing prevalence of antibiotic resistance, the stagnation of antibiotic development, and the adaptive capacity of bacteria to subvert the host immune response combine to pose significant global health concerns. Consequently, there is an urgent need to develop alternative therapeutic approaches to combat bacterial infections. Antibody-recruiting molecules (ARMs), which are bispecific small molecules that recruit endogenous antibodies to pathogenic cells or viruses, offer a promising avenue to harness the host immune system to target various diseases. In this review, we cover ARM strategies that have been developed for bacterial pathogens, including Gram-positive bacteria, Gram-negative bacteria, and mycobacteria, and we discuss the prospects and challenges of utilizing ARMs as alternatives to traditional antibiotic therapies.

Graphical Abstract

1.1. Introduction

Bacterial infections are an increasing global health concern, driven by the dual threat of antibiotic resistance and bacterial strategies to evade the host immune system. Increased antibiotic resistance is expected to cause 10 million deaths annually by 2050,¹ calling for the development of novel antibacterial agents. Additionally, elimination of pathogenic bacteria within the host is impeded by immune evasion strategies of bacteria, including neutralizing toxic host molecules, escaping cellular destruction, and altering antigenic epitopes to antibody-mediated immune recognition.² Given the rapid emergence of antibiotic-resistant bacteria, the gap in the development of new antibiotics, and the evolution of bacteria to subvert host effector functions, there is an urgent need for novel therapeutic strategies.

Immunotherapy, which enhances the body’s immune system to fight diseases, including bacterial infections, involves several modalities such as monoclonal antibodies, vaccines, T-cell therapies, cytokine modulation, and immune checkpoint inhibitors.³ Among emerging immunotherapeutic approaches, antibody-recruiting molecules (ARMs) are gaining traction as a strategy to target bacterial infections. ARMs are bispecific small molecules that graft haptens onto target cell surfaces, which leads to recruitment of anti-hapten antibodies, engagement of immune cells and the complement system, and ultimately enhanced clearance of pathogenic cells.⁴ Although ARM technology is still relatively nascent, examples of ARMs targeting cancer,^5–9 fungi,¹⁰ and viruses^11,12 have been reported, with an ongoing phase 1 clinical trial on multiple myeloma patients using a CD38-specific ARM.¹³ Prior review articles have broadly covered the ARM concept and its applications to targeting different diseases,^4,14–18 including one focused on bacteria published in 2018.¹⁹ Here, we review ARM strategies developed for combating bacteria, briefly revisiting seminal work and emphasizing recent advances using ARMs to target Gram-positive bacteria, Gram-negative bacteria, and mycobacteria.

1.2. The role of antibodies in the host immune response to bacterial infection

Antibodies are secreted by B cells in response to foreign agents like viruses, haptens, or bacteria. Before antibiotics, antibodies from animal sera were used to treat tetanus, diphtheria, pneumonia, and several other bacterial diseases.²⁰ Antibodies can be separated into two fragments: the Fab’₂ region, a bivalent fragment that binds antigen, and the Fc region, which engages with Fc receptors (FcRs) on immune cells. There are 5 antibody isotypes - IgM, IgG, IgE, IgA, and IgD – each defined by their Fc region.²¹ Although responses are isotype- and cell-dependent, antibodies promote neutralization of pathogens and toxins, complement activation, antibody-dependent cell-mediated cytotoxicity (ADCC), and increased phagocytosis.²¹

Phagocytosis plays a key role in immune responses against bacteria. Antibodies, especially IgG, opsonize bacteria for engulfment by immune cells in a process called antibody-dependent cellular phagocytosis (ADCP). Phagocytes, such as neutrophils, eosinophils, monocytes, macrophages, and dendritic cells, express opsonic receptors, like the family of Fc receptors for IgG (FcγR), which trigger phagocytosis when they are engaged.²¹ The engulfed particle is enclosed in a nascent organelle called the phagosome, where reactive oxygen species (ROS) and antimicrobial proteins create a hostile environment.²² Excluding neutrophils, acidification of the phagosome and fusion with the lysosome promotes antigen presentation to the adaptive immune system.^22,23 Since many bacteria can persist intracellularly by evading intracellular killing mechanisms, such as phagosome maturation and ROS production, increasing phagocytosis is not always beneficial.²¹ However, FcR signaling has been shown to overcome the ability of some bacteria to inhibit killing,²¹ so antibodies may be more therapeutically potent than previously thought.

Antibody therapies and antibody-antibiotic conjugates have been developed against bacteria like P. aeruginosa, E. coli, and Clostridium difficile.²⁴ Although antigen availability remains a challenge for facultative intracellular pathogens, these bacteria can survive and replicate outside of host cells and can be targeted during these extracellular stages of infection. For instance, antibodies against intracellular Mycobacterium tuberculosis have shown protection in mice.^25,26 However, bacteria can modify or conceal their antibody-recognized antigens through inducible gene expression, capsule and biofilm formation, and evolutionary processes^27,28. To overcome this, targeted epitopes must be conserved, highly expressed during infection, accessible, and distinct from human and normal microbiota.²⁹ Given these factors, the process of developing unique antibodies for bacterial infections is very challenging.

1.3. Antibody-recruiting molecules (ARMs) engage antibodies against pathogenic targets

ARMs are chimeric molecules composed of a target-binding terminus (TBT) that interacts with disease-relevant targets, an antibody-binding terminus (ABT) that binds to the Fab domain of host-endogenous antibodies, and a linker that allows optimal display of the ABT (Figure 1A).⁴ Together, the ARM, target, and endogenous antibody form a ternary complex that enhances immune clearance of the target through mechanisms described above.⁴ Common haptens within ARMs include 2,4-dinitrophenyl (DNP), galactose-α−1,3-galactose (α-Gal), and L-rhamnose, because antibodies to each exist endogenously in various isotypes (Figure 1B).⁷ In humans, the abundance of these antibodies ranges from 1% to 8%, with anti-L-rhamnose being the most common.^7,30 With respect to the TBT, the substantial molecular differences between bacterial and human cell surfaces provide an opportunity to select TBTs that are bacteria-specific ligands or substrates and are incorporated via evolutionarily conserved mechanisms to minimize the development of resistance. In this way, ARMs meet the demand for novel antimicrobials that could address the growing problem of antibiotic resistance.

Figure 1.

(A) General scheme for the ARM strategy. (B) Structures of common haptens used as the ARM ABT. Panel (A) adapted with permission from ref.³¹

1.4. ARMs targeting Gram-positive bacteria

Gram-positive bacteria encompass WHO high-priority pathogens like Vancomycin-resistant Enterococcus faecium (VRE) and methicillin-resistant Staphylococcus aureus (MRSA), as well as beneficial species such as Lactobacillus and Bifidobacterium, which play key roles in maintaining human health.^32,33 The Gram-positive bacterial envelope is characterized by a thick layer of peptidoglycan (PG), which is conserved and essential due to its roles in providing cellular integrity, rigidity, and shape.³⁴ PG has polysaccharide strands consisting of alternating units of β(1→4)-linked N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc), which are cross-linked by short stem peptides (Figure 2A). Because PG is specific to bacteria, exposed on the Gram-positive bacterial surface, and absent in humans,³⁴ researchers have capitalized on PG structure and biosynthetic pathways to develop ARMs that target Gram-positive bacteria.

Figure 2.

(A) Gram-positive cell envelope and representative structure of PG. (B and C) Examples of ARMs targeting Gram-positive bacteria. Panel (A) adapted with permission from ref.³⁴

One strategy uses antimicrobial agents with affinity for PG to graft haptens onto the surface of Gram-positive bacteria. An example is vancomycin, which binds to the terminal D-Ala-D-Ala region of a PG biosynthetic precursor.³⁵ Early research by the Whitesides group established this concept by developing a polymer combining vancomycin as the TBT and fluorescein as the ABT, which recruited exogenous anti-fluorescein antibodies to various Gram-positive bacterial species and enhanced their phagocytosis by macrophages (Figure 2B).^36,37 More recently, the Pires group developed an ARM featuring a TBT that is a fragment of the Ixodes scapularis tick antifreeze glycoprotein (IAFGP), which has a high affinity for the terminal D-Ala residue on the PG stem peptide.³⁸ The IAFGP fragment P1 was conjugated to different haptens, including fluorescein and DNP, which represented the ABT moiety. The corresponding ARMs modified and recruited antibodies to the cell surface of VRE strains of E. faecalis and E. faecium, leading to increased phagocytosis. Such ARMs based on antimicrobial agents have the potential to benefit from synergistic activity arising from the combined antibody- and antibiotic-mediated effects. A potential drawback is that mutations altering PG structure could reduce the binding affinity of the TBT.

A second strategy to target PG with ARMs uses substrate analogues of the PG biosynthetic pathway to metabolically label bacteria with haptens. PG transpeptidases, which establish cross-links during PG biosynthesis, are substrate tolerant and can incorporate unnatural D-amino acids into the PG stem peptide in live bacteria.^39,40 The absence of D-amino acids in mammals implies that such a strategy could be successfully employed to target bacteria within a host. Over the course of multiple studies, the Pires group leveraged the flexibility of PG biosynthesis to develop various Gram-positive-targeting ARMs composed of D-amino acids as the TBT, a linker, and primarily DNP as the ABT (Figure 2C).^41–43 Collectively, these studies showed that multiple D-amino acid ARM designs, including those based on monopeptides and dipeptides, successfully modified and enhanced antibody recruitment and phagocytosis of multiple Gram-positive species.^41–43 Among the most efficient of these ARMs was DK-Amide, which has an optimized linker length and a D-amino carboxamide structure designed to resist carboxypeptidase-mediated removal of the installed hapten.⁴² However, given that PG is present in all bacteria, such ARMs could potentially cause off-target effects on the host’s normal flora. To address this, the Pires group developed custom PG stem peptide mimics to enhance ARM specificity. A DNP-tetrapeptide conjugate, K(DNP)-Tetra, specifically co-opted L,D-transpeptidase PG cross-linking activity that is predominant in some pathogens, including VRE, which increased antibody recruitment to vancomycin-resistant E. faecium compared to a drug-susceptible strain.⁴⁴ Whether D-amino acid-based ARMs can selectively target Gram-positive bacteria within an infected host animal is not yet known, although the finding that a D-amino acid probe specifically labeled Listeria monocytogenes within infected macrophages points to therapeutic potential.⁴⁰

Other ARM approaches for Gram-positive bacteria have also been explored. Earlier work by the Spiegel and Pires groups developed ARMs for Gram-positive S. aureus featuring the pentapeptide motif LPXTG as a TBT, which is incorporated into PG by the transpeptidase sortase A.^45–47 A recent study by the Liu group reported a method for integrating semi-synthetic PG oligomers into bacterial cell walls.⁴⁸ Although these structures are quite complex and their mechanism of incorporation is not yet defined, they were adapted into ARMs targeting S. aureus.⁴⁹ Synthetic PG oligomers, representing the TBT, were modified through click chemistry with various ABT haptens (biotin, DNP, L-rhamnose, and α-Gal), and these ARM constructs were shown to modify bacterial cells, recruit antibodies from human serum, and enhance phagocytosis and killing of bacteria by macrophages.⁴⁹ Interestingly, similar results were obtained in both Gram-positive S. aureus and Gram-negative P. aeruginosa, despite the latter’s PG layer being obscured by an outer membrane and presumably poorly accessible to ARMs and antibodies.⁴⁹ Regardless, the ARM consisting of a PG oligomer conjugated to α-Gal was reported to nearly completely clear S. aureus from infected mice, an encouraging sign that bacteria-targeting ARMs may be effective in humans.⁴⁹ In another effort designed to improve ARM selectivity for pathogenic bacteria, Kristian et al. developed a DNA aptamer-based ARM, which consists of an α-Gal ABT conjugated to an aptamer TBT that specifically targets Group A Streptococcus (GAS) by binding to the conserved 20A24P region of the cell surface M protein.⁵⁰ Treatment of GAS with this ARM enhanced the recruitment of both mouse and human antibodies, increasing phagocytosis and bacterial killing by human neutrophils.⁵⁰ Although protein-targeting aptamers may be vulnerable to antigenic variation, novel aptamers to address resistance can be generated relatively quickly, and concerns about aptamer stability toward nucleases can possibly be addressed through chemical modification.

1.5. ARMs targeting Gram-negative bacteria

Gram-negative bacteria include WHO-recognized pathogens such as drug-resistant E. coli, A. baumannii, P. aeruginosa, and K. pneumoniae, which cause severe infections like pneumonia, sepsis, and urinary tract infections, but also comprise non-pathogenic species like Bacteroides that aid in digestion and gut health.^32,33 Gram-negative bacteria have a distinctive cell envelope characterized by a relatively thin PG layer and an outer membrane, the latter of which plays key roles in cellular integrity, bacterial adhesion, and immune evasion, among other functions.³⁴ In contrast to Gram-positive bacteria, PG in Gram-negative bacteria is concealed by the outer membrane and, consequently, is not an attractive target for ARM development due to limited antibody access. However, components of the Gram-negative outer membrane, such as the glycolipid LPS and embedded proteins (Figure 3A),³⁴ offer alternative targets that have been exploited to develop ARMs for Gram-negative bacteria.

Figure 3.

(A) Gram-negative cell envelope and representative structure of LPS. (B-D) Examples of ARMs targeting Gram-negative bacteria. Panel (A) adapted with permission from ref.³⁴

Early studies in Gram-negative bacteria invoked an ARM strategy of targeting cell surface carbohydrate-binding proteins, or lectins. In a seminal proof-of-concept, Bertozzi and Bednarski developed a biotinylated C-mannoside derivative, or BCM, which bound to the mannose receptor of E. coli and allowed sequential labeling with avidin and an anti-avidin antibody, ultimately increasing macrophage-mediated killing of E. coli (Figure 3B).⁵¹ Building on this, independent work by the Wang and Shin groups investigated ARMs featuring multivalent display of α-mannosides as the TBT and α-Gal or DNP as the ABT.^52,53 Promisingly, Shin’s ARM increased macrophage-mediated killing of FimH-expressing E. coli in the presence of antibody in a lectin-dependent manner, demonstrating selectivity for pathogenic bacteria displaying the targeted protein.⁵³ The potential improvement provided by multivalent display of the mannoside TBT in these ARMs requires additional investigation.

Metabolic labeling has also been used to deliver ARMs to Gram-negative bacterial surfaces. Dube developed a two-step, bioorthogonal click chemistry strategy to install ARMs onto Helicobacter pylori, a gastrointestinal pathogen.⁵⁴ Feeding H. pylori cells peracetylated N-azidoacetylglucosamine (Ac₄GlcNAz) generated azide-modified surface glycoproteins,⁵⁵ which was followed by Staudinger ligation-mediated attachment of phosphine-DNP reagents (Figure 3C). DNP-modified bacteria were killed more efficiently than unmodified bacteria by peripheral blood mononuclear cells in the presence of anti-DNP antibodies. This study demonstrated the first use of bioorthogonal click chemistry to deliver ARMs to bacteria, which is a potential advantage since the size of some ABTs may preclude their direct metabolic installation. Although there are concerns with exploiting a common sugar as the TBT, such as GlcNAc, in this case selectivity exists because mammalian cells are only known to incorporate GlcNAc into intracellular proteins, not surface proteins. Moreover, new strategies have been developed to metabolically label specific components of the Gram-negative cell envelope, e.g. LPS, and can potentially be adapted into ARM strategies.³⁴

Antibiotics have also been used to direct ARMs to Gram-negative bacterial surfaces. For example, the Pires group developed an ARM based on polymyxin B (PMB), an outer membrane-disrupting antibiotic with high affinity for the conserved lipid A core of LPS.⁵⁶ The ARM design utilized a TBT consisting of a PMB fragment (PMBN), which retains affinity for lipid A but does not disrupt the outer membrane (Figure 3D). A PMBN-DNP conjugate with an intermediate-sized PEG linker efficiently labeled E. coli, recruited anti-DNP antibodies from human serum, and promoted complement-dependent cytotoxicity against the bacteria. As well, these ARMs labeled other Gram-negative bacteria (P. aeruginosa, A. baumannii), consistent with the PMBN moiety binding to lipid A, which highlights their potential broader utility.

An innovative adaptation of the ligand-guided ARM approach was recently reported by the Heath group, which used their protein-catalyzed capture agent (PCC) technique⁵⁷ to discover novel macrocyclic peptides as potential ARM TBTs that specifically bind surface proteins of the Gram-negative pathogen K. pneumoniae.⁵⁸ First, a computational approach was used to mine multi-omic data for proteins with surface-exposed epitopes that are highly conserved in K. pneumoniae strains, which identified the outer membrane protein MrkA as a top candidate. Next, a combinatorial library of cyclic peptides was screened for high-potency binders to the conserved MrkA epitope, yielding a top performer, cy(LLFFF). A cy(LLFFF)-fluorophore conjugate efficiently labeled K. pneumoniae but not the Gram-negative species E. coli and Salmonella typhimurium, demonstrating exceptional selectivity. A cy(LLFFF)-DNP conjugate recruited anti-DNP antibodies to the K. pneumoniae surface and promoted phagocytosis and killing by macrophages. Together, PCC represents a powerful platform for selection of a suitable bacterial surface protein, identification of a high-affinity ligand as the TBT, and ready conjugation to ABTs to yield an ARM. In principle, this platform could rapidly generate specific ARMs against various types of bacteria.

1.6. ARMs targeting mycobacteria

Mycobacteria include intracellular pathogens that cause various infectious diseases, such as tuberculosis, leprosy, and chronic infections from nontuberculous mycobacteria (NTM). These diseases are difficult to treat, with tuberculosis alone leading to over 1.6 million deaths in 2021.⁵⁹ The mycobacterial cell envelope is distinct from canonical Gram-positive and Gram-negative envelopes, as it includes layers of PG, arabinogalactan (AG), and a unique outer membrane composed of mycolic acids, referred to as the mycomembrane (MM), which contributes to these organisms’ remarkable tolerance to stress and antibiotics.³⁴ The unique molecular components of the mycobacterial cell envelope, including glycolipids like trehalose dimycolate (TDM) (Figure 4A),³⁴ provide surface-accessible targets to exploit for the development of mycobacteria-selective ARMs that would theoretically not affect host cells or the host’s normal microbiota.

Figure 4.

(A) Mycobacterial cell envelope and representative structure of TDM. (B and C) Examples of ARMs targeting mycobacteria. Panel (A) adapted with permission from ref.³⁴

We recently reported the first ARM for mycobacteria, which centered on a metabolic labeling strategy exploiting the TDM biosynthetic pathway. Antigen 85 (Ag85) mycoloyltransferases are responsible for transferring mycolic acid lipids onto trehalose to generate TDM,³⁴ a pathway that was previously exploited to incorporate trehalose-based probes into live mycobacteria.^34,60 Therefore, as an initial ARM for mycobacteria, we designed Tre-DNP, which consists of trehalose as the TBT and DNP as the ABT (Figure 4B).³¹ Tre-DNP grafted DNP onto the surface of Mycobacterium smegmatis, but not Gram-positive B. subtilis or Gram-negative E. coli, pointing to high specificity from targeting the Ag85 pathway. M. smegmatis that was surface-modified with Tre-DNP recruited anti-DNP antibodies and was phagocytosed by macrophages more efficiently than unmodified bacteria. Subsequently, we determined that Tre-DNP incorporates into pathogenic mycobacteria, including M. tuberculosis and NTM species, and increases their phagocytosis and killing through an Fc receptor-mediated mechanism (unpublished). Since intracellular survival of mycobacteria is enhanced by uptake via common phagocytic receptors such as the mannose receptor,⁶¹ redirecting phagocytosis through an Fc-receptor-mediated route could improve recognition and clearance by host effector cells. Furthermore, we have explored a two-step, bioorthogonal ARM strategy utilizing azido trehalose⁶² labeling followed by reaction with dibenzocyclooctyne-DNP (DBCO-DNP) (Figure 4C), which significantly increases the efficiency of DNP delivery and could lead to an improved ARM strategy for mycobacteria (unpublished).

1.7. Conclusions and future research

Bacteria-targeting ARMs have demonstrated potential in enhancing immune responses against Gram-positive bacteria, Gram-negative bacteria, and mycobacteria. Strategies to develop bacteria-specific ARMs include the use of metabolic labeling, antibiotics, aptamers, surface receptor ligands, and combinations thereof. These strategies have been used to target conserved, surface-exposed bacterial components like PG, lectins, glycoproteins, LPS, and TDM, some of which are present only in particular types of bacteria and thus offer a means to increase ARM selectivity and decrease unwanted impact on the host or its native microbiota. Overall, ARMs are promising candidates for the development of specific immunotherapeutics that may help to address the worsening problem of antibiotic resistance.

Nevertheless, several challenges persist in the design and development of bacteria-targeting ARMs. For instance, native cell envelope molecules, including wall teichoic acids in Gram-positive bacteria and LPS in Gram-negative bacteria, can hinder antibody access and reduce opsonization,^46,56 making it crucial to select targets that are as surface-accessible as possible. Future efforts should concentrate on refining ARM designs to maximize antibody binding (e.g., through TBT and linker optimization), exploring multi-target strategies to increase selectivity for pathogens, and employing strategies that may increase therapeutic efficacy through more rapid, efficient, and non-toxic bacterial surface labeling, such as emerging metabolic incorporation and bioorthogonal chemistry techniques.³⁴ With respect to the aptamer and macrocyclic peptide examples, bacteria-specific ARM development was aided by high-throughput screening techniques, which should find more frequent use in the future. Additionally, refining the ABT to recruit IgG subclasses could enhance anti-bacterial responses by promoting phagocytosis and complement activation.²¹ Since anti-hapten IgG is in limited supply, synthesizing multivalent ARMs^63,64 could boost antibody recruitment to bacteria, increase avidity with Fc receptors, and lower the threshold for immune activation. Another approach would include replacing haptens as the ABT with an Fc-binding moiety.⁶⁵ For example, Sasaki and colleagues synthesized an ARM with folic acid representing the TBT and a peptide that binds to the Fc region of IgG representing the ABT.⁶⁵

The translation of ARM strategies covered herein to in vivo systems remains limited and may encounter challenges related to pharmacokinetics and biodistribution, including but not limited to sequestration, molecular stability, antigen availability, and interactions with endogenous biomolecules, all of which may affect ARM efficacy. For example, to address the issue of ARMs not reaching their intended targets due to sequestration, Manabe et al. developed ABT-photocaged ARMs, which utilize UV light to control the release of L-rhamnose,⁶⁶ and may allow for targeted spatial-temporal activation. Other factors, such as the nature of the pathogen (e.g., tissue tropism, slow-growing vs. fast-growing, length of extracellular lifestyle) and the host (e.g., endogenous anti-hapten isotype profile, FcγR expression, nutritional status) could also play a role in the efficacy of ARMs. Moving forward, advancing to in vivo studies in animal infection models is essential to test and reveal critical insights about the therapeutic potential of ARMs. It would also be valuable to understand whether ARMs demonstrate activity when combined with antibiotics, if the site of infection (e.g., lung, blood, and skin) impacts whether ARMs reduce bacterial burden, or if the intended immune response is initiated. Overall, future research to optimize ARM design, investigate mechanisms of immune enhancement, and perform in vivo evaluations will pave the way forward for ARMs as potential therapeutics for bacterial infections.