Strategies for targeting cell surface proteins using multivalent conjugates and chemical biology

Abstract

Proper function of receptors on the cell surface is essential for homeostasis. Compounds that target cell surface receptors to address dysregulation have proven exceptionally successful as therapeutic agents; however, the development of compounds with the desired specificity for receptors, cells, and tissues of choice has proven difficult in some cases. The development of compounds that can engage more than one binding site at the cell surface offers a path towards improving biological specificity or pharmacological properties. In this chapter we summarize historical context for the development of such bivalent compounds. We focus on developments in chemical methods and biological engineering to provide bivalent compounds in which the high affinity and specificity of antibodies are leveraged to create multifunctional conjugates with new and useful properties. The development of methods to meld biological macromolecules with synthetic compounds will facilitate modulation of receptor biology in ways not previously possible.

Introduction:

Proteins found on the cell surface are essential for proper responses to environmental cues. In multicellular organisms, cells communicate through the production and release of molecules that act on protein receptors found on the surface of neighbors, proximal and distal. This cell-to-cell communication is essential for coordinated biological responses in complex organisms. Dysregulation of these processes can have profound consequences and frequently results in disease. Addressing this dysregulation through the application of molecules that mimic or block the action of natural signaling molecules is a common and successful approach for therapeutic development. Efforts to target G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs), two large and important families of cell surface proteins, exemplify these efforts.

GPCRs are the largest family of cell surface proteins, with over 800 family members in humans(Hauser et al., 2017). They function through activation of guanine nucleotide-binding proteins (G-proteins), among other pathways, to regulate virtually every aspect of cell biology. Responses are induced through the binding of ligands such as small molecules, peptides, and full-size proteins to GPCRs. Over 30% of approved therapeutics target GPCRs(Hauser et al., 2017); however, several outstanding challenges remain(Wacker et al., 2017). There is substantial overlap in specificity among GPCRs and the ligands they recognize: approximately 75% of naturally occurring receptors and ligands interact with more than one receptor or ligand(Foster et al., 2019). Extraordinary efforts have been undertaken to identify synthetic small molecules that bind tightly and specifically to receptors of interest to induce desired biological responses(Griffith et al., 2020; Lyu et al., 2019; Moehle et al., 2020). Peptides and antibodies (Abs) have also been developed to modulate GPCRs, often proving effective in targeting receptors for which small molecules has proven insufficiently potent and specific(Davenport et al., 2020; Hutchings et al., 2017). RTKs, like GPCRs, are a prime target in therapeutic development, with particular relevance in the development of cancer therapeutics(Pottier et al., 2020). Most RTKs are also activated by more than one ligand, complicating approaches to make selective and potent receptor modulators(Trenker & Jura, 2020). Efforts to induce desired biological outcomes by targeting either GPCRs or RTKs is further complicated by variation in receptor expression in different tissues, sometimes as different receptor isoforms(Marti-Solano et al., 2020). Furthermore, the consequences of receptor activation or blockade can vary based on the tissue in which targeting occurs(Cheloha et al., 2015; Hao & Tatonetti, 2016).

Given these considerations, improved methods to address receptor function would be valuable. This chapter focuses on the development of multivalent compounds to target cell surface receptors. A brief overview of the historical precedence for using small molecule bivalent compounds to modulate receptor function will be presented. Next, a framework for describing different varieties of multivalent compounds will be illustrated. This framework will be used to describe recent efforts to use biomolecules to construct multivalent conjugates for targeting cell surface proteins. Special emphasis will be placed on multivalent compounds that result from the combination of chemical and biological methods (chemical biology). The methodology used to construct these conjugates will then be described. Finally, the biological and pharmacological properties of these conjugates will be summarized followed by a perspective on advances that will propel this field forward.

Discussion.

Categorization of conjugates.

Multivalent ligands have been used to target cell surface receptors to provide specialized and useful properties for decades(Newman et al., 2020). During this period, several varieties of multivalent ligands have been developed with overlapping and signature characteristics (Figure 1). Here we define bivalent (and oligovalent) ligands as those that are comprised of two (or more) moieties that target the orthosteric site of cell surface proteins. These oligovalent ligands can consist of multiple copies of the same targeting moiety (homo-oligovalent, Figure 1A) or more than one different targeting moiety (hetero-oligovalent, Figure 1B), which can target one type of receptor or more. A variation on this approach relies on the use of targeting moieties that do not bind to the orthosteric site on a receptor, but rather some other site for which binding does not activate or block signaling, here named a secondary site. The linkage of moieties that differ in the type of site they bind (orthosteric versus secondary) provides bitopic (or oligotopic) ligands (Figure 1C–D). Note that bitopic ligands differ from their bivalent counterparts in the use of a targeting moiety that binds to a secondary site. Bitopic ligands can bind to multiple, distinct sites on the same target protein (cis-bitopic, Figure 1C) or to sites on neighboring proteins (trans-bitopic, Figure 1D). These bivalent and bitopic compounds can also be used to engage targets expressed on different cells or cell populations (Figure 1E). These classes of multivalent ligands have been deployed in a variety of contexts, with distinct advantages and drawbacks, as will be discussed below.

Figure 1.

Classes of multivalent conjugates used to target cell surface receptors. Schematic of a,b, bivalent ligands selectively targeting two orthosteric binding site of the same receptor (a, homobivalent), or different receptor subtypes (b, hetreobivalent); c,d, bitopic ligands selectively targeting the primary orthosteric and a secondary binding site in a single receptor (c, cis-bitopic), or different receptor subtype (d, trans-bitopic). e, Intercellular-bitopic to target receptor subtypes expressed on two different cells.

Historical precedent with small molecules.

Some of the earliest efforts in this area focused on efforts to design bivalent ligands consisting of two copies of a pharmacophore connected by a linker, which bind to the orthosteric binding site of opioid receptors(Erez et al., 1982). This design was proposed as a way to use the binding of the first pharmacophore to reduce the entropic cost for the binding of the second ligand to its receptors. These early studies revealed that both the nature of the orthosteric site binding moieties and the length and composition of the linker connecting them were important parameters in determining bivalent ligand affinity and potency. Optimized homobivalent compounds showed potencies that exceeded those of monovalent ligands. Further studies revealed that such bivalent compounds could facilitate selective engagement of specific opioid receptor subtypes, a feat which was difficult to achieve using monovalent compounds available at that time(Portoghese et al., 1988). One contributing factor for the unique characteristics seen with bivalent ligands that target cell surface receptors is that many of these receptors oligomerize to form homodimers, heterodimers, or higher order complexes(Gomes et al., 2016). Selectively targeting cell surface protein oligomers using multivalent ligands offers the opportunity to induce biological responses only in cell types expressing each of the proteins needed to form the functional receptor oligomer. Engagement of receptor complexes has received increasing interest as a way to design new tool compounds and therapeutic candidates(Rosenbaum et al., 2020).

GPCRs often dimerize to form assemblies with properties that differ from their constituent monomers(González-Maeso, 2011; Terrillon & Bouvier, 2004). Multivalent, small molecule compounds have been designed to target these assemblies. Bivalent and bitopic compounds with high specificity towards serotonergic receptor subtypes and receptor assemblies have been reported(Soto et al., 2018; A. Zhang et al., 2007). Bivalent compounds have also proven useful for targeting assemblies of cannabinoid receptors(Nimczick & Decker, 2015; Y. Zhang et al., 2010), adrenergic receptors(Hague et al., 2006; Xu et al., 2003), adenosine receptors(Barlow et al., 2013; Soriano et al., 2009), dopamine receptors(Ågren et al., 2020; Kumar et al., 2016, 2017) and a variety of other GPCR heterodimers(Hübner et al., 2016; Toneatti et al., 2020). Bitopic compounds have also shown promise in stimulating signaling through a subset of the full repertoire of pathways engaged by standard ligands, or biased signaling(Bonifazi et al., 2019; Holze et al., 2020). There is an extensive literature on the unique and desirable properties exhibited by small molecule compounds designed to target GPCR dimers(Newman et al., 2020) and cell surface receptors more generally(Rosenbaum et al., 2020). This chapter will focus on examples in which targeting moieties besides small molecules have been applied.

Biomolecules for targeting receptors

Despite the extensive efforts invested in designing bivalent and bitopic compounds to target GPCRs and other cell surface receptors, there are many targets for which suitable small molecule compounds are not readily available. In some cases, alternative modalities such as peptides and proteins(Davenport et al., 2020), including Abs and their fragments(Heukers et al., 2019; Hutchings et al., 2017), facilitate uniquely powerful approaches for modulating target function. The utility of polypeptides for the selective targeting of receptors such as GPCRs is perhaps not surprising since a majority of GPCRs are activated by peptide ligands(Foster et al., 2019). However, natural ligands for receptors are often promiscuous, activating more than one receptor, leading to difficulties in their application as pharmacological tools or therapeutic candidates. In such cases, monoclonal Abs, or their fragments, widely applied for their high affinity and specific recognition of targets of interest, are useful. The examples discussed in detail below focus on Abs and their fragments; however, a variety of platforms relying on biomolecule oligomers for selective targeting of cell surface proteins of interest are under development(Richards, 2018).

Although Abs that recognize most proteins localized to the cell membrane are commercially available, many of these do not bind to the fully folded proteins found on live cells. Other Abs bind but do not affect the function of the bound target. Such Abs cannot be used directly to study receptor pharmacology or the corresponding biology. Approaches to specifically identify Abs that act as receptor antagonists have been published (McMahon et al., 2020; Ren et al., 2020) but they are sometimes difficult to generalize. Even rarer are examples in which Abs are converted into receptor agonists using design approaches(T. Liu et al., 2015; Ma et al., 2020). General methods to leverage the useful characteristics of Abs to directly modulate the function of cell surface receptors would be valuable.

Methodology for producing Ab conjugates

Chemical approaches and biological engineering offer a path towards equipping Abs with moieties that can directly impact the function of cell surface proteins. A comprehensive review of approaches used to modify Abs has recently been published(Walsh et al., 2021). Here we focus on examples in which conjugates are used to modulate cell surface protein function. One difficulty in making Ab conjugates to impact cell surface protein function relates to the methods used to connect receptor targeting moieties to Abs. In some cases, a bioactive polypeptide can be fused with an Ab (or Ab fragment) through recombinant expression. One complicating factor for this approach is that conventional monoclonal Abs (usually immunoglobulin-G or IgGs) are comprised of four separate polypeptide chains, two heavy chains (HCs) and two light chains (LCs), connected by disulfide bonds (Figure 2). Efforts to produce genetic fusions between IgGs and bioactive polypeptides often results in a loss of stability or folding efficiency. Extensive empirical optimization was required to identify a construct and conditions that allowed for production of a bitopic fusion between an IgG that targeted the soluble protein PCSK9 and a polypeptide ligand agonist for the type-1 glucagon-like peptide receptor (GLP1R)(Chodorge et al., 2018). Many attempts at designing such fusions provided proteins that were prone to aggregation and insolubility, were produced in low yield, or contained truncations that abrogated GLP1R agonist activity(Chodorge et al., 2018). Similar issues can plague efforts to use the Ab fragments responsible for antigen binding (Fabs, Figure 2), which are often produced by the enzymatic digestion of full size IgGs.

Figure 2.

Schematic of antibody composition, functional fragments, and sizes.

Issues of poor fusion protein behavior can be addressed to some extent by the use of single domain Abs (nanobodies, Nbs) in place of conventional Abs. Nbs are the antigen recognition domain of camelid heavy chain only Abs (Figure 2)(Cheloha, Harmand, et al., 2020). These single domain antibodies (Nbs) fold and function without HC-LC pairing and disulfide bond formation. Functionalized Nbs have been used to visualize GPCR distribution and trafficking(Heukers et al., 2019) and as a tool to assess GPCR conformation(Soave et al., 2020). A fusion between a GLP1R agonist peptide and two Nbs, one targeting GLP1R and the other targeting serum albumin to extend circulatory half-life, was readily produced by recombinant expression and effectively activated GLP1R in vitro and in mice(Pan et al., 2020) (Figure 3a). Deploying a similar approach with conventional antibodies would be complicated by mispairing between HCs and LCs. Nbs can often be used for a variety of applications in which conventional antibodies are not optimal or fail(Cheloha, Harmand, et al., 2020).

Figure 3.

Methods used in the production of Ab-ligand conjugates. A) Recombinant expression of bioactive peptide-Nb fusions. B) Undirected lysine side chain modification. C) Disulfide bond reduction with subsequent cysteine side chain conjugation. D) Unpaired cysteine residue side chain conjugation. E) Stop codon reprogramming and incorporation of unnatural amino acid with ketone functional group. F) Site-specific conversion of cysteine to formylglycine for subsequent conjugation. G) Sortase A ligation.

Abs produced using conventional methods, such as recombinant expression, can be modified with a compound containing a functional group that spontaneously reacts with an amino acid side chain group found in Abs. One common incarnation of this approach consists of modifying a small molecule of interest with an amine reactive functional group that can react with exposed lysine residues or the amine at the N-terminus (Figure 3b). Lysine is a common amino acid in natural proteins so modification via amine reactivity can result in the appendage of several copies of the small molecule cargo and heterogeneous labeling. Heavy modification of Abs can sometime cause a loss of Ab stability and function(Vira et al., 2010). A lack of control of the degree of functionalization would be predicted to be especially problematic when appending large or hydrophobic compounds. Perhaps because of this complication, there are few examples of using undirected amine conjugation to create multivalent compounds to target the cell surface. In one example, trans-bitopic (or oligotopic) conjugates were prepared from Abs modified non-site-specifically with a glycans designed to promote lysosomal trafficking of the Ab bound target(Ahn et al., 2020; Banik et al., 2020) (Figure 3b).

A more common labeling approach for creating multivalent conjugates relies on the unique reactivity of the thiol group found on the side chain of cystine (Cys) residues. Cys possesses unique reactivity among the natural amino acid side chains allowing for selective functionalization at Cys not involved in disulfide bonds. Common functional groups used for site-specific Cys labeling include maleimide- and α-halocarbonyl-functionalized compounds. Since reduced cysteine is much less common than lysine in natural proteins, tighter control of the site and degree of modification with Cys-reactive compounds is possible. For proteins that traverse the secretory pathway, such as Abs, Cys residues are often oxidized to participate in disulfide bonds, preventing their immediate functionalization. Thus, methods to reduce disulfide bonds to free the Cys side chain for functionalization have been developed. This approach was deployed to functionalize Abs with compounds that inhibit a cell surface ion pump to produce trans-bitopic conjugates that kill targeted cells (Marshall et al., 2016; Sweeny et al., 2013)(Figure 3c). Alternatively, an unpaired Cys residue, which does not engage in disulfide bond formation, can be engineered into Abs, to allow for site-specific functionalization with an inhibitor of a cell surface enzyme to produce a cis-bitopic conjugate(Figure 3d)(Cheng et al., 2018).

Site selective modification of Abs with compounds of interest can be precisely achieved through the introduction of non-natural amino acids with orthogonal reactivity through translational reprogramming or enzymatic modification of engineered peptide tags. These methods often produce homogeneous conjugates, unlike those that rely on modification of naturally occurring residues. DNA stop codon suppression methodology allows for incorporation of non-natural amino acids at specified positions in proteins of interest(Kim et al., 2013). Stop codon reprogramming was used to introduce into an Ab fragment an amino acid with a ketone functional group, which unlike canonical amino acids, readily reacts with aminoxy-containing compounds to form oxime linkages (Figure 3e)(Kim et al., 2013). This approach provided an intercellular, trans-bitopic Ab fragment-small molecule conjugate (Figure 1e) that bound to targets on T cells and tumor cells to promote T-cell-mediated tumor cell killing. Enzyme catalysis can also be used to append compounds of interest or reactive groups at short peptide tags engineered into Abs. Formylglycine generating enzyme can convert a Cys residue found within a recognition sequence into formylglycine, which can subsequently react with a variety of partners to allow for site-specific modification (Krüger et al., 2019). This approach was used to functionalize Abs with glycans to produce homogeneous trans-bitopic conjugates that direct Ab bound targets for lysosomal degradation (Figure 3f)(Ahn et al., 2020). Homogenous Ab-glycan conjugates prepared in this manner showed improved pharmacokinetic properties relative to those produced by undirected lysine modification. A different enzymatic labeling approach relies on the enzyme Sortase A, which ligates cargoes attached to peptides with N-terminal glycine residues to proteins with exposed LPETG motifs(Cheloha et al., 2019; Pishesha et al., 2018). This approach was used to create a series of homo-bitopic nanobody-ligand conjugates that activate parathyroid hormone receptor in cells and in mice (Figure 3g) (Cheloha, Fischer, et al., 2020).

Biological applications

Conjugates consisting of multiple components that target cell surface proteins can exhibit biological properties that differ from the individual components. The linkage of multiple recognition modules in which the different components bind to distinct targets on the cell surface, sometimes on different cells, has been applied in a variety of settings. This approach has been most extensively deployed in the field of bispecific Abs(Labrijn et al., 2019). Bispecific Abs are often used to improve specificity for targeting a cell type of interest or to simultaneously target multiple cell types, which will not be discussed in detail here (see Table 1 for references). A new incarnation of this approach, dubbed “RIPR” for receptor inhibition by phosphatase recruitment, was used to enforce proximity between a promiscuous cell membrane phosphatase (CD45) and RTKs by using trans bitopic fusions between Ab fragments(Fernandes et al., 2020). Ligand binding to RTKs induces phosphorylation of intracellular tyrosine residues, which can be reversed by enforced proximity with CD45. This approach was used to inhibit activation of a variety of RTKs relevant to immune cell function including PD-1, CTLA-4, TIM-3, and LAG3 (Table 1).

Table 1.

Summary of biological properties for multivalent conjugates targeting cell surface receptors consisting of antibodies and fragments.

Multivalent conjugate	Category	Targeting moiety	Proteins targeted	Signaling or 2° targeting moiety	Signaling response	Reference(s)
Established classes
Bispecific antibodies	Trans-bitopic	Ab and fragments	Various (tumor)	Ab (various)	Various (none, immune activation)	Labrijn et al., 2019
Immunocytokine	Trans-bitopic	Ab and fragments	Various (tumor)	Cytokine (IL15, IL2, IL12, IFNα, others)	Anti-tumor immune response, improved specificity	Neri, 2019
ADC (conventional)	Trans-bitopic	Ab and fragments	Various (tumor)	None (cytotoxic compound)	Induction of cell death, improved specicity	Lambert & Berkenblit, 2018
New and specific examples
Nb-synthetic peptide ligand (CLAMP)	Cis-bitopic	Nb	PTHR1	PTHR1 synthetic peptide ligand	Activation of PTHR1, blood calcium spike	Cheloha, Fischer, et al., 2020
Nb-peptide ligand genetic fusion	Cis-bitopic	Nb	GLP1R, serum albumin	GLP1 peptide ligand (genetic fusion)	Activation of GLP1R, suppression blood sugar	Pan et al., 2020
Ab-small molecule ligand	Cis-bitopic	Fab	DPP-IV	DPP-IV small molecule inhibitor	Improved potency for DPP-IV inhibition	Cheng et al., 2018
Nb-small molecule ligand	Cis-bitopic	Nb	mGluR4	Photo switchable small molecule ligand	Light controlled receptor activation	Farrants et al, 2018
Ab-small molecule ligand	Trans-bitopic	Ab	Various (tumor)	Small molecule inhibitor Na+/K+ATPase	Induction of cell death, improved specificity	Marshall et al., 2016; Sweeny et al, 2013
RIRR	Trans-bitopic	Ab and fragments	RTKs	Anti CD45 Ab or Ab fragment	Dephosphorylation of Ab-bound proximal targets	Fernandes et al., 2020
LYTACs	Trans-bitopic	Ab, peptides	EGFR, HER2, PD-L1	Glycan (synthetic compound or polymer)	Trafficking of Ab bound target to lysosome, degradation	Ahn et al, 2020; Banik et al., 2020

An alternative type of genetic fusion consists of Ab(s) (or Ab fragments) and cytokines to produce trans bitopic fusions called immunocytokines(Neri, 2019). Cytokines often act via activation of RTK signaling. Immunocytokines target tumor cells via Ab recognition and induce anti-tumor immune responses via cytokine receptor engagement. This approach has been used to deliver cytokines such as IL15(Y. Liu et al., 2018), IL2(Dougan et al., 2018), and IL12(Probst et al., 2019) to tumors or the surrounding vasculature using Abs or their fragments (Table 1). Immunocytokines sometimes suffer from off-target effects due to the high potency of cytokines relative to the targeting capacity of the Ab(Tzeng et al., 2015). This has been addressed through the use of an attenuated mutant versions of cytokines (interferon-α and leptin), which show strong activity only when directed to cell types of choice via fusion with nanobodies in trans-bitopic ligands, in an approach dubbed “activity-by-targeting”, which diminishes off-target effects (Garcin et al., 2014; Pogue et al., 2016)

Genetic fusions between Ab fragments and peptidic GPCR ligands have also been developed. There are several examples of fusions between Abs and peptide GPCR ligands in which the Ab serves only to prolong the circulatory half-life of the peptide ligand, or to bind to a soluble extracellular protein(Chodorge et al., 2018). Rarer are examples in which the Ab component also binds to a cell surface protein. In one example, a peptide ligand for GLP1R was fused to two nanobodies: one nanobody that targets a non-orthosteric site on GLP1R and the other nanobody that targets serum albumin(Pan et al., 2020). These trans-bitopic (or tritopic) conjugates stimulated enduring suppression of blood glucose levels, demonstrating strong GLP1R activation (Table 1).

The linkage of Abs with cell surface protein ligands produced using synthetic chemistry offers an avenue to improve the properties of these ligands. These efforts benefit from the large body of literature on conventional antibody-drug conjugates (ADCs)(Lambert & Berkenblit, 2018). Conventional ADCs are typically comprised of an Ab that targets a cell surface marker expressed on pathogenic cells such as tumors and a cytotoxic compound that acts on targets found in the interior of the cell. The key difference between conventional ADCs and conjugates discussed here is the cellular localization of the target: conventional ADCs require delivery of the conjugated cargo to the interior of the cell, typically through endocytosis and proteolysis. An alternative approach emphasized here consists of using Abs to deliver ligands that induce cytotoxic responses through targeting a cell surface receptor. This approach has been adopted to deliver cytotoxic small molecule inhibitors of the Na⁺/K⁺ ATPase through conjugation to Abs that target proteins expressed on tumor cells (Table 1)(Marshall et al., 2016; Sweeny et al., 2013). This approach provided trans-bitopic compounds with potent cell killing activity for cells expressing the target bound by the Ab and weak toxicity for other cells.

A variation on the theme of ADCs is to prepare a fusion between an Ab and ligand that both target the receptor of interest, or a cis-bitopic conjugate. Such conjugates have been dubbed conjugates of ligands and antibodies for membrane proteins or CLAMPs(Cheloha, Fischer, et al., 2020). The linkage of weak, synthetic peptide ligands for type-1 parathyroid hormone receptor (PTH-receptor 1, PTHR1) and nanobodies that target the same receptor at a different site provided conjugates with improved receptor activation potency relative to the original ligand (Table 1) (Cheloha, Fischer, et al., 2020). Furthermore, PTH activates both type-1 and type-2 PTH-receptor (PTHR1 and PTHR2), whereas one nanobody characterized selectively bound PTHR1. The PTH-nanobody conjugate potently activated PTHR1 but not PTHR2, showcasing the capacity of Ab targeting to impart receptor subtype specificity. A related approach was used to link an Ab inhibitor and a small molecule inhibitor specific for the cell surface localized protease DPP-IV(Cheng et al., 2018). DPP-IV is a clinically validated target for the treatment of type-2 diabetes. This cis-bitopic conjugate, structurally characterized in complex with DPP-IV, showed superior inhibition of DPP-IV relative to either the Ab or small molecule inhibitor alone (Table 1).

Conjugates between Abs and synthetic moieties can also be used to modulate receptor function in ways not possible using conventional methods. The conjugation of glycans that target cation-independent mannose-6-phosphate receptor or asialoglycoprotein receptor to stimulate lysosomal trafficking with Abs that recognize other cell surface receptors provides lysosomal targeting chimeras, or LYTACs, that stimulate lysosomal trafficking and degradation of Ab-bound targets (Table 1)(Ahn et al., 2020; Banik et al., 2020). This approach is applicable even in instances where agents that inhibit cell surface proteins are not available. LYTACs were used to stimulate degradation of a variety of targets including EGFR, HER2, and PD-L1 and to inhibit receptor-dependent cell survival. In a different approach, a small molecule agonist of the metabolic glutamate receptor (mGluR4), which undergoes isomerization across a double bond and changes agonist activity upon illumination was conjugated to a receptor binding nanobody. The resulting cis bitopic conjugate exhibits agonist activity at mGluR4 that can be toggled off and on by illumination(Farrants et al., 2018). This approach holds promise for imposing tight kinetic control of signaling, which is essential for interrogating biological processes on relevant time scales.

Conclusions and future directions.

The wide-ranging approaches used to construct and apply multivalent conjugates have provided novel tools with useful and interesting properties. Despite this progress, there are shortcomings in chemical and biological methodology that restrict facile development and application of these conjugates, particularly those with Abs as a component. It has become increasingly apparent that monovalent and site-specific modification of Abs provides homogeneous conjugates that show more consistent and predictable behavior in vivo relative to heterogenous conjugates produced by non-specific conjugation methods. Approaches to modify less common amino acids, such as methionine(Lin et al., 2017), may prove useful for these goals. Established enzymatic modification methods(Krüger et al., 2019; Pishesha et al., 2018) and newly developed alternatives(Rehm et al., 2019) will also likely be helpful in producing homogeneous conjugates.

Another limitation in developing multivalent conjugates with Abs relates to the availability of appropriately specific Abs in sufficient quantities to carry out modifications. Conventional monoclonal Abs against many targets are widely available commercially; however, they are often produced with specialized equipment and purchasing in bulk is often prohibitively expensive. Nanobodies offer a useful alternative because they can be produced from bacteria in high yield using standard research laboratory equipment(Cheloha, Harmand, et al., 2020). However, nanobodies for many cell surface proteins of interest have not yet been identified. Novel screening methods provide a pathway for identifying new nanobodies that bind to and impact the function of cell surface proteins of interest(Ma et al., 2020; McMahon et al., 2020; Ren et al., 2020). One intriguing possibility for future efforts to develop multivalent conjugates is to functionalize targeting moieties, such as Abs, with functional groups that will react when found in proximity to another targeting moiety equipped with a partner functional group. Enforced proximity can be achieved by simultaneous binding of Abs to different proteins on the cell surface to allow for conjugate production in situ(Komatsu et al., 2020). Multivalent Ab conjugates assembled in this way showed an improved ability to stimulate endocytosis relative to unlinked counterparts(Komatsu et al., 2020). This finding suggests that chemical methods could be harnessed to assemble multivalent conjugates at their site of action, avoiding the need to synthesize and purify conjugates prior to assay, which should enable accelerated identification of improved conjugates.