Avidity in antibody effector functions and biotherapeutic drug design

Abstract

Antibodies are the cardinal effector molecules of the immune system and are being leveraged with enormous success as biotherapeutic drugs. A key part of the adaptive immune response is the production of an epitope-diverse, polyclonal antibody mixture that is capable of neutralizing invading pathogens or disease-causing molecules through binding interference and by mediating humoral and cellular effector functions. Avidity - the accumulated binding strength derived from the affinities of multiple individual non-covalent interactions - is fundamental to virtually all aspects of antibody biology, including antibody-antigen binding, clonal selection and effector functions. The manipulation of antibody avidity has since emerged as an important design principle for enhancing or engineering novel properties in antibody biotherapeutics. In this Review, we describe the multiple levels of avidity interactions that trigger the overall efficacy and control of functional responses in both natural antibody biology and their therapeutic applications. Within this framework, we comprehensively review therapeutic antibody mechanisms of action, with particular emphasis on engineered optimizations and platforms. Overall, we describe how affinity and avidity tuning of engineered antibody formats are enabling a new wave of differentiated antibody drugs with tailored properties and novel functions, promising improved treatment options for a wide variety of diseases.

Fig. 1. Response kinetics governing antibody functional responses.

Avidity arising from combinations of affinity interactions are grouped in distinct tiers that integrate the common biological mechanisms of input, output and feedback. Monovalent antibody binding events, termed zero-order avidity interactions for the purpose of this Review, vary from highly transient to long-lasting, depending on affinity. This antibody scanning mode progresses to first-order avidity binding through bivalent Fab–antigen interactions and second-order avidity binding through concomitant Fab–Fab or Fc–Fc interactions. Third-order avidity is engaged when antibody oligomerization passes a threshold for Fc-mediated binding of soluble or cell-bound immune effector molecules, including configurations allowing interactions with IgG Fc receptors (FcγRs) or the complement component C1. The antibody functional response may be regulated or dampened at any avidity tier by, for example, elimination of target cells, target densities dropping below the amplification threshold or regulatory molecules expressed on either the target cell or the effector cell or recruited from plasma.

Fig. 2. Factors affecting antibody functional response activation.

a | Schematic representation of dose–response relationships for different Fc-mediated effector mechanisms. Functional responses reach the activation threshold at antibody doses that vary per effector mechanism; here illustrated in order of increasing antibody concentrations required for antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC). b | Increasing antibody doses result in increasing target occupancy and antibody densities on the target cell surface, thereby favouring distinct tiers of avidity binding; saturation may favour monovalent antibody binding. Fc-mediated effector functions are triggered at different target occupancy levels. IgG Fc receptor (FcγR)-induced ADCC involves the release of cytotoxic granules containing granzymes and perforin (an example of a natural killer (NK) cell is shown); ADCP involves the uptake and lysosomal degradation of target cells (an example of a macrophage is shown); CDC involves the triggering of an amplifiable cascade of complement proteins present in blood, terminating in the generation of a lytic membrane-attack complex. In addition to direct killing, the production of cytokines or bioactive complement fragments may contribute to additional attraction and activation of effector cells. The antibody density required for reaching the activation threshold is defined by different parameters including antibody affinity, valency and concentration; structural constraints related to epitope recognized and antibody isotype, antigen expression and distribution; and the type and presence of effector molecules and regulatory molecules.

Fig. 3. Avidity in antibody biology.

a | B cell receptors (BCRs) constitute antibodies expressed with a transmembrane region on the B cell surface. First-order avidity interactions induce BCR crosslinking and phosphorylation of the BCR-associated transducer molecules that recruit SYK and LYN tyrosine kinases, resulting in protein kinase C (PKC) activation, mitogen-activated protein kinase (MAPK) activation and calcium release. Second-order and third-order avidity binding of antibody-antigen immune complexes recruit FcγRIIB, which comprises an immunoreceptor tyrosine-based inhibitory motif (ITIM motif) that activates phosphatases and reduces BCR signalling. b | Fab-domain-mediated neutralization of pathogen structures, preventing interactions with host cells and blocking pathogen entry into the cell. Binding of a protective antibody against the repetitive P. falciparum circumsporozoite protein (PfCSP) epitope consisting of repeats of the amino acids NPNA is facilitated by second-order avidity Fab–Fab interactions between Fabs from neighbouring IgGs (bound in pairs). Neighbouring Fabs each bind a two NPNA repeat and are shifted 77° along the NPNA spiral, with five IgGs completing a full circle. The Fabs of the IgGs are oriented non-symmetrically in a repeating light chain-bottom/heavy chain-top orientation. facilitating Fab–Fab contacts (left). HIV-1 virions escape second-order avidity binding by sparse surface expression of HIV-1 envelope glycoprotein spikes. The neutralizing antibody 2G12 evolved a domain-swapped structure in which each heavy chain contacts both light chains, thereby creating a large rigid surface that facilitates avid binding of conserved carbohydrate clusters on the HIV-1 envelope spike (right). c | Clustering of immune complexes is initiated by IgG molecules that assemble into ordered hexameric structures through non-covalent Fc–Fc interactions, which facilitate third-order avidity binding and activation of C1 or intracellular signalling through recruitment of signalling molecules such as cIAP1 and TRAF2. IgG hexamer formation proceeds through recruitment of additional IgG molecules through second-order avidity interactions mediated by the Fc domains until ring closure. d | Antibodies that predominantly bind monovalently (such as those with moderate affinity or those engineered for monovalent binding) may elicit stronger effector functions because higher cell surface densities of Fc domains can be achieved. Part a adapted from ref., Springer Nature Limited.

Fig. 4. Antibody avidity engineering strategies.

a | Enhancing first-order and second-order avidity binding by dual-epitope targeting or biparatopic antibodies facilitates antibody clustering and increased functional responses (left). Targeting multiple antigens by designer polyclonals may increase antibody and antigen clustering through Fc–Fc interactions. Examples of hetero-hexamer formation between two antibodies are shown to generate assemblies for dual target-mediated C1 binding and complement activation (right). b | Multivalent/multiligand molecules such as the hexavalent tumour necrosis factor superfamily (TNFSF)–Fc (HERA) technology and multivalent antibody architectures induce tumour necrosis factor receptor superfamily (TNFRSF) member clustering and agonism (left). IgG molecules engineered for an increased ability for on-target hexamerization may trigger complement activation or act as signalling agonists by inducing cell surface receptor clustering. A dimer of an IgG engineered for enhanced self-association is shown with exemplary amino acid residues that facilitate Fc–Fc interactions (shown in red) (right). c | The activation threshold for effector functions may be decreased by affinity tuning. Exemplary combinations of amino acid residues that can be mutated to enhance C1q binding affinity are shown in red and purple. Complement activation remains conditional on first-order and second-order avidity binding (left). FcγR binding may be tuned by protein engineering and glycoengineering (right). Exemplary combinations of amino acid residues that can be mutated to enhance FcγR binding affinity are shown in red (as in tafasitamab) (Table 1) and purple (as in margetuximab) (Table 1), respectively. IgG molecules lacking a fucose residue in one or both heavy chains in the Fc domain results in increased FcγRIIIA binding and ADCC (for approved glycoengineered antibodies, see Table 1). Part b adapted with permission from ref., MDPI.