Reconciling intrinsic properties of activating TNF receptors by native ligands versus synthetic agonists

Abstract

The extracellular domain of tumor necrosis factor receptors (TNFR) generally require assembly into a homotrimeric quaternary structure as a prerequisite for initiation of signaling via the cytoplasmic domains. TNF receptor homotrimers are natively activated by similarly homo-trimerized TNF ligands, but can also be activated by synthetic agonists including engineered antibodies and Fc-ligand fusion proteins. A large body of literature from pre-clinical models supports the hypothesis that synthetic agonists targeting a diverse range of TNF receptors (including 4-1BB, CD40, OX40, GITR, DR5, TNFRSF25, HVEM, LTβR, CD27, and CD30) could amplify immune responses to provide clinical benefit in patients with infectious diseases or cancer. Unfortunately, however, the pre-clinical attributes of synthetic TNF receptor agonists have not translated well in human clinical studies, and have instead raised fundamental questions regarding the intrinsic biology of TNF receptors. Clinical observations of bell-shaped dose response curves have led some to hypothesize that TNF receptor overstimulation is possible and can lead to anergy and/or activation induced cell death of target cells. Safety issues including liver toxicity and cytokine release syndrome have also been observed in humans, raising questions as to whether those toxicities are driven by overstimulation of the targeted TNF receptor, a non-TNF receptor related attribute of the synthetic agonist, or both. Together, these clinical findings have limited the development of many TNF receptor agonists, and may have prevented generation of clinical data which reflects the full potential of TNF receptor agonism. A number of recent studies have provided structural insights into how different TNF receptor agonists bind and cluster TNF receptors, and these insights aid in deconvoluting the intrinsic biology of TNF receptors with the mechanistic underpinnings of synthetic TNF receptor agonist therapeutics.

Keywords: 41BB; CD40; OX40; TNF ligand; TNF receptor; TNF superfamily; agonist.

Figure 1

Schematic of FcγR Dependent TNFR Agonist mAb Dose Response. TNFR most commonly exist as monomers and dimers in cell membranes, and can be bound by one or both scFv domains of bivalent mAbs (A). TNFR agonist mAbs commonly require FcγR binding for TNFR activation to occur, which is dependent upon the Fc domain of the TNFR agonist mAb binding to FcγR on an ‘accessory cell’ so that multiple TNFR agonist mAbs can be displayed as an array when binding TNFR (B). An array of multiple TNFR agonist mAbs may include 4, 6, 8, etc. scFv domains in close proximity, capable of approximating the corresponding number of TNFR on a target cell if those TNFR are unoccupied. Administration of saturating concentrations of a TNFR mAb can result in a reduction in the number of free TNFR or FcγR (C). If a TNFR is bound by a TNFR agonist mAb, then that TNFR is not available to bind TNFR agonist mAbs which have been ‘arrayed’ via FcγR binding, thus reducing activation of TNFR and the corresponding pharmacodynamic response.

Figure 2

Schematic of FcγR Independent Clustering of TNFR Networks in Cell Membranes. Certain TNFR exist in cell membranes as inactive dimers, which can then assemble into trimers upon interaction with a corresponding TNF ligand. Certain TNFR agonist mAbs, may be capable of stimulating a similar response via binding particular epitopes on adjacent TNFR in cell membranes. At low antibody concentrations, TNFR agonist mAbs may bind epitopes on adjacent TNFR, and sometimes cause activation by approximating TNFR dimers or trimers into higher-order networks (A). A hypothetical maximum response is predicted to occur in this model when the molar ratio or TNFR agonist mAb is equal to the number of available binding sites on each trimer of a target TNFR (B). When this ratio is reached, every TNFR trimer is theoretically cross-linked to another TNFR trimer by the TNFR agonist mAb, thus creating a high-order network of TNFR. When the concentration of TNFR agonist mAb exceeds the number of available epitopes on TNFR dimers and trimers, then a TNFR bound by one arm of an antibody may not lead to cross-linking with a nearby TNFR, if that nearby TNFR is also bound by one or both arms of another TNFR agonist mAb (C), thus reducing receptor activation and the corresponding pharmacodynamic responses.

Figure 3

Schematic of FcγR Independent Clustering of TNFR by Low-Affinity TNFR Agonist mAbs. Both high affinity and low affinity TNFR agonist mAbs are capable of binding a TNFR monomer or dimer in a similar manner, however the low affinity antibody will have a faster ‘off-rate’ than the high affinity antibody (A). The faster off-rate of low affinity antibodies leads to an equilibrium where antibodies are rapidly binding and releasing a TNFR target in a cell membrane. In some cases, one arm of the antibody could remain bound to a TNFR while the other arm releases and then re-binds another adjacent TNFR, leading to clustering and TNFR activation (B). If the off-rate of a low affinity antibody is fast enough, then a persistent state of ‘receptor occupancy’ may not occur. This property could theoretically enable low-affinity antibodies to toggle on-and-off a TNFR target fast enough to cause cross-linking and activation even when the molar ratio of the TNFR agonist mAb is in excess to the number of TNFR binding sites (C).

Figure 4

Schematic of Bispecific Antibody Mediated Clustering of TNFR. Bispecific antibodies often target an antigen expressed by a tumor cell (commonly an immune checkpoint such as PD-L1, or a tumor specific antigen such as FAP), and contain a second arm which binds a TNFR (A). The TNFR binding arm of these antibodies is therefore monovalent. As the dose of the bispecific antibody is increased, the probability that multiple antibodies will cluster on the surface of an antigen positive tumor cell increases, and the probability that the monovalent TNFR binding arms from multiple antibodies will bind and approximate multiple nearby TNFR also increases (B). Similar to FcγR specific mAbs, when the dose of the bispecific antibody begins to exceed approximately 50% receptor occupancy on either the tumor antigen or TNFR target, the probability that any individual antibody encounters both a free tumor antigen and TNFR target decreases, leading to a decline in TNFR clustering (C).

Figure 5

Schematic of Hexavalent mAb Binding to TNFR. Tetravalent, hexavalent and decavalent antibodies each contain sufficient TNFR binding domains to facilitate TNFR clustering in the absence of cross-linking by FcγR or a tumor antigen. Each of the binding domains of these multivalent antibodies are capable of binding to a TNFR target independently from one another, and it is possible that certain domains are more ‘exposed’ to find antigen than others, as illustrated for the distal domains (A). At sub-saturating dose levels, each of the TNFR binding domains may occupy a TNFR target, and in the process cluster multiple TNFR targets into close proximity to one another, enabling activation (B). Because each binding domain on an individual antibody can interact independently with a TNFR target, the theoretical maximum pharmacodynamic effect is most likely when the number of antibodies are at a 1:4, 1:6 or 1:10 ratio to the number of TNFR targets (for tetravalent, hexavalent or decavalent antibodies, respectively). When these ratios begin to be exceeded, then independent TNFR binding domains from separate antibodies are expected to compete with one another, thus reducing the probability that an individual antibody is capable of clustering the TNFR target (C).

Figure 6

Schematic of TNF Ligand Trimer-Containing Antibody Binding to TNFR. Bispecific antibodies wherein one arm of the antibody has been replaced with a trimerized set of TNF ligand extracellular domains is capable of stimulating ligand-induced TNFR trimerization in the absence of other cross-linking or antibody clustering mechanisms, which is expected to stimulate TNFR activation even at low doses of antibody, because TNFR activation is not conditional upon clustering by the tumor antigen binding arm (A). As the dose level of the antibody increases, the probability that multiple antibodies will be clustered in close proximity to one another, thus clustering multiple trimeric TNF ligand domains also increases (B). If the quantum of TNFR activation is increased when the trimeric TNF ligand domains are clustered, via organizing TNFR in a higher-order network, a ‘peak’ pharmacodynamic effect may be observed at sub-saturating concentrations of antibody (B). When the concentration of antibody is high enough to saturate both tumor antigen and TNFR target, the probability of TNFR network formation may decrease, leading to a tailing of the pharmacodyamic response curve, but only to a level which reflects the activity of a primarily TNFR trimer pharmacodynamic response (C).

Figure 7

Schematic of a TNF Ligand Hexamer-Containing Fusion Protein Binding to TNFR. Fusion proteins containing two trimerized TNF ligand domains are expected to stimulate ligand-induced trimerization and TNFR hexamer network formation even at low doses of the fusion protein (A). As the dose of the fusion protein increases, the probability of TNFR trimer and hexamer activation is expected to increase in proportion to the dose of the fusion protein, because the TNF ligand domains are not expected to bind TNFR monomers efficiently due to the lower avidity characteristics of the interaction (B). An increasing pharmacodynamic effect is expected until the molar ratio of TNF ligand domains to trimeric TNFR is 1:1, however there is some possibility of tailing in the pharmacodynamic response if a significant proportion of the fusion proteins bind as trimers rather than hexamers, similar to the effect described in Figure 6 (C).