Identification and characterisation of anti-IL-13 inhibitory single domain antibodies provides new insights into receptor selectivity and attractive opportunities for drug discovery.

Interleukin-13 (IL-13) is a cytokine involved in T-cell immune responses and is a well validated therapeutic target for the treatment of asthma, along with other allergic and inflammatory diseases. IL-13 signals through a ternary signalling complex formed with the receptors IL-13Rα1 and IL-4Rα. This complex is assembled by IL-13 initially binding IL-13Rα1, followed by association of the binary IL-13:IL-13Rα1 complex with IL-4Rα. The receptors are shared with IL-4, but IL-4 initially binds IL-4Rα. Here we report the identification and characterisation of a diverse panel of single-domain antibodies (VHHs) that bind to IL-13 (KD 40 nM-5.5 µM) and inhibit downstream IL-13 signalling (IC50 0.2-53.8 µM). NMR mapping showed that the VHHs recognise a number of epitopes on IL-13, including previously unknown allosteric sites. Further NMR investigation of VHH204 bound to IL-13 revealed a novel allosteric mechanism of inhibition, with the antibody stabilising IL-13 in a conformation incompatible with receptor binding. This also led to the identification of a conformational equilibrium for free IL-13, providing insights into differing receptor signalling complex assembly seen for IL-13 compared to IL-4, with formation of the IL-13:IL-13Rα1 complex required to stabilise IL-13 in a conformation with high affinity for IL-4Rα. These findings highlight new opportunities for therapeutic targeting of IL-13 and we report a successful 19F fragment screen of the IL-13:VHH204 complex, including binding sites identified for several hits. To our knowledge, these 19F containing fragments represent the first small-molecules shown to bind to IL-13 and could provide starting points for a small-molecule drug discovery programme.

Identification, binding, and structural characterization of single domain anti-PD-L1 antibodies inhibitory of immune regulatory proteins PD-1 and CD80.

Programmed death-ligand 1 (PD-L1) is a key immune regulatory protein that interacts with programmed cell death protein 1 (PD-1), leading to T-cell suppression. Whilst this interaction is key in self-tolerance, cancer cells evade the immune system by overexpressing PD-L1. Inhibition of the PD-1/PD-L1 pathway with standard monoclonal antibodies has proven a highly effective cancer treatment; however, single domain antibodies (VHH) may offer numerous potential benefits. Here, we report the identification and characterization of a diverse panel of 16 novel VHHs specific to PD-L1. The panel of VHHs demonstrate affinities of 0.7 nM to 5.1 µM and were able to completely inhibit PD-1 binding to PD-L1. The binding site for each VHH on PD-L1 was determined using NMR chemical shift perturbation mapping and revealed a common binding surface encompassing the PD-1-binding site. Additionally, we solved crystal structures of two representative VHHs in complex with PD-L1, which revealed unique binding modes. Similar NMR experiments were used to identify the binding site of CD80 on PD-L1, which is another immune response regulatory element and interacts with PD-L1 localized on the same cell surface. CD80 and PD-1 were revealed to share a highly overlapping binding site on PD-L1, with the panel of VHHs identified expected to inhibit CD80 binding. Comparison of the CD80 and PD-1 binding sites on PD-L1 enabled the identification of a potential antibody binding region able to confer specificity for the inhibition of PD-1 binding only, which may offer therapeutic benefits to counteract cancer cell evasion of the immune system.

Sequence-specific 1H, 13C and 15N backbone NMR assignments for the N-terminal IgV-like domain (D1) and full extracellular region (D1D2) of PD-L1.

The co-inhibitory immune checkpoint interaction between programmed cell death-protein 1 (PD-1) and programmed cell death-ligand 1 (PD-L1) serves to regulate T-cell activation, promoting self-tolerance. Over-expression of PD-L1 is a mechanism through which tumour cells can evade detection by the immune system. Several therapeutic antibodies targeting PD-L1 or PD-1 have been approved for the treatment of a variety of cancers, however, the discovery and development of small-molecule inhibitors of PD-L1 remains a challenge. Here we report comprehensive sequence-specific backbone resonance assignments (1H, 13C, and 15N) obtained for the N-terminal IgV-like domain of PD-L1 (D1) and the full two domain extracellular region (D1D2). These NMR assignments will serve as a useful tool in the discovery of small-molecule therapeutics targeting PD-L1 and in the characterisation of functional interactions with other protein partners, such as CD80.

In Vitro Assessment of Putative PD-1/PD-L1 Inhibitors: Suggestions of an Alternative Mode of Action.

The programmed cell death protein 1 (PD-1) signaling axis is among the most important therapeutic targets in modern oncology. Aurigene Discovery Technologies Ltd. (Aurigene) has patented a series of peptidomimetic small molecules derived from the PD-1 protein sequence for use in targeting the interaction between PD-1 and its ligand, PD-L1. We evaluated three of Aurigene's most potent compounds in SPR binding assays. Our results showed that these compounds-each of which is known to be potently effective in a splenocyte recovery assay-do not directly inhibit the PD-1/PD-L1 interaction nor do they appear to bind to either of the constituent proteins, indicating that another mechanism is at play. As a result of these studies and upon consideration of structural features within the PD-1/PD-L1 complex, we hypothesize that the Aurigene molecules may interact with a currently unknown protein capable of regulating the PD-1 axis.