Presented By: Christine Rothe, Vice President of Early-Stage-Project Leadership and Data Science at Pieris Pharmaceuticals
Transcribed By: Tia Byer
Pieris Pharmaceuticals aims to develop life-changing therapies for patients suffering from cancer, respiratory, and other diseases through the innovative application of novel science and cutting-edge technologies. Pieris’ proprietary technology, based on human lipocalin proteins, hopes to do just that by specifically and selectively binding desired targets within the body to produce a therapeutic response to both cancer and respiratory diseases. At Biologics UK: Digital Day in September 2021, Pieris’ Christine Rothe presented the latest findings on the promise of bispecific antibody-Anticalin fusions programmes for immuno-oncology.
The Anticalin Protein Platform:
Anticalin proteins are novel therapeutic proteins derived from human lipocalins (human extracellular binding proteins). They are monomeric and monovalently binding, and their small and stable structure makes them ideal for inhalable delivery. Furthermore, the simple and stable structure allows for the easy generation of bi- and multispecific constructs. Pieris has built highly diverse Anticalin protein libraries which are applied in automated selection and characterization workflows to select drug candidates. Anticalin protein-based drug candidates can be tailored to different formats to best address target biology. Formats can include pure Anticalin protein-based fusion proteins or antibody-Anticalin or Anticalin-Fc fusion constructs.
Integrated Anticalin Protein Discovery Process:
Researchers at Pieris select and characterise their biologics by applying the naïve Anticalin protein libraries with their various designs and display technologies. The binders are then screened for functionality and developability before undergoing an Anticalin protein maturation performed by yeast display, allowing for the fast and parallel selection for these characteristics. To select the best pairing, candidates undergo a combination of multiple building blocks for each bispecific. Bispecifics are then funnelled into a characterisation workflow, where they are analysed for binding, functionality, and developability. The final lead is then selected and brought into the Investigational New Drug testing stage.
Antibody-Anticalin Protein Bispecifics in IO: The 4-1BB Franchise
4-1BB is a key co-stimulatory immune receptor that is mainly expressed on activated T cells and other immune cells (e.g., NK cells). 4-1BB is transiently expressed on the T cell after initial activation through TCR signalling and binds to the trimeric 4-1BB ligand expressed on antigen-presenting cells. This binding leads to 4-1BB clustering, which in turn drives the co-stimulatory activity. 4-1BB activation leads to a robust increase in T cell proliferation and cytotoxicity. Its stimulation can also promote metabolic fitness and drive memory formation, resulting in a durable anti-tumour response. Agonistic 4-1BB antibodies have shown signs of clinical efficacy but were hindered by on-target toxicity and thus a limited therapeutic window.
Pieris is designing molecules that mediate T cell activation only in the tumour microenvironment to circumvent these limitations. The lead IO asset currently is Cinrebafusp alfa (PRS-343), which consists of a 4-1BB-targeting Anticalin protein that binds with a moderate affinity of 5nM in a non-competitive mode of binding. The Anticalin protein is fused to an engineered variant of a HER2 targeting antibody.
Through binding of the antibody portion of the bispecific to HER2 expressed on tumour cells, the Anticalin protein promotes clustering of 4-1BB on the T-cell and provides the co-stimulatory signal for T-cell activation. In healthy tissues with nominal HER2 expression, the bound bispecific molecule should not lead to clustering of 4-1BB and therefore should not be able to induce co-stimulation in the periphery. Only in the tumour microenvironment, where there is a high expression of HER2, clustering and T cell activation should occur. This mode of action is intended to lead to safety advantages by reducing systemic toxicity.
By generating different fusion formats, it was possible to identify optimal bispecific geometry for 4-1BB crosslinking and T cell activation (Fig. 1). Whilst all four tested formats had similar binding kinetics to the targets, a clear difference was found upon analysing the molecules in functional assays. The heavy chain C-terminal fusion was identified as the most potent molecule. In the functional assay shown in Figure 1, HER2 expressing tumour cells were incubated with human T-cells. Signal 1 was provided by a sub-optimal concentration of an anti-CD3 antibody. Binding of the bispecific molecule to HER2 expressed on the tumour cell induced clustering of 4-1BB upon binding of the Anticalin protein, thus providing signal 2. Activation of the T-cells was measured in this assay through IL-2 secretion.
The Anticalin Protein Platform: The Future of IO
Pieris’ Anticalin protein platform reliably delivers a novel class of therapeutics with favourable drug-like properties. Cinrebafusp alfa (PRS-343) was the first 4-1BB based bispecific in the industry to enter the clinic, and was engineered to specifically activate 4-1BB in the tumour microenvironment. Studies showed that Cinrebafusp alfa (PRS-343) at dosages of up to 18mg/kg had no significant anti-HER2 or anti-4-1BB safety signal and no dose-limiting toxicity. Dose-dependent immune activation was also demonstrated with an increase in CD8+ T cells, NK cells, and cytotoxic activity in the tumour microenvironment. Increased concentrations of soluble 4-1BB (s4-1BB) in the blood further indicated target engagement of 4-1BB and activation of immune cells. Durable anti-tumour activity in a heavily pre-treated patient population showed preliminary evidence of activity among cold tumour types and HER2-low patients. As Pieris’ leading IO program, Cinrebafusp alfa (PRS-343) provides key validation of the 4-1BB franchise and follow-on programs, including PRS-344 (4-1BB/PD-L1) and PRS-342 (4-1BB/GPC3).