No abstract

Despite advances with therapies targeting the programmed cell death protein 1 (PD-1) or its ligand (PD-L1), many cancer patients are refractory to or relapse following treatment. Resistance to anti-PD-1 treatment is associated with upregulation of other checkpoint inhibitor receptors such as LAG-3 (Lymphocyte Activation Gene 3). FS118, currently being evaluated in a Phase 1 clinical trial in patients with advanced malignancies (NCT03440437), is a tetravalent bispecific antibody targeting LAG-3 and PD-L1, two immune checkpoint molecules that promote tumour escape from immune surveillance. We have characterised both in vitro and in vivo the functional activity of FS118 and find that this bispecific antibody can overcome PD-L1 and LAG-3 immune suppressive signals. We report a potential novel mechanism of action not observed with the combination of single PD-L1 and LAG-3 antibodies. Our results indicate that FS118 represents a possible novel approach to overcome some of the mechanism of resistance to PD-(L)1 blockade.

The design of proteins represents a significant challenge to modern-day structural biology. A major obstacle here is the specification of well-packed hydrophobic cores to drive the folding and stabilization of the target. Computational approaches have been used to alleviate this by testing alternate sequences prior to the production and characterization of a few proteins. Here we present the experimental counterpart of this approach. We selected stable variants from a library of ubiquitin hydrophobic-core mutants as follows. Hexahistidine-tagged proteins were displayed on the surface of phage. These proteinphage were immobilized onto Ni-coated surfaces. The bound fusion-phage were treated with protease to remove unstable or poorly folded proteins. Stable phage fusions were eluted and infected into Escherichia coli, which allowed amplification for further selection, sequencing, or protein expression. Two Ni-derivatized supports were tested: Ni-NTA chips for surface plasmon resonance (SPR) and Ni-NTA agarose beads. SPR had an advantage in that the selection process could be monitored directly. This allowed individual clones and experimental conditions to be tested rapidly prior to preparative panning of the library, which was carried out using Ni-NTA agarose beads. We demonstrate the method by selecting stable core mutants of ubiquitin, the characterization of which is described in the following paper [Finucane, M. D., and Woolfson, D. N. (1999) Biochemistry 38, XXXXX-XXXXX]. As our method selects only on the basis of structure and stability, it will be of use in improving the stabilities and structural specificities of proteins of de novo design, and in establishing rules that link sequence and structure.In de novo protein design attempts are made to construct amino acid sequences that adopt prescribed folds. There are two principal reasons for conducting such studies. First, by designing proteins we test hypotheses of how sequence relates to structure. As sequence data are accumulating much faster than structural information, it is becoming even more critical to determine rules that aid protein structure prediction and modeling. Second, successful protein designs would present routes to new proteins with ranges of structures and functions that extend beyond those found in nature.

Purpose: With the increased prevalence in checkpoint therapy resistance, there remains a significant unmet need for additional therapies for patients with relapsing or refractory cancer. We have developed FS222, a bispecific tetravalent antibody targeting CD137 and PD-L1, to induce T-cell activation to eradicate tumors without the current toxicity and efficacy limitations seen in the clinic. Experimental Design: A bispecific antibody (FS222) was developed by engineering CD137 antigen-binding sites into the Fc region of a PD-L1 IgG1 mAb. T-cell activation by FS222 was investigated using multiple in vitro assays. The antitumor efficacy, survival benefit, pharmacodynamics, and liver pharmacology of a murine surrogate molecule were assessed in syngeneic mouse tumor models. Toxicology and the pharmacokinetic/pharmacodynamic profile of FS222 were investigated in a non-human primate dose-range finding study. Results: We demonstrated simultaneous binding of CD137 and PD-L1 and showed potent T-cell activation across CD8 þ T-cell activation assays in a PD-L1-dependent manner with a CD137/PD-L1 bispecific antibody, FS222. FS222 also activated T cells in a human primary mixed lymphocyte reaction assay, with greater potency than the monospecific mAb combination. FS222 showed no signs of liver toxicity up to 30 mg/kg in a non-human primate dose-range finding study. A surrogate molecule caused significant tumor growth inhibition and survival benefit, concomitant with CD8 þ T-cell activation, in CT26 and MC38 syngeneic mouse tumor models. Conclusions: By targeting CD137 agonism to areas of PD-L1 expression, predominantly found in the tumor microenvironment, FS222 has the potential to leverage a focused, potent, and safe immune response augmenting the PD-(L)1 axis blockade.

FS102 is a HER2-specific Fcab (Fc fragment with antigen binding), which binds HER2 with high affinity and recognizes an epitope that does not overlap with those of trastuzumab or pertuzumab. In tumor cells that express high levels of HER2, FS102 caused profound HER2 internalization and degradation leading to tumor cell apoptosis. The antitumor effect of FS102 in patient-derived xenografts (PDXs) correlated strongly with the HER2 amplification status of the tumors. Superior activity of FS102 over trastuzumab or the combination of trastuzumab and pertuzumab was observed in vitro and in vivo when the gene copy number of HER2 was equal to or exceeded 10 per cell based on quantitative polymerase chain reaction (qPCR). Thus, FS102 induced complete and sustained tumor regression in a significant proportion of HER2-high PDX tumor models. We hypothesize that the unique structure and/or epitope of FS102 enables the Fcab to internalize and degrade cell surface HER2 more efficiently than standard of care antibodies. In turn, increased depletion of HER2 commits the cells to apoptosis as a result of oncogene shock. FS102 has the potential of a biomarker-driven therapeutic that derives superior antitumor effects from a unique mechanism-of-action in tumor cells which are oncogenically addicted to the HER2 pathway due to overexpression.