FS222, a CD137/PD-L1 Tetravalent Bispecific Antibody, Exhibits Low Toxicity and Antitumor Activity in Colorectal Cancer Models.

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+ 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+ 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.

Dihydroceramide desaturase and dihydrosphingolipids: debutant players in the sphingolipid arena.

Sphingolipids are a wide family of lipids that share common sphingoid backbones, including (2S,3R)-2-amino-4-octadecane-1,3-diol (dihydrosphingosine) and (2S,3R,4E)-2-amino-4-octadecene-1,3-diol (sphingosine). The metabolism and biological functions of sphingolipids derived from sphingosine have been the subject of many reviews. In contrast, dihydrosphingolipids have received poor attention, mainly due to their supposed lack of biological activity. However, the reported biological effects of active site directed dihydroceramide desaturase inhibitors and the involvement of dihydrosphingolipids in the response of cells to known therapeutic agents support that dihydrosphingolipids are not inert but are in fact biologically active and underscore the importance of elucidating further the metabolic pathways and cell signaling networks involved in the biological activities of dihydrosphingolipids. Dihydroceramide desaturase is the enzyme involved in the conversion of dihydroceramide into ceramide and it is crucial in the regulation of the balance between sphingolipids and dihydrosphingolipids. Furthermore, given the enzyme requirement for O2 and the NAD(P)H cofactor, the cellular redox balance and dihydroceramide desaturase activity may reciprocally influence each other. In this review both dihydroceramide desaturase and the biological functions of dihydrosphingolipids are addressed and perspectives on this field are discussed.

Dihydrosphingomyelin impairs HIV-1 infection by rigidifying liquid-ordered membrane domains.

The lateral organization of lipids in cell membranes is thought to regulate numerous cell processes. Most studies focus on the coexistence of two fluid phases, the liquid crystalline (l(d)) and the liquid-ordered (l(o)); the putative presence of gel domains (s(o)) is not usually taken into account. We show that in phospholipid:sphingolipid:cholesterol mixtures, in which sphingomyelin (SM) promoted fluid l(o) domains, dihydrosphingomyelin (DHSM) tended to form rigid domains. Genetic and pharmacological blockade of the dihydroceramide desaturase (Des1), which replaced SM with DHSM in cultured cells, inhibited cell infection by replication-competent and -deficient HIV-1. Increased DHSM levels gave rise to more rigid membranes, resistant to the insertion of the gp41 fusion peptide, thus inhibiting viral-cell membrane fusion. These results clarify the function of dihydrosphingolipids in biological membranes and identify Des1 as a potential target in HIV-1 infection.

Dihydroceramide intracellular increase in response to resveratrol treatment mediates autophagy in gastric cancer cells.

Resveratrol has both apoptosis and autophagy-promoting activities in different cancer cells. Dihydroceramide is the immediate precursor of the apoptotic mediator ceramide in the de novo sphingolipid synthesis pathway. Here we demonstrate that resveratrol induces autophagy in HGC-27 cells, with no sign of cell death. Autophagy occurs after an increase in dihydroceramides by inhibition of dihydroceramide desaturase. The effects of resveratrol are mimicked by a dihydroceramide desaturase inhibitor. These results demonstrate that resveratrol-induced autophagy occurs with a rise in intracellular dihydroceramide levels as the result of inhibition of dihydroceramide desaturases activity and that dihydroceramide accumulation is responsible for autophagy promotion.

Synthesis and biological activity of a novel inhibitor of dihydroceramide desaturase.

A novel mechanism-based dihydroceramide desaturase inhibitor (XM462) in which the substrate C5 methylene group is replaced by a sulfur atom is reported. Dihydroceramide desaturase inhibition occurred both in vitro and in cultured cells with IC(50) values of 8.2 and 0.78 microM, respectively, at a substrate concentration of 10 microM. In vitro experiments showed that XM462 produced a mixed-type inhibition (K(i)=2 microM, alpha=0.83). LC-MS analyses showed that accumulation of endogenous dihydroceramides occurred in cells upon treatment with XM462 in serum-free medium, whereas ceramides built up in controls. In addition, XM462 was found to be metabolised to its 1-glucosyl and 1-phosphocholine derivatives, and to the products of N-deacylation and reacylation with palmitoyl and stearoyl groups. In Jurkat A3 cells cultured in serum-free medium, viability, as the percentage of trypan blue unstained cells in total cells, was reduced upon XM462 treatment (5 microM, 24 h), but not in controls. The interest of this compound is discussed.

Co-expression of the mosquitocidal toxins Cyt1Aa and Cry11Aa from Bacillus thuringiensis subsp. israelensis in Asticcacaulis excentricus.

The cyt1Aa gene from Bacillus thuringiensis subsp. israelensis (Bti), whose product synergizes other mosquitocidal toxins, and functions as a repressor of resistance developed by mosquitoes against Bacilli insecticides, was introduced into the aquatic Gram-negative bacterium Asticcacaulis excentricus alongside the cry11Aa gene. The genes were introduced as an operon, but although mRNA was detected for both genes, no Cyt1Aa toxin was detected. Both proteins were expressed using a construct in which a promoter was inserted upstream of each gene. Recombinant A. excentricus expressing both toxins was found to be approximately twice as toxic to third instar larvae of Culex quinquefasciatus as transformants expressing just Cry11Aa.