Sialic Acid-Engineered IL4-10 Fusion Protein is Bioactive and Rapidly Cleared from the Circulation.

PURPOSE: Modulating sialylation of therapeutic glycoproteins may be used to influence their clearance and systemic exposure. We studied the effect of low and high sialylated IL4-10 fusion protein (IL4-10 FP) on in vitro and in vivo bioactivity and evaluated the effect of differential sialylation on pharmacokinetic parameters. METHODS: CHO cell lines producing low (IL4-10 FP lowSA) and high sialylated (IL4-10 FP highSA) fusion protein were generated. Bioactivity of the proteins was evaluated in an LPS-stimulated whole blood assay. Pharmacokinetics were studied in rats, analyzing plasma levels of IL4-10 FP upon intravenous injection. In vivo activity was assessed in an inflammatory pain mice model upon intrathecal injection. RESULTS: IL4-10 FP lowSA and IL4-10 FP highSA had similar potency in vitro. The pharmacokinetics study showed a 4-fold higher initial systemic clearance of IL4-10 FP lowSA, whereas the calculated half-life of both IL4-10 FP lowSA and IL4-10 FP highSA was 20.7 min. Finally, both IL4-10 FP glycoforms inhibited persistent inflammatory pain in mice to the same extent. CONCLUSIONS: Differential sialylation of IL4-10 fusion protein does not affect the in vitro and in vivo activity, but clearly results in a difference in systemic exposure. The rapid systemic clearance of low sialylated IL4-10 FP could be a favorable characteristic to minimize systemic exposure after administration in a local compartment.

New insights in Type I and II CD20 antibody mechanisms-of-action with a panel of novel CD20 antibodies.

Based on their mechanisms-of-action, CD20 monoclonal antibodies (mAbs) are grouped into Type I [complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)] and Type II [programmed cell death (PCD) and ADCC] mAbs. We generated 17 new hybridomas producing CD20 mAbs of different isotypes and determined unique heavy and light chain sequence pairs for 13 of them. We studied their epitope binding, binding kinetics and structural properties and investigated their predictive value for effector functions, i.e. PCD, CDC and ADCC. Peptide mapping and CD20 mutant screens revealed that 10 out of these 11 new mAbs have an overlapping epitope with the prototypic Type I mAb rituximab, albeit that distinct amino acids of the CD20 molecule contributed differently. Binding kinetics did not correlate with the striking differences in CDC activity among the mIgG2c mAbs. Interestingly, chimerization of mAb m1 resulted in a mAb displaying both Type I and II characteristics. PCD induction was lost upon introduction of a mutation in the framework of the heavy chain affecting the elbow angle, supporting that structural changes within this region can affect functional activities of CD20 mAbs. Together, these new CD20 mAbs provide further insights in the properties dictating the functional efficacy of CD20 mAbs.

A novel bispecific antihuman CD40/CD86 fusion protein with t-cell tolerizing potential.

Clinical trials designed to achieve tolerance in humans by selectively antagonizing one of the T-cell costimulatory pathways, CD40-CD40L or CD80/CD86-CD28, are pending. However, simultaneous blockade of both pathways synergistically prevented graft rejection and successfully induced donor-specific tolerance in animal models. Synergism is also supported in human T-cells in vitro following anti-CD86 mAb and anti-CD40 mAb blockade. Therefore, in our view the most promising clinical strategy would be to antagonize both CD40 and CD86. Fast clinical entrance of this anti-CD86 and anti-CD40 bidirectional concept is highly facilitated by a single molecule approach. In the present study, a single bispecific fusion protein was constructed that specifically binds human CD40 and CD86 and which combines the antagonistic activities of both anti-CD40 and anti-CD86 humanized mAb. The anti-CD40/86 fusion protein showed tolerance inducing potential as it prevented both allogeneic T-cell expansion and generation of cytotoxic effector T cells and induced anergic antigen specific regulatory T cells. These data provide proof of concept in successfully combining the antagonistic activity of two humanized mAb with great clinical potential in transplantation and autoimmunity, in one single molecule.

Preclinical assessment of anti-CD40 Mab 5D12 in cynomolgus monkeys.

Monoclonal antibody (Mab) 5D12 is a potent antagonist of the CD40-CD40L pathway. This cellular interaction has been validated in a large number of experimental animal models where dys-regulation of the immune system plays a role. Chimeric 5D12 (ch5D12) was constructed to reduce the potential immunogenicity and enhance the in vivo half-life when used in humans. ch5D12 is a molecularly engineered human IgG(4) antibody containing the variable domains of the heavy and light chains of the murine version of 5D12 (mu5D12). This new chimeric Mab was tested in a marmoset experimental autoimmune encephalomyelitis model and was shown to effectively prevent disease symptoms. The results of this in vivo evaluation supported clinical use of ch5D12 for immune targeted diseases. Therefore GMP material was prepared and a GLP-compliant tissue cross-reactivity study on human tissues (3 donors/37 tissues) and cynomolgus tissues (2 donors/37 tissues) was performed. ch5D12 stained on the surface of B cells and selected dendritic cells and no unexpected cross-reactivity was observed. The identical staining patterns in human and cynomolgus tissues justified the use of cynomolgus monkeys as a relevant model for humans. A GLP-compliant safety and tolerability evaluation for ch5D12 in cynomolgus monkeys was performed using the GMP produced material. Weekly administration of ch5D12 at two dose levels for 4 weeks was shown to be safe and without any side effects in all monkeys.

Cloning and expression of cDNA coding for bouganin.

Bouganin is a ribosome-inactivating protein that recently was isolated from Bougainvillea spectabilis Willd. In this work, the cloning and expression of the cDNA encoding for bouganin is described. From the cDNA, the amino-acid sequence was deduced, which correlated with the primary sequence data obtained by amino-acid sequencing on the native protein. Bouganin is synthesized as a pro-peptide consisting of 305 amino acids, the first 26 of which act as a leader signal while the 29 C-terminal amino acids are cleaved during processing of the molecule. The mature protein consists of 250 amino acids. Using the cDNA sequence encoding the mature protein of 250 amino acids, a recombinant protein was expressed, purified and characterized. The recombinant molecule had similar activity in a cell-free protein synthesis assay and had comparable toxicity on living cells as compared to the isolated native bouganin.