Development of tibulizumab, a tetravalent bispecific antibody targeting BAFF and IL-17A for the treatment of autoimmune disease.

We describe a bispecific dual-antagonist antibody against human B cell activating factor (BAFF) and interleukin 17A (IL-17). An anti-IL-17 single-chain variable fragment (scFv) derived from ixekizumab (Taltz®) was fused via a glycine-rich linker to anti-BAFF tabalumab. The IgG-scFv bound both BAFF and IL-17 simultaneously with identical stoichiometry as the parental mAbs. Stability studies of the initial IgG-scFv revealed chemical degradation and aggregation not observed in either parental antibody. The anti-IL-17 scFv showed a high melting temperature (Tm) by differential scanning calorimetry (73.1°C), but also concentration-dependent, initially reversible, protein self-association. To engineer scFv stability, three parallel approaches were taken: labile complementary-determining region (CDR) residues were replaced by stable, affinity-neutral amino acids, CDR charge distribution was balanced, and a H44-L100 interface disulfide bond was introduced. The Tm of the disulfide-stabilized scFv was largely unperturbed, yet it remained monodispersed at high protein concentration. Fluorescent dye binding titrations indicated reduced solvent exposure of hydrophobic residues and decreased proteolytic susceptibility was observed, both indicative of enhanced conformational stability. Superimposition of the H44-L100 scFv (PDB id: 6NOU) and ixekizumab antigen-binding fragment (PDB id: 6NOV) crystal structures revealed nearly identical orientation of the frameworks and CDR loops. The stabilized bispecific molecule LY3090106 (tibulizumab) potently antagonized both BAFF and IL-17 in cell-based and in vivo mouse models. In cynomolgus monkey, it suppressed B cell development and survival and remained functionally intact in circulation, with a prolonged half-life. In summary, we engineered a potent bispecific antibody targeting two key cytokines involved in human autoimmunity amenable to clinical development.

Generation and characterization of ixekizumab, a humanized monoclonal antibody that neutralizes interleukin-17A.

Interleukin (IL)-17A exists as a homodimer (A/A) or as a heterodimer (A/F) with IL-17F. IL-17A is expressed by a subset of T-cells, called Th17 cells, at inflammatory sites. Most cell types can respond to the local production of IL-17A because of the near ubiquitous expression of IL-17A receptors, IL-17RA and IL-17RC. IL-17A stimulates the release of cytokines and chemokines designed to recruit and activate both neutrophils and memory T-cells to the site of injury or inflammation and maintain a proinflammatory state. IL-17A-producing pathogenic T-cells contribute to the pathogenesis of autoimmune diseases, including psoriasis, psoriatic arthritis, rheumatoid arthritis, and ankylosing spondylitis. This study describes the generation and characterization of ixekizumab, a humanized IgG4 variant IL-17A-neutralizing antibody. Ixekizumab binds human and cynomolgus monkey IL-17A with high affinity and binds rabbit IL-17A weakly but does not bind to rodent IL-17A or other IL-17 family members. Ixekizumab effectively inhibits the interaction between IL-17A and its receptor in binding assays and potently blocks IL-17A-induced GRO or KC secretion in cell-based assays. In an in vivo mouse pharmcodynamic model, ixekizumab blocks human IL-17A-induced mouse KC secretion. These data provide a comprehensive preclinical characterization of ixekizumab, for which the efficacy and safety have been demonstrated in human clinical trials in psoriasis and psoriatic arthritis.

Therapeutic Antibody Engineering To Improve Viscosity and Phase Separation Guided by Crystal Structure.

Antibodies at high concentrations often reveal unanticipated biophysical properties suboptimal for therapeutic development. The purpose of this work was to explore the use of point mutations based on crystal structure information to improve antibody physical properties such as viscosity and phase separation (LLPS) at high concentrations. An IgG4 monoclonal antibody (Mab4) that exhibited high viscosity and phase separation at high concentration was used as a model system. Guided by the crystal structure, four CDR point mutants were made to evaluate the role of hydrophobic and charge interactions on solution behavior. Surprisingly and unpredictably, two of the charge mutants, R33G and N35E, showed a reduction in viscosity and a lower propensity to form LLPS at high concentration compared to the wild-type (WT), while a third charge mutant S28K showed an increased propensity to form LLPS compared to the WT. A fourth mutant, F102H, had reduced hydrophobicity, but unchanged viscosity and phase separation behavior. We further evaluated the correlation of various biophysical measurements including second virial coefficient (A2), interaction parameter (kD), weight-average molecular weight (WAMW), and hydrodynamic diameters (DH), at relatively low protein concentration (4 to 15 mg/mL) to physical properties, such as viscosity and liquid-liquid phase separation (LLPS), at high concentration. Surprisingly, kD measured using dynamic light scattering (DLS) at low antibody concentration correlated better with viscosity and phase separation than did A2 for Mab4. Our results suggest that the high viscosity and phase separation observed at high concentration for Mab4 are mainly driven by charge and not hydrophobicity.

Results of a phase 1 study of AME-133v (LY2469298), an Fc-engineered humanized monoclonal anti-CD20 antibody, in FcγRIIIa-genotyped patients with previously treated follicular lymphoma.

PURPOSE: AME-133v is a humanized monoclonal antibody engineered to have increased affinity to CD20 and mediate antibody-dependent cell-mediated cytotoxicity (ADCC) better than rituximab. Safety, pharmacokinetics, and efficacy were assessed in a phase 1/2 trial in patients with previously treated follicular lymphoma (FL). PATIENTS AND METHODS: AME-133v was characterized in vitro by ADCC and cell binding assays. A phase 1 study was conducted in which 23 previously treated patients with FL were assigned sequentially to one of five dose-escalation cohorts of AME-133v at 2, 7.5, 30, 100, or 375 mg/m(2) weekly × 4 doses. RESULTS: AME-133v showed a 13- to 20-fold greater binding affinity for CD20 and was 5- to 7-fold more potent than rituximab in ADCC assays. Cell binding assays showed AME-133v and rituximab competed for an overlapping epitope on the CD20 antigen, and AME-133v inhibited binding of biotinylated rituximab to CD20 in a concentration-dependent manner. AME-133v was well tolerated by patients and common related adverse events included chills and fatigue. One patient experienced a dose-limiting toxicity of neutropenia. AME-133v showed nonlinear pharmocokinetics with properties similar to rituximab. Selective reduction of B cells during and after AME-133v treatment was shown by flow cytometry of peripheral blood. A partial or complete response was observed in 5 of 23 (22%) patients and the median progression-free survival was 25.4 weeks. CONCLUSIONS: AME-133v was safe and well tolerated at the doses tested. AME-133v showed encouraging results as an anti-CD20 therapy in heavily pretreated FL patients with the less favorable FcγRIIIa F-carrier genotype.