A first-in-human, single and multiple dose study of lunsekimig, a novel anti-TSLP/anti-IL-13 NANOBODY® compound, in healthy volunteers.

Lunsekimig is a novel, bispecific NANOBODY® molecule that inhibits both thymic stromal lymphopoietin (TSLP) and interleukin (IL)-13, two key mediators of asthma pathophysiology. In this first-in-human study, we evaluated the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of lunsekimig in healthy adult participants. Participants received single ascending doses (SAD) of lunsekimig (10-400 mg intravenous [IV] or 400 mg subcutaneous [SC]) (SAD part) or multiple ascending doses (MAD part) of lunsekimig (100 or 200 mg, every 2 weeks [Q2W] for three SC doses), or placebo. Overall, 48 participants were randomized 3:1 in the SAD part and 4:1 in the MAD part for lunsekimig or placebo. The primary endpoint was safety and tolerability. The secondary endpoints included PK, antidrug antibodies (ADAs) and total target measurement. Lunsekimig was well tolerated and common treatment-emergent adverse events were COVID-19, nasopharyngitis, injection site reactions, and headache. Lunsekimig showed dose-proportional increases in exposure and linear elimination. Mean t1/2z of lunsekimig was around 10 days across all IV and SC doses of the SAD and MAD parts of the study. Increases in the serum concentration of total TSLP and IL-13 for lunsekimig versus placebo indicated target engagement. ADA of low titers were detected in four (11.1%) participants who received lunsekimig in the SAD, and seven (43.8%) in the MAD. In conclusion, lunsekimig was well tolerated in healthy participants with a linear PK profile up to single 400 mg IV and SC dose and multiple doses of 100 and 200 mg SC Q2W, with low immunogenicity.

An innovative method for characterizing neutralizing antibodies against antibody-derived therapeutics.

Detection of anti-drug antibodies (ADA) that have a neutralizing capacity is an important aspect of immunogenicity evaluation during development of biotherapeutics, but developing and validating neutralizing antibody (NAb) assays that show direct interference of a biologic function is a challenging and resource-intensive activity. In particular, the need for adequate drug and target tolerance often requires extensive pre-treatment steps that limit assay sensitivity compared with a typical bridging-format assay used to detect binding ADA. Such limitations may complicate data interpretation as a positive ADA followed by a negative NAb result could be due to the presence of non-neutralizing antibodies or could be a false-negative for NAbs due to methodology differences. To address such issues, we developed a novel assay for Nanobodies® and other antibody-derived therapeutics that solely detects ADA directed against the complementarity-determining regions (CDRs) involved in drug-target interactions. This was achieved by creating a "null variant" of the therapeutic drug, which has mutated CDRs rendering it non-functional for target binding but is otherwise identical to the drug compound. Non-CDR-binding antibodies are pre-complexed with the null variant of the Nanobody leaving only CDR-binding ADA with neutralizing potential (ANP) to be detected in this assay, which is called a NAb Epitope Characterization Assay (NECA). Method qualification results confirmed highly comparable assay characteristics (sensitivity, drug tolerance, selectivity and precision) of both the NECA and a validated ADA assay for the same Nanobody. A panel of purified neutralizing and non-neutralizing antibodies as well as non-clinical and clinical samples were used to further substantiate the fit-for-purpose and advantages of this novel assay format to detect ANP. In the clinical case study, a 20 to 40-fold difference in assay sensitivity existed between the validated ADA assay and NAb assay, which complicated data interpretation. Implementation of the NECA allowed unambiguous comparison of the levels of binding ADA and ANP in study samples which enabled us to delineate the true neutralizing capacity of the responses. Depending on the risk of the therapeutic, this method could be a valuable alternative for NAb testing by enabling earlier detection of ADA with neutralizing potential and ensuring adequate immunogenicity risk assessment.

The preclinical pharmacology of the high affinity anti-IL-6R Nanobody® ALX-0061 supports its clinical development in rheumatoid arthritis.

INTRODUCTION: The pleiotropic cytokine interleukin-6 (IL-6) plays an important role in the pathogenesis of different diseases, including rheumatoid arthritis (RA). ALX-0061 is a bispecific Nanobody® with a high affinity and potency for IL-6 receptor (IL-6R), combined with an extended half-life by targeting human serum albumin. We describe here the relevant aspects of its in vitro and in vivo pharmacology. METHODS: ALX-0061 is composed of an affinity-matured IL-6R-targeting domain fused to an albumin-binding domain representing a minimized two-domain structure. A panel of different in vitro assays was used to characterize the biological activities of ALX-0061. The pharmacological properties of ALX-0061 were examined in cynomolgus monkeys, using plasma levels of total soluble (s)IL-6R as pharmacodynamic marker. Therapeutic effect was evaluated in a human IL-6-induced acute phase response model in the same species, and in a collagen-induced arthritis (CIA) model in rhesus monkeys, using tocilizumab as positive control. RESULTS: ALX-0061 was designed to confer the desired pharmacological properties. A 200-fold increase of target affinity was obtained through affinity maturation of the parental domain. The high affinity for sIL-6R (0.19 pM) translated to a concentration-dependent and complete neutralization of sIL-6R in vitro. In cynomolgus monkeys, ALX-0061 showed a dose-dependent and complete inhibition of hIL-6-induced inflammatory parameters, including plasma levels of C-reactive protein (CRP), fibrinogen and platelets. An apparent plasma half-life of 6.6 days was observed after a single intravenous administration of 10 mg/kg ALX-0061 in cynomolgus monkeys, similar to the estimated expected half-life of serum albumin. ALX-0061 and tocilizumab demonstrated a marked decrease in serum CRP levels in a non-human primate CIA model. Clinical effect was confirmed in animals with active drug exposure throughout the study duration. CONCLUSIONS: ALX-0061 represents a minimized bispecific biotherapeutic of 26 kDa, nearly six times smaller than monoclonal antibodies. High in vitro affinity and potency was demonstrated. Albumin binding as a half-life extension technology resulted in describable and expected pharmacokinetics. Strong IL-6R engagement was shown to translate to in vivo effect in non-human primates, demonstrated via biomarker deregulation as well as clinical effect. Presented results on preclinical pharmacological properties of ALX-0061 are supportive of clinical development in RA.