Phase 2b randomized trial of OX40 inhibitor telazorlimab for moderate-to-severe atopic dermatitis.

Background: Telazorlimab is a humanized anti-OX40 monoclonal antibody being studied for treatment of T-cell-mediated diseases. Objective: This randomized, placebo-controlled, phase 2b dose-range finding study investigated efficacy, safety, pharmacokinetics, and immunogenicity of telazorlimab in subjects with atopic dermatitis. Methods: In this 2-part study (NCT03568162), adults (≥18 years) with moderate-to-severe disease were randomized to various regimens of subcutaneous telazorlimab or placebo for 16 weeks' blinded treatment, followed by 38 weeks' open-label treatment and 12 weeks' drug-free follow-up. Telazorlimab treatment groups (following a loading dose) in part 1 were 300 mg every 2 weeks; 300 mg every 4 weeks; or 75 mg every 4 weeks. Part 2 evaluated telazorlimab 600 mg every 2 weeks. The primary end point was percentage change from baseline in Eczema Area and Severity Index (EASI) at week 16. Safety assessments included incidence of treatment-emergent adverse events. Results: The study randomized 313 subjects in part 1 and 149 in part 2. At 16 weeks, the least squares mean percentage change from baseline in EASI was significantly greater in subjects receiving telazorlimab 300 mg every 2 weeks (part 1) and 600 mg every 2 weeks (part 2) versus placebo (-54.4% vs -34.2% for part 1 and -59.0% vs -41.8% for part 2, P = .008 for both). Telazorlimab was well tolerated, with similar distribution of adverse events between telazorlimab- and placebo-treated subjects in both part 1 and part 2. Conclusion: Telazorlimab, administered subcutaneously at 300 mg every 2 weeks or 600 mg every 2 weeks following a loading dose, was well tolerated and induced significant and progressive clinical improvement in adults with moderate-to-severe atopic dermatitis.

Preclinical characterization of ISB 1342, a CD38 × CD3 T-cell engager for relapsed/refractory multiple myeloma.

Although treatment of multiple myeloma (MM) with daratumumab significantly extends the patient's lifespan, resistance to therapy is inevitable. ISB 1342 was designed to target MM cells from patients with relapsed/refractory MM (r/r MM) displaying lower sensitivity to daratumumab. ISB 1342 is a bispecific antibody with a high-affinity Fab binding to CD38 on tumor cells on a different epitope than daratumumab and a detuned scFv domain affinity binding to CD3ε on T cells, to mitigate the risk of life-threatening cytokine release syndrome, using the Bispecific Engagement by Antibodies based on the TCR (BEAT) platform. In vitro, ISB 1342 efficiently killed cell lines with different levels of CD38, including those with a lower sensitivity to daratumumab. In a killing assay where multiple modes of action were enabled, ISB 1342 showed higher cytotoxicity toward MM cells compared with daratumumab. This activity was retained when used in sequential or concomitant combinations with daratumumab. The efficacy of ISB 1342 was maintained in daratumumab-treated bone marrow patient samples showing lower sensitivity to daratumumab. ISB 1342 induced complete tumor control in 2 therapeutic mouse models, unlike daratumumab. Finally, in cynomolgus monkeys, ISB 1342 displayed an acceptable toxicology profile. These data suggest that ISB 1342 may be an option in patients with r/r MM refractory to prior anti-CD38 bivalent monoclonal antibody therapies. It is currently being developed in a phase 1 clinical study.

A single mutation increases heavy-chain heterodimer assembly of bispecific antibodies by inducing structural disorder in one homodimer species.

We previously reported efficient heavy-chain assembly of heterodimeric bispecific antibodies by exchanging the interdomain protein interface of the human IgG1 CH3 dimer with the protein interface of the constant α and ß domains of the human T-cell receptor, a technology known as bispecific engagement by antibodies based on the T-cell receptor (BEAT). Efficient heterodimerization in mammalian cell transient transfections was observed, but levels were influenced by the nature of the binding arms, particularly in the Fab-scFv-Fc format. In this study, we report a single amino acid change that significantly and consistently improved the heterodimerization rate of this format (≥95%) by inducing partial disorder in one homodimer species without affecting the heterodimer. Correct folding and assembly of the heterodimer were confirmed by the high-resolution (1.88-1.98 Å) crystal structure presented here. Thermal stability and 1-anilinonaphthalene-8-sulfonic acid-binding experiments, comparing original BEAT, mutated BEAT, and "knobs-into-holes" interfaces, suggested a cooperative assembly process of heavy chains in heterodimers. The observed gain in stability of the interfaces could be classified in the following rank order: mutated BEAT > original BEAT > knobs-into-holes. We therefore propose that the superior cooperativity found in BEAT interfaces is the key driver of their greater performance. Furthermore, we show how the mutated BEAT interface can be exploited for the routine preparation of drug candidates, with minimal risk of homodimer contamination using a single Protein A chromatography step.

Single-step Protein A and Protein G avidity purification methods to support bispecific antibody discovery and development.

Heavy chain (Hc) heterodimers represent a majority of bispecific antibodies (bsAbs) under clinical development. Although recent technologies achieve high levels of Hc heterodimerization (HD), traces of homodimer contaminants are often present, and as a consequence robust purification techniques for generating highly pure heterodimers in a single step are needed. Here, we describe two different purification methods that exploit differences in Protein A (PA) or Protein G (PG) avidity between homo- and heterodimers. Differential elution between species was enabled by removing PA or PG binding in one of the Hcs of the bsAb. The PA method allowed the avidity purification of heterodimers based on the VH3 subclass, which naturally binds PA and interferes with separation, by using a combination of IgG3 Fc and a single amino acid change in VH3, N82aS. The PG method relied on a combination of three mutations that completely disrupts PG binding, M428G/N434A in IgG1 Fc and K213V in IgG1 CH1. Both methods achieved a high level of heterodimer purity as single-step techniques without Hc HD (93-98%). Since PA and PG have overlapping binding sites with the neonatal Fc receptor (FcRn), we investigated the effects of our engineering both in vitro and in vivo. Mild to moderate differences in FcRn binding and Fc thermal stability were observed, but these did not significantly change the serum half-lives of engineered control antibodies and heterodimers. The methods are conceptually compatible with various Hc HD platforms such as BEAT® (Bispecific Engagement by Antibodies based on the T cell receptor), in which the PA method has already been successfully implemented.

Immunoglobulin domain interface exchange as a platform technology for the generation of Fc heterodimers and bispecific antibodies.

Bispecific antibodies (bsAbs) are of significant importance to the development of novel antibody-based therapies, and heavy chain (Hc) heterodimers represent a major class of bispecific drug candidates. Current technologies for the generation of Hc heterodimers are suboptimal and often suffer from contamination by homodimers posing purification challenges. Here, we introduce a new technology based on biomimicry wherein the protein-protein interfaces of two different immunoglobulin (Ig) constant domain pairs are exchanged in part or fully to design new heterodimeric domains. The method can be applied across Igs to design Fc heterodimers and bsAbs. We investigated interfaces from human IgA CH3, IgD CH3, IgG1 CH3, IgM CH4, T-cell receptor (TCR) α/ß, and TCR γ/δ constant domain pairs, and we found that they successfully drive human IgG1 CH3 or IgM CH4 heterodimerization to levels similar to or above those of reference methods. A comprehensive interface exchange between the TCR α/ß constant domain pair and the IgG1 CH3 homodimer was evidenced by X-ray crystallography and used to engineer examples of bsAbs for cancer therapy. Parental antibody pairs were rapidly reformatted into scalable bsAbs that were free of homodimer traces by combining interface exchange, asymmetric Protein A binding, and the scFv × Fab format. In summary, we successfully built several new CH3- or CH4-based heterodimers that may prove useful for designing new bsAb-based therapeutics, and we anticipate that our approach could be broadly implemented across the Ig constant domain family. To our knowledge, CH4-based heterodimers have not been previously reported.

Structural analysis of the complement control protein (CCP) modules of GABA(B) receptor 1a: only one of the two CCP modules is compactly folded.

The gamma-aminobutyric acid type B (GABA(B)) receptor is a heterodimeric G-protein-coupled receptor. In humans, three splice variants of the GABA(B) receptor 1 (R1) subunit differ in having one, both, or neither of two putative complement control protein (CCP) modules at the extracellular N terminus, prior to the GABA-binding domain. The in vivo function of these predicted modules remains to be discovered, but a likely association with extracellular matrix proteins is intriguing. The portion of the GABA(B) R1a variant encompassing both of its CCP module-like sequences has been expressed, as have the sequences corresponding to each individual module. Each putative CCP module exhibits the expected pattern of disulfide formation. However, the second module (CCP2) is more compactly folded than the first, and the three-dimensional structure of this more C-terminal module (expressed alone) was solved on the basis of NMR-derived nuclear Overhauser effects. This revealed a strong similarity to previously determined CCP module structures in the regulators of complement activation. The N-terminal module (CCP1) displayed conformational heterogeneity under a wide range of conditions whether expressed alone or together with CCP2. Several lines of evidence indicated the presence of native disorder in CCP1, despite the fact that recombinant CCP1 contributes to binding to the extracellular matrix protein fibulin-2. Thus, we have shown that the two CCP modules of GABA(B) R1a have strikingly different structural properties, reflecting their different functions.