To expand the repertoire of multi-specific designs for other antibody isotypes, we present here engineering of the first heterodimeric IgA Fc with high purity, native IgA-like stability, and retained ability to bind FcαRI. This newly designed scaffold provides a first-in-class stable and manufacturable multispecific IgA platform capable of activation of neutrophils via FcαRI. A multispecific IgA platform, in turn, provides access to new biological pathways for next-generation IgA-based immunotherapies, and thus, functionalities that are untapped by IgG.

Using insights from the crystal structure of anti-Hu/Cy CD3 antibody ADI-26906 in complex with CD3 epsilon (CD3ε) and antibody engineering using a yeast-based platform, we have derived high-affinity CD3 antibody variants with very low polyreactivity and significantly improved biophysical developability. Comparison of these variants with CD3 antibodies in the clinic (as part of bi- or multispecifics) shows that affinity for CD3ε is correlated with polyreactivity. Our engineered CD3 antibodies break this correlation, forming a broad affinity range with no to low polyreactivity. 

TrYbe is a multispecific, Fc free, therapeutic antibody format. The design consideration for this fragment-based therapeutic format will be discussed, both in terms of the functional biology and the molecular properties. Data from multiple programs will be shared that exemplify a range of functional activities, demonstrate some beneficial properties of target engagement with respect to immune complex formation, and show consistent in vivo PK from albumin binding, efficient and high yielding production at manufacturing scale, and long shelf-life stability. These data will demonstrate that the TrYbe antibody format has a range of properties that make it an excellent candidate for a multi-specific, Fc free, therapeutic.  

DG motif in CDR3 can pose potential liabilities in antibodies by succinimide or iso-Asp formation, depending on the formulation pHs. This can also manifest as basic charge variant formation in accelerated or long-term stability studies. The consequence may or may not impact biological potencies. Here the authors showed that the DG motif can form succinimide in acidic formulations and had reduced potency, but can, nevertheless, be rescued by incubation at higher pHs to promote Asp/Iso-Asp formation and rescue the activities.

Bispecific anti-CD3 T cell engagers (TCEs) show promise in cancer immunotherapy through the engagement of T cells and tumor cells. TCE discovery is challenged by the need to balance tumor-killing potency, toxicity, developability, and cynomolgus monkey (cyno) cross-reactivity. This presentation demonstrates how to overcome these challenges with machine learning (ML)-driven epitope steering and mammalian-display antibody libraries to efficiently discover diverse TCE arms tuned for potency, toxicity, developability, and cyno cross-reactivity.

• TCE discovery balances potency, toxicity, and developability
• Potency and toxicity of TCE arms are dependent on combined epitopes and affinities
• ML and mammalian-display enable tuning TCE epitopes, affinities, and developability

A novel protein-drug conjugate (PDC) platform has been developed, which exploits small protein domain binders to deliver homogeneous conjugates in monospecific, biparatopic and bispecific formats. Through this innovation, differentiated PDCs have been generated which cause sustained regressions in in vivo cancer models against high-value targets including:   

• ROR1: mono- and bispecific PDCs that exploit the co-expression pattern of this target; and 
• ALPP/ALPPL2: next-generation biparatopic PDCs for the treatment of solid tumours​

Lung cancers harboring MET genetic alterations respond well to selective TKIs, but benefit only 4-5% of lung cancers. We conjugated a biparatopic METxMET antibody to a cytotoxin to generate a MET ADC (METxMET-M114) that could treat ~25% of lung cancers. Overall, our findings suggest that the MET ADC, which takes advantage of the unique trafficking properties of our antibody, is a promising candidate for the treatment of MET-overexpressing tumors. 

Comprehensive comparison of formats for the generation of robust bispecific antibodies through fusion of single-domain VHH and Fab into IgG scaffolds and a toolbox of complementary methods for in-depth analysis of key features, such as in-solution dual antigen binding, thermal stability, and aggregation propensity, to ensure high bsAb quality. Furthermore, novel set of in silico designed humanized VHH antibody phage display libraries with maximal functional diversity and CDR3 lengths from 10 to 25 aminoacids for generating fusion partners.

The preclinical manufacturability of bispecific antibodies is a multifaceted challenge that requires careful consideration of molecule design, expression systems, purification strategies, and analytical tools. Overcoming these challenges is essential for transitioning these promising therapeutic agents from discovery-based scaffold design into clinical development. In our studies we applied combinations of both molecular and purification strategies to optimize antibody production, resulting in fit-for-platform processes for ease of manufacture at larger scales.

SAIL66 is a trispecific antibody composed of an anti-CLDN6 arm and an arm for T cell recruiting. SAIL66 has a high specificity for CLDN6, despite its high similarity to CLDN3, CLDN4, and CLDN9, suggesting no undesirable off-target toxicity. This presentation will describe our screening strategy, including the optimization process, for achieving acceptable developability. Clinical studies of SAIL66 are currently underway for patients with solid cancers.

• Next generation of T cell engager targeting CLDN6
• High specificity for CLDN6 to avoid off-target toxicity
• Developability of trispecific antibody

Although many different bispecific antibody formats have been used in clinical trials for various diseases, the number of approved bispecifics is still limited, partly due to the poor developability of bispecific antibodies often observed in early development. In this talk we will share the developability of a panel of commercial and advanced clinical stage bispecific molecules to provide a benchmark for the physicochemical property and developability profiles of bispecific antibodies.