Understanding the interplay of antibody geometry with optimal paratope affinity, valency, and target epitope is critical to identifying lead multispecifics. The increased complexity and number of paratopes involved necessitates robust protein engineering strategies, empirical format screening, and biophysical characterization to overcome challenges in the design process and evaluation of multispecific antibodies. Here we present a workflow for parallel paratope optimization and high throughput antibody format screening to decrease inefficiencies.

Employing modular biotherapeutics is an effective method for maintaining pipeline productivity, particularly for smaller companies. The ADAPTIR and ADAPTIR-FLEX platform technologies allow for rapid development of novel multispecific biotherapeutics. This talk will outline isolation, characterization, and optimization of binding domains and their incorporation into our platforms to accelerate the process of drug discovery through in-human trials. I will be highlighting our clinical, Mipletamig (APVO436) and ALG.APV-527, as well as preclinical, assets.

We have developed a new generation of tumor-activated T Cell Engagers (TCEs) designed for the treatment of solid tumors, featuring an optimized balance of potency and safety. Although TCEs are potent therapeutic agents, their application in solid tumors has been limited by a narrow therapeutic window. Our Tumor Microenvironment Activated Therapeutics (T-MATE™) Platform addresses this challenge by leveraging the tumor acidity as a safety switch. T-MATEs undergo a pH-dependent conformational change that allows them to retain high activity in the acidic pH typical of the tumor microenvironment while exhibiting minimal activity at physiological pH. This technology significantly enhances the therapeutic window and enables targeting of tumor antigens that are otherwise difficult to address with conventional TCEs.

Multispecific antibodies mark the next generation of antibody based biotherapeutics. With new technologies evolving, new challenges and new solutions have been quickly introduced. Engineered cysteines were recently developed to enhance the stability of multispecific antibodies; however, expression of these “stapled” molecules results in high levels of aggregation in bioreactors. Here, we describe a multifaceted approach to fully characterize the underlying mechanism of aggregation in multispecific antibodies.

• Prioritizing parameters to screen in early discovery (format, paratopes, affinity, valency, etc.)
• Purification and characterization of multispecific antibodies and product-related impurities
• Challenges with benchmarking developability criteria for multispecific antibodies

• What Proteins/Peptides with intracellular targets are on the market?
• What are current approaches for penetrating the cell membrane?
• Pros and cons of current approaches like CCPs, nanodiscs, LNPs, pcAAVs?
• What are the major challenges in cell membrane penetration and tissue localization? 

ADCs (antibody-drug conjugates) are complex molecules that link a cytotoxic drug to a protein carrier. Fully characterizing their in vitro/in vivo behaviors is crucial for design and lead selection. In this presentation, I will provide an example to demonstrate how we utilize a combination of immunoassays and state-of-the-art mass spectrometry to assess linker stability and ADC biotransformation. Moreover, I will discuss the application of in vitro assays to evaluate linker processing and payload delivery, and how this valuable information can guide the design process.

The focus of this presentation is to describe a case study where the stability "sweet spot" of a "biosimilar" ADC was identified in the amount of time that is generally considered impossible. The results may challenge the current paradigm for ADC "developabilty" assessment, including suitable analytical strategy, as well as formulation development.

This talk will focus on two case studies of molecular characterization and unique developability challenges. First, in an HIV biotherapeutic with enhanced cross-reactivity across diverse strains. Second, in a novel anti-TIGIT antibody with optimized species cross-reactivity and manufacturability. By integrating advanced molecular modeling and biophysical validation, we demonstrate how AlphaBind enables rapid, precise optimization of complextherapeutics. This approach highlights innovative strategies for characterizing next-generation biotherapeutics using tailored approaches.

Given the drawbacks of drug carriers (lipid nanoparticles, viruses, etc), we have developed a novel macromolecular assembly called a Drug Router which engages the biophysics of tubule formation for delivery. This talk will discuss both the comparable molecular characterization to nanoparticles, but also the Router’s more tailored analysis for its contrasting components. In addition, as the Routers function by forming tubule networks, we will discuss the unique characterization of these secondary, emergent architectures.

Particle metrology, including size determination and quantification, is critical for advancing the biopharmaceutical industry. Particles in biotherapeutics can be either desired pharmaceutical agents (e.g., lipid nanoparticles, virus-like particles) or impurities (e.g., protein aggregates, extrinsic materials). NIST is developing a suite of reference materials to enable and enhance quantitative measurement of both the desired and the unwanted particulate materials. This presentation showcases recent NIST reference material developments to improve biotherapeutic particle measurements.

Lentiviral vectors (LV) are used for cell and gene therapy applications. Since 2017, nine lifesaving LV-based therapies have been approved for commercialization. Despite this progress, a few bottlenecks remain in terms of LV biomanufacturing. Here, we will propose solutions to improve LV yields, decrease costs, and streamline the biomanufacturing process. Notably, characterizing the LV particles during the production and purification process is key to improving the overall LV recovery.

I will present CLiC (Convex Lens-induced Confinement), a unique single-particle imaging platform that enables simultaneous measurements of the size, mRNA payload, and dynamic properties of nanoparticles, including vaccines, in controlled, cell-like conditions (Kamanzi et al, ACS Nano 2024 and 2021). By imaging individual confined particles during reagent exchange, such as pH changes, we emulate and explore dynamics occurring during manufacturing and within living cells. Collaborating with health scientists, our goal is to correlate these detailed multi-scale data sets with clinical outcomes, creating a comprehensive understanding of vaccine effectiveness and advancing the rational design, optimization, and quality-control of medicines.