Efficiently Identifying Bispecific Antibodies using High Throughput Single-cell BsAb Discovery Pipeline

Bispecific antibodies (BsAbs) are an emerging type of immunotherapy and have great potential to treat various diseases. Recently, researchers have developed a novel single-cell platform that can efficiently discover rare BsAb clones with unique and desirable properties that would otherwise be close to impossible to discover using other existing conventional low-throughput, biased, and trial-and-error methods. The platform also allows the discovery of generalized designed principles by analysis of the connections between sequence, structure, and function!

The Potential of Bispecific Antibodies 

Bispecific antibodies (BsAbs) are a class of engineered proteins capable of recognizing and binding to two different epitope targets on the same or different antigens. BsAbs can potentially treat various diseases, including cancer and autoimmunity, by activating and engaging immune cells, such as T cells, to kill diseased cells. This occurs because BsAbs is engineered to have one arm recognize and bind a protein on the surface of cancer cells while another arm recognizes and binds a protein on the surface of immune cells. It is known that over 50% of the BsAb preclinical and clinical pipeline corresponds to T cells activating BsAbs (TABs), which directly link T cells and tumor cells to eliminate and treat different types of cancer. 

What makes BsAbs (specifically TABs) a promising approach to disease treatment is that they can overcome many of the limitations of conventional techniques. For instance, the BsAbs approach to cancer treatment does not involve harming healthy cells as other techniques, such as chemotherapy and radiation therapy, rely on. Moreover, TABs have several advantages over the more traditional monoclonal antibodies; they have improved specificity, efficacy, toxicity, and drug resistance. Currently, over 45 CD3-based TABs are being clinically tested for the treatment of solid and hematological cancers.

Challenges of Developing Effective BsAbs Immunotherapy 

Despite the invaluable benefits of using BsAbs as immunotherapy, its development poses several challenges and entails enormous costs. Discovering BsAbs involves three steps;

1. Selecting two monoclonal antibodies with different targets
2. Fusing them to create a BsAbs 
3. Testing their functionality to look for improved binding affinity, enhanced pharmacokinetics, or reduced toxicity. 

However, several factors can affect BsAb functionality, like epitope location, antigen size and density, binding affinity, and linker length and flexibility. Therefore, assessing the functionality of BsAbs is not straightforward but rather complex. Conventionally, a microtiter plate and liquid handling system are used to discover effective BsAbs for immunotherapeutic uses. This low-throughput approach relies on trial and error, is expensive, and has limited accuracy.

A recent paper in Communications Biology by Segaliny et al. reports a high-throughput, single-cell-based functional screening pipeline to address this issue of ineffective BsAb discovery. This approach can screen many individual variants from an unbiased library to identify those with improved functionality and desirable characteristics. 

The Novel Single-Cell Discovery Pipeline & Platform and Their Uses

The high throughput BsAb discovery pipeline was assessed using an established CD19xCD3 bispecific T cell engaged (BiTE) model system. BiTEs are a class of TABs that allow T cells to kill cancer cells. They contain two single-chain fragment variables (scFvs) connected by a flexible linker. One scFv targets CD19 on B cell tumors (tumor antigen), and the other scFv targets CD3 on T cells (T cell epitope). In this recent study, the authors generated a BiTE-expressing cell library consisting of around 22,300 unique variants that were generated by combining varied scFvs, connecting linkers, and VL/VH orientations. They then used a single-cell platform to identify and screen for functional BiTEs that can induce T-cell activation and cytotoxicity.

The single-cell platform consists of various modules, including a molecular and cell engineering module for effectively generating BsAb-variant cell libraries and reporter cells, an opto-electro-mechanical module for droplet-based single-cell functional screening (identifying and sorting desired BsAb clones), and a downstream molecular analysis and validation module.

The single-cell platform consists of various modules, including a molecular and cell engineering module for effectively generating BsAb-variant cell libraries and reporter cells, an opto-electro-mechanical module for droplet-based single-cell functional screening (identifying and sorting desired BsAb clones), and a downstream molecular analysis and validation module.

The droplet microfluidic-based system of the single-cell platform separates and analyzes individual cells that produce BsAbs along with cell reporters. The functional BsAb clones stimulate and induce the cell reporters to emit fluorescence, allowing ‘positive’ droplets to be sorted from a heterogeneous population. This innovative multiplexed orthogonal assay chemistry, multi-point detection, and droplet-indexing strategy ensure screening fidelity.

What is fascinating is that the authors show that the single-cell platform can accommodate one and a half million library cells in a single run, far higher than any other existing method. The analytical performance, including screening efficiency, was characterized using a spiked library prepared with HEK293 cells expressing human CD19 at a suitable level for lymphoma cell lines and secreting a functional CD19xCD3 BiTE. The findings were remarkable – the single-cell platform can confidently isolate rare functional BsAb clones in a single run. For instance, at least one or more copies of a rare functional clone with a 0.008% abundance could be isolated with 95% confidence in a single run. In fact, 98 unique functional clones (including very rare clones of 0.001% abundance and clones bearing scFv connecting peptide linkers) were identified. 

Other than the discovery of functional BsAbs, the single-cell platform was used to identify generalizable principles and design preferences of BsAbs for functionality, such as optimal linker length, scFv orientation, and domain pairing. The platform also discovered BsAb that exhibit novel properties sequence analysis of sorted BsAb clones further validated functionality, such as the most favored orientation and the conserved AA residues for function.

Conclusion and Future Work

To conclude, this platform is adaptable and scalable, has high throughput and screening efficiency, rapid turnaround time, and reduced cost, making it a rather revolutionary system for discovering Bispecific Antibodies and transforming immunotherapy. The platform can also interrogate multiple design variables in a combinatorial, high throughput manner, in turn allowing the deciphering of the design principles of Bispecific Antibodies and obtaining an in-depth understanding of the inter-relationships among BsAb sequence structure and function. Further investigations into more clones of the CD19xCD3 BiTEs discovered from the complex library screening can potentially lead to even more optimal therapeutic candidates. In addition, further single-cell-based functional assays may enhance the platform technology’s usefulness and performance, including immune cell effector function assays capable of assessing functionality parameters such as cytokine secretion, metabolism, and/or tumor killing.

Article Source: Reference Paper

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