The treatment of solid tumors remains an area of high unmet need for patients, largely due to the biological and structural complexities of the tumor itself. These tumors present a formidable barrier to effective treatment due to their intrinsic cellular heterogeneity and the complex dynamics of the tumor microenvironment (TME). The TME, characterized by dense extracellular matrices and hypoxic conditions impedes therapeutic delivery and facilitates tumor progression. Additionally, the high adaptability of cancer cells within this environment contributes to resistance against conventional therapies. These limitations need the development of innovative therapeutic platforms which surpass the performance of traditional monoclonal antibodies. In this context, Isogenica is empowering the development of bi-specific VHH antibodies – a revolutionary class of small, modular therapeutics designed to address the intricate challenges of tumor targeting. This blog delves into the molecular underpinnings, technological advancements, and clinical implications of these groundbreaking therapeutics in oncology.

Understanding the Barriers: Tumor Complexity and Current Limitations

Three primary challenges underscore the difficulties faced by researchers:

1. Tumor Heterogeneity: The phenotypic and genotypic variability among cancer cells within a tumor significantly undermines the efficacy of single-target therapies. This heterogeneity fosters adaptive resistance and creates subpopulations of cancer cells that evade treatment, necessitating multifaceted therapeutic strategies.
2. Tumor Microenvironment: The TME comprises physical barriers such as a dense extracellular matrix and biochemical obstacles including hypoxia, acidic pH, and immunosuppressive factors. These conditions collectively impede therapeutic agents from reaching tumor cores and limit their overall efficacy.

3. Off-Target Effects: Conventional monoclonal antibodies frequently exhibit suboptimal specificity, leading to unintended interactions with non-tumor tissues. Off-target binding reduces therapeutic efficacy and increases the risk of systemic adverse effects, complicating cancer patient management.

Bi-specific antibodies (bsAbs) have emerged as a promising solution, enabling simultaneous binding to two epitopes or distinct antigens, thereby enhancing specificity and minimizing side effects. These molecules can be tailored to address tumor heterogeneity and achieve a greater antiproliferative impact. However, the first wave of mAb-based therapies has faced challenges around manufacturing efficiency, structural stability and biodistribution, as well as the risk of undesirable Fc-mediated immunological effects.

Bi-specific VHH Antibodies: Precision Meets Innovation

VHH antibodies, with their unique structural and biochemical properties offer solutions to some of these issues. Key advantages of bi-specific VHH antibodies are:

• Compact Size and Structural Stability: Approximately 15 kDa in size, they are uniquely suited for penetrating dense tumor tissues and withstanding the harsh conditions of the TME.

• Dual-Target Precision: By concurrently binding two tumor-associated antigens (TAAs), bi-specific VHH antibodies augment tumor specificity and mitigate resistance mechanisms such as antigen escape, outlined below. This ensures robust targeting of heterogeneous tumor cell populations.

• Low Immunogenicity: Incorporating pre-humanized frameworks significantly reduces the already low immunogenic potential of VHH antibodies, ensuring their compatibility with repeated dosing in clinical settings while minimizing immune-related adverse effects.

• Therapeutic Versatility: These antibodies can be tailored for T-cell engagement, immune modulation, and bi-specific antibody-drug conjugates (ADCs), making them adaptable to various oncological scenarios.

Case study: Evaluating Bi-specific VHH Antibodies in Action

Isogenica’s bi-specific VHH antibodies therapeutic potential was evaluated through proof-of-concept studies against solid tumour target PD-L1. Three individual anti-PD-L1 VHH antibodies, named VHH_01, VHH_04, and VHH_36, were selected from Isogenica’s synthetic libraries. These antibodies were expressed as single protein constructs joined to a T cell engaging (TcE) arm via a flexible linker to create bi-specific molecules, capable of simultaneous binding to targets on both immune and cancer cells.

The first step was to confirm the specificity of these molecules. Flow cytometry data demonstrated the ability of the PD-L1:TcE bispecific molecules to specifically bind both PD-L1+ tumor cells (part A) and TcE+ immune cells (part B) . They were compared to positive controls, based on the clinical anti-PD-L1 mAb durvalumab, and negative controls, which demonstrated that the killing effect of the immune cells was due specifically to binding of the anti-PD-L1 VHHs.

Specific binding of TAA:TcE bi-specific antibodies to TAA+ and TcE+ cells, validated by flow cytometry.

The tumor-killing potential of these antibodies was evaluated using a TAA+ breast cancer cell line and healthy human PBMCs. After 72 hours of co-culture, the results demonstrated effective tumor cell cytotoxicity, with potency values comparable to the clinically validated positive control. EC₅₀ values in the low pM range highlighted the performance of the molecules, with VHH_01 being the most effective, followed by VHH_04 and VHH_36.

Tumor cell cytotoxicity mediated by PD-L1xTcE bi-specifics in MDA-MB-231 breast cancer cells, assessed via LDH assay after 72 hours of co-culture with human PBMCs from healthy donors.

Then, it was essential to confirm that tumor cell killing was directly associated with the activation of TcE+ cells. To achieve this, TcE+ PBMC activation was assessed by measuring the expression of surface markers CD25 and CD69 after 72 hours of co-culture with TAA+ cells and serial dilutions of bispecific antibodies. The results showed robust activation of TcE+ cells. As expected, the negative control elicited no response, reinforcing the immune synapse as the key mechanism of action.

As shown in these early studies, Isogenica’s VHH antibodies showed strong specificity, effective tumor cytotoxicity, and robust activation of immune cells when built into bi-specific formats. This replicates the early success of fragment-based bi-specific T cell engager (BiTE) blinotumomab, as well as those currently in development.

A Benchmark in Precision Oncology

VHH antibodies offer strong binding affinities, high stability to harsh conditions, and the flexibility to be used as building blocks in more complex therapies. This allows innovative drug developers to construct therapies capable of precise tumor targeting with minimal off-target effects. Their compact size ensures superior tissue penetration, overcoming barriers in dense tumor environments, and monogenetic formats streamline manufacturing processes.

Accelerating Antibody Discovery for Modular Therapies

Isogenica’s VHH discovery platform and suite of multimerization approaches is designed to accelerate the early-stage antibody discovery, providing robust validation data to support lead selection. Through the elimination of immunization bias and the ability to screen libraries, this approach ensures that promising candidates move forward efficiently in the development pipeline. 

Unlike other antibody fragments, a huge amount of the early discovery process can be done in fast-growing bacterial expression systems – including testing of different bi-specific combinations – with early results available from just weeks from target validation.

We collaborate with biotech and pharma companies, providing a data-driven service that helps researchers and companies to navigate the complexities of antibody discovery. The preclinical results presented in this case study highlight demonstrates the strength of modular VHHs from Isogenica’s VHH libraries in building more complex therapies, expanding the possibilities for innovative biologics. 

Want to explore how our VHH technology can enhance your research? Get in touch to talk to us about how Isogenica can assist with your next discovery program.