Discovery of highly-specific TCR mimic antibodies to WT1

Navigating high homology: Discovery of TCR mimic antibodies with greater specificity to Wilms tumor 1 antigen

The development of TCR mimic (TCRm) antibodies (or TCR-like antibodies) offers a promising new approach for cancer immunotherapy but identifying TCRm antibody candidates with optimal specificity and low off-target effects can be challenging. Here, we look at an antibody discovery workflow that enabled researchers to identify TCRm candidates with higher affinity, potency, and specificity compared to three industry benchmark antibodies to Wilms Tumor 1 antigen (WT1).

TCRm antibodies offer new opportunities in immunotherapy

Current antibody therapies target only extracellular antigens. TCR mimic (TCRm) antibodies are traditional antibodies that mimic T-cell receptor functionality by recognizing unique antigens derived from intracellular proteins presented by human leukocyte antigens (pHLAs) on the surface of cancer cells. In a recent publication1, Authors Jeffrey Molldrem and Dongxing Zha discuss the challenges of targeting intracellular tumor-associated proteins in oncology, as they are typically inaccessible to standard antibody and CAR-T therapies and how recent advances in antibody engineering, particularly TCRm antibodies used in T-cell engagers and antibody-drug conjugates, enable precise targeting of cancer cells, marking a promising step forward in cancer treatment.

In addition to these advances, TCRm antibodies expand the range of targetable antigens while offering the pharmacological advantages and manufacturability of conventional antibodies, making them particularly promising for use in immunotherapies. Yet, their development faces a significant challenge in avoiding off-target recognition of pHLA complexes with high homology, which could result in unintended toxicity.

Identifying Wilms Tumor 1-specific TCRm antibodies

The Wilms Tumor 1 (WT1) protein is recognized as the National Cancer Institute’s top target for cancer immunotherapy as it is overexpressed in several solid tumors and most hematological malignancies.2 WT1 has particular significance as a target for the development of immunotherapies to treat Acute Myeloid Leukemia (AML) and myelodysplastic syndromes (MDS).Identifying antibodies with high specificity to WT1 has been challenging, with only three therapeutic antibodies commercially available. These antibodies have demonstrated the potential for cross-reactivity with M13L and PIGQ peptides presented on HLA.2

A study3 led by Dongxing Zha, PhD, was conducted to identify TCRm antibodies with high specificity for WT1/HLA-A2, important for improving the safety profile of candidate immunotherapies targeting WT1.

Explore more data from the study

Download our poster to explore advancements in highly specific anti-WT1/HLA-A2 TCR-like antibodies and their poteinal in immunotherapy.

The Keyway™ TCRm antibody discovery workflow

The research team utilized the Keyway™ TCRm Antibody Discovery Platform to identify TCRm antibodies with high specificity to WT1/HLA-A2. In this platform, antibody discovery begins with the use of ATX-Gx™ humanized mice, notable for a broad diversity of human antibody sequences and a robust immune response that maximizes the potential for development of an antibody specific to a target of interest. Antibodies produced by ATX-Gx™ mice offer broad epitopic coverage, high affinities, and require little-to-no optimization.

The lead discovery workflow began with the generation of WT1-HLA complex by Immunitrack ApS as immunogen which were used to immunize the mice at Alloy Therapeutics. Human antibody producing B-cells subsequently generated antibodies to the pHLA complex, and once titers were met, the target-specific B-cells were collected and sorted with WT1/HLA-A2 complex and counter selected with control peptide-HLA to ensure specific binding. Single B cells of antigen specific clones were sequenced to analyze the repertoire of initial discovered TCRm antibodies.

Eight lead TCRm antibodies were compared to three commercial reference antibodies targeting WT1/HLA-A2 using kinetic, cell binding, functional, and developability assessments. Lead TCRm antibodies were tested by ELISA for specificity to WT1/HLA-A2 and off-target peptides.  An Alloy Therapeutics proprietary specificity test platform using X-scan (an advanced AI/ML-enabled technology for pHLA display technology library) identified the binding pattern of TCRm antibodies to WT1/HLA-A2 and this information was used to search the human proteome for potential cross-reactive proteins. Two off-target peptides were identified (M13L and PIGQ) and peptide binding was evaluated using cell surface peptide pulse assays.

TCRm leads were then functionally assessed, first in the form of a CD3 bispecific engager measuring in vitro T-cell activation, and as a CAR-T cell in a mouse model measuring in vivo tumor killing efficacy.

Keyway™ identifies high-quality antibody leads

The Keyway™ Platform produced eight lead anti-WT1/HLA-A2 TCRm antibodies with high affinity for the target, with six showing binding affinities at the single-digit and sub-nanomolar levels (FIgure 1). All Keyway™ leads showed equivalent or improved potency compared to benchmark antibodies (Figure 1), and all demonstrated effective T-cell engagement, indicating potential for use in therapeutic applications. Keyway™ leads also showed significantly higher specificity for the target than the benchmark antibodies. Functional analysis of Keyway™ leads in the form of CD3 bispecific engagers showed target-specific T-cell activation and in a CAR-T format, complete tumor killing in vivo. Additional study data is available here.

Figure 1: Keyway™ leads showed significantly better specificity than the benchmark antibodies
 Future directions

While this study focused on WT1, the methodology and findings can be applied to other targets and diseases, expanding the potential impact of TCR mimics in immunotherapy beyond hematological cancers which account for 90% of cancer patients. TCRm antibodies are expanding beyond traditional formats into diverse therapeutic modalities, including bispecific antibodies, antibody-drug conjugates, and engineered cell therapies.4 The effectiveness of TCRm therapies could be enhanced in combination with agents like epigenetic modifiers and IFN-α that increase peptide-HLA presentation on tumor cells. 4 In addition, developing approaches that simultaneously engage co-expressed pHLA complexes or target different HLA classes may help combat resistance mechanisms.4 With the potential for high affinity, high neoantigen specificity, and lower toxicity, TCRm-based therapeutics promise to be a continued focus of immunotherapy development.5

Interested in exploring how Alloy Therapeutics’ Keyway™ TCRm Antibody Discovery Platform can help you accelerate your immunotherapy development? Contact us to discuss your project.