Targeting CTNNB1-S37F: TCR-T Cell Therapy for Solid Tumors

Targeting CTNNB1-S37F: A Public Driver Neoantigen

Engineering T Cell Therapy for Solid Tumors

Background: Neoantigens & T Cell Therapy

Tumors carry somatic mutations which can produce neoepitopes.

Most mutations (< 3%) are rarely recognized by natural T cells.

TCR-engineered T cells can be designed to target specific neoantigens.

Strategy: Shift from 'Private' (patient-specific) to 'Public' (shared) antigens.

Neoantigens: How T cells see tumors

Driver mutations are essential for cancer growth and survival.

They are uniformly expressed, preventing immune escape/antigen loss.

Predecessors: KRAS and TP53 experimental therapies show promise.

Neoantigen = peptide created by tumor mutation

T cells cannot see proteins directly; they see peptides bound to HLA

HLA type determines presentation (not every patient presents the same mutation)

<strong>Private neoantigens:</strong> Unique to one patient

<strong>Shared neoantigens:</strong> Same mutation in many patients (e.g., CTNNB1-S37F)

Why CTNNB1 / β-Catenin?

CTNNB1 encodes β-catenin, a central component of the Wnt signaling pathway.

Mutated in ~3.2% of all cancers (hotspot in exon 3).

Mechanism: Mutation prevents degradation → Constitutive oncogenic signaling.

Engineering T cells to target CTNNB1-S37F

TCR-T cells: T cells engineered to express a specific TCR

Specific TCRs identified: <br>• TCRA2-2 for HLA-A*02:01 <br>• TCRA24 for HLA-A*24:02

Derived from healthy donors to avoid immune tolerance

Precision: Only T cells with the right HLA can recognize the peptide

The Key Mutation: S37F

S37F is a recurrent hotspot mutation alongside S45F and T41A. It generates peptides predicted to bind with high affinity to common HLA alleles (HLA-A*02:01 and HLA-A*24:02).

How do we know the peptide is actually presented?

Prediction is not enough: need to confirm peptide is physically on HLA

Mass spectrometry identifies physically presented peptides on HLA molecules

CTNNB1-S37F peptides were confirmed on tumor cells

Conclusion: They are visible targets for T cells

Confirming the Target Presentation

Mass spectrometry validated S37F peptide presentation on tumor surface.

HLA-A*24:02 : SYLDSGIHF

HLA-A*02:01 : YLDSGIHFGA

Central Question

Research Question

Can a shared oncogenic mutation generate naturally presented neoantigens that can be safely and effectively targeted by TCR-T cells?

Engineering S37F-Specific TCR-T Cells

Source: T cell receptors (TCRs) identified from healthy donors.

Engineering: Transducing T cells to express these high-affinity TCRs.

Mechanism: Specific recognition of tumor cells carrying the S37F mutation.

CTNNB1-S37F peptides are presented on HLA-A02:01 & A24:02

Mass spectrometry confirmed peptides are physically bound to HLA on tumor cells

<strong>Result:</strong> Only the mutant peptide was detected (WT peptide not recognized)

This confirms that T cells have a real, specific target to see

This differentiation is critical for safety (sparing healthy cells)

In Vivo Efficacy: Complete Eradication

Models: Melanoma (natural S37F) and Endometrial Adenocarcinoma (PDX). Results demonstrated rapid tumor regression compared to control groups.

Identifying TCRs that recognize CTNNB1-S37F

Naive T cells from HLA-matched healthy donors were primed with peptide

Reactive cells were sorted to identify specific TCR sequences

<strong>Found:</strong> TCRA2-2 (for HLA-A*02:01) and TCRA24 (for HLA-A*24:02)

High sensitivity: EC50 values indicate potent recognition

<strong>Action:</strong> In vitro killing of melanoma cell lines confirmed

Why This Matters: Addressing an Unmet Need

Target for 'Undruggable' Cancers: Providing options for β-catenin-mutated tumors resistant to standard drugs.

Recurrent & Public: A single therapy design could treat multiple patients effectively.

Proof of Concept: Validates TCR-T therapy for solid tumor driver mutations.

TCR-T cells eradicate tumors in mice & organoids

Mice with melanoma or PDX tumors treated with TCRA2-2 / TCRA24

<strong>Efficacy:</strong> 100% tumor-free early; long-term survival in 6/7 mice

Organoids (human tumors in vitro) also eradicated → confirms human relevance

<strong>No Exhaustion:</strong> Biomarkers (PD-1, TIM-3) remained low

Promising safety and durability profile

Summary & Take-Home Message

CTNNB1-S37F is a prime public driver neoantigen.

High-affinity TCRs can be isolated from healthy donors.

TCR-T cells achieve complete tumor eradication in vivo.

What we learned

Limitations & Considerations

<strong>HLA Restriction:</strong> Only a subset of patients (A*02:01 / A*24:02) can benefit

<strong>Models:</strong> Mice may not fully replicate human immune system complexity

PDX/organoids help but lack whole-body response dynamics

<strong>Heterogeneity:</strong> Combination therapy may be needed for long-term success

<strong>Clinical Translation:</strong> Patient selection is key (Mutation + HLA + Performance status)

Potential in Precision Immunotherapy

<strong>Scale:</strong> ~3,885 patients/year in US with CTNNB1-S37F + correct HLA could benefit

<strong>Strategy:</strong> Future therapies could combine multiple shared driver mutation TCRs

<strong>Vision:</strong> Paves the way for "Off-the-shelf" TCR-T libraries targeting solid tumors