Multi-specific T-cell Engaging Antibodies for Selective Targeting of B-myeloid Mixed-Phenotype Acute Leukemia (MPAL)

Background

Acute leukemias encompass a diverse group of hematologic malignancies characterized by the rapid proliferation of immature blood cells. Among these, Mixed-Phenotype Acute Leukemia (MPAL) presents a unique challenge as it often eludes clear classification under traditional acute lymphoblastic leukemia or acute myeloid leukemia protocols. This ambiguity complicates risk stratification and treatment planning, contributing to poorer remission rates and higher relapse frequencies compared to single-lineage leukemias. Given the aggressive disease course and limited therapeutic guidelines, there is a critical need for treatment strategies that can accurately discriminate malignant blasts from healthy hematopoietic cells and improve patient outcomes. Currently, therapeutic approaches for MPAL largely adapt regimens designed for either ALL or AML, leading to suboptimal efficacy and pronounced toxicity. Standard chemotherapy induces severe myelosuppression, while B-cell– or myeloid-directed immunotherapies risk off-target depletion of healthy cell compartments, causing prolonged cytopenias and immunodeficiency. Single-antigen targeting agents often fail to address the dual-lineage nature of MPAL blasts, resulting in escape variants and treatment resistance. The scarcity of clinical trials and absence of lineage-specific biomarkers further inhibit the development of customized regimens, underscoring the urgent necessity for more selective and effective therapeutic modalities. In addition, some B-lineage leukemias co express CD19 and CD33, providing additional opportunities for therapeutic intervention besides MPAL.

Technology

The researchers have designed novel multi-specific T-cell engagers that combine binding domains for CD19, CD33 and CD3 to selectively recruit T cells to leukemic blasts co-expressing lymphoid and myeloid markers. By incorporating single-chain variable fragments, Fab fragments or VHH nanobodies and tuning binder affinities these constructs achieve high avidity for CD19+CD33+ cells while minimizing activity on healthy cells expressing only one antigen. Initial in vitro screens against engineered double-positive cell lines assessed binding specificity and cytotoxic potency across 53 candidates. In vivo xenograft studies with human T-cells confirmed robust tumor clearance in mixed-phenotype acute leukemia models.

This dual-targeting approach differentiates itself by requiring simultaneous engagement of both CD19 and CD33, enhancing selectivity and reducing collateral damage typical of single-antigen therapies. Fine-tuning antigen affinities exploits the unique co-expression pattern of mixed-phenotype acute leukemia, sparing normal B-cells and myeloid cells that express only one marker. The modular design enables systematic optimization of binding formats and affinities, while comprehensive preclinical data demonstrate superior safety and efficacy over conventional CD19- or CD33-directed approaches.

Benefits

• High selectivity for CD19+CD33+ leukemic blasts, sparing healthy single‐antigen cells
• Minimized off-tumor toxicity (reduced B-cell aplasia and myeloablation)
• Optimized therapeutic window through affinity-tuned binding
• Demonstrated robust in vivo tumor reduction in xenograft models
• Dual-antigen targeting overcomes limitations of single-target therapies

Additional info

Patent pending

For any questions, email kamya.rajaram@seattlechildrens.org