Abstract
Acute myeloid leukemia (AML) is a devastating blood cancer resulting from an uncontrollable proliferation of tumor cells in the bone marrow and blood. Although AML accounts for only a small fraction of all cancer diagnoses, it is considered the most common form of acute leukemia in adults. AML is associated with a poor prognosis, particularly in elderly patients or those with relapsed/refractory AML, as conventional treatments like intensive chemotherapy and hematopoietic stem cell transplantation often fail to achieve durable remission.
Over the past few decades, hematologists have increasingly explored the potential of immunotherapy and targeted therapies for AML. Among these, receptor tyrosine kinase AXL has emerged as a key regulator of AML progression and resistance to standard therapies. Moreover, its overexpression in approximately 50% of AML patients, especially after chemotherapy treatment, positions AXL as an attractive target for both therapeutic and diagnostic interventions. In this context, single-domain antibodies (sdAbs), derived from camelid heavy-chain-only antibodies, offer unique properties and advantages over traditional antibody strategies. Their small size, stability, and versatility make them particularly suited for targets that require both diagnostic and therapeutic approaches.
This PhD thesis focused on the development and characterization of AXL-specific sdAbs for dual diagnostic and therapeutic applications in AML. An alpaca was immunized with mouse/human AXL proteins to generate a diverse sdAb library, from which sdAb20 was selected as the lead compound. For the diagnostic part, sdAb20 was radiolabeled with Technetium-99m and evaluated for its ability to non-invasively detect AXL expression in preclinical AML models using SPECT/CT imaging. These imaging studies demonstrated clear visualization of AXL-positive tumors with excellent tumor-to-background contrast, validating its diagnostic potential. For therapeutic purposes, sdAb20 was engineered into an Fc-fusion construct, enhancing its efficacy, half-life, and effector functions. SdAb20-Fc significantly reduced AML cell viability and proliferation in human AML cell lines and primary patient samples, especially in combination with the chemotherapeutic agent cytarabine, which
resulted in synergistic anti-leukemic effects in vitro.
In addition to its direct effects on AML cells, we evaluated sdAb20-Fc influence on the immune system, as AXL expression on macrophages and dendritic cells has been reported before. In vivo AXL inhibition induced the upregulation of immune checkpoints PD-1/PD-L1, TIM-3 and Galectin-9 (Gal-9) on AML blasts and immune cells, possibly contributing to therapy resistance. To further address this, sdAb20-Fc was combined with an anti-Gal-9 therapeutic antibody, resulting in reduced viability of primary AML patient samples.
In conclusion, this thesis demonstrates the use of AXL-specific sdAbs as innovative tools for both diagnostic imaging and targeted therapy in AML. By addressing immune suppression and therapy resistance, and through synergistic combination strategies, this work contributes to the future development of personalized treatment approaches for AML patients
Over the past few decades, hematologists have increasingly explored the potential of immunotherapy and targeted therapies for AML. Among these, receptor tyrosine kinase AXL has emerged as a key regulator of AML progression and resistance to standard therapies. Moreover, its overexpression in approximately 50% of AML patients, especially after chemotherapy treatment, positions AXL as an attractive target for both therapeutic and diagnostic interventions. In this context, single-domain antibodies (sdAbs), derived from camelid heavy-chain-only antibodies, offer unique properties and advantages over traditional antibody strategies. Their small size, stability, and versatility make them particularly suited for targets that require both diagnostic and therapeutic approaches.
This PhD thesis focused on the development and characterization of AXL-specific sdAbs for dual diagnostic and therapeutic applications in AML. An alpaca was immunized with mouse/human AXL proteins to generate a diverse sdAb library, from which sdAb20 was selected as the lead compound. For the diagnostic part, sdAb20 was radiolabeled with Technetium-99m and evaluated for its ability to non-invasively detect AXL expression in preclinical AML models using SPECT/CT imaging. These imaging studies demonstrated clear visualization of AXL-positive tumors with excellent tumor-to-background contrast, validating its diagnostic potential. For therapeutic purposes, sdAb20 was engineered into an Fc-fusion construct, enhancing its efficacy, half-life, and effector functions. SdAb20-Fc significantly reduced AML cell viability and proliferation in human AML cell lines and primary patient samples, especially in combination with the chemotherapeutic agent cytarabine, which
resulted in synergistic anti-leukemic effects in vitro.
In addition to its direct effects on AML cells, we evaluated sdAb20-Fc influence on the immune system, as AXL expression on macrophages and dendritic cells has been reported before. In vivo AXL inhibition induced the upregulation of immune checkpoints PD-1/PD-L1, TIM-3 and Galectin-9 (Gal-9) on AML blasts and immune cells, possibly contributing to therapy resistance. To further address this, sdAb20-Fc was combined with an anti-Gal-9 therapeutic antibody, resulting in reduced viability of primary AML patient samples.
In conclusion, this thesis demonstrates the use of AXL-specific sdAbs as innovative tools for both diagnostic imaging and targeted therapy in AML. By addressing immune suppression and therapy resistance, and through synergistic combination strategies, this work contributes to the future development of personalized treatment approaches for AML patients
| Original language | English |
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| Award date | 22 Apr 2025 |
| Publication status | Published - 2025 |