Report on the Academic Paper on the Monoclonal Antibody INCA033989 for Targeting Mutated Calreticulin in Myeloid Tumors

Background: Calreticulin Mutation-Driven Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs) are hematological malignancies caused by somatic mutations occurring in multipotent hematopoietic stem cells (HSCs). These mutations predominantly affect the JAK2, CALR (calreticulin), and MPL genes. MPNs include polycythemia vera (PV), primary myelofibrosis (MF), and essential thrombocythemia (ET). According to the literature, mutations in calreticulin (mutCALR) are the second most common driver of MPNs after JAK2, occurring in approximately 35% of MF patients and 25% of ET patients. MutCALR is also associated with increased clonal expansion, higher thrombotic risk, and progression to bone marrow fibrosis.

Existing treatments for MPNs, such as the JAK1/2 inhibitor ruxolitinib, primarily alleviate symptoms or reduce splenomegaly but do not significantly reduce the mutational clone burden, meaning they fail to address the root cause of the disease. Additionally, chemotherapeutic drug resistance and intolerance are prevalent, greatly limiting efficacy. Thus, therapies capable of selectively targeting mutCALR are a critical unmet need.

To address this, a collaborative research team from Incyte Research Institute, Gustave Roussy Institute in France, and Université Paris-Saclay developed the monoclonal antibody INCA033989, targeting mutCALR, to evaluate its therapeutic potential in MPNs. This study, published in 2024, represents the first comprehensive elucidation of INCA033989’s mechanisms and therapeutic potential through in vitro and in vivo tests.

---

Study Design and Methodology

Overall Research Design

This study utilized various experimental approaches to comprehensively evaluate INCA033989’s efficacy in vitro and in vivo: 1. Antibody screening and characterization: Screening from an antibody library to identify high-affinity binding to mutant calreticulin. 2. In vitro pharmacological evaluation: Testing INCA033989’s selective binding and anti-cancer activity. 3. In vivo model validation: Using mouse competitive transplantation models to examine therapeutic efficacy and molecular mechanisms.

Antibody Screening and Characterization

The research team employed surface plasmon resonance (SPR) to identify INCA033989’s binding specificity to the mutant neoepitope on CALR’s C-terminal. The dissociation constants (Kd) were 1.75 nM for CALRdel52 and 6.78 nM for CALRins5, indicating superior binding affinity to CALRdel52. Specificity analysis confirmed that INCA033989 selectively binds mutCALR, with no binding to wild-type calreticulin (wtCALR).

In Vitro Cell Line and Primary Cell Experiments

Testing was performed on two hematopoietic cell lines (Ba/F3 and UT-7), engineered to express mutant CALR or act as controls. Key results include: - INCA033989 dramatically inhibited the signaling activation of tpoR/CALR mutant complexes via pathways like pSTAT3, pSTAT5, etc. - Proliferation assays demonstrated dose-dependent selective killing of CALRdel52- or CALRins5-mutant cells, with no impact on wild-type cells.

Furthermore, primary human hematopoietic stem and progenitor cells (CD34+ HSPCs) from patients exhibiting CALR mutations were tested. Results showed that INCA033989 effectively killed mutant clone-derived cells, inhibited pathological differentiation into megakaryocytes, and preserved normal hematopoiesis.

In Vivo Competitive Transplantation Model

To further evaluate INCA033989, a tamoxifen-induced CALRdel52 mouse model was used in a competitive hematopoietic transplantation experiment: - Blood and bone marrow analyses revealed that INCA033989 significantly reduced the proportion of mutant-type platelets to 36% versus 96% in the control group. No relapse of induced mutant clones was observed after treatment discontinuation. - Bone marrow analysis showed that INCA033989 selectively reduced mutCALR-positive long-term hematopoietic stem cells (LT-HSCs) without affecting wild-type HSCs.

These results suggest that INCA033989 exerts its therapeutic effects by selectively clearing mutant clones while preserving the normal hematopoietic system.

---

Key Findings and Mechanisms

Research Results and Contributions

The study established the action mechanisms and therapeutic potential of INCA033989: 1. Mechanism of Action: INCA033989 targets the mutCALR/tpoR complex, promoting endocytosis and disrupting disease-driving signaling pathways. 2. Selectivity: The drug selectively clears mutCALR-positive pathogenic HSCs without harming normal hematopoiesis. 3. Clinical Potential: Unlike existing non-selective treatments, INCA033989 specifically reduces mutCALR clonal burden and offers a pathway to potential disease eradication.

Scientific Highlights

The academic and clinical novelty of this research can be summarized as follows: 1. First mutCALR-targeting antibody: A groundbreaking therapeutic approach for CALR mutation-driven MPNs. 2. Validated across multiple models: Demonstrated efficacy in patient-derived CD34+ HSPCs and animal models. 3. Non-conventional anti-cancer mechanism: Achieves precision therapy through antibody-mediated endocytosis rather than immune effector cell recruitment. 4. Disease driver intervention: Successfully targets mutant HSCs, offering hope for curative solutions in MPNs.

---

Implications and Future Directions

This study reveals INCA033989 as a potential disease-modifying therapy, paving the way for tailored treatments in CALR mutation-driven MPNs. In particular, this drug demonstrates significant therapeutic promise for high-risk or advanced-stage patients.

Phase 1 clinical trials for INCA033989 will focus on its safety and efficacy in ET and MF patients. This research also sets a framework for developing targeted antibodies against other mutations (e.g., JAK2, MPL) in MPN subtypes.

Summary

INCA033989, the first monoclonal antibody specifically targeting mutated calreticulin, shows significant anti-tumor efficacy and potential for clinical translation. This research lays a strong foundation for personalized therapy in MPNs and promises to transform the treatment landscape.