A Novel Fc-Engineered Antibody to CD33 with Enhanced ADCC Activity for Treatment of AML

Acute myeloid leukemia (AML), the most frequent form of adult acute leukemia, is a heterogeneous disease with respect to presentation and clinical outcome. Although there has been progress made in treatment for some subgroups of AML patients, there is still a high medical need to develop new anti-leukemic therapies. CD33 is a myeloid lineage-specific antigen which is expressed on the cell surface of normal myeloid cells and AML blasts. CD33 has been investigated as a target for antibody-based therapies for over a decade, leading to transient approval of Mylotarg^®, an antibody-calicheamicin drug conjugate. Mylotarg^®has been shown to substantially improve outcome of patients with AML in combination with chemotherapy, however is associated with additional toxicity. Here we describe a novel, unconjugated Fc-engineered antibody to CD33, mAb 33.1, which mediates potent antibody dependent cellular cytotoxicity (ADCC) against AML derived cell lines and malignant AML blasts.

MAb 33.1 is a fully human, Fc-engineered, IgG1-type of antibody which has increased affinity to human Fc-gamma receptor IIIa and binds CD33 with low nanomolar affinity as determined by FACS scatchard analysis. ADCC of mAb 33.1 was determined on a panel of myeloid cell lines and primary AML cells. ADCC assays were performed using peripheral blood mononuclear cells (PBMC) from healthy donors as effector cells and cytotoxicity was determined by LDH release or FACS quantification of 7-AAD-positive cells. Internalization kinetics was assessed by FACS analysis using fluorochrome labelled antibody. Epitope mapping of mAb 33.1 was performed by direct competition experiments and hydrogen/deuterium exchange mass spectrometry (HXMS).

Antibody mAb 33.1 is able to mediate potent ADCC on a panel of AML derived cell lines and primary AML blasts in vitro. Direct comparison of mAb 33.1 to the humanized CD33 mAb lintuzumab (HuM195) demonstrates superior cytolytic activity on AML cell lines and primary AML cells. ADCC activity of mAb 33.1 is also evident on AML cell lines and primary AML blasts that do not respond to lintuzumab treatment in vitro. In contrast to lintuzumab, mAb 33.1 shows significantly slower internalization kinetics on HL60 cells, leading to prolonged exposure of antigen/antibody complexes on the cell surface. In direct competition experiments mAb 33.1 is competing with lintuzumab for antigen binding. HXMS experiments revealed that mAb 33.1 recognizes a novel epitope on CD33 different to that of lintuzumab.

MAb 33.1 represents a novel, Fc-engineered antibody specific for CD33 which shows decelerated internalization compared to lintuzumab, and which recognizes a novel epitope on CD33 different to that of lintuzumab. MAb 33.1 displays strongly enhanced ADCC against leukemia cell lines and primary AML cells, resulting in significantly higher cytolysis than lintuzumab. MAb 33.1 is considered as a promising candidate for treatment of patients with CD33-positive myeloid malignancies and phase I clinical trials are currently under preparation.

Heider:Boehringer Ingelheim: Employment. Konopitzky:Boehringer Ingelheim: Employment. Ostermann:Boehringer Ingelheim: Employment. Lamche:Boehringer Ingelheim: Employment. Kuepcue:Boehringer Ingelheim: Employment. Koessl:Boehringer Ingelheim: Employment. Adam:Boehringer Ingelheim: Employment. Borges:Boehringer Ingelheim: Employment.