Evaluation Of CD33 Expression and Functional Analysis Of The CD33/CD3 Bispecific BiTE® Antibody AMG 330 In Primary AML Samples

Abstract

Antibody-based immunotherapy represents a promising strategy to specifically target and eliminate chemoresistant leukemic cells in acute myeloid leukemia (AML). We evaluated a single-chain bispecific CD33/CD3 BiTE® antibody (AMG 330) for its suitability as immunotherapy in AML. A prerequisite for successful immunotherapeutic approaches using this molecule is the expression of CD33 on AML blasts including leukemic stem cells (LSCs). Therefore, we quantified CD33 expression on AML blasts and LSCs by flow cytometry (mean fluorescence intensity, MFI) and correlated expression intensity with cytogenetic and molecular disease characteristics in order to identify patient cohorts possibly most suited for CD33-targeted therapies. CD33 expression was detected in >99% of patient samples (n=621, MFI ratio ≥ 1.5) although highly variable. A strong correlation between high CD33 expression levels and NPM1 mutations (p<0.001) was found. In contrast, low CD33 expression levels were significantly associated with complex karyotypes and t(8,21) translocations (p<0.001). Furthermore, LSCs within the CD34+/CD38- compartment displayed CD33 at higher levels than healthy donor stem cells (p=0.047). Importantly, colony formation of CD34+/Lin-cells from healthy donors was not affected after pre-incubation with AMG 330 and T-cells. A major hurdle for measuring cytotoxic effects on AML blasts has long been that primary AML patient samples show progressive cell death within a few days ex-vivo. To simulate the natural setting of target and T-cells in AML patients, we developed a long-term culture system for AML blasts that allowed us to observe these co-cultures for up to 5 weeks. Thus, we were able to show effective elimination of AML blasts within primary samples by AMG 330-activated and expanded residual CD3+/CD45RA-/CCR7+ memory T-lymphocytes. While the functional relevance of CD33 expression levels was shown by faster lysis kinetics of CD33BRIGHT vs. CD33DIM AML cell lines in an in-vitro cytotoxicity assay potent anti-leukemic activity on primary AML blasts was observed irrespective of CD33 expression level. At low effector to target ratios (up to 1:79), the recruited T-cells lysed autologous blasts completely in the majority of samples. Further T-cell analysis showed that naive T-cells (CD45RA+/CCR7+) were not expanded by AMG 330; neither were terminally differentiated T-cells (CD45RA+/CCR7-), probably due to their poor proliferative capacity. We did not observe an increase in percentage of CD3+/CD4+/CD25+/FoxP3+regulatory T-cells in the presence of AMG 330, suggesting that these cells may not have impacted AMG 330-mediated T-cell activity in our experiments. Compared to control cultures, T-cells were shown to up-regulate the activation markers CD25, PD-1, TIM3 and LAG3 upon response to AMG 330, which was partially reversible after complete target cell elimination. However, PD-1 up-regulation did not correlate with an up-regulation of PD-L1 on AML blasts despite substantial INFγ secretion by activated T-cells. This study provides the first correlation of CD33 expression levels to a comprehensive genotype analysis in adult AML patients. While CD33 expression may vary by AML biologic subgroup, AMG 330 exposure led to lysis of AML blasts even in samples with low levels of expression. Targeting CD33 using AMG 330 in primary AML samples led to efficient T-cell activation and expansion as expected from the mechanism of action of BiTE® antibodies. The remarkable ex-vivo activity of AMG 330 supports further development of AMG 330 as an immunotherapy for patients with AML. Disclosures: Kufer: AMGEN Research (Munich) GmbH: Employment; AMGEN Inc.: Equity Ownership. Kischel:AMGEN Research (Munich) GmbH: Employment; AMGEN Inc.: Equity Ownership. Zugmaier:Amgen Research (Munich) GmbH: Employment; Amgen Inc.: Equity Ownership. Baeuerle:AMGEN Research (Munich) GmbH: Employment; AMGEN Inc.: Equity Ownership. Riethmüller:AMGEN Inc.: Equity Ownership.

Novel Therapies in AML: Reason for Hope or Just Hype?

“…41 In vitro studies of AMG-330 with primary AML cells showed potent cytotoxicity regardless of the degree of CD33 expression. 42 Of note, AMG-330 did not reduce surface expression of CD33, and its cytotoxic effects were potentiated by panobinostat and azacitidine. 43 MGD006 represents another approach to combined antibody targeting and T-cell directed killing of AML cells.…”

Immunotherapy in acute myeloid leukemia

“…Ex vivo CD33/CD3 BiTE antibody therapy (AMG330) demonstrated redirected, targeted lysis of AML blasts and stem cells, whereas an in vivo murine experiment produced decreased tumor growth . Ex vivo primary human AML samples treated with AMG330 exhibited lysis of AML blasts with increased T‐cell activation . Another AML BiTE antibody, which combines CD3 with WT1 and HLA‐0201 in a murine model, demonstrated undetectable leukemic growth by day 14 with MRD by day 18 .…”