Pre-Clinical Efficacy of the Anti-CD38 Monoclonal Antibody (mAb) Isatuximab in Acute Myeloid Leukemia (AML)

Background: Although many patients with AML respond to induction chemotherapy, refractory disease is common, and relapse represents the major cause of treatment failure. Furthermore, the outcomes in older patients who are unable to receive intensive chemotherapy without unacceptable side effects remain dismal. Thus, the identification of novel, less toxic and effective drugs in AML is warranted. Novel mAbs have been successfully investigated in various hematological malignancies but not in AML. That notwithstanding, anti-CD38 mAbs which have demonstrated remarkable efficacy in multiple myeloma (MM), could have a potential role in AML since CD38 expression has been observed in blasts from AML patients. However, to the best of our knowledge, there are almost no data about the use of anti-CD38 mAbs in AML.

Aims: To define CD38 expression in AML, and to determine the mechanism of action and preclinical efficacy of isatuximab (SAR650984), an IgG1 anti-CD38 mAb under clinical development in MM.

Results: We used multidimensional 8-color flow cytometry to evaluate the expression of CD38 in bone marrow blasts from 111 newly diagnosed elderly AML patients. Overall, 93% of AML patients expressed CD38, with 35% of cases had bimodal (heterogenous) reactivity for CD38 whereas the remaining 58% of patients showed homogenous CD38 positive expression. Interestingly, we observed a significant correlation between the stage of blasts maturation arrest and reactivity for CD38, as the percentage of CD38+ patients progressively increased from minimally differentiated AML into subtypes without and with maturation (35% vs. 51% and 71%; p=.03).

After demonstrating that CD38 is homogenously expressed by blasts from more than half of AML patients, we measured CD38 expression in 7 AML cell lines. We selected KG-1 and MOLM-13 as cell lines representative of negative vs. bright CD38 expression, respectively. We started by analyzing if isatuximab had a direct effect on AML blasts, but after treatment with increasingly higher doses of Isatuximab (range: 0,01µg/mL - 100µg/mL) for 24h, there was no impact on cell proliferation or viability. Furthermore, while isatuximab binds to C1q, CD38 receptor density in AML blasts was insufficient to trigger complement activation on tumor cells based on the absence of C3 deposition. However, when we co-cultured CD38^bright MOLM-13 blasts plus isatuximab (10µg/mL for 24h) with human leukocytes to measure ADCC and ADCP, we found that in the presence of human leukocytes, isatuximab induced a 40% increment in blast cell death; furthermore, by using sensitive FACS sorting to remove key immune cell populations from the culture, we demonstrated that the presence of NK cells was critical for the efficacy of isatuximab, whereas depletion of macrophages and T-cells had minimal impact on cell killing. Subsequently, we cultured KG-1 and MOLM-3 with NK cells isolated from six donors in the presence of isatuximab (10µg/mL for 24h) and confirmed significant (p=.03) and selective cell killing of CD38^bright MOLM-13 but not CD38^neg KG-1 AML blasts.

Since the efficacy of isatuximab was dependent on CD38 levels, we decided to investigate a potential synergism with all-trans retinoic acid (ATRA), as it has been described to up-regulate CD38 expression and has been investigated in non-APL AML. First, we confirmed that pre-incubation of AML cell lines with ATRA (30ng/ml for 24h) induced a 3-fold increment in CD38 expression without any effect on the viability of KG-1 and MOLM-3 cells. Then we cultured both AML lines with NK cells plus isatuximab (10µg/mL for 24h) after pre-treatment with ATRA, and observed, with selected donors, an increment in the percentage of MOLM-13 and KG-1 cell killing. Finally, we tested isatuximab (10ug/ml) ex vivo in primary samples from 11 AML patients and observed significant cell killing (median 20% of tumor lysis after 24h, p=.002) in ten out of eleven patients. Furthermore, there was a trend for higher percentage of tumor lysis in CD38+ patients compared to cases with heterogeneous CD38 expression (33% vs. 12,5%; p=0.12).

Conclusions: Using a comprehensive panel of assays, cell lines, and primary patient samples, we showed for the first time that the anti-CD38 mAb isatuximab has activity in preclinical AML models, with NK-cell mediated ADCC as the most relevant mechanism of action. CD38 may therefore represent an important and novel therapeutic target in CD38+ AML patients.