In Vivo Induction of Leukemia-Specific Adaptive and Innate Immune Cells by Treatment of AML-Diseased Rats and Therapy-Refractory AML Patients with Blast Modulating Response Modifiers

Abstract

There is a high medical need to develop new strategies for the treatment of patients with acute myeloid leukemia (AML) refractory to conventional therapy. In vitro, the combinations of the blast-modulatory response modifiers GM-CSF + Prostaglandin E1, (summarized as Kit M) have been shown to convert myeloid leukemic blasts into antigen-presenting dendritic cells of leukemic origin (DC_leu) that were able to (re-)activate the innate and adaptive immune system, direct it specifically against leukemic blasts, and induce memory cells. This study aimed to investigate the immune modulatory capacity and antileukemic efficacy of Kit M in vivo. Brown Norway rats suffering from AML were treated with Kit M (twofold application). Blasts and immune cells were monitored in peripheral blood (PB) and spleen. Upon the observation of promising immune modulatory effects in the treated animals, two patients with AML refractory to multiple lines of therapy were offered treatment with Kit M on an individualized basis. Safety, as well as immunological and clinical effects, were monitored. Samples obtained from a third patient in similar clinical conditions not receiving Kit M were used as controls for immune monitoring tests. Animal experiments: Drugs were well tolerated by the treated animals. After 9 days of treatment, DC_leu and memory-like T cells increased in the peripheral blood, whereas regulatory T cells, especially blasts, decreased in treated as compared to untreated control animals. Clinical courses: No severe side effects were observed. In patient 1482, PB blasts remained under the detection threshold during 27 days of treatment, thrombocytes were normalized, and (leukemia specific) immune effector cells of the adaptive and innate immune system increased up to 800-fold compared to the start of treatment. Patient 1601 responded with a 12% reduction in blasts in PB immediately after Kit M treatment. Several subtypes of (leukemia-specific) immune effector cells in PB increased up to four-fold during the 19 days of treatment. In contrast, immune-reactive cells decreased under mild chemotherapy in the PB of control patient 1511 with comparably refractory AML. Within the limitation of low numbers in both animal experiments and clinical applications, our data suggest that Kit M treatment of AML-diseased rats and patients is feasible and may induce leukemia-specific immune reactions and clinical improvement. A larger series and a prospective clinical trial will be required to confirm our observations. Beyond optimized doses and schedules of the applied compounds, the combination with other antileukemic strategies or the application of Kit M in less proliferative stages of the myeloid diseases need to be discussed. If effects are confirmed, the concept may add to the armamentarium of treatments for highly aggressive blood cancer.

Keywords: AML; AML immunotherapy; immune-monitoring; kits; leukemia-derived dendritic cells; leukemia-specific cells; new AML drugs.

Figure 1

Treatment of leukemia-diseased rats with Kit M induced DC/DC_leu and DC/DC_leu-activated immune-reactive and memory-like T cells and reduced regulatory T cells and blasts, preferentially in PB/spleen. Cell subtypes are given in Supplementary Table S1.

Figure 2

Clinical course of disease of P1482 following low-dose chemotherapy and Kit M treatment. Chemotherapy (hydroxycarbamide, cytarabine) was given from the start of observation to day 11. Kit M treatment was between day 11 and 38; no treatment was from day 38 to the end of observation. Blood cells (thrombocytes, hemoglobin, neutrophils, blasts) in peripheral blood (PB) and frequencies of BM blasts and leukocytes/white blood cells (WBCs) are given. ↑ Timepoints of erythrocyte transfusions; ↓ timepoints of thrombocyte transfusions. Immune monitoring at defined timepoints showed, in contrast to the samples obtained before treatment and from the patient without Kit M treatment, a continuous increase in DCs and proliferating CD8⁺ T cells, of T_non-naive (and a decrease in T_naive) of the CD8 and CD4-lines, of Th₁+ and Th₁₇+, CD4⁺ T cells, and of Bcell_memory over the 4-week Kit M treatment. The same was true for T_cm and T_em of the CD8 and CD4-lines and for frequencies of NK cells (either CD161⁺ or CD56⁺), of CIK cells (either CD161⁺ or CD56⁺), Can be removed and iNKT cells (of the NK- as well as the CD3-type) (Figure 5A, P1482).

Figure 3

Clinical course of disease of P1601 during Kit M treatment ± chemotherapy. No treatment was given between day 1 and 9, Kit M treatment was from day 9 to 26, and chemotherapeutical treatment (hydroxycarbamide) was from day 19 to 29. Patients’ pneumonia was additionally treated daily by prednisolone on day 12–14. Blood cells (thrombocytes, hemoglobin, neutrophils, blasts) in peripheral blood (PB) and leukocytes/white blood cells (WBCs) are given. ↑ Timepoints of erythrocyte transfusions; ↓ timepoints of thrombocyte-transfusions; pleural punctures.

Figure 4

Clinical course of disease of P1511 during chemotherapy (control without Kit M). Chemotherapy (cytarabine, midostaurin) was given from the start to the end of observation. Blood cells (thrombocytes, hemoglobin, blasts) in peripheral blood (PB) and leukocytes/white blood cells (WBCs) are given. ↑ Timepoints of erythrocyte transfusion; ↓ timepoints of thrombocyte transfusion.

Figure 5

Immune monitoring under the treatment of therapy refractory patients (P1482, P1601) with Kit M compared to a control patient (P1511). All values in the course of the disease are given as ‘fold change’ values, referring to the value at the beginning of observation. P1511: chemotherapy during the whole observation time. P1482: low-dose chemotherapy from day 1 to 11, Kit M treatment between day 11 and 38, no treatment from day 38 to the end of observation. P1601: no treatment between day 1 and 9, Kit M treatment from day 9 to 26, chemotherapy from day 19 to 29. * Application of prednisolone from day 12 to 14. Details and abbreviations on cellular subtypes are given in Supplementary Table S2. Details on individual treatments are provided in Supplementary Table S3. (A) Effects of Kit M treatment on patients’ DCs, cells of adaptive immunity, memory cells, and cells of innate immunity in the course of the disease. (B) Effects of Kit M treatment (P1482, P1601) (vs. no Kit M treatment (P1511)) on the provision of leukemia-specific cells.

Figure 5

Immune monitoring under the treatment of therapy refractory patients (P1482, P1601) with Kit M compared to a control patient (P1511). All values in the course of the disease are given as ‘fold change’ values, referring to the value at the beginning of observation. P1511: chemotherapy during the whole observation time. P1482: low-dose chemotherapy from day 1 to 11, Kit M treatment between day 11 and 38, no treatment from day 38 to the end of observation. P1601: no treatment between day 1 and 9, Kit M treatment from day 9 to 26, chemotherapy from day 19 to 29. * Application of prednisolone from day 12 to 14. Details and abbreviations on cellular subtypes are given in Supplementary Table S2. Details on individual treatments are provided in Supplementary Table S3. (A) Effects of Kit M treatment on patients’ DCs, cells of adaptive immunity, memory cells, and cells of innate immunity in the course of the disease. (B) Effects of Kit M treatment (P1482, P1601) (vs. no Kit M treatment (P1511)) on the provision of leukemia-specific cells.