NK cells from an AML patient have recovered in remission and reached comparable cytolytic activity to that of a healthy monozygotic twin mediated by the single-chain triplebody SPM-2

Abstract

BackgroundThe capacity of patient’s Natural Killer cells (NKs) to be activated for cytolysis is an important prerequisite for the success of antibody-derived agents such as single-chain triplebodies (triplebodies) in cancer therapy. NKs recovered from AML patients at diagnosis are often found to be reduced in peripheral blood titers and cytolytic activity. Here, we had the unique opportunity to compare blood titers and cytolytic function of NKs from an AML patient with those of a healthy monozygotic twin. The sibling’s NKs were compared with the patient’s drawn either at diagnosis or in remission after chemotherapy. The cytolytic activities of NKs from these different sources for the patient’s autologous AML blasts and other leukemic target cells in conjunction with triplebody SPM-2, targeting the surface antigens CD33 and CD123 on the AML cells, were compared.MethodsPatient NKs drawn at diagnosis were compared to NKs drawn in remission after chemotherapy and a sibling’s NKs, all prepared from PBMCs by immunomagnetic beads (MACS). Redirected lysis (RDL) assays using SPM-2 and antibody-dependent cellular cytotoxicity (ADCC) assays using the therapeutic antibody RituximabTM were performed with the enriched NKs. In addition, MACS-sorted NKs were analyzed for NK cell activating receptors (NCRs) by flow cytometry, and the release of TNF-alpha and IFN-gamma from blood samples of both siblings after the addition of the triplebody were measured in ELISA-assays.ResultsPatient NKs isolated from peripheral blood drawn in remission produced comparable lysis as NKs from the healthy twin against the patient’s autologous bone marrow (BM) blasts, mediated by SPM-2. The NCR receptor expression profiles on NKs from patient and twin were similar, but NK cell titers in peripheral blood were lower for samples drawn at diagnosis than in remission.ConclusionsPeripheral blood NK titers and ex vivo cytolytic activities mediated by triplebody SPM-2 were comparable for cells drawn from an AML patient in remission and a healthy twin. If these results can be generalized, then NKs from AML patients in remission are sufficient in numbers and cytolytic activity to make triplebodies promising new agents for the treatment of AML.

Dual-targeting triplebody 33-16-123 (SPM-2) mediates effective redirected lysis of primary blasts from patients with a broad range of AML subtypes in combination with natural killer cells

“…Blasts from patients with a FAB M1 subtype often display a very immature maturation state and express only low levels of CD33, or are CD33 negative, and are often difficult to treat with agents mono-specific for CD33. 60 Cells from patients 2 to 9, 11 to 16, 19, 20, 22, 23 and 25 to 28 with intermediate and adverse ELN genetic subtypes (Table 2) were also lysed efficiently by SPM-2 with EC 50 values ranging from 10.3 – 1078 pM (Figure 2B-D). These data strongly support the prediction 8 that a dual-targeting agent with specificity for CD33 and CD123 acting together with NK cells should be able to eliminate blasts from almost all AML patients with a very broad range of disease subtypes.…”

“…For in vitro cytolysis assays the target cells were labeled with calcein. 60,68 Peripheral blood mononuclear cells (PBMCs) from an unrelated healthy donor were expanded in culture for 20 d in the presence of IL-2. These cells, called lymphokine-activated killer cells (LAK cells), consisted of approx.…”

“…A number of therapeutic agents with similar molecular formats as those described for CD33 have been developed, including immunoglobulins, 47–49 a radio-immunoconjugate, 50 ADCs and a fusion protein between the cytokine IL-3 and a fragment of diphtheria toxin, 51–54 bi-specific T cell-recruiting agents (BiTEs) and Dual-Antigen Recruiting T-cell engagers (DARTs), 55–58 dual-targeting triplebodies, 59,60 and CAR-transfected T cells. 61–64 Expression of CD123 is largely restricted to hematopoietic cells, but expression on endothelial cells has been reported.…”

“…8 Larger studies in patients with many different subtypes of the disease are needed to investigate, whether new dual-targeting agents simultaneously addressing both antigens on the same cell 59,60 or combinations of corresponding mono-targeting agents are promising for clinical applications for a broad group of AML patients. In a first large study pursuing this goal, the expression of CD33 and CD123 alone and in combination was investigated on cellular samples from 319 AML patients.…”

“…60 Here we describe the generation of the clinical candidate SPM-2, an optimized variant carrying humanized and disulfide-stabilized single-chain variable fragments (scFvs) as antigen binding sites, plus additional mutations favoring its clinical development. We anticipate that SPM-2 will be able to discriminate at least to a degree between AML-LSCs and remaining normal HSCs of a patient, due to its dual-targeting capacity and the greater combined surface density of CD33 and CD123 on AML-LSCs than on normal HSCs.…”