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.

A number of agents designed for immunotherapy of Acute Myeloid Leukemia (AML) are in preclinical and early clinical development. Most of them target a single antigen on the surface of AML cells. Here we describe the development and key biological properties of a tri-specific agent, the dual-targeting triplebody SPM-2, with binding sites for target antigens CD33 and CD123, and for CD16 to engage NK cells as cytolytic effectors. Primary blasts of nearly all AML patients carry at least one of these target antigens and the pair is particularly promising for the elimination of blasts and leukemia stem cells (LSCs) from a majority of AML patients by dual-targeting agents. The cytolytic activity of NK cells mediated by SPM-2 was analyzed in vitro for primary leukemic cells from 29 patients with a broad range of AML-subtypes. Blasts from all 29 patients, including patients with genomic alterations associated with an unfavorable genetic subtype, were lysed at nanomolar concentrations of SPM-2. Maximum susceptibility was observed for cells with a combined density of CD33 and CD123 above 10,000 copies/cell. Cell populations enriched for AML-LSCs (CD34pos and CD34pos CD38neg cells) from 2 AML patients carried an increased combined antigen density and were lysed at correspondingly lower concentrations of SPM-2 than unsorted blasts. These initial findings raise the expectation that SPM-2 may also be capable of eliminating AML-LSCs and thus of prolonging survival. In the future, patients with a broad range of AML subtypes may benefit from treatment with SPM-2.

CD19-specific triplebody SPM-1 engages NK and γδ T cells for rapid and efficient lysis of malignant B-lymphoid cells.

Triplebodies are antibody-derived recombinant proteins carrying 3 antigen-binding domains in a single polypeptide chain. Triplebody SPM-1 was designed for lysis of CD19-bearing malignant B-lymphoid cells through the engagement of CD16-expressing cytolytic effectors, including NK and γδ T cells.SPM-1 is an optimized version of triplebody ds(19-16-19) and includes humanization, disulfide stabilization and the removal of potentially immunogenic sequences. A three-step chromatographic procedure yielded 1.7 - 5.5 mg of purified, monomeric protein per liter of culture medium. In cytolysis assays with NK cell effectors, SPM-1 mediated potent lysis of cancer-derived B cell lines and primary cells from patients with various B-lymphoid malignancies, which surpassed the ADCC activity of the therapeutic antibody Rituximab. EC50-values ranged from 3 to 86 pM. Finally, in an impedance-based assay, SPM-1 mediated a particularly rapid lysis of CD19-bearing target cells by engaging and activating both primary and expanded human γδ T cells from healthy donors as effectors.These data establish SPM-1 as a useful tool for a kinetic analysis of the cytolytic reactions mediated by γδ T and NK cells and as an agent deserving further development towards clinical use for the treatment of B-lymphoid malignancies.

Chip-based platform for dynamic analysis of NK cell cytolysis mediated by a triplebody.

Cancer therapy via redirected lysis mediated by antibodies and antibody-derived agents relies on the availability of substantial numbers of sufficiently active immune effector cells. To monitor antitumor responses before and during therapy, sensitive methods are needed, capable of quantitating specific lysis of target cells. Here we present a chip-based single-cell cytometric assay, which uses adherent human target cells arrayed in structured micro-fields. Using a fluorescent indicator of cell death and time-lapse microscopy in an automated high-throughput mode, we measured specific target cell lysis by activated human NK cells, mediated by the therapeutic single chain triplebody SPM-2 (33-16-123). This antibody-derived tri-specific fusion protein carries binding sites for the myeloid antigens CD33 and CD123 and recruits NK cells via a binding site for the Fc-receptor CD16. Specific lysis increased with increasing triplebody concentration, and the single-cell assay was validated by direct comparison with a standard calcein-release assay. The chip-based approach allowed measurement of lysis events over 16 hours (compared to 4 hours for the calcein assay) and required far smaller numbers of primary cells. In addition, dynamic properties inaccessible to conventional methods provide new details about the activation of cytolytic effector cells by antibody-derived agents. Thus, the killing rate exhibited a dose-dependent maximum during the reaction interval. In clinical applications ex vivo monitoring of NK activity of patient's endogenous cells will likely help to choose appropriate therapy, to detect impaired or recovered NK function, and possibly to identify rare subsets of cancer cells with particular sensitivity to effector-cell mediated lysis.

Dual-targeting triplebody 33-3-19 mediates selective lysis of biphenotypic CD19+ CD33+ leukemia cells.

Simultaneous targeting of multiple tumor-associated antigens (TAAs) in cancer immunotherapy is presumed to enhance tumor cell selectivity and to reduce immune escape.The combination of B lymphoid marker CD19 and myeloid marker CD33 is exclusively present on biphenotypic B/myeloid leukemia cells. Triplebody 33-3-19 binds specifically to both of these TAAs and activates T cells as immune effectors. Thereby it induces specific lysis of established myeloid (MOLM13, THP-1) and B-lymphoid cell lines (BV173, SEM, Raji, ARH77) as well as of primary patient cells. EC50 values range from 3 pM to 2.4 nM. In accordance with our hypothesis, 33-3-19 is able to induce preferential lysis of double- rather than single-positive leukemia cells in a target cell mixture: CD19/CD33 double-positive BV173 cells were eliminated to a significantly greater extent than CD19 single-positive SEM cells (36.6% vs. 20.9% in 3 hours, p = 0.0048) in the presence of both cell lines. In contrast, equivalent elimination efficiencies were observed for both cell lines, when control triplebody 19-3-19 or a mixture of the bispecific single chain variable fragments 19-3 and 33-3 were used. This result highlights the potential of dual-targeting agents for efficient and selective immune-intervention in leukemia patients.

T cell-recruiting triplebody 19-3-19 mediates serial lysis of malignant B-lymphoid cells by a single T cell.

Triplebody 19-3-19, an antibody-derived protein, carries three single chain fragment variable domains in tandem in a single polypeptide chain. 19-3-19 binds CD19-bearing lymphoid cells via its two distal domains and primary T cells via its CD3-targeting central domain in an antigen-specific manner. Here, malignant B-lymphoid cell lines and primary cells from patients with B cell malignancies were used as targets in cytotoxicity tests with pre-stimulated allogeneic T cells as effectors. 19-3-19 mediated up to 95 % specific lysis of CD19-positive tumor cells and, at picomolar EC50 doses, had similar cytolytic potency as the clinically successful agent Blinatumomab. 19-3-19 activated resting T cells from healthy unrelated donors and mediated specific lysis of both autologous and allogeneic CD19-positive cells. 19-3-19 led to the elimination of 70 % of CD19-positive target cells even with resting T cells as effectors at an effector-to-target cell ratio of 1 : 10. The molecule is therefore capable of mediating serial lysis of target cells by a single T cell. These results highlight that central domains capable of engaging different immune effectors can be incorporated into the triplebody format to provide more individualized therapy tailored to a patient's specific immune status.

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.

BACKGROUND: The 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. METHODS: Patient 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. RESULTS: Patient 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. CONCLUSIONS: Peripheral 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.