CD16xCD33 bispecific killer cell engager (BiKE) activates NK cells against primary MDS and MDSC CD33+ targets.

Myelodysplastic syndromes (MDS) are stem cell disorders that can progress to acute myeloid leukemia. Although hematopoietic cell transplantation can be curative, additional therapies are needed for a disease that disproportionally afflicts the elderly. We tested the ability of a CD16xCD33 BiKE to induce natural killer (NK) cell function in 67 MDS patients. Compared with age-matched normal controls, CD7(+) lymphocytes, NK cells, and CD16 expression were markedly decreased in MDS patients. Despite this, reverse antibody-dependent cell-mediated cytotoxicity assays showed potent degranulation and cytokine production when resting MDS-NK cells were triggered with an agonistic CD16 monoclonal antibody. Blood and marrow MDS-NK cells treated with bispecific killer cell engager (BiKE) significantly enhanced degranulation and tumor necrosis factor-α and interferon-γ production against HL-60 and endogenous CD33(+) MDS targets. MDS patients had a significantly increased proportion of immunosuppressive CD33(+) myeloid-derived suppressor cells (MDSCs) that negatively correlated with MDS lymphocyte populations and CD16 loss on NK cells. Treatment with the CD16xCD33 BiKE successfully reversed MDSC immunosuppression of NK cells and induced MDSC target cell lysis. Lastly, the BiKE induced optimal MDS-NK cell function irrespective of disease stage. Our data suggest that the CD16xCD33 BiKE functions against both CD33(+) MDS and MDSC targets and may be therapeutically beneficial for MDS patients.

An exploratory analysis of mitochondrial haplotypes and allogeneic hematopoietic cell transplantation outcomes.

Certain mitochondrial haplotypes (mthaps) are associated with disease, possibly through differences in oxidative phosphorylation and/or immunosurveillance. We explored whether mthaps are associated with allogeneic hematopoietic cell transplantation (HCT) outcomes. Recipient (n = 437) and donor (n = 327) DNA were genotyped for common European mthaps (H, J, U, T, Z, K, V, X, I, W, and K2). HCT outcomes for mthap matched siblings (n = 198), all recipients, and all donors were modeled using relative risks (RR) and 95% confidence intervals and compared with mthap H, the most common mitochondrial haplotypes. Siblings with I and V were significantly more likely to die within 5 years (RR = 3.0; 95% confidence interval [CI], 1.2 to 7.9; and RR = 4.6; 95% CI, 1.8 to 12.3, respectively). W siblings experienced higher acute graft-versus-host disease (GVHD) grades II to IV events (RR = 2.1; 95% CI, 1.1 to 2.4) with no events for those with K or K2. Similar results were observed for all recipients combined, although J recipients experienced lower GVHD and higher relapse. Patients with I donors had a 2.7-fold (1.2 to 6.2) increased risk of death in 5 years, whereas few patients with K2 or W donors died. No patients with K2 donors and few patients with U donors relapsed. Mthap may be an important consideration in HCT outcomes, although validation and functional studies are needed. If confirmed, it may be feasible to select donors based on mthap to increase positive or decrease negative outcomes.

Tim-3 is an inducible human natural killer cell receptor that enhances interferon gamma production in response to galectin-9.

NK-cell function is regulated by the integration of signals received from activating and inhibitory receptors. Here we show that a novel immune receptor, T-cell Ig and mucin-containing domain-3 (Tim-3), is expressed on resting human NK cells and is up-regulated on activation. The NK92 NK-cell line engineered to overexpress Tim-3 showed a marked increase in IFN-γ production in the presence of soluble rhGal-9 or Raji tumor cells engineered to express Gal-9. The Tim-3(+) population of low-dose IL-12/IL-18-activated primary NK cells significantly increased IFN-γ production in response to soluble rhGal-9, Gal-9 presented by cell lines, and primary acute myelogenous leukemia (AML) targets that endogenously express Gal-9. This effect is highly specific as Tim-3 Ab blockade significantly decreased IFN-γ production, and Tim-3 cross-linking induced ERK activation and degradation of IκBα. Exposure to Gal-9-expressing target cells had little effect on CD107a degranulation. Reconstituted NK cells obtained from patients after hematopoietic cell transplantation had diminished expression of Tim-3 compared with paired donors. This observation correlates with the known IFN-γ defect seen early posttransplantation. In conclusion, we show that Tim-3 functions as a human NK-cell coreceptor to enhance IFN-γ production, which has important implications for control of infectious disease and cancer.

Heterodimeric bispecific single-chain variable-fragment antibodies against EpCAM and CD16 induce effective antibody-dependent cellular cytotoxicity against human carcinoma cells.

A heterodimeric bispecific biological recombinant drug was synthesized by splicing DNA fragments from two fully humanized single-chain variable-fragment (scFV) antibody fragments forming a novel drug simultaneously recognizing the CD16 natural killer (NK) cell marker and the cancer marker epithelial cell adhesion molecule (EpCAM). The drug precipitously enhanced the killing of human carcinomas of the prostate, breast, colon, head, and neck even at very low effector:target ratios. The drug EpCAM16 rendered even nonactivated NK cell-proficient killers and activated them to kill via degranulation and cytokine production. Studies show that bispecific antibodies can be used to induce proficient killing of the carcinoma targets that ordinarily are resistant to NK-mediated killing. Apparently, the innate immune system can be effectively recruited to kill cancer cells using the bispecific antibody platform and EpCAM targeting.

Targeting natural killer cells to acute myeloid leukemia in vitro with a CD16 x 33 bispecific killer cell engager and ADAM17 inhibition.

PURPOSE: The graft versus leukemia effect by natural killer (NK) cells prevents relapse following hematopoietic stem cell transplantation. We determined whether a novel bispecific killer cell engager (BiKE) signaling through CD16 and targeting CD33 could activate NK cells at high potency against acute myelogenous leukemia (AML) targets. EXPERIMENTAL DESIGN: We investigated the ability of our fully humanized CD16 × CD33 (CD16 × 33) BiKE to trigger in vitro NK cell activation against HL60 (CD33(+)), RAJI (CD33(-)), and primary AML targets (de novo and refractory) to determine whether treatment with CD16 × 33 BiKE in combination with an ADAM17 inhibitor could prevent CD16 shedding (a novel inhibitory mechanism induced by NK cell activation) and overcome inhibition of class I MHC recognizing inhibitory receptors. RESULTS: NK cell cytotoxicity and cytokine release were specifically triggered by the CD16 × 33 BiKE when cells were cultured with HL60 targets, CD33(+) de novo and refractory AML targets. Combination treatment with CD16 × 33 BiKE and ADAM17 inhibitor resulted in inhibition of CD16 shedding in NK cells, and enhanced NK cell activation. Treatment of NK cells from double umbilical cord blood transplant (UCBT) recipients with the CD16 × 33 BiKE resulted in activation, especially in those recipients with cytomegalovirus reactivation. CONCLUSION: CD16 × 33 BiKE can overcome self-inhibitory signals and effectively elicit NK cell effector activity against AML. These in vitro studies highlight the potential of CD16 × 33 BiKE ± ADAM17 inhibition to enhance NK cell activation and specificity against CD33(+) AML, which optimally could be applied in patients with relapsed AML or for adjuvant antileukemic therapy posttransplantation.

Bispecific and trispecific killer cell engagers directly activate human NK cells through CD16 signaling and induce cytotoxicity and cytokine production.

This study evaluates the mechanism by which bispecific and trispecific killer cell engagers (BiKEs and TriKEs) act to trigger human natural killer (NK) cell effector function and investigates their ability to induce NK cell cytokine and chemokine production against human B-cell leukemia. We examined the ability of BiKEs and TriKEs to trigger NK cell activation through direct CD16 signaling, measuring intracellular Ca²âº mobilization, secretion of lytic granules, induction of target cell apoptosis, and production of cytokine and chemokines in response to the Raji cell line and primary leukemia targets. Resting NK cells triggered by the recombinant reagents led to intracellular Ca²âº mobilization through direct CD16 signaling. Coculture of reagent-treated resting NK cells with Raji targets resulted in significant increases in NK cell degranulation and target cell death. BiKEs and TriKEs effectively mediated NK cytotoxicity of Raji targets at high and low effector-to-target ratios and maintained functional stability after 24 and 48 hours of culture in human serum. NK cell production of IFN-γ, TNF-α, granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-8, macrophage inflammatory protein (MIP)-1α, and regulated and normal T cell expressed and secreted (RANTES) was differentially induced in the presence of recombinant reagents and Raji targets. Moreover, significant increases in NK cell degranulation and enhancement of IFN-γ production against primary acute lymphoblastic leukemia and chronic lymphocytic leukemia targets were induced with reagent treatment of resting NK cells. In conclusion, BiKEs and TriKEs directly trigger NK cell activation through CD16, significantly increasing NK cell cytolytic activity and cytokine production against tumor targets, showing their therapeutic potential for enhancing NK cell immunotherapies for leukemias and lymphomas.