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Bispecific and Trispecific Killer Cell Engagers Directly Activate Human NK Cells Through CD16 Signaling and Induce Cytotoxicity and Cytokine Production

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Bispecific and Trispecific Killer Cell Engagers Directly Activate Human NK Cells Through CD16 Signaling and Induce Cytotoxicity and Cytokine Production Published OnlineFirst October 17, 2012; DOI: 10.1158/1535-7163.MCT-12-0692 Molecular Cancer Preclinical Development Therapeutics Bispecific and Trispecific Killer Cell Engagers Directly Activate Human NK Cells through CD16 Signaling and Induce Cytotoxicity and Cytokine Production Michelle K. Gleason1, Michael R. Verneris2, Deborah A. Todhunter3, Bin Zhang1, Valarie McCullar1, Sophia X. Zhou1, Angela Panoskaltsis-Mortari2, Louis M. Weiner4, Daniel A. Vallera3, and Jeffrey S. Miller1 Abstract 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 þ and TriKEs to trigger NK cell activation through direct CD16 signaling, measuring intracellular Ca2 mobili- zation, 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 þ reagents led to intracellular Ca2 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-g, TNF-a, granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-8, macrophage inflam- matory protein (MIP)-1a, 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-g 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 immu- notherapies for leukemias and lymphomas. Mol Cancer Ther; 11(12); 2674–84. Ó2012 AACR. Introduction fragment variable (bscFv and tscFv) recombinant re- With more than 70,000 cases anticipated in the United agents, most of these undesired limitations are avoided States for 2012, non-Hodgkins lymphoma (NHL) is the while eliciting high effector function as they lack the Fc most common adult hematologic malignancy, of which portion of whole antibodies and have a targeted specific- 85% of cases are of B-cell origin (1, 2). Monoclonal ity for CD16 (5–7). As a result, recombinant reagents are antibodies (mAb), such as rituximab, have shown to be attractive for clinical use in enhancing natural killer (NK) therapeutic for the treatment of NHL (3). Despite this cell immunotherapies. success, there are limitations that decrease the overall The ability of NK cells to recognize and kill targets is efficiency of mAb therapies (4). With the development regulated by a sophisticated repertoire of inhibitory and of CD16-directed bispecific and trispecific single-chain activating cell surface receptors. NK cell cytotoxicity can occur by natural cytotoxicity, mediated via the natural cytotoxicity receptors (NCR), or by antibodies, such as rituximab, to trigger antibody-dependent cell-mediated Authors' Affiliations: 1Adult and 2Pediatric Divisions of Hematology, Oncology, and Transplantation and 3Section on Molecular Cancer Ther- cytotoxicity (ADCC) through CD16, the activating low- apeutics, Department of Therapeutic Radiology-Radiation Oncology, Uni- affinity Fc-g receptor for immunoglobulin G (IgG) highly versity of Minnesota Masonic Cancer Center, Minneapolis, Minnesota; and dim 4Department of Oncology, Lombardi Comprehensive Cancer Center, Geor- expressed by the CD56 subset of NK cells (8–11). getown University, Washington, DC Natural cytotoxicity is triggered via NCRs by de novo D.A. Vallera and J.S. Miller contributed equally to this work. expression of NK cell activating receptor ligands on target cells. In the absence of cytokine stimulation, these recep- Corresponding Author: Jeffrey S. Miller, University of Minnesota Masonic Cancer Center, MMC 806, Harvard Street at East River Road, tors inefficiently elicit a cytotoxic or cytokine response Minneapolis, MN 55455. Phone: 612-625-7409; Fax: 612-626-3941; independently, but together they are able to function E-mail: [email protected] synergistically to activate a resting NK cell and promote doi: 10.1158/1535-7163.MCT-12-0692 effector function (9, 12). In contrast, ADCC is mediated Ó2012 American Association for Cancer Research. when CD16 binds to opsonized targets through Fc 2674 Mol Cancer Ther; 11(12) December 2012 Downloaded from mct.aacrjournals.org on September 26, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst October 17, 2012; DOI: 10.1158/1535-7163.MCT-12-0692 BiKEs and TriKEs Enhance NK Cell Effector Function engagement and signals through immunoreceptor tyro- (NM3E2; ref. 6), a 20-amino acid segment of human sine-based activation motifs (ITAM) of the associated muscle aldolase, the VH and VL regions of humanized FceRIg chain and CD3z chain subunits (13). The signal anti-CD22 (14), humanized anti-CD19 (14), and a XhoI delivered via CD16 is potent and induces both a cytotoxic restriction site. The resultant gene fragment was spliced and cytokine response in the absence of cytokine stimu- into the pET21d expression vector, and DNA sequencing lation that can further be enhanced by coengagement of analysis (Biomedical Genomics Center, University of other activating receptors (12). Minnesota) was used to verify that the gene was correct In this study, we show the ability of a CD16/CD19 BiKE in sequence and cloned in frame. Genes encoding the and a CD16/CD19/CD22 TriKE to trigger NK cell acti- BiKEs and monospecific scFV controls were created in vation through direct signaling of CD16 and induce the same manner. For bacterial protein expression and directed secretion of lytic granules and target cell death. purification by ion exchange and size-exclusion chro- Furthermore, we show for the first time the ability of these matography, methods were used as previously des- reagents to induce NK cell activation that leads to cytokine cribed (14). and chemokine production. Proliferation assay Materials and Methods The Burkitt lymphoma Raji cell line (American Type Cell isolation and purification Culture Collection) was cultured at 37 C with 5% CO2 in Peripheral blood mononuclear cells (PBMC) were iso- RPMI-1640 medium (Gibco) supplemented with 10% FBS lated from adult blood (Memorial Blood Center, Minnea- (Gibco). Then, 2 Â 104 Raji cells were treated with varying polis, MN) by centrifugation using a Histopaque gradient concentrations of CD16/CD19/CD22 or nonspecific con- (Sigma-Aldrich), and NK cells were purified by removing trol CD16/EpCAM and 1 nmol/L of a targeted diphtheria T cells, B cells, stem cells, dendritic cells, monocytes, toxin-CD19/CD22 (DT-CD19/CD22) was added and a granulocytes, and erythroid cells via magnetic beads 3H-thymidine proliferation was carried out as previously according to the manufacturer’s protocol (Miltenyi Bio- described (14). tec). Primary acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic lymphobla- Cytokine/chemokine production, CD107a, and sitc leukemia (CLL) samples were obtained from the caspase-3 assay Leukemia MDS Tissue Bank (LMTB) at the University of PBMC or purified NK cells were incubated overnight at Minnesota (Minneapolis, MN). All samples were obtained 37C, 5% CO in basal medium (RPMI supplemented with after informed consent and in accordance with the Dec- 2 10% fetal calf serum). Cells were washed, treated with laration of Helsinki, using guidelines approved by the bscFv CD16/CD19, tscFv CD16/CD19/CD22, rituximab Committee on the Use of Human Subjects in Research at (Genentech), CD22 parental antibody (derived from the the University of Minnesota. RFB4 hybridoma), CD19 parental antibody (derived from the HD37 hybridoma), scFv anti-CD16 or scFv CD19/ Flow cytometry CD22 (negative controls), and CD107a and intracellular Cell suspensions were stained with the following IFN-g assays were conducted as previously described mAbs: PE/Cy7-conjugated CD56 (HCD56; BioLegend), (15). For evaluation of caspase-3 activation, Raji target ECD-conjugated CD3 (UCHT1; Beckman Coulter), FITC- cell cleaved caspase-3 expression was evaluated by fluo- conjugated CD16 (3G8; BD Biosciences), PE-conjugated rescence-activated cell sorting (FACS) analysis using a À þ CD19 (SJ25C1; BD Biosciences), APC-conjugated CD20 live forward/side scatter gate and a CD56 /CD20 gate (2H7; BioLegend), FITC-conjugated CD22 (H1B22; BioLe- to determine cleaved caspase-3–positive Raji popula- gend), PerCP/Cy5.5-conjugated anti-human CD107a tions relative to an isotype control (rabbit IgG; Cell (LAMP-1; H4A3; BioLegend), Pacific Blue-conjugated Signaling Technology). For Luminex analysis of cyto- anti-human IFN-g (4S.B3; BioLegend), and AF488-conju- kines/chemokines, NK cells, Raji cells, and NK cells gated cleaved caspase-3 (9669; Cell Signaling Technolo- cocultured with Raji cells were treated with the BiKE, gy). Phenotypic
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