Trispecific Killer Engager Research Articles

189 - Trispecific Killer Engager CD16xIL15xCD33 Enhances Alloreactivity of NK Cells Against Aberrant Mast Cells of Patients with Systemic Mastocytosis

189 - Trispecific Killer Engager CD16xIL15xCD33 Enhances Alloreactivity of NK Cells Against Aberrant Mast Cells of Patients with Systemic Mastocytosis

CD16-IL15-CD33 Trispecific Killer Engager (TriKE) Overcomes Cancer-Induced Immune Suppression and Induces Natural Killer Cell-Mediated Control of MDS and AML Via Enhanced Killing Kinetics

Although adoptive transfer of NK cells with IL-2 can induce complete remissions in 30-50% of patients with refractory AML, efficacy is limited by IL-2 mediated induction of Tregs and by lack of antigen specificity. Thus we generated a 161533 trispecific killer engager (TriKE) molecule containing an anti-CD16 scFv to engage NK cells, an anti-CD33 scFv to engage myeloid targets (including myelodysplastic syndrome [MDS]), and a modified IL-15 linker. We have previously shown that this molecule is superior to a 1633 bispecific killer engager (BiKE) in killing of AML targets and that it promotes enhanced survival and in vivo expansion of NK cells. We questioned the mechanism for the increased potency of the 161533 TriKE and if the TriKE could activate the dysfunctional and suppressed NK cells found in patients with MDS. Cryopreserved mononuclear cells (obtained from the NMDP Sample Repository) collected from 8 patients with advanced MDS were tested to investigate how the TriKE might enhance functionality in this setting. Previously we reported that MDS patients have significantly decreased frequencies of NK cells due to increased CD33+ myeloid derived suppressor cells. Our 161533 TriKE enhanced the function of NK cells derived from MDS patients against acute promyelocytic CD33+ leukemia HL-60 tumor targets (Figure 1A), when compared to 1633 BiKE, in a flow cytometry assay measuring NK cell degranulation (% CD107a: 41.8±3.8 vs. 30.3±3.2, p=0.004), and inflammatory cytokine production (% IFNg: 40.7±5.0 vs. 30.0±4.9, p=0.009; % TNFa: 36.9±5.5 vs. 28.4±4.8, p=0.009). State of the art microchip-based live cell imaging was then employed to evaluate NK cell function and contact-to-target dynamics (Figure 1B). Briefly, resting NK cells and HL-60 target cells were stained with distinct dyes and then co-incubated in the presence of BiKE or TriKE in microwells at 37°C and 5% CO2. Cells were then imaged for 12 hours using a Zeiss 880 microscope and analyzed by Matlab. In contrast to those incubated with BiKE, NK cells cultured in the presence of TriKE had augmented cytotoxicity (37%±6% vs. 59%±6%, p=0.02) and killed their targets remarkably faster (time to first target kill = 148±30 min vs. 75±26 min, p< 0.0001). In addition, NK cell serial killers were more common in the presence of TriKE compared to BiKE (number of killed targets ≥3: 18%±7% vs. 9%±1%, p=0.04). Having shown the robust killing dynamics of TriKE primed NK cells, we next designed an in vivo dose escalation study to evaluate HL-60 tumor control in our xenogeneic mouse model. NSG (NOD scid gamma) mice were conditioned with a sub-lethal dose of radiation (275 cGy) and engrafted with 750,000 HL-60luc cells, which allow for tracking of tumor growth using bioluminescent imaging (BLI). Mice were infused with 1 million fresh healthy donor human NK cells and treated daily with recombinant IL-15 (5 ug/injection), 20-200 ug of 161533 TriKE, or left untreated. Higher doses of TriKE provided better tumor control (6.3x109±2.9x109 [200 ug 1615133] vs. 1.6 x1010±2.7x109 [20 ug 161533] p/sec/cm2/sr) demonstrating dose responsiveness at days 14 and 21 This was not due to increased proliferation of effectors (NK cell numbers) induced by the IL-15 segment of the TriKE as the absolute PBNK cell counts in mice were not different across the TriKE concentrations. However, the day 14 and 21 absolute PBNK counts were higher in all of the TriKE groups when compared to the IL-15 treated mice, including in the 20 ug 161533 TriKE group which best matched the 5 ug IL-15 dose. These data indicated that the TriKE molecule mediates superior NK cell expansion or maintenance in vivo. Taken together the in vivo data suggests that 161533 TriKE not only mediates tumor control by inducing NK cell proliferation and survival in a methodology that exceeds signals provided by IL-15 alone, but also increases tumor killing through enhanced killing kinetics. In summary, we have shown that the 161533 TriKE can rescue dysfunctional NK cells suppressed in patients with MDS and can mediate potent in vivo tumor killing presumably through better NK cell maintenance and enhanced killing kinetics This is of translational importance and demonstrates that the cancer induced immune suppression is reversible by the activating signals induced by the TriKE molecule. The 161533 TriKE represents a promising modality to maximizing NK cell based immunotherapies against MDS and AML and will be in phase I clinical testing the first half of 2017. [Display omitted] DisclosuresCooley:Fate Therapeutics: Research Funding. Vallera:Oxis Biotech: Consultancy, Membership on an entity's Board of Directors or advisory committees. Miller:Fate Therapeutics: Consultancy, Research Funding; Oxis Biotech: Consultancy, Other: SAB.

IL15 Trispecific Killer Engagers (TriKE) Make Natural Killer Cells Specific to CD33+ Targets While Also Inducing Persistence, In Vivo Expansion, and Enhanced Function.

The effectiveness of NK cell infusions to induce leukemic remission is limited by lack of both antigen specificity and in vivo expansion. To address the first issue, we previously generated a bispecific killer engager (BiKE) containing single-chain scFv against CD16 and CD33 to create an immunologic synapse between NK cells and CD33(+) myeloid targets. We have now incorporated a novel modified human IL15 crosslinker, producing a 161533 trispecific killer engager (TriKE) to induce expansion, priming, and survival, which we hypothesize will enhance clinical efficacy. Reagents were tested in proliferation and functional assays and in an in vivo xenograft model of AML. When compared with the 1633 BiKE, the 161533 TriKE induced superior NK cell cytotoxicity, degranulation, and cytokine production against CD33(+) HL-60 targets and increased NK survival and proliferation. Specificity was shown by the ability of a 1615EpCAM TriKE to kill CD33-EpCAM(+) targets. Using NK cells from patients after allogeneic stem cell transplantation when NK cell function is defective, the 161533 TriKE restored potent NK function against primary AML targets and induced specific NK cell proliferation. These results were confirmed in an immunodeficient mouse HL-60-Luc tumor model where the 161533 TriKE exhibited superior antitumor activity and induced in vivo persistence and survival of human NK cells for at least 3 weeks. Off-the-shelf 161533 TriKE imparts antigen specificity and promotes in vivo persistence, activation, and survival of NK cells. These qualities are ideal for NK cell therapy of myeloid malignancies or targeting antigens of solid tumors. Clin Cancer Res; 22(14); 3440-50. ©2016 AACRSee related commentary by Talmadge, p. 3419.

CD16-IL15-CD33 Trispecific Killer Engager (TriKE) induces NK cell expansion, persistence, and myeloid blast antigen specific killing.

Abstract NK cell infusions induce complete remissions in 30–50% of patients with refractory AML when combined with lymphodepleting chemotherapy and IL-2. NK cell therapy is limited by lack of antigen specificity and by IL-2 mediated induction of Tregs. To overcome these limitations we developed a 161533 trispecific killer engager (TriKE) molecule containing an anti-CD16 scFv, to engage NK cells, an anti-CD33 scFv, to engage AML targets, and an IL-15 molecule. We have shown previously that a 1633 bispecific killer engager (BiKE) is capable of creating an immunologic synapse between NK cells and AML targets leading to target killing. These molecules generate higher affinity interactions than natural CD16 binding to Fc portions of therapeutic antibodies, resulting in increased NK cell functionality. Addition of IL-15 in the TriKE molecule is meant to enhance NK cell priming, proliferation, and maintenance without inducing Tregs. Cytotoxicity assays and cytokine secretion assessment of NK cells incubated with CD33+ HL-60 target cells indicated that the TriKE induced greater NK cell activation than the BiKE or no molecule control. The TriKE was able to induce potent NK cell proliferation and survival when compared to the BiKE. Interestingly, when compared to recombinant IL-15 the TriKE induced less proliferation of T cells while inducing similar proliferation of NK cells from healthy donors, indicating specificity. The TriKE was capable of rescuing NK cell function of post-transplant patients. In vivo functionality was tested in an NSG xenogeneic model engrafted with HL-60-luc targets and NK cells. TriKE treatment resulted in reduced tumor burden and NK cell maintenance at day 21, displaying the immunotherapeutic potential of the molecule.

Generation of BiKEs and TriKEs to Improve NK Cell-Mediated Targeting of Tumor Cells.

Cancer immunotherapies have gained significant momentum over the past decade, particularly with the advent of checkpoint inhibitors and CAR T-cells. While the latter personalized targeted immunotherapy has revolutionized the field, a need for off-the-shelf therapies remains. The ability of NK cells to quickly lyse antibody-coated tumors and potently secrete cytokines without prior priming has made NK cells ideal candidates for antigen-specific immunotherapy. NK cells have been targeted to tumors through two main strategies: mono-specific antibodies and bi/tri-specific antibodies. Mono-specific antibodies drive NK cell antibody-dependent cell-mediated cytotoxicity (ADCC) of tumor cells. Bi/tri-specific antibodies drive re-directed lysis of tumor cells through binding of a tumor antigen and direct binding and crosslinking of the CD16 receptor on NK cells, thus bypassing the need for binding of the Fc portion of mono-specific antibodies. This chapter focuses on the generation of bi- and tri-specific killer engagers (BiKEs and TriKEs) meant to target NK cells to tumors. BiKEs and TriKEs are smaller molecules composed of 2-3 variable portions of antibodies with different specificities, and represent a novel and more versatile strategy compared to traditional bi- and tri-specific antibody platforms.

Trispecific Killer Engagers (TriKEs) that contain IL-15 to make NK cells antigen specific and to sustain their persistence and expansion

Natural killer (NK) cells are capable of immune surveillance mediated by a balance of activating and inhibitory receptors. We have shown that adoptive transfer of NK cells can induce complete remissions in patients with refractory AML when combined with lymphodepleting chemotherapy and IL-2 to stimulate survival and in vivo expansion of the NK cells. Using this approach, 30-50% of patients with refractory attain clinical remissions. We hypothesize that clinical benefit is limited by the lack of antigen specificity and because IL-2 induces regulatory T cells (Treg) that inhibit NK cell proliferation. We have developed trispecific killer engagers (TriKE) to overcome these limitations. We have previously shown that bispecific killer engagers (BiKEs) are capable of creating immunologic synapses between NK cells and CD33 antigens on AML and MDS targets leading to NK cell signaling through the highly potent CD16 (FcγRIII) receptor. We observed that although CD16 engagement leads to enhanced killing and cytokine production by NK cells, there is no effect on proliferation (designated 1633 in Figure, Panel B). Because IL-15 is the homeostatic factor for NK cell proliferation, survival, activation, and development and because unlike IL-2, it does not stimulate CD4+ CD25+ Treg, we developed a TriKE that includes a modified human IL-15 crosslinker sandwiched between single chain Fv against CD16 and CD33 (designated 161533). The final target gene was spliced into pET21d vector expression plasmid and transformed into the Escherichia coli and compared to a 1633 BiKE with the identical Fv regions as above. TriKEs were recovered from inclusion bodies, refolded, and purified. The addition of the IL-15 crosslinker to the molecule reduced its isoelectric point by two pH units creating more favorable conditions for purification and enhancing yield. Despite identical amounts of starting inclusion bodies, the final yield of 161533 TriKE was twice the yield of 1633 BiKE indicating more favorable purification dynamics. To establish the selectivity of the anti-CD33 binding moiety, BiKEs and TriKEs were analyzed using flow cytometry and both bound to CD16 and CD33 selectively without interference from the IL-15 crosslinker. To determine the capacity of BiKE and TriKE reagents to mediate NK cell killing, activity was compared in a 4-hour chromium release assay (representative of 3 separate experiments) against CD33+ HL60 targets (Figure Panel A) or CD33- HT29 (not shown). The highest specific activity is with the 161533 TriKE containing a modified IL-15 linker as control samples of anti-CD16 and anti-CD33 alone did not augment cytotoxicity while 1633 BiKE mediating killing was intermediate. CD33- HT29 was not killed. We next tested BiKE and TriKE in a proliferation assay using CSFE labeled primary NK cells. NCI produced recombinant human IL-15 was used as a positive control. The TriKE exhibited potent proliferation of primary NK cells compared to BiKE (Figure Panel B). As target cell induced NK cell production of cytokines is an important aspect of the anti-leukemia response, we compared the BiKE and TriKE for their ability to mediate cytokine production with primary NK cells and HL60 targets. TriKE induced greater target cell specific TNFα, INFγ, IL-6, MIP1α, GM-CSF and IL-8 compared to BiKE. We then tested the activity of the TriKE in a xenogeneic model of human NK cell adoptive transfer where NSG mice are given 0.75 million HL60-Luc cells followed by 250 cGy XRT ± 1 million NK cells 3 days later from a CD3 and CD19 depleted clinical product and daily IP injection of 50 mcg/kg TriKE. Significant anti-tumor activity without toxicity was seen in the NK and TriKE treated mice (P<0.002). In addition, the 161533 TriKE had potent in vivo IL-15 activity to support the in vivo persistence and expansion of NK cells while BiKE did not (data not shown). In summary, we have shown that a novel TriKE construct can mediate CD16 directed cytotoxicity against CD33 targets and this is specifically maintained when adding a modified IL-15 linker. The IL-15 activity is fully preserved in vitro and in vivo and unexpectedly enhances the purification product. This will allow us to redirect NK cells to malignant targets while providing cytokine stimulation with an easily exportable off-the-shelf non-gene therapy strategy using a drug that also maintains NK cell persistence, survival and in vivo expansion. Drug production is in progress for clinical testing in 2016. [Display omitted] DisclosuresMiller:Coronado: Speakers Bureau; BioSciences: Speakers Bureau; Celegene: Speakers Bureau.

NK cells in therapy of cancer.

Natural killer (NK) cells recognize targets stressed by malignant transformation or infection and can be long-lived. They become educated by interacting with major histocompatibility antigen (MHC) class I molecules to gain function to kill targets and produce cytokines. In the clinic, haploidentical NK cells can be adoptively transferred to treat cancer. Persistence and in vivo expansion of NK cells depends on lymphodepleting chemotherapy to make space and induce release of endogenous IL-15. In vivo expansion is also enhanced by cytokine administration but IL-2 has the down side of stimulating CD25hi regulatory T cells (Tregs). Other limitations to NK-cell therapy include poor in vivo survival and lack of specificity. Bispecific or trispecific killer engagers that target CD16 on NK cells to enhance recognition of tumor antigens, and desintegrin and metalloproteinase 17 (ADAM17) inhibition that prevents CD16 shedding after NK-cell activation should promote enhanced killing of cancer with specificity. These are exciting times; more than 35 years after NK cells were initially described, we are exploiting their capacity for clinical therapy.