Purpose 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 IL-15 crosslinker, producing a 161533 trispecific killer engager (TriKE) to induce expansion, priming, and survival, which we hypothesize will enhance clinical efficacy. Methods Reagents were tested in proliferation and functional assays and in an in vivo xenograft model of AML. Results When compared to 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 anti-tumor activity and induced in vivo persistence and survival of human NK cells for at least 3 weeks. Conclusions 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.
Tumor-induced immune defects can weaken host immune response and permit tumor cell growth. In a systemic model of murine acute myeloid leukemia (AML), tumor progression resulted in increased regulatory T cells (Treg) and elevation of program death-1 (PD-1) expression on CD8 cytotoxic T cells (CTLs) at the tumor site. PD-1 knockout mice were more resistant to AML despite the presence of similar percentage of Tregs compared with wild type. In vitro, intact Treg suppression of CD8 T-cell responses was dependent on PD-1 expression by T cells and Tregs and PD-L1 expression by anti-gen-presenting cells. In vivo, the function of adoptively transferred AML-reactive CTLs was reduced by AML-associated Tregs. Anti-PD-L1 monoclonal antibody treatment increased the proliferation and function of CTLs at tumor sites, reduced AML tumor burden, and resulted in long-term survivors. Treg depletion followed by PD-1/PD-L1 blockade showed superior efficacy for eradication of established AML. These data demonstrated that interaction between PD-1 and PD-L1 can facilitate Treg-induced suppression of T-effector cells and dampen the antitumor immune response. PD-1/PD-L1 blockade coupled with Treg depletion represents an important new approach that can be readily translated into the clinic to improve the therapeutic efficacy of adoptive AML-reactive CTLs in advanced AML disease. (Blood. 2010;116(14):2484-2493)
The development of 3D in vitro models capable of recapitulating native tumor microenvironments could improve the translatability of potential anticancer drugs and treatments. Here, 3D bioprinting techniques are used to build tumor constructs via precise placement of living cells, functional biomaterials, and programmable release capsules. This enables the spatiotemporal control of signaling molecular gradients, thereby dynamically modulating cellular behaviors at a local level. Vascularized tumor models are created to mimic key steps of cancer dissemination (invasion, intravasation, and angiogenesis), based on guided migration of tumor cells and endothelial cells in the context of stromal cells and growth factors. The utility of the metastatic models for drug screening is demonstrated by evaluating the anticancer efficacy of immunotoxins. These 3D vascularized tumor tissues provide a proof‐of‐concept platform to i) fundamentally explore the molecular mechanisms of tumor progression and metastasis, and ii) preclinically identify therapeutic agents and screen anticancer drugs.
This study evaluates the mechanism by which bispecific and trispecific killer cell engagers (BiKEs and TriKEs) act to trigger human 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 Ca2+ 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 Ca2+ mobilization through direct CD16 signaling. Co-culture 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 (E:T) ratios and maintained functional stability after 24 and 48 hours of culture in human serum. NK cell production of IFN-γ, TNF-α, GM-CSF, IL-8, MIP-1α and 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 ALL and CLL 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, demonstrating their therapeutic potential for enhancing NK cell immunotherapies for leukemias and lymphomas.
Graft-versus-host disease (GVHD), in which immunocom
Purpose The graft versus leukemia (GVL) effect by Natural Killer (NK) cells prevents relapse following hematopoietic stem cell transplantation. We determined whether a novel bi-specific killer cell engager (BiKE) signaling through CD16 and targeting CD33 could activate NK cells at high potency against AML targets. Experimental Design We investigated the ability of our fully humanized CD16x33 BiKE to trigger in vitro NK cell activation against HL60 (CD33+), RAJI (CD33−), and primary AML targets (de novo, refractory and post transplant) to determine whether treatment with CD16x33 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 CD16x33 BiKE when cells were cultured with HL60 targets, CD33+ de novo and refractory AML targets. Combination treatment with CD16x33 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 CD16x33 BiKE resulted in activation, especially in those recipients with CMV reactivation. Conclusion CD16x33 BiKE can overcome self inhibitory signals and effectively elicit NK cell effector activity against AML. These in vitro studies highlight the potential of CD16x33 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 anti-leukemic therapy post-transplantation.
Key Points NK cells and their expression of FcRγIII (CD16) are decreased in MDS and inversely correlate with a substantial increase in MDSCs. CD16xCD33 BiKE potently activates blood and marrow MDS-NK cells at all diseases stages to lyse CD33+ MDS and CD33+ MDSC targets.
A number of clever recombinant methodologies have been developed that recapitulate the valencies of IgG’s (bivalent) and IgA’s (tetravalent). Although, higher synthetic valencies have been achieved by conjugation of either monoclonal antibodies or single-chain antibodies to nanoparticles and liposomes, a method for the preparation of recombinant antibodies with valencies similar to IgM’s (decavalent) but considerably less than what is generally found after antibody particle conjugation has yet to be devised. Recently, we have developed a methodology for the design of bivalent Chemically Self-Assembled Antibody Nanorings (CSANs). We now report the crystal structure of the nanoring subunit composed of the E. coli DHFR dimer and a methotrexate dimerizer (MTX2-C9) containing visible nine methylene linker and a protocol for the preparation of CSANs from this subunit with valencies similar to IgMs, ranging from 8–10 single chain antibodies (scFvs). The multivalent CSANs were reversibly assembled from a fusion protein dihydrofolate reductase (DHFR)-DHFR-antiCD3 scFv containing a single glycine linker between the two DHFR scaffolding proteins. We also demonstrate that, similar to the parental bivalent anti-CD3 monoclonal antibody (MAB), anti-CD3 CSANs selectively bind to CD3+ leukemia cells, and undergo rapid internalization through a caveolin-independent pathway that requires cholesterol, actin polymerization and protein tyrosine kinase activation. While treatment with the monoclonal antibody leads to T-cell activation and nearly complete loss (i.e. 90%) of surface displayed T-cell receptor (TCR), only 25–30% of the TCR down regulate and no significant T-cell proliferation is observed after treatment of peripheral blood mononuclear cells (PBMCs) with anti-CD3 CSANs. Consistent with the proliferation findings, 15–25 % less CD25 (IL-2 receptor) was found on the surface of PBMCs treated with either the polyvalent or bivalent anti-CD3 CSANs, respectively, than on PBMCs treated with the parental MAB. Comparative experiments with F(ab')2 derived from the MAB confirm that the activation of the T-cells by the MAB is dependent on the Fc domain, and thus interactions of the PBMC T-cells with accessory cells, such as macrophages. Taken together, our results demonstrate that anti-CD3 CSANs with valencies ranging from 2 to 8 could be employed for radionuclide, drug or potentially oligonucleotide delivery to T-cells without, as has been observed for other antibody conjugated nanoparticles, the deleterious affects of activation observed for MAB. Further the CSAN construct may be adapted for the preparation of other multivalent scFvs.
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