Adoptive infusion of natural killer (NK) cells is being increasingly explored as a therapy in patients with cancer, although clinical responses are thus far limited to patients with hematological ...malignancies. Inadequate homing of infused NK cells to the tumor site represents a key factor that may explain the poor anti-tumor effect of NK cell therapy against solid tumors. One of the major players in the regulation of lymphocyte chemotaxis is the chemokine receptor chemokine (C-X-C motif) receptor 3 (CXCR3) which is expressed on activated NK cells and induces NK cell migration toward gradients of the chemokine (C-X-C motif) ligand (CXCL9, 10 and 11). Here, we show that ex vivo expansion of human NK cells results in a tenfold increased expression of the CXCR3 receptor compared with resting NK cells (
p
= 0.04). Consequently, these NK cells displayed an improved migratory capacity toward solid tumors, which was dependent on tumor-derived CXCL10. In xenograft models, adoptively transferred NK cells showed increased migration toward CXCL10-transfected melanoma tumors compared with CXCL10-negative wild-type tumors, resulting in significantly reduced tumor burden and increased survival (median survival 41 vs. 32 days,
p
= 0.03). Furthermore, administration of interferon-gamma locally in the tumor stimulated the production of CXCL10 in subcutaneous melanoma tumors resulting in increased infiltration of adoptively transferred CXCR3-positive expanded NK cells. Our findings demonstrate the importance of CXCL10-induced chemoattraction in the anti-tumor response of adoptively transferred expanded NK cells against solid melanoma tumors.
Adoptive natural killer (NK) cell transfer is being increasingly used as cancer treatment. However, clinical responses have so far been limited to patients with hematological malignancies. A ...potential limiting factor in patients with solid tumors is defective homing of the infused NK cells to the tumor site. Chemokines regulate the migration of leukocytes expressing corresponding chemokine receptors. Various solid tumors, including renal cell carcinoma (RCC), readily secrete ligands for the chemokine receptor CXCR2. We hypothesize that infusion of NK cells expressing high levels of the CXCR2 chemokine receptor will result in increased influx of the transferred NK cells into tumors, and improved clinical outcome in patients with cancer.
Blood and tumor biopsies from 14 primary RCC patients were assessed by flow cytometry and chemokine analysis. Primary NK cells were transduced with human CXCR2 using a retroviral system. CXCR2 receptor functionality was determined by Calcium flux and NK cell migration was evaluated in transwell assays.
We detected higher concentrations of CXCR2 ligands in tumors compared with plasma of RCC patients. In addition, CXCL5 levels correlated with the intratumoral infiltration of CXCR2-positive NK cells. However, tumor-infiltrating NK cells from RCC patients expressed lower CXCR2 compared with peripheral blood NK cells. Moreover, healthy donor NK cells rapidly lost their CXCR2 expression upon in vitro culture and expansion. Genetic modification of human primary NK cells to re-express CXCR2 improved their ability to specifically migrate along a chemokine gradient of recombinant CXCR2 ligands or RCC tumor supernatants compared with controls. The enhanced trafficking resulted in increased killing of target cells. In addition, while their functionality remained unchanged compared with control NK cells, CXCR2-transduced NK cells obtained increased adhesion properties and formed more conjugates with target cells.
To increase the success of NK cell-based therapies of solid tumors, it is of great importance to promote their homing to the tumor site. In this study, we show that stable engineering of human primary NK cells to express a chemokine receptor thereby enhancing their migration is a promising strategy to improve anti-tumor responses following adoptive transfer of NK cells.
BackgroundNatural killer (NK) cells hold great promise as a source for allogeneic cell therapy against hematological malignancies, including acute myeloid leukemia (AML). Current treatments are ...hampered by variability in NK cell subset responses, a limitation which could be circumvented by specific expansion of highly potent single killer immunoglobulin-like receptor (KIR)+NKG2C+ adaptive NK cells to maximize missing-self reactivity.MethodsWe developed a GMP-compliant protocol to expand adaptive NK cells from cryopreserved cells derived from select third-party superdonors, that is, donors harboring large adaptive NK cell subsets with desired KIR specificities at baseline. We studied the adaptive state of the cell product (ADAPT-NK) by flow cytometry and mass cytometry as well as cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq). We investigated the functional responses of ADAPT-NK cells against a wide range of tumor target cell lines and primary AML samples using flow cytometry and IncuCyte as well as in a mouse model of AML.ResultsADAPT-NK cells were >90% pure with a homogeneous expression of a single self-HLA specific KIR and expanded a median of 470-fold. The ADAPT-NK cells largely retained their adaptive transcriptional signature with activation of effector programs without signs of exhaustion. ADAPT-NK cells showed high degranulation capacity and efficient killing of HLA-C/KIR mismatched tumor cell lines as well as primary leukemic blasts from AML patients. Finally, the expanded adaptive NK cells had preserved robust antibody-dependent cellular cytotoxicity potential and combination of ADAPT-NK cells with an anti-CD16/IL-15/anti-CD33 tri-specific engager led to near-complete killing of resistant CD45dim blast subtypes.ConclusionsThese preclinical data demonstrate the feasibility of off-the-shelf therapy with a non-engineered, yet highly specific, NK cell population with full missing-self recognition capability.
Abbreviations NK Natural killer RCC Renal cell carcinoma ccRCC clear cell Renal cell carcinoma DNAM-1 DNAX accessory molecule-1 PVR Poliovirus receptor CRISPR/Cas9 Clustered regularly interspaced ...short palindromic repeats associated protein 9 KIR Killer cell immunoglobulin-like receptors MHC Major histocompatibility complex PBMC Peripheral blood mononuclear cell NCR Natural Cytotoxicity Receptor TIGIT T cell immunoreceptor with Ig and ITIM domains KO Knock out WT Wild type TCGA The cancer genome atlas PMN Polymorphonuclear MDSC Myeloid-derived suppressor cells CBLB Casitas B-Lineage Lymphoma Proto-Oncogene B Gas6 Growth arrest-specific protein 6 TAM Tyro3, Axl, and Mer COX-2 Cyclooxygenase-2 Arg-1 Arginase-1 iNOS Inducible nitric oxide synthase t-SNE t-distributed stochastic neighbor embedding Dear Editor, The activating receptor DNAX accessory molecule-1 (DNAM-1) plays an important role in T and natural killer (NK) cell-mediated cytotoxicity via the interaction with its ligands poliovirus receptor (PVR, CD155) and Nectin-2 (CD112). Analysis of The Cancer Genome Atlas (TCGA) data revealed that high DNAM-1 expression is associated with increased overall survival and progression-free survival in patients with RCC. Abbreviations: RCC, renal cell carcinoma; DNAM-1, DNAX accessory molecule-1; NK, natural killer; PVR, poliovirus receptor; MFI, mean fluorescence intensity; WT, wild type; KO, knockout; eNK, experienced NK; t-SNE, t-distributed stochastic neighbor embedding; PBMC, peripheral blood mononuclear cell; GO, gene ontology; KEGG, Kyoto encyclopedia of genes and genomes; CBLB, Casitas B-Lineage Lymphoma Proto-Oncogene B. The expression of PVR has shown to be a negative prognostic marker in several cancers, including urothelial carcinoma, hepatocellular carcinoma, and head and neck squamous cell carcinoma. Consistent with this study, we found that warfarin inhibited PVR-mediated downregulation of DNAM-1 in NK cells cultured with WT Caki-1 cells (Figure 1I), suggesting that DNAM-1 downregulation might be due to ubiquitin-mediated proteolysis. Since CBLB knockout or knockdown has been shown to increase NK cell cytotoxicity against tumor cells, targeting CBLB may be a potential way to enhance NK cell antitumor activities 7, 8.
Anaplastic thyroid carcinoma (ATC) is one of the most aggressive forms of cancer with no curative therapies available. To date, strategies to target ATC by immunotherapy have not been evaluated. We ...investigated whether ATC would be a suitable target for natural killer (NK) cell-based immunotherapy.
We first established seven new cell lines from ATC tumors, three from papillary thyroid carcinoma tumors and analyzed them together with eight additional ATC cell lines. Cells were analyzed for sensitivity to lysis by NK cells and their ability to chemoattract and regulate the activity of NK cells. In addition, fresh tumor samples and peripheral blood from six patients with ATC were analyzed for NK cell infiltration and phenotype.
We observed that ATC cell lines are sensitive to lysis by ex vivo expanded NK cells and that the lysis was abrogated upon blockade of NKG2D. Sensitivity of thyroid cancer cell lines to NK cell-mediated lysis correlated with surface expression of UL16-binding protein 2 on tumor cells. Moreover, ATC cell lines produced high levels of CXCL10 and stimulated migration of expanded NK cells and ATC tumors were enriched for NK cells expressing the cognate chemokine receptor CXCR3. However, compared with NK cells in peripheral blood, ATC tumor-derived NK cells displayed a suppressed phenotype with a downregulated expression of NKG2D. In vitro, suppression of NK cell-mediated lysis and NKG2D expression by ATC cells was restored upon neutralization of prostaglandin-E2.
ATC cell lines are sensitive to NK cell-mediated lysis via ULBP2/5/6 and chemoattract CXCR3-positive NK cells. Patients with ATC may benefit from NK cell-based immunotherapy.
Natural killer (NK) cells play a key role in tumor immunosurveillance due to their ability to induce apoptosis of tumor cells and produce pro-inflammatory cytokines, without prior immune ...sensitization. In particular, NK cells demonstrate potent anti-tumor immune responses against metastases and hematological malignancies. These characteristics make NK cells attractive for cancer immunotherapy. In contrast to hematological malignancies, however, adoptive transfer of NK cells has so far not provided clinical benefit in patients with solid tumors. Critical barriers in targeting solid tumors include inefficient homing of infused cells to tumor sites and immunosuppression in the tumor microenvironment. In this thesis, I have investigated strategies to improve NK cell migration to solid tumors and explored the immune landscape in patients with renal cell carcinoma (RCC), focusing on NK cells. In the first part of this thesis, local production of ligands for the chemokine receptor CXCR3 was induced in the microenvironment of melanoma tumors, which enhanced intratumoral localization of infused a Viva expanded human NK cells in mouse xenograft models, resulting in superior anti-tumor immunity (paper I). Subsequently, we genetically engineered human NK cells to express the chemokine receptor CXCR2, which conferred them with the ability to specifically migrate to recombinant and RCC tumor-derived CXCR2 ligands, enabling improved targeting of tumor cells in vitro (paper II). In the second part, we performed a comprehensive analysis of immune cell and soluble factor profiles in blood and tumor biopsies of 14 patients with primary RCC, identifying factors important for NK cell migration, activation, and immunosuppression (paper III). We found profound changes in intratumoral NK cell phenotypes compared with those in peripheral blood, with downregulation of the activation receptor DNAM-1 possibly representing a tumor immune escape mechanism. Moreover, we identified low expression of DNAM-1 and PD-1 on intratumoral and circulating NK cells, respectively, as potential biomarkers of disease progression. In summary, approaches to improve NK cell homing to tumors and a greater understanding of the RCC immune landscape provided in this thesis, will advance the use and increase the success of NK cell-based therapies in patients with solid tumors.
Natural killer (NK) cells hold great promise as a source for allogeneic cell therapy against hematological malignancies, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). NK ...cell recognition of allogeneic tumors is strictly regulated by inhibitory killer cell immunoglobulin-like receptors (KIR) that bind to groups of HLA class I alleles. However, KIR expression on NK cells is highly diverse due to variation in gene content, polymorphism and copy number in combination with stochastic expression of the protein in individual cells. As a consequence, the number of efficacious allogeneic NK cells within a product isolated and expanded from random donors can vary a great deal and potentially be negligible. Our group has defined a repertoire of NK cells that is uniquely found in individuals with prior exposure to cytomegalovirus (CMV). Interestingly, these cells were shown to share many attributes usually reserved for adaptive immune cells including increased longevity, memory, and serial killing. We have previously described a 14-day protocol to enrich for adaptive NKG2C+CD57+ NK cells from CMV sero-positive donors with a homogenous expression of one single self-HLA specific KIR (self KIR). Here, we present new data on the GMP-transfer and clinical scale-up of this protocol, providing a route to off-the-shelf adaptive NK cell therapy for refractory high-risk AML/MDS. By screening >250 healthy donors, we first established the prerequisites for robust expansion of adaptive NK cells from peripheral blood of CMV+ donors and found that donors with >15% pre-existing adaptive NK cells showed efficient expansion of adaptive NK cells (Figure 1A-B). Apheresis products from a pool of pre-screened third-party donors are currently being collected for GMP freezing and use in an off-the-shelf setting intended for HLA mismatched patients to maximize alloreactivity by “missing” self. The GMP compatible protocol led to a robust expansion of clinical doses of self-KIR+ adaptive NK cells, with an average frequency of 60% self-specific KIR+ cells in the end product (Figure 1C-D). Based on the expression of self-KIR the expanded cells were educated, showing large dense-core granules and high levels of granzyme B. Further characterization in CyTOF using 36 phenotypic and functional markers revealed a highly activated state with high expression of DNAM-1 and CD2, which are critical for NK cell adhesion and function (Figure 1E). Notably, the expanded adaptive NK cells were negative for the HLA-E binding inhibitory receptor NKG2A, which is a major check point for T- and NK-cell based therapies. A microchip single-cell imaging platform revealed high serial killing capacity of the expanded adaptive NK cells. In flow cytometry-based killing assays and long-term killing assays this enhanced capacity for serial killing correlated with highly efficient targeting of mismatched PHA blasts (Figure 1F), tumor cell lines (Figure 1G), and MDS blasts. These pre-clinical data demonstrate the feasibility of off-the-shelf therapy with a non-engineered and yet highly specific NK cell population, representing the first route to clinical testing of missing self-recognition as it was originally defined over thirty years ago.
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Valamehr:Fate Therapeutics Inc.: Employment. Alici:Vycellix: Consultancy, Equity Ownership, Patents & Royalties, Research Funding; Intellia: Membership on an entity's Board of Directors or advisory committees. Ljunggren:Fate Therapeutics: Patents & Royalties; Vycellix: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Malmberg:Fate Therapeutics Inc.: Consultancy, Research Funding.
Chemotherapeutic agents such as paclitaxel applied in ultra-low, non-cytotoxic doses were previously shown to stimulate dendritic cell activity and anti-tumor immune responses upon vaccination in ...mouse transplantable tumor models. However, the mechanisms of these alterations-termed chemoimmunomodulation or chemomodulation-are still not clear. This study investigated the effect of paclitaxel applied in ultra-low, non-cytotoxic doses on the efficiency of immunization of healthy C57BL/6 mice with the peptide derived from tyrosinase related protein (TRP)-2 as a model melanoma antigen. Using an IFNγ ELISPOT assay, it was found that administration of 1 mg paclitaxel/kg in combination with the peptide vaccination strongly increased the frequencies of TRP-2 specific spleen T-cells as compared to levels due to the vaccination alone. This was associated with a significant decrease in the levels of regulatory T-cells (Treg) and immature myeloid cells (known as a counterpart of myeloid derived suppressor cells MDSC in healthy mice). Such impairments of potential immunosuppressive cells were found to correlate with a strong increase in the amount of effector CD8+ and CD4+ T-cells in the bone marrow and spleen. Furthermore, in paclitaxel-treated mice, a significant augmentation of natural killer (NK) cell numbers in the bone marrow and their ability to produce IFNγ were observed. In addition, the level of NK-T-cells in the lymph nodes was also increased. It is suggested that paclitaxel applied in ultra-low, non-cytotoxic doses may potentially enhance the efficacy of anti-tumor vaccinations by neutralizing immunosuppressive Treg and MDSC populations in tumor-bearing hosts.
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