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In Vivo Tracking of Adoptively Transferred Natural Killer Cells in Rhesus Macaques Using ⁸⁹Zirconium-Oxine Cell Labeling and PET Imaging.

ABSTRACT: PURPOSE:Trials of adoptive natural killer (NK)-cell immunotherapy for hematologic malignancies have thus far shown only marginal effects, despite the potent in vitro antitumor activity of these cells. Homing of infused cells to tumor microenvironments is critical for efficacy, but has not been well characterized. We established a novel method to track and quantify the distribution of adoptively transferred NK cells using rhesus macaques (RM) as a clinically relevant preclinical model. EXPERIMENTAL DESIGN:RM NK cells were expanded ex vivo for 14-21 days, labeled with ⁸⁹Zr-oxine complex, and assessed for phenotype, function, and survival. Trafficking of ⁸⁹Zr-labeled ex vivo-expanded NK cells infused into RMs was monitored and quantitated by serial positron emission tomography (PET)/CT (n = 3, 2.05 ± 0.72 MBq, 23.5 ± 2.0 × 10⁶ NK cells/kg) and compared with that of ⁸⁹Zr-labeled nonexpanded NK cells, apoptotic NK cells, and hematopoietic stem and progenitor cells (HSPC). RESULTS:NK cells retained sufficient levels of ⁸⁹Zr for accurate in vivo tracking for 7 days. ⁸⁹Zr labeling did not alter cellular phenotype, viability, or function. PET/CT showed NK cells initially localized in the lungs, followed by their migration to the liver, spleen, and, at low levels, bone marrow. One day following transfer, only 3.4% of infused NK cells localized to the BM versus 22.1% of HSPCs. No clinical side effects were observed, and dosimetry analysis indicated low organ radioexposures of 6.24 mSv/MBq (spleen) or lower. CONCLUSIONS:These data support translation of this technique to humans to track the distribution of adoptively infused cells and to develop novel techniques to improve immune cell homing to tumor microenvironments.

SUBMITTER: Sato N

PROVIDER: S-EPMC7269806 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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<i>In Vivo</i> Tracking of Adoptively Transferred Natural Killer Cells in Rhesus Macaques Using <sup>89</sup>Zirconium-Oxine Cell Labeling and PET Imaging.

Sato Noriko N Stringaris Kate K Davidson-Moncada Jan K JK Reger Robert R Adler Stephen S SS Dunbar Cynthia C Choyke Peter L PL Childs Richard W RW

Clinical cancer research : an official journal of the American Association for Cancer Research 20200207 11

<h4>Purpose</h4>Trials of adoptive natural killer (NK)-cell immunotherapy for hematologic malignancies have thus far shown only marginal effects, despite the potent <i>in vitro</i> antitumor activity of these cells. Homing of infused cells to tumor microenvironments is critical for efficacy, but has not been well characterized. We established a novel method to track and quantify the distribution of adoptively transferred NK cells using rhesus macaques (RM) as a clinically relevant preclinical mo ...[more]

PMID: 32034075

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Project description:BackgroundChimeric antigen receptor (CAR) T cell therapy is an exciting cell-based cancer immunotherapy. Unfortunately, CAR-T cell therapy is associated with serious toxicities such as cytokine release syndrome (CRS) and neurotoxicity. The mechanism of these serious adverse events (SAEs) and how homing, distribution and retention of CAR-T cells contribute to toxicities is not fully understood. Enabling in vitro methods to allow meaningful, sensitive in vivo biodistribution studies is needed to better understand CAR-T cell disposition and its relationship to both effectiveness and safety of these products.MethodsTo determine if radiolabelling of CAR-T cells could support positron emission tomography (PET)-based biodistribution studies, we labeled IL-13Rα2 targeting scFv-IL-13Rα2-CAR-T cells (CAR-T cells) with 89Zirconium-oxine (89Zr-oxine) and characterized and compared their product attributes with non-labeled CAR-T cells. The 89Zr-oxine labeling conditions were optimized for incubation time, temperature, and use of serum for labeling. In addition, T cell subtype characterization and product attributes of radiolabeled CAR-T cells were studied to assess their overall quality including cell viability, proliferation, phenotype markers of T-cell activation and exhaustion, cytolytic activity and release of interferon-γ upon co-culture with IL-13Rα2 expressing glioma cells.ResultsWe observed that radiolabeling of CAR-T cells with 89Zr-oxine is quick, efficient, and radioactivity is retained in the cells for at least 8 days with minimal loss. Also, viability of radiolabeled CAR-T cells and subtypes such as CD4 + , CD8 + and scFV-IL-13Rα2 transgene positive T cell population were characterized and found similar to that of unlabeled cells as determined by TUNEL assay, caspase 3/7 enzyme and granzyme B activity assay. Moreover, there were no significant changes in T cell activation (CD24, CD44, CD69 and IFN-γ) or T cell exhaustion (PD-1, LAG-3 and TIM3) markers expression between radiolabeled and unlabeled CAR-T cells. In chemotaxis assays, migratory capability of radiolabeled CAR-T cells to IL-13Rα2Fc was similar to that of non-labeled cells.ConclusionsImportantly, radiolabeling has minimal impact on biological product attributes including potency of CAR-T cells towards IL-13Rα2 positive tumor cells but not IL-13Rα2 negative cells as measured by cytolytic activity and release of IFN-γ. Thus, IL-13Rα2 targeting CAR-T cells radiolabeled with 89Zr-oxine retain critical product attributes and suggest 89Zr-oxine radiolabeling of CAR-T cells may facilitate biodistribution and tissue trafficking studies in vivo using PET.

Project description:The encapsulation of Glucocorticoids (GCs) into long-circulating liposomes (LCLs) is a proven strategy to reduce the side effects of glucocorticoids and improve the treatment of inflammatory diseases, such as rheumatoid arthritis (RA). With the aim of supporting the development of GC-loaded LCLs, and potentially predict patient response to therapy clinically, we evaluated a direct PET imaging radiolabelling approach for preformed GC-LCLs in an animal model of human inflammatory arthritis. Methods: A preformed PEGylated liposomal methylprednisolone hemisuccinate (NSSL-MPS) nanomedicine was radiolabelled using [89Zr]Zr(oxinate)4 (89Zr-oxine), characterised and tracked in vivo using PET imaging in a K/BxN serum-transfer arthritis (STA) mouse model of inflammatory arthritis and non-inflamed controls. Histology and joint size measurements were used to confirm inflammation. The biodistribution of 89Zr-NSSL-MPS was compared to that of free 89Zr in the same model. A therapeutic study using NSSL-MPS using the same time points as the PET/CT imaging was carried out. Results: The radiolabelling efficiency of NSSL-MPS with [89Zr]Zr(oxinate)4 was 69 ± 8 %. PET/CT imaging of 89Zr-NSSL-MPS showed high uptake (3.6 ± 1.5 % ID; 17.4 ± 9.3 % ID/mL) at inflamed joints, with low activity present in non-inflamed joints (0.5 ± 0.1 % ID; 2.7 ± 1.1 % ID/mL). Importantly, a clear correlation between joint swelling and high 89Zr-NSSL-MPS uptake was observed, which was not observed with free 89Zr. STA mice receiving a therapeutic dose of NSSL-MPS showed a reduction in inflammation at the time points used for the PET/CT imaging compared with the control group. Conclusions: PET imaging was used for the first time to track a liposomal glucocorticoid, showing high uptake at visible and occult inflamed sites and a good correlation with the degree of inflammation. A subsequent therapeutic response matching imaging time points in the same model demonstrated the potential of this radiolabeling method as a theranostic tool for the prediction of therapeutic response - with NSSL-MPS and similar nanomedicines - in the treatment of inflammatory diseases.

Project description:BACKGROUND:The peculiar multiple myeloma microenvironment, characterized by up-regulated levels of several inflammatory chemokines, including the CXCR3 receptor ligands CXCL9 and CXCL10, limits NK cell positioning into the bone marrow by interfering with CXCR4 function. It is still unclear if the consequent reduced influx of transferred cells into the tumor represents a potential limiting factor for the success of NK cell-based adoptive therapy. We hypothesize that inhibition of CXCR3 function on NK cells will result in increased tumor clearance, due to higher NK cell bone marrow infiltration. METHODS:Since different activation protocols differently affect expression and function of homing receptors, we analyzed the bone marrow homing properties and anti-tumor efficacy of NK cells stimulated in vitro with two independent protocols. NK cells were purified from wild-type or Cxcr3-/- mice and incubated with IL-15 alone or with a combination of IL-12, IL-15, IL-18 (IL-12/15/18). Alternatively, CXCR3 function was neutralized in vivo using a specific blocking antibody. NK cell functional behavior and tumor growth were analyzed in bone marrow samples by FACS analysis. RESULTS:Both activation protocols promoted degranulation and IFN-γ production by donor NK cells infiltrating the bone marrow of tumor-bearing mice, although IL-15 promoted a faster but more transient acquisition of functional capacities. In addition, IL-15-activated cells accumulated more in the bone marrow in a short time but showed lower persistence in vivo. Targeting of CXCR3 increased the bone marrow homing capacity of IL-15 but not IL12/15/18 activated NK cells. This effect correlated with a superior and durable myeloma clearance capacity of transferred cells in vivo. CONCLUSIONS:Our results demonstrate that in vitro activation affects NK cell anti-myeloma activity in vivo by regulating their BM infiltration. Furthermore, we provided direct evidence that CXCR3 restrains NK cell anti-tumor capacity in vivo according to the activation protocol used, and that the effects of NK cell-based adoptive immunotherapy for multiple myeloma can be improved by increasing their bone marrow homing through CXCR3 inhibition.

Project description:BackgroundAdoptive T cell transfer (ACT) therapy improves outcomes in patients with advanced malignancies, yet many individuals relapse due to the infusion of T cells with poor function or persistence. Toll-like receptor (TLR) agonists can invigorate antitumor T cell responses when administered directly to patients, but these responses often coincide with toxicities. We posited that TLR agonists could be repurposed ex vivo to condition T cells with remarkable potency in vivo, circumventing TLR-related toxicity.MethodsIn this study we investigated how tumor-specific murine CD8+ T cells and human tumor infiltrating lymphocytes (TILs) are impacted when expanded ex vivo with the TLR9 agonist CpG.ResultsHerein we reveal a new way to reverse the tolerant state of adoptively transferred CD8+ T cells against tumors using TLR-activated B cells. We repurposed the TLR9 agonist, CpG, commonly used in the clinic, to bolster T cell-B cell interactions during expansion for ACT. T cells expanded ex vivo from a CpG-treated culture demonstrated potent antitumor efficacy and prolonged persistence in vivo. This antitumor efficacy was accomplished without in vivo administration of TLR agonists or other adjuvants of high-dose interleukin (IL)-2 or vaccination, which are classically required for effective ACT therapy. CpG-conditioned CD8+ T cells acquired a unique proteomic signature hallmarked by an IL-2RαhighICOShighCD39low phenotype and an altered metabolic profile, all reliant on B cells transiently present in the culture. Likewise, human TILs benefitted from expansion with CpG ex vivo, as they also possessed the IL-2RαhighICOShighCD39low phenotype. CpG fostered the expansion of potent CD8+ T cells with the signature phenotype and antitumor ability via empowering a direct B-T cell interaction. Isolated B cells also imparted T cells with the CpG-associated phenotype and improved tumor immunity without the aid of additional antigen-presenting cells or other immune cells in the culture.ConclusionsOur results demonstrate a novel way to use TLR agonists to improve immunotherapy and reveal a vital role for B cells in the generation of potent CD8+ T cell-based therapies. Our findings have immediate implications in the clinical treatment of advanced solid tumors.

Dataset Information

In Vivo Tracking of Adoptively Transferred Natural Killer Cells in Rhesus Macaques Using ⁸⁹Zirconium-Oxine Cell Labeling and PET Imaging.

Publications

<i>In Vivo</i> Tracking of Adoptively Transferred Natural Killer Cells in Rhesus Macaques Using <sup>89</sup>Zirconium-Oxine Cell Labeling and PET Imaging.

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In Vivo Tracking of Adoptively Transferred Natural Killer Cells in Rhesus Macaques Using 89Zirconium-Oxine Cell Labeling and PET Imaging.

Publications

<i>In Vivo</i> Tracking of Adoptively Transferred Natural Killer Cells in Rhesus Macaques Using <sup>89</sup>Zirconium-Oxine Cell Labeling and PET Imaging.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

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In Vivo Tracking of Adoptively Transferred Natural Killer Cells in Rhesus Macaques Using ⁸⁹Zirconium-Oxine Cell Labeling and PET Imaging.