Dose IL-2 Research Articles

Role of Adamts-1 in Pleomorphic Xanthoastrocytoma Tumor Cells Progression.

To analyze the expression of ADAMTS-1, NF-?B, and STAT3 in human pleomorphic xanthoastrocytoma specimens, and their correlation with glioma advancement. Pleomorphic xanthoastrocytoma tumor cell lines were treated with low and high doses of cytokines at 24 and 48 hours (h) to replicate the inflammatory environment. The effects of IL-1 were assessed with the scratch wound-healing assay, and the expression levels of ADAMTS-1, NF-?B, and STAT3 of the groups were determined by western blot analysis. Cytokine treatment significantly increased the migration of PXA glioma cells after scratching at 24h and 48h time points. Similarly, 10 and 30 ng/mL IL-1 induced 1.86 and 1.94 fold increases, respectively, in ADAMTS-1 expression after 24h, and 3 and 3.27 fold increases, respectively, after 48h, compared with the non-treatment control group.10 and 30 ng/mL IL-1 doses caused 2.5 and 2.6 fold increase, in NF-?B protein levels after 24h, and 3.16 and 3.41 fold increases after 48h, compared with the non-treatment group. The protein levels of STAT3 after 24h were 2.62 and 2.43 fold higher, and 3.78 and 3.84 fold higher after 48 hours, with 10 and 30 ng/mL IL-1, compared with the non-treatment group. The proliferation and progression of glioma cells were proportional to the increased expression levels of ADAMTS-1, NF-?B, and STAT3. Our findings indicate that the proteolytic function of ADAMTS-1 may be associated with the malignant transformation of low-grade gliomas.

IMMU-16. INTRA-TUMOURAL IL-12 DELIVERY ENABLES CAR T-CELL IMMUNOTHERAPY FOR HIGH-GRADE GLIOMA

Treatment with T-cells redirected to tumour specificity with a chimeric antigen receptor (CAR) may be well suited to treat intracranial tumours due to the ability of T-cells to access the central nervous system and migrate to infiltrative sites of disease. In adult glioblastoma, a case report of local and distant eradication of intracranial and spinal tumour deposits following intraventricular infusion of IL13Ra2-CAR T-cells indicates the potential of this approach. However, in contrast to the sustained complete remissions observed in haematological malignancies, in the majority of patients with glioblastoma CAR T-cell therapy has not resulted in clinical benefit. Tumour heterogeneity and the highly immune inhibitory tumour microenvironment (TME) are likely key barriers to achieving durable anti-tumour immunity. Here use intra-tumoural administration of IL-12 to enable CAR T-cell immunity. We employed CAR-T cells targeting the tumour-specific epidermal growth factor variant III (EGFRvIII). In an immunocompetent orthotopic mouse model of high-grade glioma, we show that CAR-T cells alone failed to control fully established tumour, but when combined with a single, locally delivered dose of IL-12, durable antitumor responses were achieved. IL-12 not only boosted cytotoxicity of CAR T-cells, but also reshaped the TME driving increased infiltration of proinflammatory CD4+ T-cells, decreased numbers of regulatory T-cells (Tregs) and activation of the myeloid compartment. Critically, immunotherapy enabling benefits of IL-12 were achieved with minimal systemic effects. Our findings show that local delivery of IL-12 is an effective adjuvant for CAR-T cell therapy for high-grade glioma. Assessment of application in high-risk childhood brain tumours is ongoing.

Low dose IL-2 in patients with steroid-dependent dysimmune manifestations associated with myelodysplastic syndromes: a three-case report

Systemic inflammatory and autoimmune diseases can be associated with myelodysplastic syndromes. Current treatments (steroids, immunosuppressive agents, biologics) are unsatisfactory because of their low response rate, dependence or adverse events. We aimed at evaluating the effects of low doses of IL-2 (ld-IL2) as a regulatory T-cell inducer in this context. We treated three patients with ld-IL2 with myelodysplastic syndromes and an associated dysimmune disorder (polymyalgia rheumatic, relapsing polychondritis associated with Sweet's syndrome and vasculitis with cutaneous and joint involvement, respectively). All three patients were dependent on steroids and refractory to biologics or azacitidine. They received doses of 1-1.5 million units of proleukin/day during 5 days and then every fortnight. The treatment led to a clinical improvement and steroid sparing in 2/3 patients with no serious adverse events, and no progression of the disease. Our results support the investigation of ld-IL2 in MDS associated with immune disorders in controlled clinical studies.

IL-2 and Anti-TGF-β Promote NK Cell Reconstitution and Anti-tumor Effects after Syngeneic Hematopoietic Stem Cell Transplantation.

Simple SummaryHematopoietic stem cell transplantation (HSCT) causes early immune deficiency and susceptibility to both opportunistic infections and cancer relapse. In this study, using a mouse model where donor cells can be tracked over time, we have observed that the combination of IL-2 (a cytokine which activates the immune system) combined with the blockade of TGF-β (a cytokine which suppresses the immune system) increased immune recovery and resulted in greater anti-tumor efficacy. The combination of IL-2 and anti-TGF-β accelerated NK cell and myeloid cell reconstitution after HSCT.The failure of autologous hematopoietic stem cell transplantation (HSCT) has been associated with a profound immunodeficiency that follows shortly after treatment, which renders patients susceptible to opportunistic infections and/or cancer relapse. Thus, given the additional immunosuppressive pathways involved in immune evasion in cancer, strategies that induce a faster reconstitution of key immune effector cells are needed. Natural killer (NK) cells mediate potent anti-tumor effector functions and are the first immune cells to repopulate after HSCT. TGF-β is a potent immunosuppressive cytokine that can impede both the development and function of immune cells. Here, we evaluated the use of an immunotherapeutic regimen that combines low dose of IL-2, an NK cell stimulatory signal, with TGF-β neutralization, in order to accelerate NK cell reconstitution following congenic HSCT in mice by providing stimulatory signals yet also abrogating inhibitory ones. This therapy led to a marked expansion of NK cells and accelerated NK cell maturation. Following HSCT, mature NK cells from the treated recipients displayed an activated phenotype and enhanced anti-tumor responses both in vitro and in vivo. No overt toxicities or adverse effects were observed in the treated recipients. However, these stimulatory effects on NK cell recovery were predicated upon continuous treatment as cessation of treatment led to return to baseline levels and to no improvement of overall immune recovery when assessed at later time-points, indicating strict regulatory control of the NK cell compartment. Overall, this study still demonstrates that therapies that combine positive and negative signals can be plausible strategies to accelerate NK cell reconstitution following HSCT and augment anti-tumor efficacy.

Low-Dose IL-2 for Treating Moderate to Severe Alopecia Areata: A 52-Week Multicenter Prospective Placebo-Controlled Study Assessing its Impact on T Regulatory Cell and NK Cell Populations

Low-Dose IL-2 for Treating Moderate to Severe Alopecia Areata: A 52-Week Multicenter Prospective Placebo-Controlled Study Assessing its Impact on T Regulatory Cell and NK Cell Populations

WITHDRAWN: The effect of anti IL-2/IL-2 complex versus stand-alone low dose of IL-2 on imiquimod induced psoriasis like skin inflammation model

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Abstract 584: TGN1412 induces dose and time dependent human cytokines in a humanized PBMC mouse model for cytokine release syndrome

Abstract There have been remarkable progresses in the development of immune therapies for cancer. However, the promise of immune therapy comes with significant challenges of a variety of adverse effects. Due to the lack of predictive preclinical model to evaluate antibody mediated adverse effects, TGN1412, a humanized anti-CD28 monoclonal antibody which acts as a superagonist of CD28 to activate T cells, unfortunately caused life-threatening cytokine release syndrome (CRS) during the first-in-man trial. Therefore there is a critical need for translational protocols that could predict immune toxicity more accurately. We have developed a sensitive and reproducible PBMC-humanized mouse model that can measure cytokine release induced by bispecifics or checkpoint inhibitors in vivo. In this study the utility of this platform was demonstrated using TGN1412 as a model. NSG™ mice or derivative strains such as SGM3 mice were engrafted with human PBMCs for 6 days before being treated with TGN1412 at different concentrations (0.2, 1.0 and 2 mg/kg). Mouse blood was collected at 1, 2, 4, 6, 24 and 48 hours post dose for flow cytometry and cytokine analysis. We observed dose and time dependent increase of human IFN-γ, TNFα, IL-2, IL-4, IL-6 and IL-10 levels in the serum following TGN1412 treatment with TNFα peaking at 1 h and the others peaking at 2-4 h post dose. A significant decrease in hCD45+ cells was observed in the peripheral blood collected at 6h post treatment. TGN1412 was tested in the humanized NSG™ mice with PBMCs from 7 different donors, and a significant decrease in hCD45+ cell population in the peripheral blood was observed in all donors. We have demonstrated that the novel mouse model using PBMC-humanized NSG™ can successfully detect CRS induced by TGN1412 in a dose and time dependent manner, suggesting that indeed CRS could be a strong adverse effect associated with TGN1412 treatment in human. Our data supports that our newly developed mouse model for CRS assessment may be a valuable tool to assess in vivo safety of experimental human therapeutic antibodies including immune checkpoint inhibitors and bispecifics with better reliability than an in vitro assay. Citation Format: Hongyuan Yang, Jing Jiao, Danying Cai, Mingshan Cheng, Michael Brehm, Dale Grainer, Leonard Shultz, James Keck. TGN1412 induces dose and time dependent human cytokines in a humanized PBMC mouse model for cytokine release syndrome [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 584.

IL-7/αIL-7 mAb M25 immunocomplexes expand CD8+ T cells but paradoxically abrogate the antitumor activity of CTLA-4 and PD-1 blockage

Supraphysiological levels of IL-7 induce increase counts of pre-B cells, naive T cells and memory phenotype CD8+ T cells. Immunocomplexes of IL-7 and αIL-7 mAb M25 (IL-7/M25) were described as IL-7 superagonist in vivo. Thus, treatment of mice with IL-7/M25 remarkably increases the size of the T cell pool. We decided to use IL-7/M25 in order to expand the T cell population prior to the administration of αCTLA-4 and αPD-1 mAbs in tumor-bearing mice and in turn boost the immunotherapy based on a combination of CTLA-4 and PD-1 blockage. We found that just four doses of IL-7/M25 increased the absolute numbers of splenocytes approximately fivefold and significantly shifted the CD4+:CD8+ T cell ratio in favor of CD8+ T cells. There was also a substantive increase in relative counts of memory phenotype CD8+ T cells (approximately threefold) within CD8+ T cells but a significant decrease (approximately 30%) in relative counts of Treg cells within CD4+ T cells. All these data suggest that IL-7/M25 offer a suitable approach to potentiate tumor immunotherapy through CTLA-4 and PD-1 blockage. Unexpectedly, IL-7/M25 significantly abrogated the antitumor activity of αCTLA-4 plus αPD-1 mAbs in the following mouse tumor models: MC-38 and CT26 colon carcinoma and B16F10 melanoma. This paradoxical effect of IL-7/M25 on the antitumor activity of CTLA-4 and PD-1 blockage was not mediated via either increased levels of IL-10 or TGF-β in the sera or increased counts of IL-10-producing B or T cells in the spleen of mice injected with IL-7/M25. Thus, our work shows that caution should be exercised when combining two immunotherapy approaches together.

Abstract B03: A phase I study of cetuximab followed by activated and expanded NK cells for refractory nasopharyngeal cancer

Abstract Background: Natural killer cells (NK) are innate immune cells that can kill oncogenically transformed cells. In nasopharyngeal cancer (NPC), epidermal growth factor receptor (EGRF) is highly expressed, and targeted therapy using cetuximab (a chimeric monoclonal based antibody against EGFR) has been used to treat NPC. In this trial, we sought to enhance cetuximab-mediated antibody dependent cellular cytotoxicity (ADCC) by administering it in combination with activated NK cells, which bind the Fc component of the antibody via their surface CD16 receptor. To this end, we applied a method that allows vigorous expansion of activated CD16+ NK cells ex vivo by stimulation with irradiated K562-mb15-41BBL cells. We hypothesized that administration of activated NK cells after cetuximab can increase its anti-NPC activity. Aim: To determine the safety and toxicity profile of sequential cetuximab therapy, followed by concurrent cetuximab with autologous NK cells in patients with refractory nasopharyngeal cancer. Methods: A phase 1, leading on phase 2 study (NCT 02507154) was conducted in patients with recurrent/metastatic nasopharyngeal cancer who had failed at least 2 lines of prior chemotherapy. These patients were scheduled to first receive cetuximab monotherapy for 6-8 weeks, and then cetuximab followed by infusion of expanded and activated autologous NK cells, with 6 doses of subcutaneous IL-2 administered over 1 week to promote NK cell persistence. The primary end point was safety of this combination therapy; secondary endpoint was objective response of measurable disease determined by the RECIST criteria. Results: Seven patients (receiving 10 cetuximab-NK infusions in total) completed the phase 1 trial. The first 3 patients received 1 × 106 NK cells/kg; the dose was escalated to 1 × 107 NK cells/kg and administered to the remaining 4 patients. NK cell expansion, performed over 10 days, produced NK cell numbers that exceeded those planned for infusion in all 7 patients; median number of NK cell recovery was 27,400% of input NK cells (n = 10). ADCC with cetuximab against the cell line SUNE2 was tested 24 hours after NK cell administration and showed median cell killing of 87% (range 56%-95%) in a 4-hour assays at an effector:target ratio of 2:1. All patients tolerated the NK infusion well; no grade 3-4 toxicities were observed. Skin rash was the commonest side effect with cetuximab monotherapy (grade 1-2), which was not aggravated after the administration of cetuximab with NK cells. With a median follow-up of 9 months, we observed 1 partial response, 2 stable diseases, and 4 progressive diseases. Persistence of the quantity and function of NK cells beyond 8 weeks following NK therapy correlated with improved response. Conclusion: Administration of cetuximab with autologous activated CD16+ NK cells at 1 × 107 NK cells/kg is well tolerated in previously heavily treated NPC patients. Grade 1-2 skin toxicity was the predominant side effect, which did not result in termination of this combination therapy. Citation Format: Chwee Ming Lim, Anthony Liou, Michelle Poon, Liang Piu Koh, Lip Kun Tan, Noriko Shimasaki, Dario Campana, Boon Cher Goh. A phase I study of cetuximab followed by activated and expanded NK cells for refractory nasopharyngeal cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr B03.

Dose-dependent pro- or anti-fibrotic responses of endometriotic stromal cells to interleukin-1\u03b2 and tumor necrosis factor \u03b1

Endometriosis are characterized by dense fibrous tissue. Numerous studies have investigated roles of inflammation on the pathophysiology of endometriosis. However, the interplay of inflammation and fibrosis remains to be clarified. Here we show that low levels of interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNFα) promoted a fibrotic phenotype, whereas high levels of IL-1β and TNFα inactivated the fibrotic phenotype of endometriotic stromal cells (Ectopic-ES). IL-1β 10 pg/mL and TNFα 100 and 1,000 pg/mL had minimal effects, whereas the highest dose of IL-1β (100 pg/mL) significantly decreased collagen gel contraction in Ectopic-ES. Furthermore, in Ectopic-ES, low levels of IL-1β (1 pg/mL) and/or TNFα 10 pg/mL significantly increased Col I mRNA expression, whereas higher doses of IL-1β (10 and/or 100 pg/mL) and/or TNFα (100 and/or 1,000 pg/mL) significantly decreased Col I and/or αSMA mRNA expression and the percentage of cells with Col I + and/or αSMA + stress fibers. In contrast, in either menstrual endometrial stromal cells of patients with endometriosis or those of healthy women, varying doses of IL-1β and/or TNFα had no significant effects on either Col I or αSMA mRNA/protein expression. The present findings bring into question whether we should still continue to attempt anti-inflammatory treatment strategies for endometriosis.

Long-term follow up of lifileucel (LN-144) cryopreserved autologous tumor infiltrating lymphocyte therapy in patients with advanced melanoma progressed on multiple prior therapies.

10006 Background: Treatment options are limited for patients with advanced melanoma who have progressed on checkpoint inhibitors and targeted therapies. Adoptive cell therapy using tumor-infiltrating lymphocytes (TIL) leverages and enhances the body’s natural defense against cancer and has shown durable responses in heavily pretreated melanoma patients. Methods: C-144-01 is a global Phase 2 open-label, multicenter study of efficacy & safety of lifileucel in patients with unresectable metastatic melanoma who have progressed on checkpoint inhibitors and BRAF/MEK inhibitors, if BRAFv600 mutant. We report on Cohort 2 (N = 66) patients who have received TIL. Tumors were resected at local institutions, processed in central GMP facilities for TIL production, manufactured, cryopreserved & shipped back to sites in a 22-day process. Therapy consisted of one week of lymphodepletion, a single lifileucel infusion, and up to 6 IL-2 doses. ORR was based on RECIST v1.1 by investigator assessment. Data cutoff was Feb 2, 2020. Results: Baseline characteristics: 3.3 mean prior therapies (anti-PD1 100%; anti-CTLA-4 80%; BRAF/MEK inhibitor 23%), high baseline tumor burden (106 mm mean target lesion sum of diameters), 44% liver/brain lesions, 40.9% LDH > ULN. ORR by investigator was 36.4% (2 CR, 22 PR) and DCR was 80.3%. Mean time to response was 1.9 months (range: 1.3-5.6). After a median study follow-up of 17.0 months, median DOR (mDOR) was still not reached. Six responders have progressed, 2 have died and 2 started other anti-cancer therapy without progression. The adverse event profile was consistent with the underlying advanced disease and the lymphodepletion and IL-2 regimens. Additional follow-up data will be available for presentation. Conclusions: Lifileucel treatment results in a 36.4% ORR and mDOR was not reached at 17.0 months of median study follow up in a heavily pretreated metastatic melanoma patients with high baseline disease burden who progressed on multiple prior therapies, including anti-PD1 and BRAF/MEK inhibitors, if BRAFv600 mutant. Clinical trial information: NCT02360579.

Partial T cell defects and expanded CD56bright NK cells in an SCID patient carrying hypomorphic mutation in the IL2RG gene.

X-linked severe combined immunodeficiency (X-SCID) caused by full mutation of the IL2RG gene leads to T- B+ NK- phenotype and is usually associated with severe opportunistic infections, diarrhea, and failure to thrive. When IL2RG hypomorphic mutation occurs, diagnosis could be delayed and challenging since only moderate reduction of T and NK cells may be present. Here, we explored phenotypic insights and the impact of the p.R222C hypomorphic mutation (IL2RGR222C ) in distinct cell subsets in an 8-month-old patient with atypical X-SCID. We found reduced CD4+ T cell counts, a decreased frequency of naïve CD4+ and CD8+ T cells, and an expansion of B cells. Ex vivo STAT5 phosphorylation was impaired in CD4+ CD45RO+ T cells, yet compensated by supraphysiological doses of IL-2. Sanger sequencing on purified cell subsets showed a partial reversion of the mutation in total CD3+ cells, specifically in recent thymic emigrants (RTE), effector memory (EM), and CD45RA+ terminally differentiated EM (EMRA) CD4+ T cells. Of note, patient's NK cells had a normal frequency compared to age-matched healthy subjects, but displayed an expansion of CD56bright cells with higher perforin content and cytotoxic potential, associated with accumulation of NK-cell stimulatory cytokines (IL-2, IL-7, IL-15). Overall, this report highlights an alteration in the NK-cell compartment that, together with the high disease-phenotype variability, should be considered in the suspicion of X-SCID with hypomorphic IL2RG mutation.

Abstract B108: Hypoxia induces phenotypic and functional changes in NK cells which negatively impacts antitumor immunity in the solid tumor microenvironment

Abstract Natural killer (NK) cells are key effectors in cancer immunosurveillance, and they can eliminate tumor cells through the release of cytotoxic granules triggered by interactions with natural ligands or through antibody-dependent cellular cytotoxicity (ADCC). NK cell-based treatment has had therapeutic success for hematologic malignancies, but strategies to treat solid tumors have been more limited. One of the biggest barriers to NK cell immunotherapy is immunosuppression within the tumor microenvironment. An important aspect of this immunosuppression is the highly hypoxic environment created by proliferating tumor cells. To further study the effect of hypoxia on NK cell function, our lab has been collaborating with XCell Biosciences to use their novel incubators that precisely control oxygen and pressure levels. We incubated NK cells isolated from peripheral blood mononuclear cells (PBMCs) at three different oxygen (O2) levels: 12% O2 to mimic the peripheral blood, 5% O2 to mimic the bone marrow, and 1% O2 to mimic the solid tumor microenvironment. All comparisons were made against a standard incubator condition. NK cells were incubated for three different time points—24 hours, 72 hours, and 7 days—and activated with low dose IL-15. We conducted a mass cytometry analysis to assess phenotype of PBMCs and enriched NK cells incubated at the different oxygen conditions. NK cells from the 1% O2 condition express fewer activating receptors (NKG2D, Nkp30, Nkp46, DNAM-1) and less perforin and granzyme than NK cells from the higher oxygen conditions. However, the NK cells from 1% O2 conditions express high levels of the activating receptors CD69 and CD16 (which triggers ADCC). This altered activation state is reflected in cell-killing assays using an IncuCyte machine. Here, fluorescently labeled target cells and NK cells are incubated together and images are taken for 48 hours to measure target cell death. We observed that natural cytotoxicity against K562 targets is severely impaired in the NK cells at the lowest oxygen condition but ADCC responses using the antibody rituximab against Raji targets remain intact. We observe a sharp decrease in proliferation, as measured by CellTrace dilution and Ki-67 expression in the NK cells in the 1% O2 condition. We also detect a decrease in glycolytic and mitochondrial metabolism, as measured by a Seahorse Analyzer, as the oxygen levels decrease. Supernatants collected from NK cells in the lower oxygen conditions (12% O2 and 5% O2) contain more inflammatory cytokines (IFNγ, TNFα, GM-CSF) and chemokines (CCL3, CCL4, CCL5) than NK cells in high oxygen conditions, indicating a shift from cytotoxic to a more immunomodulatory state. These results indicate that hypoxia changes the phenotype and function of NK cells. Overall, the insights gained from these experiments can help overcome hypoxia-induced immune suppression in the tumor microenvironment and enhance NK cell-based immunotherapy for solid tumors. Citation Format: Upasana Sunil Arvindam, Pippa Kennedy, Bri Ettestad, Peter Hinderlie, Jeffrey S. Miller, Martin Felices. Hypoxia induces phenotypic and functional changes in NK cells which negatively impacts antitumor immunity in the solid tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B108.

Development and Evaluation of Interleukin-2-Derived Radiotracers for PET Imaging of T Cells in Mice.

Recently, N-(4-18F-fluorobenzoyl)-interleukin-2 (18F-FB-IL2) was introduced as a PET tracer for T cell imaging. However, production is complex and time-consuming. Therefore, we developed 2 radiolabeled IL2 variants, namely aluminum 18F-fluoride-(restrained complexing agent)-IL2 (18F-AlF-RESCA-IL2) and 68Ga-gallium-(1,4,7-triazacyclononane-4,7-diacetic acid-1-glutaric acid)-IL2 (68Ga-Ga-NODAGA-IL2), and compared their in vitro and in vivo characteristics with 18F-FB-IL2. Methods: Radiolabeling of 18F-AlF-RESCA-IL2 and 68Ga-Ga-NODAGA-IL2 was optimized, and stability was evaluated in human serum. Receptor binding was studied with activated human peripheral blood mononuclear cells (hPBMCs). Ex vivo tracer biodistribution in immunocompetent BALB/cOlaHsd (BALB/c) mice was performed at 15, 60, and 90 min after tracer injection. In vivo binding characteristics were studied in severe combined immunodeficient (SCID) mice inoculated with activated hPBMCs in Matrigel. Tracer was injected 15 min after hPBMC inoculation, and a 60-min dynamic PET scan was acquired, followed by ex vivo biodistribution studies. Specific uptake was determined by coinjection of tracer with unlabeled IL2 and by evaluating uptake in a control group inoculated with Matrigel only. Results:68Ga-Ga-NODAGA-IL2 and 18F-AlF-RESCA-IL2 were produced with radiochemical purity of more than 95% and radiochemical yield of 13.1% ± 4.7% and 2.4% ± 1.6% within 60 and 90 min, respectively. Both tracers were stable in serum, with more than 90% being intact tracer after 1 h. In vitro, both tracers displayed preferential binding to activated hPBMCs. Ex vivo biodistribution studies on BALB/c mice showed higher uptake of 18F-AlF-RESCA-IL2 than of 18F-FB-IL2 in liver, kidney, spleen, bone, and bone marrow. 68Ga-Ga-NODAGA-IL2 uptake in liver and kidney was higher than 18F-FB-IL2 uptake. In vivo, all tracers revealed uptake in activated hPBMCs in SCID mice. Low uptake was seen after a blocking dose of IL2 and in the Matrigel control group. In addition, 18F-AlF-RESCA-IL2 yielded the highest-contrast PET images of target lymph nodes. Conclusion: Production of 18F-AlF-RESCA-IL2 and 68Ga-Ga-NODAGA-IL2 is simpler and faster than that of 18F-FB-IL2. Both tracers showed good in vitro and in vivo characteristics, with high uptake in lymphoid tissue and hPBMC xenografts.

Lack of cell movement impairs survival of peripheral blood IL-2-stimulated natural killer cells originating from solid cancer and promotes red blood cells to induce their switch toward a regulatory phenotype

BackgroundRed blood cells (RBCs) can have a modulatory effect on immune cells; so changes in their dynamism could considerably influence their physiology, and consequently the immune activities of neighbouring cells, like natural killer (NK) cells. Herein, we studied the effect of both RBCs and lack of cell movement on the proliferation, survival and regulation of peripheral IL-2-stimulated NK cells from normal and solid malignant conditions. MethodsExperiments were conducted on twelve cell culture groups, including NK cells from patients with solid malignant tumor or healthy controls, cultured alone or with autologous or nonautologous RBCs under shaking or no shaking conditions. ResultsNK cells from neoplastic patients behaved differently depending on the culture conditions including shaking and/or RBCs presence. Therefore, NK cells survival was downregulated in the absence of shaking; whereas, shaking have not only upregulated cell survival, but also downregulated the levels of p53-related apoptosis. Moreover, RBCs enhanced NK cells proliferation; while, this effect was modulated by shaking. Furthermore, RBCs can generate opposite effects on the production and modulation of protumoral or immunosuppressive cytokines, depending on the origin of NK cells, i.e., whether they derive from healthy or solid malignant tumor conditions. Finally, NK cells become able to express Foxp3 regulatory marker when combining three main conditions that include (i) treatment with high dose of IL-2, (ii) presence of RBCs, and (iii) absence of shaking. ConclusionsOur outcomes showed for the first time that cell stagnation would be markedly involved in peripheral NK cell apoptosis, as well as in switching toward a regulatory phenotype-induced Foxp3. Cell movement may be one of ex vivo potential approaches in boosting the activities and survival of such cells during solid cancer.

Sustained Immunogenic and Clinical Effects of Low-Dose Interleukin-2 Therapy with an Intermittent Maintenance Method for Refractory Chronic Graft-Versus-Host Disease: Results of Phase1/2a LDIL2-01 Study

Introduction and Objectives Regulatory T cells (Treg) play a central role in the maintenance of immune tolerance. We previously reported that the altered Treg homeostasis in the prolonged lymphopenia after HSCT is one of the major pathogenesis of impaired Treg recovery leading to the onset of chronic GVHD and low-dose IL-2 can restore normal Treg homeostasis by a PD-1-dependent mechanism, resulting in the improvement of clinical GVHD symptoms. In these years, the tolerogenic effects of IL-2 have been tested for various autoimmune diseases by clinical trials. However, the schedules of IL-2 dosing in each trial are different and the optimal strategy to expand Treg is still unclear. Our murine study has shown that daily administration of IL-2 seems to be optimal for the initial expansion of Treg but less frequent dosing within the threshold could be preferable for the following maintenance of expanded Treg. Methods Based on the findings, we conducted a phase1/2a clinical trial of low-dose IL-2 for steroid-refractory chronic GVHD with a distinctive dosing schedule consisting of an initial induction phase (IL-2 dosing everyday for the first 4 weeks) and a subsequent maintenance phase (IL-2 dosing 3 times a week for the following 8 weeks). Patients who obtained clinical benefit by the 3 months could proceed to the extended therapy up to total 12 months. The appropriate single dose of IL-2 was examined by a three-step dose escalation design. Results A total of 12 patients were enrolled. None had progression of chronic GVHD or recurrent hematologic malignancy. Dose-limiting toxicity did not occur even at the maximum dose. All patients were evaluable for response, and 7 had clinically significant responses involving multiple sites. Response was observed most often in the joints (67%). One patient with oral GVHD and another patient with gastrointestinal GVHD achieved complete response. One patient with severe lung GVHD markedly improved pulmonary functional test (%FEV1; from 24.8% to 64.8 %) and activity of daily life (from bedridden to return to work). Treg were preferentially increased in all patients, with a peak median value almost 10 times the baseline value (P Conclusion These results indicate that the sustained efficacy of IL-2 against chronic GVHD does not necessarily require that Treg is maintained at a high level by continuing daily IL-2 administration over the long term. Our data provide important information for reconsidering IL-2 dosing methods from the viewpoint of long-term safety and possible combination with other treatments.

NK Cell Impairment after Haploidentical Allogeneic Hematopoietic Cell Transplant in Patients Receiving Post-Transplant Cyclophosphamide (PTCy)

Haploidentical hematopoietic cell transplantation (haplo-HCT) with post-transplant cyclophosphamide (PTCy) as graft versus host disease (GVHD) prophylaxis is frequently used for patients who do not have HLA-identical donors. However, the effect of PTCy on immune reconstitution (IR) has not been studied extensively in large prospective cohorts, despite a high incidence of early viral infections. We quantified IR in 60 patients after haplo-HCT with PTCy, mycophenolate mofetil and tacrolimus (TAC) and compared results to 34 patients with 8/8 HLA matched related or unrelated donors (MD) receiving TAC and methotrexate for GVHD prophylaxis. Both groups received reduced intensity conditioning for hematologic malignancies. Samples were prospectively collected at 1, 2, 3, 6, 9, 12, 18, 24 and 30 months after HCT and analyzed using multi-color flow cytometry to characterize distinct lymphocyte populations. Peripheral blood NK cells 1 month after PTCy were characterized using CyTOF and a panel of 35 metal-labeled monoclonal antibodies and compared to age-matched healthy donor NK cells. Recovery of all T cell subsets (CD4Tcon, CD4Treg, CD8) was significantly reduced in the PTCy cohort compared to MD early after HCT (Figure 1A, B, C). Despite delayed CD4Treg recovery, the Treg:Tcon ratio was significantly higher early after HCT in the PTCY cohort due to relatively deeper depression in CD4Tcon (0.18 vs 0.06, p<0.0001 at 1 month, 0.12 vs 0.06, p=0.0004 at 3 months, Figure 1D). Absolute NK cells were lower at 1 month after PTCy (Figure 1E), entirely due to reduced numbers of CD56^dimCD16⁺ NK cells (Figure 1G). Subsequently, CD56^dim NK cells remained significantly lower in the PTCy group until 3 months after HCT. In contrast, recovery of CD56^brightCD16⁻ NK cells was significantly increased in the PTCy cohort at 2, 3 and 6 months after HCT (p<0.01. Figure 1F). The distribution of NK subsets 1 month post-HCT was also significantly different between two cohorts due to a higher frequency of immature NK cells in the PTCY cohort (Figure 1H). CyTOF analysis performed 1 month after haplo-HCT showed that NK cells expressed higher level of activating receptor NKp46, CD95, granzyme-B and inhibitory receptor NKG2A and lower expression of CD57 compared to HD (Figure 2). Functional assays showed that PTCy NK cells displayed reduced degranulation and release of IFNγ after activation compared to HD. These functions were rescued after priming with low dose IL-15 in vitro. In summary, the effect of PTCy on IR was most pronounced at 1 month with significantly delayed recovery of CD3, CD4Tcon, CD4Treg, CD8 and CD56^dim NK cells. After PTCy, NK cells display an immature phenotype and functional impairment that is at least partly corrected by priming with low dose IL-15. Impairment of NK and T cell recovery early after PTCy-based HCT underscores the need to adopt novel strategies to overcome immune defects induced by this platform.

Interleukin-2 chronotherapy for metastatic renal cell carcinoma: Results of a phase I-II study

BackgroundInterleukin-2 (IL-2) was the cornerstone treatment for metastatic renal cell carcinoma (RCC) until the advent of tyrosine kinase inhibitors, but it still has therapeutic value. As a single bolus of IL-2 causes toxicity, there is interest in administration regimens with better tolerability and efficacy. Chronotherapy is the administration of therapy according to the circadian rhythm’s influence on the immune and hormonal systems. This phase I-II trial evaluated the safety of IL-2 chronotherapy in metastatic RCC patients and determined the maximum tolerated dose. The secondary objective was to identify prognostic factors for survival. MethodsThree chronomodulation schedules (5:00–13:00, 13:00–21:00, and 21:00–5:00) were tested. Each schedule was an 8-h IL-2 infusion, with a Gaussian distribution of drug concentration peaking at 4 h. To identify the maximum tolerated dose, the dose for different patients was escalated from 2 MIU/m2 (level I) to 18.6 MIU/m2 (level VI). ResultsThirty patients were enrolled and completed treatment. Two patients were treated at 5:00–13:00, 15 at 13:00–21:00, and 13 at 21:00–5:00. Nine cases of grade 3 toxicity occurred in 7 patients at the highest dose (18.6 MIU/m2); no grade 4 toxicity occurred. The maximum tolerated dose was 14.0 MUI/m2. Patients were followed for a median of 16 months (range, 2–107). One patient was lost to follow-up, 3 patients were alive at last contact, and 26 patients died. Six patients achieved long-term survival (≥48 months). There was one complete response, four partial responses, 11 cases of stable disease and 14 of progressive disease. The response rate was 16% (5/30) and disease-control rate was 53% (16/30). Median progression-free survival was 4.5 months, and median overall survival was 14.5 months. Kaplan-Meier analyses revealed significant associations between overall survival and ECOG performance score (0 vs. 1–2), MSKCC score (0–2 vs. ≥ 3), IMDC risk score (0–2 vs. ≥ 3), IL-2 dose level (IV-VI vs. I-III), and prolactin (increase vs. no increase), and but not for chronotherapy schedule. ConclusionIL-2 chronotherapy appears to be safe, moderately toxic and active in metastatic RCC. It may represent a new modality of IL-2 administration for these patients.

Radiotherapy as an Immunological Booster in Patients with Metastatic Melanoma or Renal Cell Carcinoma Treated with High-Dose Interleukin-2: Final Data

Abstract Background High-dose interleukin-2 (HDIL-2) represents a curative treatment option in metastatic melanoma (MM) and renal cell carcinoma (RCC) patients, although still in need of improving its therapeutic efficacy. Radiotherapy (XRT) strongly synergizes with immunotherapy and may boost the effects of HDIL-2 allowing for less toxic schedules. Methods In this proof-of-principle phase II study, biological markers of immunological response were considered as primary endpoints in previously treated MM and RCC patients. The treatment consisted of 3 daily doses of XRT (6-12 Gy) delivered to one lesion before the first and third IL-2 infusion (18 MIU/m2/day by IV infusion for 72 h), repeated every 3 weeks for a maximum of 6 cycles. In a first stage, 4 out of 7 enrolled patients showed an immunological response allowing the recruitment of a further 12 patients according to the minimax two-stage Simon design. The immunological efficacy of the combined XRT/HDIL-2 treatment was assessed measuring the proportion of circulating immune effectors specific for tumor antigens known to be expressed in MM and in RCC (i.e. Tyrosinase, gp100, Mart-1, 5T4, CAIX/G250, EGFR, survivin, MAGEA3 and NYESO1) by means of INF-γ Elispot assay. Moreover, the predictive value of pre-treatment serum biomarkers was also assessed. Results Since September 2012 a total of 19 patients have been enrolled (9 RCC and 10 MM patients of which 6 uveal, 2 mucosal and 2 cutaneous) with a median age of 55 years. Assessed clinical responses were 4 PR (3 RCC and 1 MM), 6 SD (2 RCC and 4 MM) and 9 PD. Median PFS was 3.2 and 2.9 months and median OS was 8.7 and 6.7 months, for RCC and MM respectively. According to CTCAE 4.0, the majority of toxicities was grade 1-2. IL-2 dose was never reduced, nor was the infusion interrupted. Interestingly, we found an enhancement greater than 10% of antigen-specific spot-forming cells (SFCs) for at least one tumor antigen after treatment in 16 out of 19 patients. Moreover, we detected much lower serum basal levels of IL-8 and IL-1b in responders (CR/PR/SD=10) compared to non responders (PD = 9). Conclusion HDIL2/XRT combined treatment is well tolerated and fairly active in the MM and RCC settings. Clinical trial identification EudraCT: 2012-001786-32. Legal entity responsible for the study The authors. Funding Italian Ministry of Health as part of the “Ricerca Finalizzata 2009 - Direzione Generale della Ricerca Scientifica e Tecnologica” Call for Project Proposals. Disclosure All authors have declared no conflicts of interest.

T Regulatory Cells From Non-obese Diabetic Mice Show Low Responsiveness to IL-2 Stimulation and Exhibit Differential Expression of Anergy-Related and Ubiquitination Factors.

Foxp3+ Regulatory T cells (Tregs) are pivotal for the maintenance of tolerance. Alterations in their number and/or function have been proposed to occur in the autoimmune-prone non-obese diabetic (NOD) mouse. Comparing the frequencies and absolute numbers of CD4+Foxp3+CD25+ Tregs among 4 to 6-week old NOD, B6, and BALB/c mice, we observed differences in counts and Foxp3 expression in Tregs from secondary lymphoid organs, but not in the thymus. Upon TCR and IL-2 stimulation, NOD Tregs showed lower responses than Tregs from B6 and BALB/c mice. Indeed, NOD Tregs responded with less proliferation and with smaller increments in the expression of CD25, LAP-1, CD39, PD-1, PD-L1, and LAG-3, when in vitro cultured for 3 days with anti-CD3/CD28 in the absence or presence of IL-2, Tregs from NOD mice showed to be highly dependent on IL-2 to maintain Foxp3 expression. Moreover, NOD Tregs become producers of IL-17 and INF-gamma more easily than Tregs from the other strains. In addition, NOD Tregs showed lower responsiveness to IL-2, with significantly reduced levels of pSTAT5, even at high IL-2 doses, with respect to B6 and BALB/c Tregs. Interestingly, NOD Tregs exhibit differences in the expression of SOCS3, GRAIL, and OTUB1 when compared with Tregs from B6 and BALB/c mice. Both, at steady state conditions and also after activation, Tregs from NOD mice showed increased levels of OTUB1 and low levels of GRAIL. In addition, NOD Tregs had differences in the expression of ubiquitin related molecules that play a role in the maintenance of Foxp3 cellular pools. Indeed, significantly higher STUB1/USP7 ratios were detected in NOD Tregs, both at basal conditions and after stimulation, compared to in B6 and BALB/c Tregs. Moreover, the addition of a proteasome inhibitor to cell cultures, conferred NOD Tregs the ability to retain Foxp3 expression. Herein, we provide evidence indicating a differential expression of SOCS3, GRAIL, and STUB1/USP7 in Tregs from NOD mice, factors known to be involved in IL-2R signaling and to affect Foxp3 stability. These findings add to the current knowledge of the immunobiology of Tregs and may be related to the known insufficiency of Tregs from NOD mice to maintain self-tolerance.