IL-2 Therapy Research Articles

Abstract 2261: Understanding the mechanism underlying resistance to immunotherapy in TNBC

Abstract Triple Negative Breast Cancer (TNBC) accounts for 15-20% of all breast cancer cases. TNBC patients have the worst outcome amongst all breast cancers; and do not respond well to conventional therapies. Although TNBC patients have a high amount of tumor infiltrating lymphocytes, and express higher level of PD-L1 compared to other breast cancer subtypes, they do not all respond to PD-1 blockade therapy. IL-12 is a cytokine produced by dendritic cells (DCs), and other APCs, known to induce the activation of natural killer cells, cytotoxic T-cells, and induce a Th1 phenotype. Although the immune activating potential of Il-12 are known, its systemic uses have been limited due to high toxicity. Using three different tumor models (PDXs: BCM 4913, MC1 in humanized mice and E0771 syngeneic model C57/Bl), we are investigating the response of combining intratumoral injection of Adenovirus IL-12 (ad.Il-12) and anti-PD1 therapy. Materials/Methods: E0771, a mice TNBC cell line was injected in the mammary fat pad of C57BL/6 mice. The same procedure was followed using MC1 and BCM 4913, in humanized NSG-MGM3 mice. The mice received weekly intratumoral injection of ad.IL-12; a replication defective adenoviral vector containing mIL-12 (mouse) and hIL-12 (PDX) cDNA under the transcriptional control of Rous sarcoma virus long terminal repeat (received from Dr. Chen). Anti-PD1 (InVivoMab anti-mouse PD-1 CD279) was administered 3 times a week, Pembrolizumab (once a week). Post treatment, INF-gamma level were measured from blood and tumor samples, in addition, tumor sizes were compared between treatment groups (Control/ad.IL-12/anti-PD1/ad.IL-12 + anti-PD1), as well as survival curves. Immunohistochemistry was used to assess levels on TILs in the tumors. Results: Ad.Il-12 and PD-1 blockade therapy combination was more effective at reducing tumor growth in BCM4913, while in E00771 and MC1 combination treatment showed no difference compared to single agent. In all the models, levels of INF-gamma in the tumor were significantly upregulated in the combination group compared to control and single agents, while no difference was found between treatment groups from blood samples. Tumor growth in the combination group and the treated group were slower compared to the controls in BCM 4913. IHC data indicate a higher level of TILs in the treatment groups, with a statistically significant difference in the combination group compared to the others. Conclusion: Our preliminary data suggest that intratumoral injection of IL-12 confine the release of INF-gamma in the tumor microenvironment, and mitigate the toxicity associated with systemic admission of ad.IL-12. Treatment response is not achieved in every model, suggesting an underlying mechanism which confers resistance to the treatment. We are currently investigating different models in the hope to identify markers of responders versus non responders. Citation Format: Ann C. Anselme, Wei Qian, Anthony Kozielski, Roberto R. Rosato, Jenny C. Chang. Understanding the mechanism underlying resistance to immunotherapy in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2261.

Abstract 1553: Collagen affinity improves safety and efficacy of antibody and cytokine cancer immunotherapies

Abstract Cancer immunotherapy with immune checkpoint inhibitors (CPI), interleukin (IL)-2, and IL-12 have demonstrated clinical antitumor efficacy but is frequently accompanied with severe side-effects caused by excessive and systemic immune system activation. Here, we addressed this need by targeting both the CPI antibodies anti-CTLA4 (αCTLA4) + anti-PD-L1 (αPD-L1) and the cytokines IL-2 and IL-12 to tumors via conjugation (for the antibodies) or recombinant fusion (for the cytokines) to a collagen-binding domain (CBD) derived from the blood protein von Willebrand factor (VWF) A3 domain. This approach harnesses the exposure of tumor stroma collagen to blood components due to the leakiness of the tumor vasculature. We show that intravenously (i.v.) administered CBD protein accumulated mainly in tumors, with 57% of total injected dose depositing in an orthotopic MMTV-PyMT breast tumor model. CBD conjugation or fusion decreased the systemic toxicity of both αCTLA4+αPD-L1 combination therapy and IL-2/IL-12. CBD-conjugation to CPI abolished T cell infiltration into the liver and eliminated hepatotoxicity as well as decreased systemic cytokine release in the blood serum. CBD-fusion to IL-2 ameliorated capillary leak syndrome and pulmonary edema. CBD-fusion to IL-12 significantly decreased hepatotoxicity and systemic IFNg concentration. These side-effects have been reported in the clinic. CBD-CPI and CBD-IL-2 significantly suppressed tumor growth compared to their unmodified forms in B16F10 melanoma, CT26 colon carcinoma and MMTV-PyMT slow growing breast cancer models, and both CBD-CPI and CBD-IL-2 increased tumor-infiltrating CD8+ T cells; increases in the ratio of effector CD8+ T cells to T regulatory cells were observed. In an orthotopic breast tumor model, combination treatment with CPI and IL-2 eradicated tumors in 9/13 animals with the CBD-modified drugs, whereas it did so in only 1/13 animals with the unmodified drugs. CBD-IL-12 therapy induced remission of B16F10 melanoma and EMT6 breast cancer, along with increased intratumoral IFNg concentration compared to normal IL-12 therapy. Our data suggest that the A3 domain of VWF can be used to engineer immunotherapies (e.g. CPI and cytokines) with high translational promise as systemically-administered tumor targeting drugs with improved safety and efficacy compared to their native forms. Unlike other active tumor-targeting approaches, CBD-based targeting is novel in a sense that it exploits abnormal tumor vessel structure; this characteristic affords binding to the ubiquitous extracellular matrix protein in the tumor, while limiting exposure to other tissues. CBD-immunotherapeutics may efficiently activate tumor-antigen-specific T cells and increase intratumoral downstream cytokines while maintaining systemic immune homeostasis by avoiding influencing non-tumor antigen specific T cells and systemic cytokine release. Citation Format: Jun Ishihara, Ako Ishihara, Aslan Mansurov, Koichi Sasaki, Steve S. Lee, John-Michael Williford, Lambert Potin, Peyman Hosseinchi, Laura T. Gray, Kiyomitsu Katsumata, Stephen J. Kron, Melody A. Swartz, Jeffrey A. Hubbell. Collagen affinity improves safety and efficacy of antibody and cytokine cancer immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1553.

​obesity and metastatic melanoma: A national perspective from the pre-immune checkpoint inhibitor era.

e21032 Background: There is an ongoing debate about correlation of obesity with favorable outcomes in patients with metastatic melanoma receiving treatment. Our study measured the differences in in-hospital outcome parameters such as length of stay (LOS), cost and mortality in patients with metastatic melanoma with respect to obesity. Methods: We used National Inpatient Sample (NIS) dataset from 2008 to 2014 to identify the diagnosis of Metastatic melanoma (ICD-9 code:172.9) among hospitalized patients. Data was matched and weighted to obtain a nationally representative sample for analysis. The AHRQ comorbidity measure of obesity was utilized to identify obesity as a coexisting medical condition that is not directly related to the principal diagnosis. Categorical and continuous variables were tested using Chi-square test and Student t-test respectively. Multivariate hierarchical regression was used for the analysis of primary outcomes of interest. Results: We identified a total of 1,809 cases of metastatic melanoma from 2008 to 2014. Among them, 908 (50.19%) were obese and 901 (49.80%) non-obese. 195 out of the 1,809 (10.77%) patients were treated with high dose infusion of IL-2. Overall, the mean LOS (≈6.03 days [non-obese] vs ≈5.72 days [obese], p = 0.949) and mean cost of hospitalization (16720.28$ vs 17639.95$, p = 0.428; mean difference of 919.67$) was not significantly different based on obesity as a comorbid condition. The mortality rate was found to be significantly lower in obese patients (41% vs. 59%, p = 0.017), but did not reach statistical significance on logistic regression analysis (OR:0.95, p = 0.130). Further subgroup analysis of patients with metastatic melanoma who received high dose IL-2, we found no significant differences in mean LOS (5.03 vs 7.03, p = 0.14) as well as mean cost of hospitalization (27432.72$ vs 41881.13$, p = 0.485) between non-obese and obese patients. However, the in-hospital mortality for patients undergoing high dose IL-2 therapy could not be studied given no cases of in-hospital mortality occurred in this subgroup. Conclusions: Our study demonstrates that there are no significant differences in in-hospital outcomes for patients with metastatic melanoma with respect to obesity, even in the subgroup of patients treated with IL-2 immunotherapy. Thus, there is a need for further studies to adequately understand the exact biological mechanism of obesity in metastatic melanoma, the influence of obesity on immunotherapy outcomes and its translation into patient care outcomes.

Expansion of regulatory T cells using low-dose interleukin-2 attenuates hypertension in an experimental model of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a chronic multisystem autoimmune disorder that is characterized by prevalent hypertension, renal injury, and cardiovascular disease. Numerous studies have reported a low prevalence and/or impaired function of regulatory T (TREG) cells in both patients with SLE and murine models of the disease. Evidence suggests that TREG cell dysfunction in SLE results from a deficiency in IL-2. Recent studies have reported that low-dose IL-2 therapy expands TREG cells in mouse models of SLE, but whether expanding TREG cells protects against hypertension and renal injury during SLE is unclear. To examine this question, female SLE (NZBWF1) and control (NZW) mice were injected with vehicle or recombinant mouse IL-2 three times in 24 h followed by single maintenance doses every 5 days for 4 wk. Treatment with IL-2 effectively expanded TREG cell populations in the peripheral blood, spleen, and kidneys. Circulating levels of anti-dsDNA IgG autoantibodies, a marker of SLE disease activity, were higher in SLE mice compared with control mice but were unaffected by IL-2 treatment. As previously reported by our laboratory, mean arterial pressure, measured in conscious mice by a carotid catheter, was higher in SLE mice than in control mice. Mean arterial pressure was significantly lower in IL-2-treated SLE mice compared with vehicle-treated SLE mice, suggesting that expanding TREG cells using low-dose IL-2 attenuates the development of hypertension. While the mechanism for the protection against hypertension is unclear, it does not appear to be related to the delay of SLE disease progression.

IL-2 therapy preferentially expands adoptively transferred donor-specific Tregs improving skin allograft survival.

Regulatory T cells (Tregs) have unique immunosuppressive properties and are essential to ensure effective immunoregulation. In animal models, Tregs have been shown to prevent autoimmune disorders and establish transplantation tolerance. Therefore, the prospect of harnessing Tregs, either by increasing their frequency or by conferring allospecificity, has prompted a growing interest in the development of immunotherapies. Here, employing a well‐established skin transplant model with a single major histocompatibility complex mismatch, we compared the therapeutic efficacy of adoptively transfer Treg with or without donor specificity and the administration of IL‐2 to promote in vivo expansion of Treg. We showed that IL‐2 treatment preferentially enhances the proliferation of the allospecific Tregs adoptively transferred in an antigen‐dependent manner. In addition, donor‐specific Tregs significantly increased the expression of regulatory‐related marker, such as CTLA4 and inducible costimulator (ICOS), in the skin allograft and draining lymph nodes compared to endogenous and polyclonal transferred Tregs. Importantly, by combining IL‐2 with donor‐specific Tregs, but not with polyclonal Tregs, a synergistic effect in prolonging skin allograft survival was observed. Altogether, our data suggest that this combination therapy could provide the appropriate conditions to enhance the immunoregulation of alloimmune responses in clinical transplantation.

Overall responses with coordinated pembrolizumab and high dose IL-2 (5-in-a-row schedule) for therapy of metastatic clear cell renal cancer: A single center, single arm trial.

657 Background: Ligation of IL-2 receptor or blockade of PD-1 receptor may change lymphocytes to induce regression of cancers with diverse histology or site of origin. Single agent objective response rates of 14-25% have been reported for IL-2 therapy of metastatic clear cell RCC (ccRCC). A response rate of 33% was observed in pembrolizumab treated ccRCC patients. Nivolumab treated ccRCC patients were observed to have early intratumoral migration of lymphocytes. A case report of IL-2 induced major regression right after no change on nivolumab therapy suggested that combining the two means of lymphocyte stimulation could be effective. Other trials combining IL-2 receptor agonists (NKTR-214) and PD-1 blockade have also reported regression of ccRCC. Distinctive attributes of high dose IL-2 therapy are the required inpatient stay and the durability of the complete responses. Methods: This single-institution, single arm study addresses the safety and feasibility of the combination of IL-2 and pembrolizumab in the treatment of metastatic ccRCC. Subjects are treated on four nine-week blocks, as follows: Pembrolizumab is given on weeks 1, 4, and 7 of each block. Patients are admitted for 5 doses of high dose IL-2 (given over ~ 33 hours/3 days) on weeks 2, 3, 5, and 6 of blocks 2 and 3. Safety is monitored by a Pocock boundary of .05 likelihood of 0.15 dose limiting toxicity rate. The Simon 2-stage alternative hypothesis for the sample size was a 45+% major response rate vs null hypothesis < 20%, at alpha = 0.10, 90% power. Results: No accrual stop for safety was triggered. Thirteen of the first 18 patients responded, substantially exceeding the requirement of 8+/24 combination-treated patients to reject the null. Seven patients responded after receiving pembrolizumab alone, six after starting combination therapy in block 2. Accrual is completed; at 27. Kaplan-Meier analysis projects ORR of 69%, with ORR 90%-lower confidence bound of 55%. Conclusions: The combination of high dose IL-2 and pembrolizumab is feasible, with a high response rate, justifying further exploration of this dual immune treatment of metastatic ccRCC. Clinical trial information: NCT02964078.

Therapy with high-dose Interleukin-2 (HD IL-2) in metastatic melanoma and renal cell carcinoma following PD1 or PDL1 inhibition

BackgroundMetastatic melanoma (mM) and renal cell carcinoma (mRCC) are often treated with anti-PD-1 based therapy, however not all patients respond and further therapies are needed. High dose interleukin-2 (HD IL-2) can lead to durable responses in a subset of mM and mRCC patients. The efficacy and toxicity of HD IL-2 therapy following anti-PD-1 or anti-PD-L1 therapy have not yet been explored.MethodsReports on mM and mRCC patients who had received HD IL-2 after PD-1 or PD-L1 inhibition were queried from the PROCLAIMSM database. Patient characteristics, toxicity and efficacy were analyzed.ResultsA total of 57 patients (40 mM, 17 mRCC) were treated with high dose IL-2 after PD-1 or PD-L1 inhibition and had data recorded in the PROCLAIM database. The best overall response rate to HD IL-2 was 22.5% for mM (4 complete response (CR), 5 partial responses (PRs)) and 24% for mRCC (2 CRs, 2 PRs). The toxicity related to HD IL-2 observed in these patients was similar to that observed in patients treated with HD IL-2 without prior checkpoint blockade. One patient who had received prior PD-L1 blockade developed drug induced pneumonitis with HD IL-2 requiring steroid therapy.ConclusionIn this retrospective analysis, HD IL-2 therapy displayed durable antitumor activity in mM and mRCC patients who progressed following treatment with PD-1 and PD-L1 inhibition. The toxicities were generally manageable and consistent with expectations from HD IL-2 but physicians should watch for immune related toxicities such as pneumonitis. This analysis supports the development of randomized prospective trials to assess the proper sequencing and combination of immune checkpoint blockade and cytokine therapy.

A Pilot Phase II Trial of Combining Extracorporeal Photopheresis (ECP) and Low Dose IL-2 for Treatment of Sclerodermatous Steroid Refractory Chronic Graft-Versus-Host Disease (cGvHD)

Chronic GVHD remains as the major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation. Systemic corticosteroids are the first line of therapy, but outcomes are poor in steroid refractory (SR) patients. Previous reports show that in SR patients, IL-2 therapy results in partial response (PR) associated with increase in regulatory T cells (Tregs). ECP has been used for cGVHD with skin response rates of 40-100%. Based on non-overlapping toxicities and, we prospectively studied a combination of low dose IL-2 + ECP to treat SR-cGVHD. Patients with SR-cGVHD, were treated with low dose IL-2 (1 × 106 U/m2, daily) + ECP for 12 weeks. IL-2 was provided by Prometheus Laboratories. ECP was administered 2 times/week for 4 weeks followed by 2 treatments every 2 weeks for the next 8 weeks. The NIH consensus criteria was used to assess cGVHD severity. PBMC and plasma samples were banked at baseline and on 3-week intervals to assess regulatory T cell (Tregs) and reactive oxygen species (ROS). Of the 12 consented patients, 9 completed the study (2 were not treated due to rapid disease progression before enrollment and 1 died from sepsis at week 11). All enrolled patients (n=10) had scleroderma as the predominant cGVHD manifestation. Median age was 45.6 years (range: 23-66). Median prior lines of therapy were 4.2(range 3-6), 5 patients had prior exposure to ruxolitinib and 4 were on ECP at the enrollment. Donors were fully matched unrelated (n=8), matched sibling (n=1) or mismatched unrelated (n=1). Conditioning regimen was myeloablative (n=5) or reduced intensity (n=4). GVHD prophylaxis was tacrolimus/Sirolimus (tac/sir) (n=6), tac/methotrexate (MTX) (n=2), or Tac/sir/MTX (n=1). Of the 10 enrolled patients, 9 responded to IL-2 + ECP combination (all classified as PR). A 37% dose reduction in steroid dose was noted among 7 patients. The remaining 3 patients were on stable steroid dose at end of study. Only 1 patient had progressive cGVHD in form of scleroderma. ECP+IL-2 therapy was well tolerated with no drug related serious adverse events (SAE). Grade 3-4 AEs included grade 3 metabolic and nutrition (n=8), grade 3 anemia (n=3), and grade 4 atrial flutter (n=1). Infectious complications were noted in 3 patients (one grade 4 [sepsis]). There was a robust increase in Tregs from the baseline mean of 0.755% (range 0.08-1.88) to end of treatment 6.999% (range 0.78-17.13) [p=0.02] Fig 1. Increment in Tregs was seen in patients who were on ECP therapy prior to enrollment (n=4) and in one patient who progressed while on therapy. No correlation was found between the treatment response and ROS levels. In conclusion, the combination of ECP +IL-2 is well tolerated in patients with SR-cGVHD with no apparent increase in infectious complications or other toxicities. Clinical response correlates with a robust increase in Tregs, but no correlation was found with ROS levels.

Right Time and Place for IL12: Targeted Delivery Stimulates Immune Therapy.

Systemic IL12 therapy has potent antitumor effects, but clinical delivery of this potent cytokine has been complicated by systemic toxicity. A novel strategy to deliver IL12 to the tumor microenvironment appears promising in a first-in-human study, appearing to stimulate tumor-specific adaptive immune responses with minimal systemic toxicity.See related article by Strauss et al., p. 99.

Antitumor activity of interleukin-18 on A549 human lung cancer cell line.

In this study, we investigated the antitumor activity of interleukin (IL)-18 on A549 human lung cancer cell line and evaluated the potential of IL-18 therapy in lung cancer. We generated a human IL-18 lentiviral expression vector and examined three groups of A549 cells, including nontransduced cells and cells transduced with either IL-18 or an empty lentiviral expression vector. IL-18 expression, cell proliferation, and apoptosis were examined using Western blotting, methylthiazolyldiphenyl-tetrazolium bromide assay, and flow cytometry, respectively. The expression of the T helper 1 (Th1) cytokine interferon-γ (IFN-γ) and Th2 cytokine IL-4 was analyzed by enzyme-linked immunosorbent assay (ELISA). Compared to the other groups of cells, A549 cells transduced with the IL-18 lentiviral expression vector exhibited significant increases in IL-18 expression, apoptosis, and the fraction of cells in G0 and G1 phases of the cell cycle and significant decrease in proliferation. Furthermore, ELISA results showed that IFN-γ expression increased significantly and IL-4 decreased in A549 cells transfected with IL-18 lentivirus expression vector. Using a lentiviral expression vector, IL-18 was expressed stably and efficiently in A549 cells, which showed attenuated proliferation and tumor cell growth, and enhanced tumor cell apoptosis. IL-18 expression also induced the secretion of IFN-γ, while decreasing the production of IL-4, therefore restoring the balance between Th1/Th2 cell subsets. These findings further demonstrated the antitumor activity of IL-18 and might open new therapeutic avenues for the prevention and treatment of lung cancer.

Low-dose IL-2 therapy for autoimmune diseases.

Low-dose IL-2 therapy for autoimmune diseases.

Low-Dose Interleukin-2 Therapy Enhances Cytotoxicity of CD56bright NK Cells in Patients with Chronic Gvhd

Introduction: Natural killer (NK) cells play an important role in defense against infections and cancer. Two major subsets of mature NK cells have been described. CD56bright CD16- NK cells, which normally represent only 5-10% of circulating NK cells, are thought to exhibit less cytolytic activity and greater immune regulatory functions than CD56dim CD16+ NK cells. However, recent studies have reported that cytolytic activity of CD56bright NK cells increases when these cells are stimulated with IL-15or the combination of IL-12, IL-15 and IL-18. Previous studies from our center have shown that daily administration of low-dose IL-2 in patients with chronic graft-versus-host disease (cGVHD) induces selective expansion of CD4+FoxP3+Helios+ regulatory T cells and CD56bright NK cells and improves clinical manifestations of cGVHD. The function of CD56bright NK cells expanded by low-dose IL-2 has not previously been studied.Methods: Single cell mass cytometry (CyTOF) with a panel of 35 metal tagged antibodies was performed on cryopreserved peripheral blood mononuclear cells (PBMC) from 10 adult patients with active cGVHD receiving daily low-dose IL-2 therapy. Patients in this clinical trial received extracorporeal photopheresis (ECP) for 8 weeks prior to starting daily low dose IL-2. ECP therapy (twice weekly) was continued when patients began low-dose IL-2 (1x106 IU/M2/day x 8 weeks). The analytic panel included 26 cell surface markers to identify distinct lymphocyte subsets and 9 intracellular markers to measure functional status and activation of specific signaling pathways. viSNE was used to visualize of high-dimensional data on a two-dimensional map and quantify single cell mass cytometry data. NK cytolytic activity was measured in flow cytometry-based cytotoxicity assays. CD56bright and CD56dim NK cells from 8 adult patients were purified from cryopreserved PBMC by cell sorting and incubated with labeled K562 cells for 4 hours followed by staining with 7-AAD and Annexin-V. E:T ratio of 1:1 was used for incubation with K562 targets.Results: No changes in extracellular or intracellular NK cell markers or quantitative changes in NK cells were observed during the initial 8 week ECP treatment period. Selective expansion of CD56bright NK cells was noted after 1 week of IL-2 therapy (9W) and continued during 8 weeks of daily IL-2 therapy. Increased expression of NKp30, Nkp46, NKG2D, HLA-DR and Ki67 occurred in expanded CD56bright NK cells with peak expression at 1 week after starting IL-2 (9W). At later time points during IL-2 therapy, expression of NKG2D, HLA-DR and Ki67 returned to baseline (Figure 1A). Expression of CD56, CD122 and NKG2A continued to increase during IL-2 treatment. In contrast, expression of CD25 by expanded CD56bright NK cells decreased during IL-2 treatment. Expression of phosphorylated signaling proteins did not change in any NK cell subset during IL-2 treatment. Cytolytic activity was measured in CD56bright and CD56dim NK cell subsets at different times during ECP and IL-2 therapy. After thawing, flow-sorted NK cell subsets were cultured for 16-20 hours with IL-2 (100 IU/ml). Cells were then washed and incubated with tumor targets (K562) for 4 hours and % killing was assessed by flow cytometry. Compared to pre IL-2 treatment, cytolytic activity of CD56bright NK cells increased during IL-2 treatment while cytotoxicity of CD56dim NK cells did not change. Notably, cytotoxicity of CD56bright NK cells became significantly higher than CD56dim NK cells during IL-2 therapy (Figure 1B).Conclusion: Single cell mass cytometry revealed that daily low dose IL-2 therapy induces selective expansion, activation and increased expression of activating NK receptors in CD56bright NK cells. CD56dim NK cells were not affected by IL-2 therapy. In vitro assays revealed that cytolytic activity of CD56bright NK cells increased during IL-2 treatment and exceeded the cytotoxicity of CD56dim NK cells. CD56bright NK cells, traditionally considered to be minimally tumor-responsive, are effectively stimulated by daily low dose IL-2 exposure to enable potent cytotoxicity in response to tumor targets. In patients receiving low-dose IL-2 after allogeneic HSCT, expanded CD56bright NK cells may contribute to graft versus leukemia (GVL) and help prevent relapse after transplant. DisclosuresNikiforow:Kite Pharma: Consultancy. Ho:Jazz Pharmaceuticals: Consultancy. Antin:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Soiffer:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

FBXO38 mediates PD-1 ubiquitination and regulates anti-tumour immunity of T cells.

Dysfunctional T cells in the tumour microenvironment have abnormally high expression of PD-1 and antibody inhibitors against PD-1 or its ligand (PD-L1) have become commonly used drugs to treat various types of cancer1-4. The clinical success of these inhibitors highlights the need to study the mechanisms by which PD-1 is regulated. Here we report a mechanism of PD-1 degradation and the importance of this mechanism in anti-tumour immunity in preclinical models. We show that surface PD-1 undergoes internalization, subsequent ubiquitination and proteasome degradation in activated T cells. FBXO38 is an E3 ligase of PD-1 that mediates Lys48-linked poly-ubiquitination and subsequent proteasome degradation. Conditional knockout of Fbxo38 in T cells did not affect T cell receptor and CD28 signalling, but led to faster tumour progression in mice owing to higher levels of PD-1 in tumour-infiltrating T cells. Anti-PD-1 therapy normalized the effect of FBXO38 deficiency on tumour growth in mice, which suggests that PD-1 is the primary target of FBXO38 in T cells. In human tumour tissues and a mouse cancer model, transcriptional levels of FBXO38 and Fbxo38, respectively, were downregulated in tumour-infiltrating T cells. However, IL-2 therapy rescued Fbxo38 transcription and therefore downregulated PD-1 levels in PD-1+ T cells in mice. These data indicate that FBXO38 regulates PD-1 expression and highlight an alternative method to block the PD-1 pathway.

GEN-1 immunotherapy for the treatment of ovarian cancer.

GEN-1 is a gene-based immunotherapy, comprising a human IL-12 gene expression plasmid and a synthetic plasmid delivery system, delivered intraperitoneally (ip.) to produce local and persistent levels of a pleiotropic immunocytokine, IL-12, at the tumor site in patients with advanced ovarian cancer. The goal of local and persistent IL-12 delivery is to remodel the highly immunosuppressive tumor microenvironment to favor immune stimulation while avoiding serious systemic toxicities, a major limitation of recombinant IL-12 therapy. Safe and sustained local production of IL-12 and related immunocytokines at the tumor site could produce potentially more favorable immunological changes in the tumor microenvironment and antitumor responses than a bolus systemic delivery of recombinant IL-12. Treatment safety, clinical benefits and biological activity of GEN-1 ip. in patients with ovarian cancer and in representative animal models are described.

Autoimmune haemolytic anaemia and autoimmune thrombocytopenia in childhood-onset systemic lupus erythematosus: updates on pathogenesis and treatment.

Autoimmune haemolytic anaemia (AIHA) and autoimmune thrombocytopenia are common complications of childhood-onset lupus, which may be life-threatening. A greater understanding of the pathogenesis of these haematologic manifestations will enhance our understanding of the biology of systemic lupus erythematosus (SLE) and inform the identification of novel treatments. The mechanisms underlying AIHA and autoimmune thrombocytopenia are incompletely understood and likely multifactorial. Although the development of auto-antibodies is central to the disease process, recent studies have demonstrated the importance of cytokines in the underlying pathologic process. In-vitro and in-vivo evidence points to a role for IL17 in the pathogenesis of AIHA, which involves loss of tolerance to red cell auto-antigens and the development of autoantibodies. Sirolimus, an mTor inhibitor, has benefited patients with primary autoimmune cytopenias, possibly by stimulating T regulatory cells, and may also have efficacy for SLE-associated cytopenias. Similarly, low-dose recombinant human IL-2 therapy has shown promising results for improving platelet counts in patients with autoimmune thrombocytopenia, possibly by restoring the balance between T regulatory, T helper and Th17 cells. The emergence of new agents directed at restoring immune dysregulation hold promise for the treatment of AIHA and autoimmune thrombocytopenia and should provide better tolerated alternatives to high-dose corticosteroids.

A method for expansion of T cells from cynomolgus monkey (Macaca fascicularis)

T cells have been successfully applied to cancer immunotherapy. However, a challenge is an expansion of T cells from cynomolgus monkey, which is a valuable non-human primate model for T cell therapy transferring to the clinic. Here, we compared several strategies to expand cynomolgus monkey T cell and developed an appropriate method. Our study demonstrated that 100ng/ml CD3 mAb + 1% PHA+ 1000U/ml IL2 therapy significantly expanded peripheral blood CD3+ T cells without compromising T cell phenotype in vitro. The results of this study could be used for T cell remodeling, which has significant therapeutic potential in Chimeric Antigen Receptor-T (CAR-T) cell immunotherapy.

Clinical Relevance of Autoantibodies against Interleukin-2 in Patients with Systemic Lupus Erythematosus.

Background:Increased serum autoantibodies against interleukin-2 (anti-IL-2 autoantibodies) were reported in patients with systemic lupus erythematosus (SLE) and in patients receiving IL-2 therapy. This study aimed to explore the clinical relevance of serum anti-IL-2 autoantibodies and the interactions between low-dose IL-2 therapy and serum anti-IL-2 autoantibodies.Methods:Serum samples were collected from 152 SLE patients and 100 age- and gender-matched healthy controls (HCs). Among them, 75 SLE patients were followed up for 10 weeks, and all of them were treated with corticosteroids, antimalarials, and/or immunosuppressants. Forty-six out of the 75 SLE patients received low-dose IL-2 therapy additionally. Clinical and laboratory parameters were collected at baseline and week 10. Serum anti-IL-2 autoantibodies were determined by enzyme-linked immunosorbent assay.Results:Compared with HCs, median levels and positive rates of serum anti-IL-2 autoantibodies were higher in SLE patients (32.58 [23.63, 45.23] arbitrary unit [AU] vs. 37.54 [27.88, 60.74] AU, P = 0.006, and 5.0% vs. 18.4%, P = 0.002, respectively). Compared to those without the corresponding disorders, serum anti-IL-2 autoantibody was increased in patients with alopecia (49.79 [36.06, 64.95] AU vs. 35.06 [25.40, 58.46] AU, P = 0.033), but it was decreased in those with lupus nephritis (31.71 [22.60, 43.25] AU vs. 44.15 [31.43, 68.52] AU, P = 0.001). Moreover, serum anti-IL-2 autoantibody was positively correlated with serum IgA (r = 0.229, P = 0.005), total IgG (r = 0.327, P < 0.001), and total IgM (r = 0.164, P = 0.050). Treatment with exogenous IL-2 was not significantly associated with serum anti-IL-2 autoantibody. In addition, no significant difference was found in serum anti-IL-2 autoantibody between responders and nonresponders to low-dose IL-2 therapy.Conclusions:Serum anti-IL-2 autoantibody was increased and associated with disease severity in SLE. Exogenous low-dose IL-2 did not significantly induce anti-IL-2 autoantibody production.

Abstract CT146: First-in-human phase I combination of the IL-15 receptor super agonist complex ALT-803 with a therapeutic (anti-CD20) monoclonal antibody (mAb) for patients with relapsed or refractory indolent non-Hodgkin lymphoma (iNHL)

Abstract Immunotherapy is an effective modality for cancer treatment. IL-15 is a cytokine that stimulates CD8+ T cell and NK cell survival, metabolism, and anti-tumor immunity. We hypothesized that IL-15 therapy will augment anti-cancer immune responses in concert with tumor-targeting therapeutic mAbs. Pre-clinical studies have shown that the IL-15 receptor super agonist complex, ALT-803, enhanced the anti-tumor effects of anti-CD20 mAb against iNHL in vitro and in vivo (Rosario M et al., Clin Ca Res, 2016). Here, we conducted the first clinical study combining ALT-803 with a tumor-targeting mAb, in patients with rel/ref iNHL. A total of 21 iNHL patients [N=16 FL (FLIPI low: 4, int: 4, high: 8), N=4 MZL, N=1 SLL] were treated. Median age was 64 years (range 54-81), median number of prior therapies was 2 (range 1-18), and 5 patients were refractory to prior anti-CD20 mAb therapy. Treatment included an induction cycle of 4 weekly doses of rituximab (375 mg/m2 intravenously, IV) and ALT-803. The ALT-803 dose levels were 1, 3, and 6 mcg/kg IV followed by 6, 10, 15, and 20 mcg/kg subcutaneously (SC, N=3 per dose level). In patients with SD, PR or CR, consolidation therapy with ALT-803 (at the same dose level) and rituximab was then administered every 8 weeks for 4 cycles. No dose limiting toxicities were observed, and the recommended phase 2 dose was 20 mcg/kg SC. Overall ALT-803 was well tolerated with no grade 4 or 5 adverse events. Grade 3 AEs (regardless of attribution to ALT-803) included transient hypertension (14%), anemia (5%), nausea (5%), chills (5%), fever (5%), neutropenia (5%), and hyperglycemia (5%). Patients who received ALT-803 SC had a self-limited injection site reaction. To date, best ORR was 10/21 (48%), with 7 CR, 3 PR, 8 SD (all with decreased tumor volume), and 3 PD. Of the 12 patients treated with SC ALT-803, only 1 patient had PD, with the remainder receiving ongoing consolidation. In the 5 rituximab-refractory patients we observed 1 CR, 2 SD (45% and 36% tumor volume decrease), and 2 PD. PK analysis showed that SC ALT-803 administration resulted in a lower Cmax and prolonged blood levels compared to IV. At the 15 and 20 mcg/kg SC dose levels of ALT-803, NK cells were significantly increased by percentage (mean peak 30+5%, 6-fold, P&amp;lt;0.001) and number (mean peak 687+347/uL, 10-fold, P&amp;lt;0.001). This was accompanied by a marked increase in proliferation (Ki67, 97+0.5%, P&amp;lt;0.001) and activation (CD69, 10-fold, P&amp;lt;0.01) in vivo. CD8+ T cell number (2-fold, P&amp;lt;0.01) and proliferation (Ki67, 56+6%, P&amp;lt;0.001) were also significantly increased, without increases in CD4+ or regulatory T cell numbers. Patient NK cells exhibited enhanced functional responses (degranulation and IFN-γ production) against anti-CD20 mAb coated NHL targets after ALT-803 administration, compared to pre-ALT-803 samples. NK cells (P&amp;lt;0.05) and CD8 T cells (P&amp;lt;0.05) were also increased at the 6 and 10 mcg/kg SC ALT-803 levels. Thus, the immunostimulatory IL-15-based agent ALT-803 may be safely combined with a therapeutic mAb to significantly enhance NK and CD8+ T cell number and function in patients with iNHL. Phase 2 testing is underway in iNHL. Clinical investigations combining ALT-803 with additional therapeutic mAbs, complementary types of immunotherapy, and in additional malignancies are warranted. Citation Format: Todd A. Fehniger, Brian T. Hess, Veronika Bachanova, Michelle Becker-Hapak, Ethan McClain, Melissa Berrien-Elliott, Julia Wagner, Nancy L. Bartlett, Brad Kahl, Neha Mehta-Shah, Amanda F. Cashen, Feng Gao, Kyle Conradi, Amy D. Rock, Emily K. Jeng, Liza Hernandez, Jack O. Egan, Peter R. Rhode, Hing C. Wong. First-in-human phase I combination of the IL-15 receptor super agonist complex ALT-803 with a therapeutic (anti-CD20) monoclonal antibody (mAb) for patients with relapsed or refractory indolent non-Hodgkin lymphoma (iNHL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT146.

Glycogen synthase kinase 3 inhibition lowers PD-1 expression, promotes long-term survival and memory generation in antigen-specific CAR-T cells

Successful remission in hematological cancers by CAR-T cell immunotherapy has yet to be replicated in solid tumors like GBM. A significant impediment of CAR-T immunotherapy in solid tumors is poor exposure of T cells to tumor antigens resulting in suboptimal CAR-T cell activation, which ultimately fails to induce a robust anti-tumor immune response. Costimulatory moieties in advanced-generation CARs, along with additional IL2 therapy has been shown to be insufficient to overcome this hurdle and have its cytotoxic limitations. GSK3 is constitutively active in naïve T cells and is transiently inactivated during T cell activation resulting in rapid T cell proliferation. Pharmacologic inhibition of GSK3 in GBM-specific CAR-T cells reduced FasL expression, increased T cell proliferation and reduced exhaustion by lowering PD-1 levels resulting in the development of CAR-T effector memory phenotype. Treatment with GSK3-inhibited CAR-T cells resulted in 100% tumor elimination during the tumor-rechallenge experiment in GBM-bearing animals and increased accumulation of memory CAR-T cells in secondary lymphoid organs. These adjuvant-like effects of GSK3 inhibition on activated CAR-T cells may be a valuable adjunct to a successful implementation of CAR-T immunotherapy against GBM and other solid tumors.

IL-2 antibodies in type 1 diabetes and during IL-2 therapy.

IL-2 antibodies in type 1 diabetes and during IL-2 therapy.