Dose IL-2 Research Articles

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.

Results of a Phase 1 Trial of Gda-201, Nicotinamide-Expanded Allogeneic Natural Killer Cells (NAM-NK) in Patients with Refractory Non-Hodgkin Lymphoma (NHL) and Multiple Myeloma (MM)

Background: NK cells have the capacity to kill tumor targets, representing a novel immunotherapeutic approach to cancer. We have shown promising clinical activity in AML with a previous NK cell preparation. Limitations of NK therapies have included specificity, persistence after infusion, and potential for maximal activity of NK cells in vivo. GDA-201 is a cellular product composed of natural killer (NK) cells from healthy donors expanded ex vivo with nicotinamide (NAM) and IL-15; this is a unique ex vivo activation strategy to induce persistence of potent anti-tumor activity. Prior in vitro studies and pre-clinical models demonstrated that NAM-exposed NK cells exhibited augmented resistance against exhaustion and improved killing function, proliferation, and organ retention. We now report safety and efficacy from a phase 1 clinical trial of GDA-201 in patients (pts) with relapsed or refractory (R/R) NHL or MM. Methods: Following donor apheresis, CD3-depleted mononuclear cells were cultured for 14-16 days with NAM (5mM) and IL-15 (20ng/ml), resulting in a 40-fold increase in NK cells and increased expression of CD62L from 2.9% to 21%. GDA-201 contained ~98% NK cells, and CD3 content was maintained at <0.5% (<5x105/kg/dose). Pts with R/R CD 20-positive NHL or refractory MM received cyclophosphamide (400mg/m2 IV x 3d) and fludarabine (30 mg/m2 /d IV x 3d), followed by two doses of GDA-201 (Days 0 and 2) and low-dose IL-2 (6 million units sc). Pts with NHL or MM received rituximab (375 mg/m2 x 4 weekly or elotuzumab (10 mg/kg x 3 weekly), respectively, to enhance NK cell targeting through antibody-dependent cellular cytotoxicity (ADCC). Results: 20 pts were enrolled: 7 with NHL (4 follicular, 2 transformed, 1 diffuse large cell lymphoma) and 13 with MM, in 3 cohorts of escalating GDA-201 dose; 11 pts received the maximum target dose (median 1.7 x 108 cells/kg, range 1.6-2.0 x 108 cells/kg). There were no dose limiting toxicities. The most common grade 3/4 adverse events were neutropenia and thrombocytopenia, febrile neutropenia (n=2), increased creatinine, hyponatremia, pulmonary edema; all events were transient. One pt had grade 2 cytokine release syndrome at day 18, presenting with fever, hypoxemia and hypotension, responding to tocilizumab; pt later died of E Coli sepsis. There were no neurotoxic events, GVHD or marrow aplasia. Among 7 NHL pts, there were 3 CR and 2 PR with overall response rate of 71%. Median duration of response was 12 months (CR patients) and 5 months (PR patients). Figure 1A illustrates a 57-year-old man with history of CLL and Richter's transformation (large cell lymphoma), pre- GDA-201 and 6 months post therapy; the pt had continued response with 80% tumor shrinkage at 6 months. In MM patients, 1 patient with extramedullary disease had CR and 4 had SD with median duration 2.5 months. In our previous study using overnight-activated NK cells, persistence 7 days after adoptive transfer was limited. Using GDA-201, flow cytometry confirmed persistence of donor NAM-NK in peripheral blood up to day 7-10 (day 7 range 2-55% donor NK cells; Figure 1B), as well as enhanced in vivo proliferation (median Ki67 99%). Conclusions: Cellular therapy using GDA-201 with monoclonal antibodies was safe, and demonstrated early evidence of clinical activity in heavily pre-treated pts with advanced NHL and MM. The recommended dose of GDA-201 for phase 2 is 2.0 x 108 cells/kg. The clinical responses showed that NK cell targeting through ADCC can be efficacious and increase response. Laboratory studies showed that GDA-201 had better persistence than observed in our previous studies using overnight activated cytokine alone stimulated NK cells. This study demonstrated that GDA-201 has an efficacy signal, and larger phase II studies are warranted. Disclosures Bachanova: Incyte: Research Funding; Gamida Cell: Research Funding; Novartis: Research Funding; GT Biopharma: Research Funding; Celgene: Research Funding; Kite: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees. McKenna:Fate Therapeutics: Research Funding; Magenta Therapeutics: Research Funding; CIBMTR BMT CTN (NIH): Other: Medical Monitor; Icahn School of Medicine, New York, New York: Consultancy; National Eye Institute (NIH): Other: DSMB (2); Gamida: Research Funding; NMDP: Other: Donor and Patient Safety Monitoring Advisory Group; Intima: Patents & Royalties: Royalities, Research Funding. Brachya:Gamida Cell: Employment, Equity Ownership. Peled:Gamida Cell: Employment, Equity Ownership. Miller:GT BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CytoSen: Membership on an entity's Board of Directors or advisory committees; Dr. Reddys Laboratory: Membership on an entity's Board of Directors or advisory committees; Moderna: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc: Consultancy, Research Funding; OnKImmune: Membership on an entity's Board of Directors or advisory committees.

A Case of Acute Lymphocytic Leukaemia with t(3;13) and Central Nervous System Leukemia after Allogenic Cord Blood Transplantation

Background: Acute lymphoblastic leukemia (ALL) is a neoplastic cancer characterized by clonal expansion of leukemic cells in lymph organs and bone marrow. Lots of kinds of different chromosomal translocation can be found in those leukemic cells. However, the role of the abnormal chromosomals and genes in leukemogenesis is not yet fully understood. Identifying new chromosomal translocation can facilitate a better understanding of pathogenesis of this disease. Case presentation: We report a rare case of acute lymphocytic leukaemia with t(3;13)(q29,q21). The patient was diagnosed pre-B-ALL with no abnormal chromosomal or gene fusion and achieved complete remission (CR) after induction chemotherapy. Ten months later, she relapsed in the consolidation with cytogenetics test showing 46,XX,t(3;13)(q29,q21). Given no CR after 2 chemotherapy regimens, she received salvage cord blood transplantation. Regular intrathecal methotrexate was applied to prevent central nervous system leukemia. Good graft-versus-leukemia (GVL) effect was induced by daily injection of low dose of IL-2 two months post transplantation. Minimal residual disease (MRD) negativity was maintained until central nervous system leukemia was found 8 months after transplantation. A whole exome sequencing was performed. Nine driver mutation genes and seven tumor genes were found. Conclusions: We highly suspect that the relapse in central nervous system after transplantation is associated with the rare chromosomal translocation. Disclosures No relevant conflicts of interest to declare.

Influenza Vaccination Primes Human Myeloid Cell Cytokine Secretion and NK Cell Function.

Cytokine-induced memory-like (CIML) NK cells generated in response to proinflammatory cytokines in vitro and in vivo can also be generated by vaccination, exhibiting heightened responses to cytokine stimulation months after their initial induction. Our previous study demonstrated that in vitro human NK cell responses to inactivated influenza virus were also indirectly augmented by very low doses of IL-15, which increased induction of myeloid cell-derived cytokine secretion. These findings led us to hypothesize that IL-15 stimulation could reveal a similar effect for active influenza vaccination and influence CIML NK cell effector functions. In this study, 51 healthy adults were vaccinated with seasonal influenza vaccine, and PBMC were collected before and up to 30 d after vaccination. Myeloid and lymphoid cell cytokine secretion was measured after in vitro PBMC restimulation with low-dose IL-15, alone or in combination with inactivated H3N2 virus; the associated NK cell response was assessed by flow cytometry. PBMC collected 30 d postvaccination showed heightened cytokine production in response to IL-15 compared with PBMC collected at baseline; these responses were further enhanced when IL-15 was combined with H3N2. NK cell activation in response to IL-15 alone (CD25) and H3N2 plus IL-15 (CD25 and IFN-γ) was enhanced postvaccination. We also observed proliferation of less-differentiated NK cells with downregulation of cytokine receptors as early as 3 d after vaccination, suggesting cytokine stimulation in vivo. We conclude that vaccination-induced "training" of accessory cells combines with the generation of CIML NK cells to enhance the overall NK cell response postvaccination.

Low dose of IL-2 combined with rapamycin restores and maintains the long-term balance of Th17/Treg cells in refractory SLE patients

BackgroundThe development of Systemic lupus erythematosus (SLE) has been associated with the balance of Th17 and Treg cells. IL-2 and rapamycin can influence the populations of both Th17 and Treg cells. However, it is unclear whether low dose of IL-2 and rapamycin can relieve the symptoms of SLE patients and what is the mechanisms. In this study, we aim to analyze the effect of low dose of IL-2 plus rapamycin on the number of Tregs, Th17 cells and the ratio of Th17/Treg cells, as well as to evaluate its therapeutic efficacy in refractory SLE patients.ResultFifty refractory SLE patients and 70 healthy controls were enrolled and followed up for 24 weeks. We found that compared with HC, the refractory SLE patients had a lower number of Tregs, a similar number of Th17 cells, but an increased ratio of Th17/Treg. After the treatment, the number of Tregs of the patients at 12th and 24th week was significantly increased. While the number of Th17 cells was unchanged, the ratio of Th17/Treg was significantly decreased at both 6 weeks and 24 weeks. After 6, 12 and 24 weeks of treatment, the SLEDAI score was significantly reduced. The prednison dosage at 6th,12th and 24th week post treatment was significantly decreased.ConclusionOur results support that the reduction of Tregs and the imbalance of Th17/Treg cells were correlated with the occurrence and development of refractory SLE. Low dose of IL-2 combined with rapamycin was able to restore the number of Tregs and the balance of Th17/Treg cells. As a result, this approach was able to induce immune tolerance and promote disease remission, allowing for the reduction in prednisone dosage.Trial registrationChiCTR-IPR-16009451 Registration date: 2016/10/16

Abstract 2810: Exploring T cell- repertoire in the tumor microenvironment during check-point inhibition in patients with metastatic solid tumors

Abstract Background: Immunomodulation has improved anti-tumor activity. However, this treatment modality is only effective in a proportion of patients. Hence, analyzing changes in the tumor microenvironment is essential for understanding the variety of response patterns seen among patients undergoing the treatment. It also provides clinicians with valuable knowledge of possible new therapeutic targets. Aim: Assessment of the repertoire of T-cells (tumor-infiltrating lymphocytes -TIL) in the tumor microenvironment during check-point inhibition in patients with metastatic solid tumors. Material and methods: 35 patients with different metastatic solid tumors were included in the study at our Unit for Experimental Treatment at Rigshospitalet, Denmark. Patients were given treatment with check-point inhibitors targeting PD-1/PD-L1/CTLA-4 either in monotherapy or combined with other agents. Fresh tumor biopsies were taken at two time-points, namely, at baseline and on-treatment (50 days interval). T-cells were expanded in vitro with high doses of IL2 (6,000IU/mL IL2), harvested and cryopreserved for later use. Samples were analyzed using a multicolor flow cytometry panel. Response to the treatment was assessed radiologically using to RECIST criteria 1.1 and patients were divided in responder and non-responders. Results: Expansion was successful in seventeen patients and five other follow-up samples are still in culture at the moment. The current data analysis was run based on the seventeen cryopreserved samples. Among responders, we observed that baseline samples showed a statistically significant high percentage of effector CD8+(CD45RO+CCR7-) and CD8+naïve T cells(CD62L+), with p value 0.034 and 0.009, respectively. The presence of a high proportion of naïve CD8+Tcells in tumor microenvironment of responders could be explained by the fact that there is a high proportion of T cells surviving and becoming memory T cells and eventually effector T cells. No statistical difference was seen at the baseline expression of PD-1, either in the CD8 (p 0.66) or in the CD4 compartment (p 0.88). For follow-up samples, CD4+ effector memory T cells seemed to be more present among responders (p 0.009). Interestingly, NK-cells expressing CD56 (p 0.046) and co-expressing CD137 (p 0.005), known as an activation marker, appeared to be more present in samples from the responder group. CD4+T cells deriving form follow-up samples were also co-stained for LAG-3 revealing that responders had a low percentage of TILs expressing both markers (p 0.016). Paired-sample test will be run as the expansion phase for the remaining 5 samples still in culture is over. Conclusion: The preliminary findings of our study suggest that response achieved during check-point inhibition is reliant on the subset of T cells present in the tumor microenvironment and on how its composition changes over time. Citation Format: Vinicius Araujo Barbosa de Lima, Morten Hansen, Kristoffer Staal Rohrberg, Iben Spangaard, Inge Marie Svane, Ulrik Lassen. Exploring T cell- repertoire in the tumor microenvironment during check-point inhibition in patients with metastatic solid tumors [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 2810.

Abstract 1106: Co-administration of the iRGD tumor-penetrating peptide improves the tumor immunostimulatory effects of low-dose IL-2

Abstract Background: Recombinant IL-2 at high doses is an effective immunotherapy treatment for various types of solid tumors. However, its clinical utility has been limited by serious mechanism-based side-effects. The clinical-stage iRGD peptide specifically targets tumors and, via activation of the ‘CendR’ trans-tissue transport pathway, increases tumor penetration and accumulation of various types of co-administered drugs. By selectively increasing the drug delivery into tumors, but not to normal tissues, iRGD potentiates the pharmacological activity of various anti-cancer agents within tumor tissue, while reducing their toxicities. In this study we investigate whether co-administration of iRGD with low-dose IL-2 can improve the tumor immune microenvironment by increasing in the ratios of tumor-killing effector CD8 cells vs. the immunosuppressive T-regulatory cells. Methods: Subcutaneous breast tumors (4T1) were generated in immunocompetent mice. Mice were treated with either vehicle control, iRGD, IL-2, or IL-2 + iRGD for 5 days. Tumors were preserved for immunohistochemistry (IHC) or enzymatically digested for fluorescence activated cell sorting (FACS) 16 hours after the last dosing. The FACS and IHC panels were designed to detect the percentage of total T cells, CD4 and CD8 T cells, and Treg cells. Results: Low doses of IL-2 alone were found to increase Tregs within the tumor but had no effect in CD4 or CD8 effector T cells, compared to vehicle treatment. However, when Low dose IL-2 was co-administered with iRGD, we observed a significantly lower percentage of T regs and an increase in CD4 effector T cells. More importantly the ratio of CD8 / Treg cells was increased by at least 10-fold, compared to low dose IL-2 alone. Conclusions: The combination treatment with iRGD and low doses of IL-2 is capable of favorably altering the tumor immune microenvironment such that immunosuppressive T reg cells were reduced, while there was an increase in the effector CD4 and CD8 T-cell populations. The tumor-selective IL-2 pharmacology benefit obtained with iRGD co-administration may provide new options for the use of the well-validated IL-2 in solid tumor patients, including a strategy to overcome the primary resistance to PD-1 blockade. Citation Format: Harri Jarvelainen, Gregory P. Botta, Tatiana Hurtado De Mendoza, Erkki Ruoslahti. Co-administration of the iRGD tumor-penetrating peptide improves the tumor immunostimulatory effects of low-dose IL-2 [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 1106.

Low-Dose Interleukin 2 in Children with Recently Diagnosed Type 1 Diabetes: A Phase 1/2 Randomised, Double-Blind, Placebo-Controlled Dose Finding Study

Background: Low-dose interleukin 2 (ld-IL2) selectively activates and expands regulatory T cells (Tregs) and thus has the potential to skew the regulatory/effector T (Treg/Teff) cell balance towards improved regulation. Regimens of ld-IL2 are actively investigated as a treatment for various autoimmune diseases, including type 1 diabetes (T1D). We previously reported a dose-dependent specific activation and expansion of Tregs in adults with established T1D receiving a short 5-day treatment with ld-IL2. We report results from the DF-Child trial, in which we investigated which low doses of IL2 would more effectively activate Treg cells during a 1-year treatment in children with recently diagnosed T1D. Methods: DF-Child was a multicentre, double-blinded, phase 1/2 clinical trial: 24 children (7-14 years-old) with T1D diagnosed within the previous 3 months were randomized in a 1:1:1:1 ratio to s.c. injection of placebo or IL2, at doses of 0.125, 0.250, or 0.500 MIU/m2, given daily for a 5-day course and then fortnightly for 1 year. The primary outcome was change in Treg cells expressed as a percentage of CD4+ T cells at day-5, and pre-specified that an increased on Tregs ≥60% from baseline would identify Treg high responders. This trial is registered with ClinicalTrials.gov, NCT01862120. Findings: Twenty-four patients were randomized between June 27, 2013, and Jan 1, 2016. IL2 was well tolerated at all doses, with no serious adverse events. Non-serious adverse events were transient and mild to moderate. Ld-IL2 induced a dose dependent increase in the proportion of Tregs. Although the mean Treg responses of all dose-groups were significantly different from placebo, the individual Treg responses to IL-2 were variable and fluctuated over time. All treated subjects showed an increase in Tregs, and we identified 7 Treg high responders among those treated with the 0.250 and 0.500 MIU/m2/day doses (3 and 4, respectively). There was no significant change in glycaemic control in any of the dose-groups compared to placebo, and no differences reported in insulin doses. However, there was a trend for an improved maintenance of induced C-peptide production at one year in the 7 Treg high responders. Interpretation: The good safety profile at all doses, the dose-dependent effects on Tregs and the observed variability of the Treg response to ld-IL2 in newly diagnosed T1D children call for using the highest dose in future developments. The better preservation of insulin production in Treg high responders supports the potential of Tregs in regulating autoimmunity in T1D and warrants pursuing the investigation of ld-IL2 for its treatment and prevention. Trial Registration Number: This trial is registered with ClinicalTrials.gov, NCT01862120. Funding: Assistance Publique-Hopitaux de Paris, Investissements d’Avenir programme (ANR-11-IDEX-0004-02, LabEx Transimmunom and ANR-16-RHUS-0001, RHU iMAP) and European Research Council Advanced Grant (FP7-IDEAS-ERC-322856, TRiPoD). Declaration of Interest: DK, MR and CB are co-inventor of a patent entitled “IL 2 based therapy” (OEB 11 305269.0) and DK and MR are shareholders in ILTOO pharma, the exclusive licensee of this patent. Ethical Approval: The study was approved by the institutional review board of Pitie-Salpetriere Hospital, and conducted in accordance with the Declaration of Helsinki and good clinical practice guidelines. Written informed consent was obtained from all participants before enrolment in the study.

PS1215 SAFETY AND TOLERABILITY OF ALLOGENEIC, OFF‐THE SHELF PLACENTAL NATURAL KILLER CELLS (PNK 007) IN PHASE 1 MULTIPLE MYELOMA AND ACUTE MYELOID LEUKEMIA STUDIES (NCT02955550 AND NCT02781467)

Background:PNK‐007 is an allogeneic, off‐the‐shelf cell therapy enriched for CD56+/CD3‐ NK cells expanded from placental CD34+ cells. PNK‐007 exhibits cytotoxicity against various cancer cell types.Aims:Characterize the safety and tolerability of PNK‐007 single‐infusion in subjects (subj) with relapsed/refractory (r/r) AML (completed) and with r/r and Newly Diagnosed (ND) MM (active; not recruiting) subj within 28 days dose‐limiting toxicity period (DLT).Methods:After lymphodepletion in AML study, single PNK‐007 infusion of 1, 3, or 10 million (M) cells/kg followed by subcutaneous recombinant human IL‐2 (IL2) at 6 M units every other day, starting on the day of PNK‐007 infusion, for up to 6 doses to facilitate PNK‐007 expansion was administered. In the MM study, single PNK‐007 infusion following ASCT with 10 M cells/kg Day (D) 14 with or without IL2; 30 M cells/kg D14 with IL2; or 30 M cells/kg D7 with IL2 after variable pre‐ASCT induction therapy was administered. Treatment‐emergent adverse events (TEAE) were extracted from clinical dataset for the 28‐day DLT period. The safety analysis included TEAE assessment using Medical Dictionary for Regulatory Activities (MedDRA) System Organ Class (SOC) and Preferred Terms (PT) order by severity, grading, seriousness, events of interest, and PNK‐007 relatedness per the investigators’ assessments.Results:In the AML study (n = 10), subj aged 30–70 years received a median of 3 prior lines of therapy. The most common SOC was Metabolism disorders with hypokalemia reported in 7 subj. Within cardiac disorders SOC, a grade (Gr) 5 unrelated cardiac arrest was an event of interest in 1 subj (10%). This subject experienced respiratory distress (day 14), dialysis (day 17), and died of cardiac arrest on day 17; not attributed to PNK‐007. In Gastrointestinal SOC, 4 subj (40%) reported nausea, 1 PNK‐007 related. 6 subj experienced hypotension (60%) with 1 Gr 3 PNK‐007 related. Infections noted were sinusitis, Klebsiella infection, Cytomegalovirus, and serious sepsis/Pneumonia/Clostridium difficile/Bacteremia; all PNK‐007 unrelated. 5 subj reported serious adverse event (SAE); one attributed to PNK‐007. This related event was noted as a DLT of cytokine release syndrome (CRS) which occurred 14 days after PNK‐007 infusion and was managed with tocilizumab.In the MM study (n = 15; aged 44–69 years), 12 subj were ND and 3 were r/r MM. No Gr ≥ 3 events were reported under Cardiac or Nervous System disorders SOC. The most common SOC noted was Gastrointestinal disorders with TEAE in 12 subj (80%). Within General disorders SOC, pyrexia was reported in 7 subj (47%); all unrelated to PNK‐007. Anaemia was noted in 5 subj (33%); 1 PNK‐007 related. 1 subj experienced a Gr 2 capillary leak syndrome on the day of 6th IL2 dose (day 10) along with nephrotic syndrome on the same day; both attributed to IL2. 4 subj (26%) experienced maculo‐papular rash (2 Gr 3); unrelated. Infections noted were oral candidiasis (2 Gr 2 and 1 Gr 3), Gr 2 soft tissue infection, 1 Gr 3 serious staphylococcal abscess, and 1 Gr 3 serious streptococcal pneumonia, all unrelated. 4 subj reported SAEs; all unrelated to PNK‐007.Summary/Conclusion:PNK‐007 has a favorable safety and tolerability profile. Overall, CRS incidence was 1 out of 25 subj with no fatality. No GvHD, Infusion related toxicity, Cardiac, or Neurotoxicity were reported. All noted Infections SOC events were unrelated to PNK‐007. The safety profile of PNK‐007 merits evaluation for therapeutic use in hematological malignancies and solid tumors.image

A phase II study of autologous tumor infiltrating lymphocytes (TIL, LN-144/LN-145) in patients with solid tumors.

TPS2648 Background: Adoptive cell therapy (ACT) with tumor infiltrating lymphocytes (TIL) has demonstrated durable complete responses in immunogenic tumors with high mutational burden in metastatic melanoma patients who had not received prior immune checkpoint inhibitors (ICI); CR rate 24%. Pembrolizumab is an approved agent for the treatment of metastatic melanoma and head & neck cancers, among others. Further, ICI have been reported to potentially enhance the efficacy of TIL therapy. One aim of this study is to improve the efficacy response for early line patients by combining TIL with anti-PD-1 in ICI-naïve patients with metastatic melanoma (Cohort 1) and head & neck cancers (Cohorts 2). In Cohort 3, TIL therapy alone is offered to NSCLC patients who have received prior systemic therapy, including ICI. Methods: IOV-COM-202 is a prospective, Phase 2 multicenter, open-label study in which 36 patients (12 per cohort) are to be enrolled in one of three cohorts; Cohorts 1 and 2: TIL therapy in combination with pembrolizumab, or Cohort 3: TIL therapy alone. Patients will have tumors resected at local centers and shipped to a central GMP facility to undergo a 22-day manufacturing process that yields cryopreserved infusion product (LN-144/LN-145) that is shipped back to treating center. All patients receive TIL therapy consisting of 1 week of preconditioning cyclophosphamide/fludarabine, followed by a single infusion of LN-144/LN-145 (Day 0) and up to 6 doses of IL-2 (600,000 IU/kg). Patients in Cohorts 1 and 2 also receive pembrolizumab on Day -1 and then Q3W for up to 2 years or until disease progression or acceptable toxicity. Co-primary endpoints for each cohort are objective response rate (ORR) per RECIST 1.1, and safety (grade ≥ 3 TEAE). Eligibility criteria: Cohorts 1 (melanoma) and 2 (head & neck): patients must not have received prior ICI (eg, anti-PD-1, anti-CTLA-4) and may have received up to 3 lines of prior systemic therapy, Cohort 3 (NSCLC): patients must have received 1-3 prior lines of systemic therapy including ICI. After tumor resection for TIL manufacturing, patients must have additional measurable disease for assessment per RECIST 1.1. Adequate bone marrow/organ function and ECOG PS of 0 or 1 is required. Clinical trial information: NCT03645928.

Safety and efficacy of cryopreserved autologous tumor infiltrating lymphocyte therapy (LN-144, lifileucel) in advanced metastatic melanoma patients who progressed on multiple prior therapies including anti-PD-1.

2518 Background: Treatment options are limited for patients with advanced melanoma who have progressed on checkpoint inhibitors and targeted therapies such as BRAF/MEK inhibitors (if BRAF-V600E mutated). Adoptive cell therapy utilizing tumor-infiltrating lymphocytes (TIL) has shown antitumor efficacy with durable long-term responses in heavily pretreated melanoma patients. Safety and efficacy of lifileucel (LN-144), a centrally manufactured autologous TIL therapy are presented. Methods: C-144-01 is a global Phase 2 open-label, multicenter study of the efficacy and safety of lifileucel in patients with unresectable metastatic melanoma. We report on Cohort 2 (N = 55) patients who received cryopreserved lifileucel. Tumors resected at local institutions were processed in central GMP facilities for TIL production in a 22-day process. Final TIL infusion product was cryopreserved and shipped to sites. Patients received one week of cyclophosphamide/fludarabine preconditioning lymphodepletion, a single lifileucel infusion, followed by up to 6 doses of IL-2. Results: In 55 patients with Stage IIIC/IV unresectable melanoma, 3.1 mean prior therapies (anti-PD1 100%; anti-CTLA-4 80%; BRAF/MEK inhibitor 24%), high baseline tumor burden (110 mm mean target lesion sum of diameters), ORR was 38% (2 CR, 18 PR, 1 uPR). Of 21 responders, 4 have progressed to date with median follow up of 7.4 months. Overall disease control was 76%. Improved responses in some patients were observed with longer follow up. Most (54) patients progressed on prior anti-PD1 and those with PD-L1 negative status (TPS < 5%) were among responders. Mean cells infused was 28 x109. Median IL-2 doses administered was 6.0. Adverse events resolved to baseline, 2 weeks post TIL infusion, a potentially important benefit of one-time TIL therapy. Conclusions: Lifileucel treatment results in 38% ORR in heavily pretreated metastatic melanoma patients with high baseline disease burden who received prior anti-PD1 and BRAF/MEK inhibitor if BRAF mutated. Based on these data, a new Cohort 4 in C-144-01 has been initiated to support lifileucel registration. Clinical trial information: NCT02360579.

Safety and efficacy of adoptive cell transfer using autologous tumor infiltrating lymphocytes (LN-145) for treatment of recurrent, metastatic, or persistent cervical carcinoma.

2538 Background: There is a high unmet medical need for effective treatments for patients with recurrent, metastatic, or persistent cervical cancer. Most patients are young and survival rates are poor. ORR for second line therapies is between 4 and 14% for chemotherapy and recently approved immunotherapy. Adoptive cell transfer using tumor infiltrating lymphocytes (TIL) have demonstrated durable responses in some patients with recurrent cervical cancer thus offering the potential for long-term disease control. Methods: Study C-145-04 is an ongoing, open-label, multicenter Phase 2 clinical trial evaluating the safety and efficacy of LN-145 TIL therapy in patients with advanced cervical cancer who have undergone at least one prior line of chemotherapy. Prior checkpoint inhibitor therapy is an exclusion criterion. The primary endpoint is ORR per RECIST 1.1; secondary endpoints include duration of response (DOR), disease control rate (DCR), and LN-145 safety. Tumors surgically harvested at local institutions are shipped to central GMP facilities for TIL generation in a 22-day manufacturing process. Final LN-145 TIL product is cryopreserved and shipped to sites. Patients receive one week of preconditioning lymphodepletion (cyclophosphamide, fludarabine), a single LN-145 infusion, followed by up to 6 doses of IL-2 (600,000 IU/kg). Results: As of 4 Feb 2019, 27 efficacy-c patients have received Gen 2 of LN-145, with a mean age of 47 years and 2.6 mean prior lines of therapy. Preliminary efficacy results: ORR was 44% (1 CR, 9 PR, 2 uPR), DCR was 89% at 3.5-month median study follow-up with 11/12 patients maintaining their response. Improved responses were observed in 4 patients with longer follow-up. Mean TIL cells infused was 28x109. Median IL-2 doses administered was 6.0. The adverse event profile was generally consistent with the underlying advanced disease and the profile of the lymphodepletion and IL-2 regimens. Conclusions: LN-145 results in 44% ORR in previously treated cervical cancer patients with acceptable safety and efficacy profile. LN-145 offers patients a viable therapeutic option warranting further investigation. Clinical trial information: NCT03108495.

Adoptive transfer of tumor-infiltrating lymphocytes in patients with sarcomas, ovarian, and pancreatic cancers.

TPS2650 Background: Adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TIL) has a long history of efficacy in metastatic melanoma, and is being increasingly considered across other solid tumors. Preclinical data generated at MD Anderson Cancer Center has demonstrated the ability to grow TIL from a variety of tumor types including various types of sarcomas, ovarian and pancreas cancers. We are testing the efficacy of TIL across multiple tumor types using two different manufacturing protocols. Methods: We are conducting two ongoing investigator initiated basket TIL therapy trials. The first (NCT03449108) includes cohorts with poorly differentiated soft tissue and bone sarcomas, osteosarcoma, and platinum resistant ovarian cancer. The TIL product used in this trial is an investigational cell product (LN-145, Iovance Biotherapeutics, Inc.). The second trial (NCT03610490) includes cohorts of osteosarcoma, platinum resistant ovarian cancer, and pancreatic cancer (who have progressed on, or received maximal benefit from, front-line therapy). For this trial, TIL are manufactured at MD Anderson Cancer Center using a protocol that includes the use of urelumab (an agonistic anti-CD137 antibody) combined with T cell receptor activation during TIL expansion. In both trials eligible subjects undergo tumor harvest using a surgical excisional biopsy of the tumor for TIL manufacturing, receive a modified cyclophosphamide and fludarabaine lymphodepletion regimen and up to six doses of IL-2 (600,000 IU/kg) following TIL infusion. No intervening therapy is allowed between tumor harvest and initiation of lymphodepletion. The primary endpoint for each cohort is ORR as assessed by investigators using RECIST 1.1 criteria. The Simon’s two stage design is used to monitor the efficacy of each cohort independently. In the first stage, 10 patients will be treated per cohort. If there is no confirmed response in these 10 evaluable patients, the cohort will be terminated. If the cohort moves forward to Stage II, an additional 8 patients will be treated leading to a total of 18 patients. Three or more responders out of 18 treated patients for the cohort will be considered clinically relevant to justify further investigation. Enrollment is ongoing in all cohorts in both trials. An accrual update will be provided at the annual meeting. Clinical trial information: NCT03449108, NCT03610490.

Fc engineered anti-CD33mAb potentiates cytotoxicity of mbIL-21 expanded NK-cells against primary AML pre-treated with decitabine

Background & Aim Natural killer (NK) cells are limited in number and require cytokines for in vivo expansion. Recipient NK cells are shown to have both qualitative and quantitative defects in acute myeloid leukemia (AML) patients, providing rationale for infusion of donor-derived NK cells. We showed that decitabine (DAC) enhances expression of NKG2D ligands in AML blasts and renders the blasts susceptible to killing by NK cells (Vasu et al. 2016). We conducted a phase I trial giving 5-day DAC in relapsed, refractory AML patients followed by haploidentical NK cells on day 0 and six doses of IL-2, where we demonstrated short term engraftment of donor-derived NK cells (up to 48 hours) can be achieved by fludarabine and DAC. Here, we report preclinical studies using membrane bound IL-21 (mbIL-21) expanded NK cells, which can be given without extraneous cytokine support. Methods, Results & Conclusion NK cells from healthy donors were expanded for 14 days using irradiated K562 feeder cells displaying mbIL-21 and 4-1BBL (CSTX002) to derive mbIL-21 NK cells. These cells have minimal senescence despite robust proliferation with the feasibility to infuse multiple NK cell doses of 1 × 108/kg (Denman et al. 2012). We investigated if mbIL-21 NK cells exhibit natural killer activity alone or in the presence of Fc-engineered anti-CD33 antibody [antibody-dependent cellular cytotoxicity (ADCC)] against primary AML samples from patients who received DAC, pre-NK cell infusion (day-1) and at 24 days post haplo-NK infusion (Post-NK infusion). Upon incubation with primary AML blasts, mbIL-21 NK cells displayed an activation profile as evidenced by surface CD107a induction (p=0.0051) and variable donor-dependent intra-cellular IFNγ production, which was further increased with CD33mAb coated AML cells (p=0.0018). ADCC assays using chromium-51 (Cr51) labelled primary AML blasts revealed mbIL-21 NK cells lysed primary AML blasts alone which was significantly enhanced with CD33mAb coated target cells (p=0.0004). Importantly, CD33mAb dependent enhanced-cytotoxicity by mbIL-21 NK cells was maintained in AML cells from patients even 24 days post DAC treatment. Further, in-vivo infusion of mbIL-21 NK cells in AML patient derived xenograft (AML PDX) mice, treated with CD33mAb, reduced the tumor burden. These data show the therapeutic utility of mbIL-21 NK cells that can be further potentiated by addition of Fc engineered anti-CD33 antibody in AML.

In vivo induction of T regulatory cells with recombinant mouse IL-2_anti-mouse IL-2 antibody complex

Abstract T regulatory cells (Tregs) control peripheral tolerance, and their immunosuppressive function is essential to maintain a balanced immune system. Tregs constitutively express IL-2Rα (CD25) and the transcription factor FoxP3. IL-2 is a pleiotropic cytokine and is vital for the development of Tregs. IL-2 binds to the trimeric receptor formed by IL-2Rα, IL-2Rβ (CD122), and the common gamma chain IL-2Rg (CD132). In this study we induced in vivo Treg (CD25bright, FoxP3+, CD4+) expansion by injecting a complex made of recombinant mouse IL-2 and anti-mouse CD25 (clone JES6-1A12). The mice were injected for four consecutive days with different concentrations of IL-2_JES6-1A12 complex, as well as IL-2_JES6-1A12 complex plus an excess of IL-2, or IL-2 alone. There was a significant induction of Tregs in mice that received the complex as compared to animals that received only a comparable dose of IL-2 alone. The ratio Tregs/effector cells (defined as CD8+,CD44+, CD122+) was approximately two fold in mice that received the complex. Mice injected with the complex plus an excess of IL-2 showed similar proportions of Tregs and effector cells. Previous studies showed that the use of a different IL-2 neutralizing clone (clone S4B6, recognizes and block CD122) preferentially induced effector cells. This experimental model will facilitate the study of diseases or immune mechanisms where Tregs mediate an immune response or immune system dysregulation.

Innate lymphoid cells support regulatory T cells in the intestine through interleukin-2.

Interleukin (IL)-2 is a pleiotropic cytokine that is necessary to prevent chronic inflammation in the gastrointestinal tract1–4. The protective effects of IL-2 involve the generation, maintenance and function of regulatory T cells (Tregs)4–8, and low-dose IL-2 has emerged as a potential therapeutic strategy in inflammatory bowel disease (IBD) patients9. However, the cellular and molecular pathways that control the production of IL-2 in the context of intestinal health are undefined. Here we identify that IL-2 is acutely required to maintain Tregs and immunologic homeostasis throughout the gastrointestinal tract. Strikingly, lineage-specific deletion of IL-2 in T cells did not recapitulate these phenotypes in the small intestine. Unbiased analyses revealed that group 3 innate lymphoid cells (ILC3) are the dominant cellular source of IL-2 in the small intestine, which is selectively induced by IL-1β. Macrophages produce IL-1β in the small intestine and activation of this pathway involves MyD88- and Nod2-dependent sensing of the microbiota. Loss-of-function studies defined that ILC3-derived IL-2 is essential to maintain Tregs, immunologic homeostasis and oral tolerance to dietary antigens uniquely in the small intestine. Furthermore, ILC3 production of IL-2 was significantly reduced in the small intestine of Crohn’s disease patients, and this correlated with diminished Tregs. Collectively, these results reveal a previously unappreciated pathway whereby a microbiota- and IL-1β-dependent axis promotes ILC3 production of IL-2 to orchestrate immune regulation in the intestine.

Shp-2 is critical for ERK and metabolic engagement downstream of IL-15 receptor in NK cells

The phosphatase Shp-2 was implicated in NK cell development and functions due to its interaction with NK inhibitory receptors, but its exact role in NK cells is still unclear. Here we show, using mice conditionally deficient for Shp-2 in the NK lineage, that NK cell development and responsiveness are largely unaffected. Instead, we find that Shp-2 serves mainly to enforce NK cell responses to activation by IL-15 and IL-2. Shp-2-deficient NK cells have reduced proliferation and survival when treated with high dose IL-15 or IL-2. Mechanistically, Shp-2 deficiency hampers acute IL-15 stimulation-induced raise in glycolytic and respiration rates, and causes a dramatic defect in ERK activation. Moreover, inhibition of the ERK and mTOR cascades largely phenocopies the defect observed in the absence of Shp-2. Together, our data reveal a critical function of Shp-2 as a molecular nexus bridging acute IL-15 signaling with downstream metabolic burst and NK cell expansion.

The outcome and toxicity profile of checkpoint inhibitor immunotherapy subsequent to high-dose IL-2 in the treatment of metastatic melanoma and renal cell carcinoma.

14 Background: Approved treatments for metastatic melanoma (MM) and metastatic renal cell carcinoma (mRCC) include targeted agents, high dose interleukin-2 (HD IL-2) and checkpoint inhibitors (CI). A subset of responders to HD-IL2 can achieve long term durable remissions (7-10%). Recently, data suggests that durable remissions are possible with CI. Thus, despite increased toxicity, first-line immunotherapy with HD-IL2 is a reasonable consideration in carefully selected patients with clear cell mRCC and MM followed by CI upon relapse. Our study explores the utility and safety of CI subsequent to HD-IL2 in patients (pts) in this population. Methods: We conducted a single institution retrospective analysis of pts with MM or mRCC who received HD-IL2 and subsequent CI from 2008-2017. Pts treated with prior targeted therapy were included. Statistical analysis was performed using Fischer's exact tests, log-rank test for KM analysis and non-parametric Wilcoxon Rank Sum tests to compare the groups. Results: We identified 34 unique pts (19 MM, 15 mRCC) from our pre-specified cohort. Pts were male (73%), Caucasian (88%), and median age=53. mRCC pts received more cancer related treatments than MM prior to CIs after HD-IL2 was given (2 vs. 1, p=0.002), had more total CI cycles administered (12 vs. 7, p=0.10) but less IL-2 doses than MM pts (20 vs. 24, p=0.26). mRCC pts tended to record higher HD-IL2 toxicity grade compared to MM pts (exact =0.04). 26% (9/34) of pts experienced a grade 2 or higher CI toxicity. Pts had higher HD IL-2 toxicities than CI toxicities during therapy but these two measures were not significantly correlated (r=0.07, p=0.73); furthermore, there was no survival difference between pts with reported grade 2 or higher CI toxicity compared to pts without any CI toxicity ( p=0.14). Conclusions: Our study suggests that CI therapy after HD-IL2 is feasible and not associated with more frequent toxicity or less clinical efficacy in pts with MM or mRCC.

IRGD in combination with IL-2 reprograms tumor immunosuppression.

55 Background: Fibrotic solid tumors have lagged in immunotherapy efficacy. Although breast cancer (BC) shows modest single-agent activity, pancreatic ductal adenocarcinoma (PDAC) immunotherapy has repeatedly failed in clinical trials. A protective desmoplastic, inflammatory reaction encapsulates BC and PDAC where it can make up to 80% of the tumor microenvironment (TME). Reports in BC and PDAC patients and mouse models suggest that tumoricidal effector CD8+ T-cells are indeed present, yet suppressed by regulatory CD4+/CD25+/FoxP3+ T-cells (Tregs) and myeloid cells. Long-term PDAC and BC survivors harbor CD8+ T-cells by immunohistochemistry (IHC), and although inactivated (CD107a-), they are not terminally exhausted (PD-1low). Further, when CD8+ T-cells were found in large quantities within a treatment-naïve biopsy, that patient had an increased overall survival. We hypothesize that low doses of the T-cell proliferative cytokine IL-2 can class switch the TME T-cells if specifically concentrated within the tumor by the stroma-penetrating peptide iRGD. Methods: Subcutaneous BC tumors (4T1) and PDAC (KPC ) tumors were formed in immunocompetent mouse models. Mice were treated with either vehicle, iRGD, IL-2, or both in combination. Tumors were preserved for IHC or enzymatically digested for FACS. CD45+ live cells were fluorescently labeled for effector T-cells (CD4+/CD44+ or CD8+/CD44+), Tregs (CD4+/CD25+/FoxP3+), or cytotoxic T-cells (CD8+/CD44+/Granzyme B+/IL-2+). Results: Versus normal controls, tumors from BC and PDAC both showed a subsequent increase in bulk CD3+ T-cells within the tumor microenvironment (14.3% ± 5 vs. 27.6% ± 8). A significant increase in CD3+ T-cells within all tumors (10%) occured with iRGD only, IL-2 only, or iRGD + IL-2 treatment. Whereas sub-populations of T-cells in the iRGD only and IL-2 only treatment groups was overwhelming composed of Tregs (5%) at CD4/Treg and CD8/Treg ratios of 1.75 and 0.5 respectively, the combination of iRGD + IL-2 shifted the T-cell sub-populations away from Tregs (1.5%) and towards increased CD4/Treg and CD8/Treg ratios (4 and 12 respectively). Conclusions: The combination of iRGD + IL-2 is capable of reprogramming the immunosuppressive Treg tumor microenvironment, increasing effector CD4+ and CD8+ T-cells.

Safety and efficacy of cryopreserved autologous tumor infiltrating lymphocyte therapy (LN-144, lifileucel) in advanced metastatic melanoma patients previously treated with at least one prior systemic therapy.

136 Background: While immunotherapies including checkpoint inhibitors and targeted therapies (BRAF/MEK inhibitors) are options for patients with metastatic melanoma, many patients still develop progressive disease. These patients have few treatment options available including high dose IL-2 and chemotherapy with reported second line response rates of 4-10%. Adoptive cell therapy utilizing tumor-infiltrating lymphocytes (TIL) is recognized as an effective treatment in metastatic melanoma, able to elicit durable and complete responses in even heavily pretreated patients. We provide preliminary data for lifileucel TIL (LN-144) in heavily pre-treated metastatic melanoma patients who progressed on multiple checkpoint and BRAF/MEK inhibitors. Methods: C-144-01 is an ongoing global Phase 2, open-label, multicenter study of efficacy and safety of lifileucel in patients with unresectable metastatic melanoma. We report on Cohort 2 (N = 47) patients who received cryopreserved lifileucel. Tumors resected at local institutions were processed at central GMP facilities in a 22-day manufacturing process. The final product was cryopreserved and shipped to sites. Patients received a one week cyclophosphamide/fludarabine preconditioning lymphodepletion regimen, a single lifileucel infusion, followed by up to 6 doses of iv IL-2 (600,000 IU/kg). Results: Patients with Stage IIIC/IV melanoma had 3.3 mean prior therapies (range: 1-9) and high baseline tumor burden, reflected by a mean sum of diameters of target lesions of 112 mm. Preliminary efficacy results: ORR = 38% (1 CR, 13 PR, 4 uPR), DCR = 77%, and median DOR 6.4 mo (range: 1.3 to 13.7) with median follow-up 6.0 mo. Longer follow-up led to improved responses in some patients including the CR. Frequency of AEs decreased over time, a potentially important benefit of one-time TIL treatment. Conclusions: Preliminary data support lifileucel TIL as an efficacious and well-tolerated therapeutic option for patients with metastatic melanoma who have failed multiple lines of prior therapies including checkpoint inhibitors and BRAF/MEK inhibitors. Clinical trial information: NCT02360579.