High-dose IL-2 Research Articles

Abstract 3518: AB248 is a CD8+ T cell selective IL-2 designed for superior safety and anti-tumor efficacy

Abstract High dose IL-2 was the first modern immunotherapy to show complete responses in a subset of cancer patients. The liabilities of IL-2 prompted development of second-generation molecules, which signal through the IL-2Rβγ (“not-α” IL-2 and IL-15 variants). Although these molecules avoid vascular leak syndrome, their clinical efficacy appears to be suboptimal compared to high dose IL-2 [1]. Furthermore, not-α IL-2s show biased expansion of NK cells over CD8+ T cells in patients due to their high expression of IL-2Rβ on NK cells, which can act as a sink and contribute to toxicity, and these molecules do not eliminate IL-2Rβγ-mediated activation of Tregs [2, 3]. To maximize the therapeutic potential of IL-2, we developed AB248, a cis-targeted IL-2 fusion protein that selectively signals on CD8+ T-cells with limited activity on the highly IL-2/IL-15-sensitive NK cells and immunosuppressive Tregs. We have previously shown that selective expansion of CD8+ T cells over NK cells and Tregs is achieved in vivo using muAB248, AB248’s murine surrogate. Furthermore, a single dose of muAB248 elicits strong efficacy in multiple tumor models without body weight loss. Here, we detail the properties of AB248, a fusion of a human CD8-targeting antibody that selectively binds to CD8+ T cells over NK cells, and an IL-2 mutein with reduced affinity to IL-2Rα and IL-2Rβ. In STAT5 assays, AB248 showed approximately 1000-fold preference for the activation of CD8+ T cells over NK cells and Tregs. AB248 mediated a dose-dependent selective expansion of CD8 T cells over NK and Tregs in non-human primates. Furthermore, AB248 reproduced the expected in vitro gene signatures of an IL2Rβγ agonist, demonstrating that AB248 recapitulates native IL-2Rβγ signaling selectively on CD8+ T cells.We previously demonstrated in mice that while the efficacy of not-α IL-2 was mediated by CD8+ T cells and not NK cells, toxicity as measured by body weight loss was dependent upon NK cells but not CD8+ T cells. Here, we show that human NK cells may also drive IL-2βγ agonist-induced toxicities. Both IL-2 and not-α IL-2 induced IFN-γ secretion from human PBMCs, whereas AB248 did not. Strikingly, depletion of CD56+ NK cells eliminated IL-2-induced cytokine secretion, demonstrating that human NK cells are capable of spontaneously secreting IFN-γ in response to IL-2 signaling. In the context of TCR stimulation, AB248 induced robust secretion of effector cytokines from CD8+ T cells, but no cytokine secretion was seen in absence of TCR activation. Our data suggest that while AB248 can induce antigen-independent expansion of CD8+ T cells, it only induces effector cytokine secretion in the context of an additional activating signal via the TCR. Collectively, these data show that AB248 demonstrates an improved safety and efficacy profile as compared to not-α IL-2 and is a promising immuno-oncology therapeutic. Ref: 1. Overwijk, Ann. Rev. Med., 2020; 2. Janku, AACR, 2021; 3. Italiano, JCO, 2021 Citation Format: Kelly D. Moynihan, Danielle Pappas, Terrence Park, Wei Chen, Irene Ni, Paul Bessette, Henry Nguyen, David Liu, Mike Chin, Ruth Lan, Aaron Arvey, Ton N. Schumacher, Yik Andy Yeung, Ivana Djuretic. AB248 is a CD8+ T cell selective IL-2 designed for superior safety and anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3518.

Abstract 561: CAR-targeted IL-2 drives selective CAR-T cell expansion and improves anti-tumor efficacy

Abstract Chimeric antigen receptor T cell (CAR-T) therapies have transformed the treatment of some hematological malignancies and are showing promising preliminary results in solid tumors. Recent studies have shown that in vivo expansion and persistence of CAR-Ts are correlated with improved therapeutic outcomes in patients. Administration of IL-2 enhances CAR-T engraftment, persistence, and functionality in preclinical models. However, the clinical potential of IL-2 stimulation with cell therapies is limited using current molecules due to severe toxicity of high-dose IL-2 and the inadequate selectivity of existing engineered IL-2 variants which expand multiple endogenous cells in addition to CAR-T cells. To address this challenge, we have applied our cis-targeting technology to develop CAR-T selective IL-2 fusion molecules that specifically activate CAR-Ts, while exhibiting minimal activity on CAR-negative cells. Cis-targeted IL-2 fusions are comprised of a fusion between a targeting antibody and an IL-2 mutein having attenuated binding to IL2R⍺ and IL2Rβ. The attenuated cytokine selectively binds and activates IL-2 receptors on CAR-Ts via the avidity provided by the targeting arm. We engineered two cis-targeted CAR-T selective IL-2 fusions, CAR-IL2 or EGFRt-IL2. CAR-IL2 targets the FMC63 CAR directly without blocking CD19 antigen recognition, enabling targeting of approved anti-CD19 CAR-T products, and EGFRt-IL2 targets the EGFRt tag co-expressed with the CAR. Molecules were characterized in vitro using primary human CAR-transduced T cells. The in vivo activity was examined using tumor bearing NSG mice infused with human CAR-Ts. The specificity of the CAR-IL2 or EGFRt-IL2 molecules was demonstrated by their ability to selectively induce pSTAT5 signaling resulting in >1000-fold preferential STAT5 activity in CAR-expressing cells compared to CAR-negative cells. The ability of the fusion proteins to selectively expand CAR-Ts in vivo was shown through a substantial and specific expansion of the infused CAR-Ts. The CAR-T fraction increased from approximately 50% of infused T cells, to over 93% of T cells, demonstrating a nearly 40-fold expansion in vivo. Re-expansion of CAR-Ts was demonstrated after allowing the CAR-Ts to rest in vivo >100 days; re-dosing resulted in significant and selective re-expansion of CAR-T cells. Furthermore, treatment with the CAR-IL2 molecule enhanced tumor regression of CD19+ lymphoma-bearing mice infused with suboptimal doses of CAR-Ts. Together, these data demonstrate that our cis-targeted IL-2 molecules selectively activate CAR-Ts in vitro and enhance CAR-T expansion and anti-tumor efficacy in vivo. Cis-targeted IL-2 fusion molecules directed by anti-CAR or anti-tag antibodies represent a promising approach to confer enhanced CAR-T activity in a specific and temporally controlled manner. Citation Format: Nathan D. Mathewson, Kelly D. Moynihan, Sara Sleiman, Wei Chen, Paul Bessette, Christopher Kimberlin, Eric Wigton, Danielle Pappas, Terrence Park, Ton N. Schumacher, Saar I. Gill, Yik Andy Yeung, Ivana Djuretic. CAR-targeted IL-2 drives selective CAR-T cell expansion and improves anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 561.

Abstract 5581: A genetically modified tumor-infiltrating lymphocytes product (GT202) exhibits enhanced persistence and anti-tumor efficacy against solid tumor in vivo

Abstract Adoptive cell therapy by tumor-infiltrating lymphocytes (TILs) has demonstrated significant clinical benefits for patients with solid tumors. However, the toxicities related to high-dose IL-2 that is required to support TIL survival and the exhaustion status of the final TIL product after rapid expansion in culture largely limits the clinical application and long-term efficacy of TIL therapy. Grit Biotechnology designed a genetically engineered TIL product (GT202) to express a membrane-bound cytokine that is critical for T cell activation and effector function, leading to enhanced cytotoxicity as well as persistence in vitro and in vivo. Multiple designs of the transmembrane domain of GT202 were compared and the best design was selected based on stable and efficient expression of GT202 on the membrane of TILs. GT202 demonstrated significantly increased cytotoxicity and cytokine production when cocultured with tumor cells in vitro than conventional TILs and better tumor control ability during a serial killing assay. In the meanwhile, GT202 TILs showed enhanced long-term survival and better preservation of TCR clonality without IL-2 in vitro, as well as persisted longer in multiple tissues without IL-2 over conventional TILs + IL-2 in NGS mice. Notably, GT202 TILs achieved better anti-tumor efficacy with low IL-2 in NSG mice inoculated with HLA-matched tumor cells than conventional TILs, corelated with their better infiltration and proliferation in spleen and tumor. In summary, GT202 demonstrated enhanced anti-tumor efficacy and persistence as well as decreased IL-2 dependency in vitro and in vivo. Currently, an investigator-initiated trial of GT202 is ongoing in China. Citation Format: Jingwei Sun, Jiahui Jin, Yongchao Tan, Lingyun Chen, Quanwei Wang, Yarong Liu. A genetically modified tumor-infiltrating lymphocytes product (GT202) exhibits enhanced persistence and anti-tumor efficacy against solid tumor in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5581.

DELTA-1: A global, multicenter, phase 2 study of ITIL-168, an unrestricted autologous tumor-infiltrating lymphocyte (TIL) cell therapy, in adult patients with advanced cutaneous melanoma.

TPS9594 Background: Patients (pts) with advanced (unresectable or metastatic) cutaneous melanoma and persistent disease after checkpoint inhibitor therapy have poor outcomes and limited treatment options, highlighting a significant unmet medical need (Schadendorf D et al. Lancet. 2018;392:971-984). Investigational autologous TIL cell therapies have shown promise in this population, partly attributable to their intrinsic and patient-specific antitumor activity (Borch TH et al. J Immunother Cancer. 2020;8:e000668). Made from each patient’s digested and cryopreserved tumor, ITIL-168 is an autologous TIL cell therapy manufactured to offer an unrestricted T-cell receptor repertoire. A single-center, compassionate use clinical series demonstrated the feasibility and clinical utility of an earlier version of ITIL-168, with a high overall response rate among pts previously treated with PD-1 inhibitor (PD-1i) therapy (58%, n = 12; Pillai M et al. Ann Oncol. 2021;32[suppl 5]:S882). DELTA-1 (NCT05050006) is a global, multicenter phase 2 study to evaluate efficacy and safety of ITIL-168 in pts with cutaneous melanoma relapsed or refractory to a PD-1i, pts intolerant to a PD-1i, and pts whose current best response to a PD-1i is stable disease. Methods: Pts aged ≥18 years with histologically confirmed advanced cutaneous melanoma, ECOG performance status 0-1, and adequate organ function will be enrolled in 1 of 3 cohorts. Cohort 1 (n≈80) will include pts who relapsed after or were refractory to ≥1 prior line of systemic therapy, including a PD-1i and, if BRAF-mutated, a BRAFi ± MEKi. Cohorts 2 and 3 (n≈25 each) will include pts intolerant to PD-1i and those with stable disease after ≥4 doses of PD-1i, respectively. After tumor resection for TIL harvest, pts must have ≥1 remaining measurable lesion per RECIST 1.1. Pts with uveal, acral, or mucosal melanoma, prior allogeneic transplant or cell therapy, and with central nervous system (CNS) disorder or symptomatic and/or untreated CNS metastases are ineligible. Pts will receive 5 days of lymphodepleting chemotherapy (cyclophosphamide ×2 days overlapping with fludarabine ×5 days) followed by a single ITIL-168 infusion (≥5×109 cells) and supportive short-course, high-dose IL-2. The primary endpoint is objective response rate (ORR) per central review. Secondary endpoints include duration of response, progression-free survival, overall survival, disease control rate, TIL persistence, and safety. Hypothesis testing of ORR will be performed for cohort 1. The primary analysis will occur when all pts in the cohort 1 modified intent-to-treat population have been followed for ≥6 months after the first posttreatment disease assessment. DELTA-1 opened for enrollment in September 2021. Updated site information will be given at the time of presentation. Clinical trial information: NCT05050006.

Detailed immunogenomic analysis of high dose IL-2 pharmacodynamic effects: A benchmark for next-generation IL-2-based immunotherapies.

e16501 Background: High-dose interleukin-2 (HD IL-2) was the first approved immune-oncology (IO) agent based on proven clinical efficacy in renal cell carcinoma (RCC) and metastatic melanoma, but its use was limited due to significant toxicities. Multiple next-generation IL-2 agents designed to retain efficacy while improving tolerability are in development. Yet, understanding of genomic markers that define optimized target pharmacology is incomplete. Newer evaluation techniques not available at the time of previous HD IL-2 experience may allow for identification of pertinent genomic markers. In this retrospective study, we report for the first time, RNA sequencing (RNAseq) of peripheral blood mononuclear cell (PBMC) samples from metastatic (mRCC) patients before, during and after HD IL-2 treatment. Detailed immunogenomic responses to HD IL-2 treatment with insight into traditional flow cytometry (FC) are presented. Methods: PBMC samples were collected for n = 23 HD IL-2 treated mRCC patients between 2009 and 2015 on Day 1, 3 and 5. Patient samples underwent prior FC analysis described elsewhere (Foureau et al, 2014; Cancer Immunol. Immunother. 63(12)). RNAseq was performed using NovaSeq6000 (Illumina) paired end sequencing on bulk PBMC samples from immediately before (Day 1), during (Day 3) and post-treatment (Day 5) Cycle 1 and/or Cycle 2 of the first course of HD IL-2. Individual genes and signatures for immune and proliferation differences were analyzed. Results: RNAseq analysis of PBMC samples demonstrated that CD8+ T cells transiently decreased in the blood during treatment but recovered to baseline on Day 5, and CD4 Treg cells increased on Day 3 and Day 5, which confirmed prior FC findings. TCR clonality was unchanged which indicates that T cell recovery and expansion is widespread and not driven by a specific subset of T cells. Additional notable insights included increased monocytes at Day 3, with the opposite for NK and B cells (Day 3 decrease and Day 5 expansion). Using a novel GeneCentric proliferation signature, increased expression was noted on Day 3 and Day 5 vs. Day 1, reflecting pronounced IL-2 induced proliferation. Conclusions: Immunogenomics provided further detail regarding IL-2 activity in addition to recapitulating several parameters from FC. Expression analysis of minimally invasive PBMC samples demonstrates the importance of this novel genomic approach to understand the pharmacology of IL-2 and related derivatives.

Efficacy and safety of autologous expanded tumor infiltrating lymphocytes (TILs) in multiple solid tumors.

2536 Background: TIL therapy has been used extensively in metastatic melanoma patients for many years, now with ongoing efforts to commercialize treatment. The efficacy of TIL outside of melanoma is largely unknown thus we designed and implemented a trial using TIL manufactured at a single academic center for treatment refractory metastatic colorectal (CRC), pancreas (PDAC) and ovarian (OVA) cancers. Methods: Patients with CRC, PDAC and OVA refractory to standard therapies with ECOG PS 0-1 and normal organ function were eligible for TIL harvest. Ex vivo TIL expansion and manufacturing was conducted at the MD Anderson TIL lab under conditions that included IL2 and 41BB stimulation (using urelumab). All patients received a lymphodepletion regimen consisting of cyclophosphamide 60mg/kg days -7 and -6 and fludarabine 25mg/m2 days -5 through day -1, followed by infusion of pooled ex-vivo expanded TIL. Patients received up to 6 doses of high dose IL-2 (600,000 IU/kg) after TIL infusion. The primary endpoint was evaluation of the objective response rate (ORR) using RECIST 1.1 criteria with secondary endpoints including disease control rate, duration of response, PFS, OS and safety. Results: A total of 17 patients underwent TIL harvest and 16 were treated on protocol; including 8 CRC, 5 PDAC and 3 OVA. Median age was 57.5 (range 33-70) and 50% were females. Median number of lines of prior therapy was 2 (range 1-8). Median number of TIL infused was 76 X 109 (range 20.3 x 109-150 x 109). Median doses of cyclophosphamide and fludarabine administered were 2 (range, 2-2) and 3 (range, 1-5), respectively. Median doses of IL-2 administered was 6 (range, 1-6). There were no responders. Best response included prolonged SD in a patient with PDAC lasting until 18 months. Grade 3 or higher toxicities attributable to therapy was seen in 14 subjects (87.5%; 95% CI: 61.7 – 98.4) with the majority of toxicities representing expected pancytopenia from lymphodepletion. Infusion product analysis showed the presence of effector memory cells with high expression of CD39 irrespective of tumor type. Early on-treatment biopsy of PDAC patient with prolonged SD showed presence of proliferating (KI67+) CD4+ and CD8+ TIL. Conclusions: Generation of TIL at a single academic center for CRC, PDAC and OVA is feasible and treatment is associated with no new safety signals. For these tumor types, further research is required to identify host factors associated with resistance to TIL therapy and optimize manufacturing processes to create more effective TIL cell therapy. Clinical trial information: NCT03610490.

Antitumor activity of T cells expressing a novel anti-folate receptor alpha (FOLR1) costimulatory antigen receptor (CoStAR) in a human xenograft murine solid tumor model and implications for in-human studies.

2535 Background: ITIL-306 is an autologous tumor-infiltrating lymphocyte (TIL) therapy that integrates T-cell receptor (TCR)-specific antigen recognition (Signal 1) with robust costimulation via the novel CoStAR transgene upon engagement with FOLR1 (Signal 2; Sukumaran, et al. JITC. 2021;9:198). Here, we assessed IL-2 independent effector function by anti-FOLR1 CoStAR T cells in vitro and evaluated activity in a novel, more representative murine solid tumor model. Methods: For in vitro studies, healthy donor T cells were manufactured to express anti-FOLR1 CoStAR or left nontransduced (NTD). Product cells were stimulated with a single (Day [D]0; ±IL-2) or serial (D0, 7, 14, 21; no IL-2) addition of target cells expressing membrane-anchored OKT3 (Signal 1) and FOLR1 (Signal 2). T-cell activation and proliferation were measured. For in vivo studies, the carcinoembryonic antigen (CEA)-positive (Signal 1) H508 cell line was engineered to express FOLR1 (Signal 2) and injected into 6 mice/group (D–21). NTD or healthy donor T cells expressing HLA-A*02-restricted anti-CEA TCR only, anti-FOLR1 CoStAR only, or dual TCR+CoStAR were given intravenously (D0), and compared with PBS control. Tumor growth, survival, and T-cell expansion were assessed in 2 donors to D96. Results: After single and serial stimulation, only anti-FOLR1 CoStAR T cells showed sustained proliferation without exogenous IL-2. PD-1 positivity was low ( < 10%) on anti-FOLR1 T cells up to D24. In vivo, T-cell persistence on D14 was increased in dual TCR+CoStAR vs anti-CEA TCR only mice ( P<.001) and all other groups ( P<.01-.001). Tumor growth up to D58 was significantly lower in dual TCR+CoStAR ( <.5 cm3) vs all other groups (each > 2 cm3; P<.0001). Survival was significantly longer in dual TCR+CoStAR ( P<.01) vs all other groups; only dual TCR+CoStAR mice were alive at D58, with 5/6 alive at D96. The second donor showed similar tumor control irrespective of exogenous IL-2 in responder mice, with > 50% alive at D96, and robust T-cell expansion in dual TCR+CoStAR mice. Conclusions: When combined with TCR-specific binding, CoStAR significantly enhanced T-cell proliferation, persistence, and antitumor activity in vivo vs TCR alone, resulting in tumor control and prolonged survival. Effects were not observed with CoStAR alone, underscoring that signaling through CoStAR alone does not induce T-cell effector function. The sustained proliferation of anti-FOLR1 CoStAR T cells without exogenous IL-2 support in vitro and in vivo supports a clinical TIL regimen free of high-dose IL-2. These results suggest that CoStAR may improve the clinical performance and benefit/risk profile of TILs, whereby fewer toxicities are expected with the removal of post-TIL IL-2 support. This will be explored in an upcoming first-in-human clinical study with ITIL-306.

A phase 1 adoptive cell therapy using drug-enhanced, tumor-infiltrating lymphocytes, DeTIL-0255, in adults with advanced malignancies.

TPS5602 Background: Tumor-infiltrating lymphocytes (TIL) are a heterogeneous population of T cells that recognize multiple endogenous tumor antigens but may have developed an exhausted phenotype due to the tumor microenvironment. While existing TIL therapies produce durable responses in patients with melanoma, cervical, and head and neck cancers, poor in vitro cell expansion, limited short-lived in vivo persistence, and diminishing potency restrict this approach’s broader application. DeTIL-0255 (drug-enhanced TIL [DeTIL]) is an autologous adoptive cell therapy (ACT) derived from a patient’s tumor and expanded ex vivo with NX-0255, a small-molecule inhibitor of the E3 ligase, Casitas B-lineage lymphoma proto-oncogene B (CBL-B). CBL-B is expressed in T cells, where it functions as a regulator of immune cell activation, in part by requiring CD28 co-stimulation in addition to T-cell receptor activation. Desirable properties enhanced by DeTIL-0255 when compared with TIL include increased number of stem-like CD39-/CD69-, and CD8+ T cells associated with persistence, as well as enhanced cytolytic function. ACT with T cells expanded ex vivo using NX-0255 demonstrated increased anti-tumor activity, longer survival, increased stem-like phenotype, and persistence of tumor antigen-specific T cells in mouse tumor models. Adoptive cell transfer of DeTIL-0255, therefore, may exhibit broader functional activity than conventional TIL, potentially conferring improved anti-tumor activity and response. Methods: NX-DeTIL-0255-201 is a Phase 1 multicenter, open-label study of DeTIL-0255 administered with systemic high-dose IL-2 following nonmyeloablative lymphodepleting chemotherapy in patients with advanced gynecological malignancies for whom standard therapy with proven clinical benefit does not exist, is no longer effective, or is inappropriate. The primary objectives are to evaluate the safety, tolerability, and preliminary antitumor activity of DeTIL-0255. A safety run-in will consist of 3 to 6 patients treated with DeTIL-0255 and evaluated for dose-limiting toxicity (DLT). The DLT period starts with DeTIL-0255 infusion and ends after a total of 28 days. The safety run-in will investigate DeTIL-0255 at a dose range of 1 to 150 x 109 CD3+ T cells (exact dose varying based on expansion potential of DeTIL-0255 from tumor biopsies). Following the safety run-in, cohort expansion will further evaluate the safety and antitumor activity of DeTIL-0255 in patients with recurrent/persistent platinum-resistant epithelial ovarian cancer, cervical carcinoma, and endometrial cancer. Key eligibility criteria include measurable disease, a resectable lesion for TIL harvest, ≥2 prior lines of therapy, and an Eastern Cooperative Oncology Group performance status 0 or 1. The study is expected to enroll ̃54 patients in ̃10 sites across the United States. Clinical trial information: NCT05107739.

Low Dose Interleukin-2 Ameliorates Sjögren's Syndrome in a Murine Model.

Sjögren’s syndrome (SS) is a systemic autoimmune disease with no efficient treatment, and it is associated with dysregulated immune cells and impaired interleukin (IL)-2 signaling. IL-2 is critical for the development and maintenance of Treg cells. The use of low dose of IL-2 (LDIL-2) in the treatment of autoimmune diseases is promising, but the efficacy and mechanism in SS therapy are still to be confirmed. This study aims to investigate the therapeutic effect of LDIL-2 on SS in NOD (non-obese diabetic) mice. NOD mice (female, 8 weeks old) were randomly assigned into three groups (n = 8). Low dose of IL-2 (LDIL-2), high dose of IL-2 (HDIL-2), and isometric sterile water (control) were administered subcutaneously daily from week 8 to week 16. LDIL-2 administration significantly recovered the reduction in saliva flow and suppressed lymphocyte inflammation of the submandibular glands (SMGs) when compared with those treated with sterile water as controls (p < 0.05). SS related biomarkers including ANA, Anti-SSA/Ro, and Anti-SSB/La also declined (p < 0.05). In the low dose of IL-2 treated group, the proportion of CD4+CD25+Foxp3+Tregs in both spleen and cervical-lymph-node were higher than control mice (p < 0.05). Furthermore, CD4+Bcl-6+PD-1+CXCR5+Tfh cells, CD4+IFN-γ+Th1 cells, and CD4+IL-17A+Th17 cells were significantly reduced in LDIL-2 group (p < 0.05). Analysis of the SMGs biopsies showed significantly decreased inflammation scores after LDIL-2 administration and an increase of Tregs with immunohistochemical staining. Our findings provide in vivo evidence that LDIL-2 was an effective therapeutic intervention for SS observed in NOD mice and may restore immune balance through the promotion of Treg and suppression of germinal center (GC) B cells and effector T cells.

Human IL-2Rɑ subunit binding modulation of IL-2 through a decline in electrostatic interactions: A computational and experimental approach.

Although high-dose IL-2 has clear antitumor effects, severe side effects like severe toxicity and activation of Tregs by binding of IL-2 to high-affinity IL-2R, hypotension, and vascular leak syndrome limit its applications as a therapeutic antitumor agent. Here in this study, a rational computational approach was employed to develop and design novel triple-mutant IL-2 variants with the aim of improving IL-2-based immunotherapy. The affinity of the mutants towards IL-2Rα was further computed with the aid of molecular dynamic simulations and umbrella sampling techniques and the obtained results were compared to those of wild-type IL-2. In vitro experiments by flow cytometry showed that the anti-CD25 mAb was able to bind to PBMC cells even after mutant 2 preincubation, however, the binding strength of the mutant to α-subunit was less than of wtIL-2. Additionally, reduction of IL-2Rα subunit affinity did not significantly disturb IL-2/IL2Rβγc subunits interactions.

TIL expansion with high dose IL-2 or low dose IL-2 with anti-CD3/anti-CD28 stimulation provides different quality of TIL-expanded T cell clones

Tumor infiltrating lymphocytes (TILs) are cells that are present inside the tumor environment, of which include T cells, B cells and natural killer (NK) cells. At present, TILs are used for immunotherapy in various cancers. Knowledge on adoptive transfer of TILs in ovarian cancer is still limited, especially regarding TIL expansion methods. Therefore, the aim of our study was to compare the quality of T cell clones between two expansion methods for ovarian cancer TILs. We show that TILs stimulated with the mitogenic stimulation method (low dose IL-2 with anti-human CD3/CD28) and the standard stimulation method (high dose IL-2 only) both increased total number of T cells. TCR repertoire analyses revealed different TCR repertoire patterns between TIL-expanded T cells that were stimulated with the standard stimulation method (high dose IL-2 only) and the mitogenic stimulation method (low dose IL-2 with anti-human CD3/CD28). Regardless, when TILs were expanded using the standard stimulation method (high dose IL-2 only), the predominant T cell receptor beta variable (TRBV) chains that were used in both TIL-expanded clones of the CD4+ and CD8+ subpopulations were similar. In addition, there were also TIL-expanded CD4+ and CD8+ T cell clones that were dominant in only one or the other subpopulations. These results reveal the bias in TIL quality after being stimulated with different protocols. Further studies are required to understand the selection of TIL expansion, in order for a more efficacy adoptive transfer treatment.

Targeting Ovarian Cancer with IL-2 Cytokine/Antibody Complexes: A Summary and Recent Advances.

Interleukin (IL)-2 was first identified as a potent T cell growth factor in 1976 [1] and cloned in 1983 [2]. In the late 1990s, IL-2 gained further attention as the first immunotherapy demonstrating clinical efficacy against metastatic cancer, as high dose IL-2 was approved by the United States Food & Drug Administration (FDA) for the treatment of metastatic renal cell carcinoma in 1992 and metastatic melanoma in 1998.

Abstract LB150: Clinical Feasibility and Treatment Outcomes with Unselected Autologous Tumor Infiltrating Lymphocyte Therapy in Patients with Advanced Cutaneous Melanoma

Abstract Introduction: A minority of patients (pts) with advanced melanoma achieve long-term survival with immunotherapy and those who relapse following immune checkpoint inhibition (ICI) or BRAFi have limited treatment options. The intrinsic anti-tumor activity and broad neoantigen-specific TCR repertoire of unselected autologous TIL may provide advantages over other treatments in solid tumors, including ICI-refractory melanoma. Methods: This is a retrospective analysis of a single-center experience of TIL for treatment of advanced cutaneous melanoma. Unselected autologous young TIL derived from digested tumor tissue were manufactured under a MHRA Manufacturing Specials license. Pts with advanced cutaneous melanoma and no other treatment options received non-myeloablative lymphodepleting chemotherapy (cyclophosphamide 60 mg/kg/d x 2d, fludarabine 25 mg/m2/d x 5 d [Cy/Flu]), followed by TIL infusion and post-TIL high-dose (HD) IL-2 (600,000-720,000 IU/Kg) on a compassionate use basis. Efficacy for 15 imaging-evaluable pts was reported by investigator assessment of CT/MRI per RECIST 1.1; 6 additional pts were followed using non-RECIST 1.1 imaging (PET) and clinical monitoring. Clinically significant adverse events (AEs) with onset post-TIL infusion were reported for all treated pts. Data cutoff date: 31-DEC-2019. Results: Between OCT-2011 and AUG-2019, 21 pts with advanced cutaneous melanoma were treated with Cy/Flu, TIL (median 31.9 x109 cells infused), and HD IL-2 (median 8 doses). All had high-risk metastatic disease (median 4 sites, stage IV M1d [38%]), an average of 3 prior therapies (any ICI [91%], PD-1 [57%], BRAFi [52%], and MEKi [24%]). With a median follow up of 52.2 mos, the response rate in imaging-evaluable pts (n=15) was 53% with a CR rate of 13%; DCR was 73% including 3 (20%) pts with stable disease. Additional durable responses were observed in the 6 pts followed by PET and clinical monitoring. Responses were generally consistent across subgroups including age, number of disease sites, tumor burden, brain metastases, number of prior lines of therapies, prior PD-1 blockade, prior BRAFi, and prior MEKi. For all treated pts, the median survival was 21.3 mos. Toxicity was generally self-limited and consistent with Cy/Flu and HD IL-2. Common AEs (≥20%, any grade) were thrombocytopenia (62%), pyrexia (57%), rigors (43%), neutropenia (29%), tachycardia (29%), pulmonary edema (24%), and vascular leak (24%); no treatment-related deaths were observed. Conclusion: The high response rate observed in this series exceeds the 41% ORR estimated for TIL in advanced cutaneous melanoma (meta-analysis by Dafni et al. Ann Oncol., 2019) and highlights the successful bench-to-bedside application of unselected autologous TIL to address unmet medical need in advanced melanoma. Use of tumor digests as starting material for manufacturing of TIL demonstrates feasibility of this approach. A multicenter Phase 2 trial of this therapy in advanced melanoma is planned for 2021. Citation Format: Robert E. Hawkins, Yizhou Jiang, Paul C. Lorigan, Fiona C. Thistlethwaite, Manon Pillai, Martine Thomas, Natalia Kirilova, John S. Bridgeman, Gray Kueberuwa, Ryan S. Guest, Zachary J. Roberts. Clinical Feasibility and Treatment Outcomes with Unselected Autologous Tumor Infiltrating Lymphocyte Therapy in Patients with Advanced Cutaneous Melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB150.

Abstract 1512: OrthoCARs: Engineered human IL-2/IL-2Rb orthogonal pairs selectively enhance CAR T cell antitumor efficacy by driving T cell expansion and fitness

Abstract CAR T cell therapy (CAR) has demonstrated remarkable clinical efficacy in hematological malignancies. However, barriers such as poor T cell effector function, lack of proliferation, and limited persistence prevent CARs from reaching their full curative potential. IL-2 is a potent stimulator of T cell proliferation, survival, and cytotoxic function, making it an attractive cytokine to support CARs. However, therapeutic use of IL-2 is limited by systemic toxicity due its promiscuous activation of undesired immune cell populations. To facilitate selective expansion and activation of CARs we have developed a human orthogonal ligand/receptor system consisting of a pegylated IL-2 mutein (STK-009) that does not significantly activate the wild type IL-2 receptor and a mutated IL-2 Receptor Beta (hoRb) that is fully activated by STK-009, but does not respond to the native IL-2 ligand. This system enables in vivo IL-2 signaling in CARs engineered to express hoRb while avoiding signaling bystander T cells and NK cells. Here, we demonstrate the ability of the STK-009/hoRb receptor pair to selectively enhance the anti-tumor efficacy of hoRb expressing CD19 CARs (SYNCAR-001) in preclinical lymphoma mouse models at will. We also demonstrate, in non-human primates (NHP), that STK-009 is selective for hoRb expressing cells. SYNCAR-001 + STK-009 can lead to complete responses in subcutaneous Raji mouse models, even with SYNCAR-001 dosed at sub-efficacious levels (400,000 CAR-Ts/mouse). Subcutaneous dosing of STK-009 expands SYNCAR-001 systemically and drives infiltration of SYNCAR-001 into tumors. UMAP analysis of STK-009 treated SYNCAR-001 reveal a small PD1+LAG3+ subpopulation during tumor rejection which disappeared once tumors are controlled. The majority of T cells showed no exhaustion markers. Further, an IL-7R+ population arises and remains after tumor control, indicating long term memory development. Nanostring analysis confirms upregulation of IL-7R and other critical genes involved in cytotoxic activity and persistence in SYNCAR-001 when treated with STK-009. These data demonstrate that STK-009 treatment expands SYNCAR-001 and delivers a gene signature indicative of enhanced T cell fitness and activity. Subcutaneous administration of STK-009 in NHP shows no evidence of toxicity or physiological IL-2 mediated activity on immune cells, including Teffs, Tregs, and NK cells. Pharmacokinetic analysis of STK-009 shows stable exposure with minimal clearance, demonstrating the selectivity of STK-009. These findings validate an orthogonal platform that selectively drives potent T cell effector functions of engineered cells without the toxicities mediated by NK cells or non-tumor specific T cells associated with high dose IL-2 therapy. These results demonstrate the ability of this orthogonal platform to improve the efficacy and durability of CARs. Citation Format: Paul-Joseph P. Aspuria, Sandro Vivona, Michele Bauer, Marie Semana, Navneet Ratti, Scott McCauley, Romina Riener, Rene de Waal Malefyt, Deepti Rokkam, Jan Emmerich, Patrick J. Lupardus, Rob A. Kastelein, Martin Oft. OrthoCARs: Engineered human IL-2/IL-2Rb orthogonal pairs selectively enhance CAR T cell antitumor efficacy by driving T cell expansion and fitness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1512.

Abstract 1744: STK-012, an alpha/beta selective IL-2 mutein for the activation of the antigen-activated T cells in solid tumor

Abstract Interleukin-2 (IL-2) is a potent stimulator of T and NK cell proliferation, survival, and cytotoxic function. High dose IL-2 induces complete responses as a single agent in certain cancers. Its use is limited due to toxicities such as severe hypotension and vascular leak syndrome (VLS). A better understanding of the mechanisms of IL-2 efficacy and toxicity and development of more selective therapies are needed to improve the use of IL-2. Next generation IL-2 therapeutics have been engineered to bias activity towards the dimeric form of the IL-2 receptor, which consists of IL-2Rβ (CD122) and IL-2Rγ (CD132), and away from the high affinity trimeric form of the receptor, which also includes IL-2Rα (CD25). This approach is designed to stimulate NK cells and naïve effector T-cells, which express the dimeric, and not the trimeric, form of the receptor. However, this approach largely misses antigen-activated and tumor-specific T-cells, which have high expression of both CD25 and CD122. In addition, this approach expands peripheral NK cells, which have been shown in mice to cause VLS (1-3).To specifically stimulate antigen-activated CD25+ effector T cells in cancer patients and avoid systemic NK and naïve T cell activation, we have developed a PEGylated, CD25/CD122-selective IL-2 mutein (STK-012) and its mouse surrogate (STK-014). Here we present efficacy and safety of STK-014 compared to wild type IL-2 (wtIL-2) and a CD122/CD132 biased IL-2 (βγIL-2). We also present safety of STK-012 in non-human primates (NHP). STK-012/STK-014 selectively induced STAT5 phosphorylation and proliferation in antigen activated T cells but not in NK cells and naïve T cells. In mice, STK-014 showed reduced toxicity compared to wtIL-2 and βγIL-2. In particular, wtIL-2 and βγIL-2 induced VLS while STK-014 did not. Moreover, STK-014 demonstrated complete responses both as single agent and in combination with a PD-1 antibody in syngeneic tumor models. In general, STK-014 demonstrated improved efficacy compared to wtIL-2 and βγIL-2. STK-014 dramatically increased intratumoral T cells and intratumoral cytotoxic activity compared wtIL-2 and βγIL-2 while avoiding T cell activation in the spleen. STK-014 treatment drastically increased the CD8+ T cell to Treg ratio within the tumor when compared to control tumors (25-fold), but also in comparison to wtIL-2 (2.75-fold) and to βγIL-2 (5.2-fold).In NHP, STK-012 was well tolerated at doses supra-efficacious in mice. Weekly dosing of STK-012 led to continuous elevated serum concentrations, demonstrating selectivity for CD25/122+ T cells. STK-012 selectively induced memory T cell expansion, including CD28+ CD95+ CD8 T cells. In summary, STK-012 avoids IL-2 mediated toxicity and may enable the specific expansion of antigen activated memory T cells in cancer patients, leading to durable tumor response. 1 Peace DJ and Cheever MA. JEM, 1989.2 Gatley MK et al. JI, 1988.3 Assier E et al. JI; 2004. Citation Format: Jan Emmerich, Michele Bauer, Marie Semana, Bhargavi Jayaraman, Sandro Vivona, Scott McCauley, Romina Riener, Rene De Waal Malefyt, Paul-Joseph Aspuria, Deepti Rokkam, Patrick Lupardus, Rob Kastelein, Martin Oft. STK-012, an alpha/beta selective IL-2 mutein for the activation of the antigen-activated T cells in solid tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1744.

Abstract 1448: Combination therapy of anti-sense oligonucleotide targeting TGF-beta2 (TASO) and IL-2 (Proleukin) has anti-cancer effect in solid cancer

Abstract Background: TGF-Β (transforming growth factor-beta) is an essential cytokine for tumor proliferation and metastasis. The expression of TGF-Β correlates with malignancy of various cancers and involves immunosuppression and angiogenesis of a tumor. IL-2 is a major cytokine to proliferate T cells and NK cells which are major players of cancer immunity. However, the toxicity of high dose IL-2 limits its use in cancer therapy. Combination treatment of TGF-Β inhibitor and IL-2 would have an anti-tumor effect by immune cells through diminishing immunosuppression by TGF-Β and enforcement of immune cells by IL-2. Trabedersen is an anti-sense oligonucleotide targeting human TGF-Β mRNA. It is shown that Trabedersen is well tolerable in cancer patients and effective reagent to treat pancreatic cancer, melanoma, and glioblastoma. Proleukin is the only approved IL-2 reagent to treat Renal cell carcinoma and Melanoma. Methods: Trabedersen and Proleukin activated human PBMC (Peripheral Blood Mononuclear Cell) are treated to several solid cancer cell lines, such as Breast cancer, Pancreatic cancer, Melanoma, Lung cancer, and Colon cancer to see the cytotoxicity effect of combination therapy of Trabedersen and Proleukin. NSG mouse (NOD Scid Gamma mouse, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) which are humanized by human PBMC engraftment and the tumor growth of Melanoma and TNBC (Triple Negative Breast Cancer cell) cell line are monitored. Results: The combination treatment of Trabedersen and low dose Proleukin decreased cancer cell viability in vitro experiment in solid cancer cell lines. Melanoma and TNBC tumor growth was delayed in humanized NSG mouse model by Trabedersen and low dose IL-2 combination therapy and tumor growth delay was statistically significant to Trabedersen alone or IL-2 alone group. Tumor infiltrating lymphocyte population was increased in Trabedersen treated group and FoxP3+ regulatory T cell population in blood and tumor microenvironment was decreased by treatment of Trabedersen. Conclusion: TGF-Β inhibitor (Trabedersen) and low dose IL-2 (Proleukin) combination treatment is expected to be an effective regimen in solid cancer treatment than individual treatment by alteration of tumor environment. Modulation of the dose of Proleukin expects to help reduce the toxicity of IL-2 and increase the anti-cancer effect by combination with Trabedersen. Citation Format: Jun-Eui Park, Jihye Koo, Hong-Kyu Lee, Tae Hun Kim. Combination therapy of anti-sense oligonucleotide targeting TGF-beta2 (TASO) and IL-2 (Proleukin) has anti-cancer effect in solid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1448.

Early and Profound Exhausted Phenotype of Central Memory and Memory Stem T Cells in the Bone Marrow of AML Patients Characterizes and Predicts Relapse after Allo-HSCT

Background: Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) is the only cure for high-risk acute myeloid leukemia (AML). Unfortunately, relapse remains the major cause of death after HSCT. We investigated if T-cell dysfunction is associated to post-transplant relapse.Patients and Methods: We longitudinally analysed T-cell dynamics in bone marrow (BM) and peripheral blood (PB) of 32 AML patients receiving HSCT from HLA-matched (20 pts) or HLA haploidentical (Haplo, 12 pts) donors. BM and PB samples were collected 60 days after HSCT and at relapse (median 237 days; 16 pts) or, when complete remission was maintained, at 1 year (CR; 16 pts). 10 healthy donors (HD) were used as controls. Samples were analysed by polychromatic flow cytometry for the expression of inhibitory receptors (IR) on CD4 and CD8 T-cell subsets defined by CD45RA, CD62L and CD95 expression. Results were also analysed with BH-SNE, an unbiased dimensionality reduction algorithm. To evaluate T-cell function and specificity, CD107a expression, cytokine profiles and killing of autologous blasts were quantified.Results: After Haplo-HSCT, PD-1, CTLA-4, 2B4 and Tim-3 were expressed in BM and PB T cells at higher percentage when compared to HD and independently from the clinical outcome. Conversely, after HLA-matched HSCT, patients who relapsed displayed a higher frequency of BM-infiltrating T cells expressing PD-1, CTLA-4 and Tim-3 than CR pts (p<0.05) and HD (p<0.01). When investigating each T-cell subset, in both HD and CR pts the IR expression was confined to effector memory and effectors; at relapse, instead, PD-1, 2B4, KLRG1 and Tim-3 were more commonly expressed in BM-infiltrating central memory (TCM) and memory stem T cells (TSCM, p<0.01). Interestingly, at relapse leukemia expressed PD-L1 (9/9 cases) and Galectin-9 (6/9) and the latter linearly correlated with Tim-3+ BM CD8 T-cell percentages, suggesting a preferential role for this immunomodulatory axis after HSCT. Based on phenotype similarities, the BH-SNE algorithm separated HD and HSCT samples in bi-dimensional maps and identified T cell clusters that associated with relapse (32) and were characterized by T cells co-expressing high levels of multiple IRs.Consistent with the exhausted phenotype, BM CD8 T-cells at relapse displayed lower degranulation ability and IL-2 production compared to CR (p<0.05). The ability to degranulate and to produce pro-inflammatory cytokines of BM-T cells expressing at least 1 inhibitory receptor (IR+) could be recovered in five patients upon polyclonal stimulation in vitro with high doses of IL-2, suggesting that T-cell dysfunction can be efficiently reversed. Upon challenge with autologous leukemic blasts, higher proportion of IR+ lymphocytes upregulated HLA-DR (p<0.05), granzyme A and B (p<0.001) and killed blasts (p<0.05) compared to IR- T cells. In addition, IR+ but not IR- cells were efficiently expanded by stimulation with matched Leukemic Antigen-Presenting Cells (fold increase 146 and 3, respectively).Interestingly, we observed that, at day 60 after HLA-matched HSCT, BM CD8 TSCM and TCM cells from patients who shall subsequently relapse already expressed PD-1, Tim-3 and LAG3 at higher percentage when compared to patients who shall maintain long-term CR (p<0.05). Co-expression of at least 3 IR on TSCM and TCM T-cells, but not on effectors, or TEM, at this early time-point was associated with significantly decreased disease free survival.Conclusions: After HSCT, the molecular signature of exhausted CD8 cells in relapsing patients includes PD-1, CTLA-4, 2B4 and Tim-3, expressed on early differentiated T cells. Exhausted BM-T cells are preferentially enriched in specificities directed against leukemic blasts. Taken together, these results highlight a wide, though reversible, immunological dysfunction mediated by AML relapsing blasts. Interestingly, the analysis on early time points suggests that the exhausted phenotype may be predictive of leukemia relapse. [Display omitted] DisclosuresBondanza:TxCell SA: Research Funding; Menarini Biotech: Consultancy; Molmed Spa: Research Funding. Bonini:MolMed: Consultancy.

Functional Characteristics of FAS-L Resistant and IL-2 Deprived Regulatory T Cells (Treg A) in Idiopathic Aplastic Anaemia

IntroductionAplastic anemia (AA) is an immune mediated bone marrow failure syndrome characterized by reduced numbers and dysfunctional regulatory T cells (Tregs). Using deep phenotyping, we have previously identified an immune signature that predicts clinical response to immunosuppression (IST) (Kordasti et al., Blood 2016). The so-called Treg A (CD4+CD25hiCD127loCD45RAhiCD95-CCR4lo) were higher in IST non-responders, whereas responder patients had a higher proportion of Treg B (CD4+CD25hiCD127loCD45RAloCD95+CCR4hi). The aims of the current study were to investigate the reasons for the reduction of specific Treg subset, the ex vivo expandability of the remaining Tregs in response to IL-2, their function and stability following expansion. The efficacy and longevity of these cells were tested in an animal model, with the aim of using autologous ex vivo expanded Tregs as a potential treatment for AA.Methods and ResultsFAS-L-induced apoptosis leads to reduction of Treg B, this effect can be reduced by low-dose IL-2Given the role of FAS-L positive T cells in the pathophysiology of AA pathophysiology and the fact that CD95 (FAS) is one of the significant non-redundant markers that defines Treg B, we tested the FAS-L sensitivity of these cells. After 5 hours culture with FAS-L (5 mg/ml), the number of apoptotic cells was significantly higher in Treg B compared to Treg A (3 replicates, p < 0.02). The effect was prevented by the addition of low-dose IL-2 (5 U/mL) to the culture.Treg expansion and their functionTotal Tregs from AA and healthy donors (HDs) are sensitive to low dose IL-2 as shown by STAT5 phosphorylation (p < 0.001, n = 6 AA and 2 HDs). Moreover, Tregs from AA and HDs can be expanded polyclonally at a comparable rate (fold increase 33x vs 21x, p = n.s. n = 6 AA and 8 HDs) in a Treg promoting culture (stimulation with anti CD3/CD28 beads and IL-2 1,000 IU/ml for 4 weeks with all-trans retinoic acid and rapamycin). Although Treg A had lower level of p-STAT5 in the presence of low-dose IL-2, this reached to a similar level to Treg B following culture with higher doses of IL-2. To investigate the potential differences between Treg A and B in terms of function and expandability, Treg A and B were sorted from 6 HDs and 4 AA patients, and then expanded for 4 weeks (as above). Treg A and B expanded in HDs (Treg A 45x, and B 33x fold increase, p = n.s.), whereas Treg A expanded at a higher rate compared to Treg B in AA (Treg A 2120x, and B 90x fold increase, p = 0.03). The in vitro expanded Treg A and B both suppress proliferation of conventional T cells (Tconv) (n = 6, p < 0.001, no difference between A and B, figure a). Treg stability was assessed by DNA methylation analysis using deep amplicon bisulfite sequencing of the Treg-specific demethylated region (TSDR) . TSDR CpG sites in the expanded Treg A and Treg B populations from HD and AA patients are 2% and 13% methylated respectively, confirming their stability.Treg function in vivoTo test the suppressive ability of these Tregs, we transplanted NSG-SGM3 mice with either HDs PBMCs alone (n = 3), or HDs ex vivo expanded Tregs alone (n = 3) or HDs PBMCs co-injected with HDs ex vivo expanded Tregs (n = 3). Mice injected with both PBMCs and Tregs received a further dose of Tregs at day 16 and then weekly injections until day 28. Mice injected with PBMCs only had worst overall survival compared to the mice with PBMCs + Tregs or Tregs only (13 versus 35 days, p < 0.0001) and developed acute GVHD during the first 10 days following the injection (day 13 PBMCs mice 17.36 g versus PBMCs + Tregs mice 22.7 g, p = 0.0052, figure b). There was no weight difference between PBCs + Tregs mice and Tregs only mice.ConclusionsIn summary, although the FAS-L sensitive Tregs, which are the most functional Tregs, are reduced in AA, the remaining Tregs in these patients are IL-2 responsive and can be expanded in vitro . We were able to sort Treg subpopulations (Treg A and B) from AA patients and show that they are expandable, stable and functional up to 8:1 ratio with Tconv. We have also tested the efficacy of ex vivo expanded Tregs in preventing GVHD in a mouse model. Overall, the current study suggests that in AA there may be an increased exposure to FAS-L and an insufficient level of IL-2, which selectively induce apoptosis of Treg B. Moreover, the ex vivo expansion of stable and functional AA Treg subsets is feasible. The expanded Tregs are likely to be able to control the inflammatory response in these patients, hence pave the way toward a novel cellular therapy for AA. [Display omitted] DisclosuresKordasti:Celgene: Research Funding.

Combination of radiation therapy, bempegaldesleukin, and checkpoint blockade eradicates advanced solid tumors and metastases in mice

BackgroundCurrent clinical trials are using radiation therapy (RT) to enhance an antitumor response elicited by high-dose interleukin (IL)-2 therapy or immune checkpoint blockade (ICB). Bempegaldesleukin (BEMPEG) is an investigational CD122-preferential...

Expansion of tumor-infiltrating lymphocytes (TIL) from penile cancer patients

Penile cancer is a rare but highly lethal cancer, and therapeutic options for patients presenting with lymph nodal disease are very limited. Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) was shown to provide durable objective response in patients with metastatic melanoma and TIL have been expanded from solid tumors at rates between 70 and 90% depending on the specific diagnosis. We evaluated whether TIL could be expanded from surgical specimens of patients with penile cancer. Tumor samples from metastatic lymph nodes obtained at the time of inguinal lymph node dissection were collected, minced into fragments, placed in individual wells of a 24-well plate, and propagated in high dose IL-2 for four weeks. The phenotype of expanded TILs was assessed by flow cytometry and their anti-tumor reactivity was assessed by IFN-γ ELISA. TIL were expanded from 11 out of 12 (91.6%) samples of metastatic lymph nodes. Expanded TIL were predominantly CD3+ (mean 67.5%, SD 19.4%) with a mean of 46.8% CD8+ T cells (SD 21.1%). Five out of 11 samples (45.4%) from expanded TIL secreted IFN-γ in response to autologous tumor. TIL expansion and phenotype of expanded T cell lymphocytes were independent of previous HPV infection and treatment with neoadjuvant chemotherapy. This is the first report demonstrating successful expansion of tumor-reactive TIL from penile cancer patients, which support development of ACT strategies using TIL for the treatment of advanced and recurrent penile cancer.