High-dose IL-2 Research Articles

Phase I results of human endogenous retrovirus type-E (HERV-E) TCR transduced T-cells in patients (pts) with metastatic clear cell renal cell carcinoma (mccRCC).

2549 Background: The CT-RCC HERV-E is expressed in the majority of ccRCC tumors with no expression in normal tissues. T-cells transduced to express a HERV-E TCR are cytotoxic to ccRCC & mediate tumor regression of human ccRCC in a murine model. We developed a method to manufacture HERV-E TCR transduced T-cells (HERV-E TCR) for clinical development. Methods: We conducted a phase I dose-escalation trial (DL1:1x106, DL2:5x106, DL3:1x107 & DL4:5x107cells/kg) to evaluate the safety of adoptively infused HERV-E TCR in mccRCC pts. HERV-E TCR contain T-cells engineered to express an HLA-A*11 restricted HERV-E TCR and a truncated CD34 cassette for in vivo monitoring. Eligible HLA-A*11 pts had mccRCC, ECOG 0-1 & prior antiangiogenic & checkpoint inhibitors therapy unless contraindicated/unavailable. Cyclophosphamide & fludarabine were given followed by HERV-E TCR infusion & IL-2 administration. Results: 14 HLA-A*11+ pts (median age 56) were treated including 3 pts in each DLs 1-3 & 5 pts in DL4. 86% had ≥ 3 prior treatment lines (range 1-7): 36% had high-dose IL2, 57% Ipilimumab-Nivolumab & 50% ≥ 3 lines of anti-VEGFR therapy. Bone, liver & brain metastases were present in 71%, 43%, and 14% of pts. All HERV-E TCR products met sterility, purity, and potency criteria for release and infusion (Table). No HERV-E TCR dose-limiting toxicities or treatment-related deaths occurred. Pts received a mean: 12 IL-2 doses (IQR 11-14). 57% pts had G3-4 febrile neutropenia & 7% G3-4 capillary leak syndrome. 1 pt experienced G2 skin rash possibly related to HERV-E TCR. Best response included 7% pts with partial response & 29% with stable disease ≥ 8 weeks. The mPFS: 62 days (IQR 31,90). At DL4, TCR transgene expression was detectable in PBMCs at a mean 6.3% (range (r) 0.02-25), 12.3% (r 0.01-61.2), 3.45% (r 0.00-13.7) & 2.89% (r 0.00-11.5) on days 4, 7, 14, & 21. HERV-E TCR were detected in the malignant pleural effusion of 1 pt. Conclusions: This is the first trial evaluating TCR-engineered T-cells targeting a human endogenous retrovirus in ccRCC. The manufacturing method utilized produced large numbers of highly purified CD8+ HERV-E reactive T-cells that were not associated with any dose limiting toxicities when given at doses up to 5x107 cells/kg. For pts in DL4, HERV-E TCR were observed to proliferate in vivo, traffic to a metastatic site, and induce tumor regression in one mccRCC pts. Clinical trial information: NCT03354390 . [Table: see text]

A pilot trial of autologous tumor infiltrating lymphocytes (lifileucel, LN-144) for patients with asymptomatic melanoma brain metastases.

TPS9606 Background: Melanoma brain metastases (MBM) are a leading cause of morbidity and mortality for patients with advanced melanoma. Modern systemic therapies are insufficient at controlling MBM, and median overall survival (OS) for pts with MBM is < 1 year. Adoptive T cell therapy using tumor infiltrating lymphocytes (TIL) has demonstrated efficacy in advanced melanoma. Lifileucel (LN-144), an autologous TIL product, was recently shown to be safe and effective for patient with PD-1 refractory melanoma. Patients with active MBM were excluded from lifileucel trials to date. Methods: This single-center pilot trial (NCT05640193) is enrolling up to 10 pts with asymptomatic MBM from non-uveal melanoma to receive lifileucel. Patients must have ≥1 intracranial lesion measuring 5-30mm visible on MRI to be used as a target lesion for modified (m)RECIST measurement, progression on prior anti-PD-1 therapy (with or without anti-CTLA-4), progression on targeted therapy if BRAF V600E/K-mutated), ECOG PS ≤1, ≥1 resectable lesion(s) (≥1.5 cm), recovered from prior surgery/anticancer treatment-related AEs (grade ≤1). Patients are not eligible if they have symptomatic MBM and/or require corticosteroids of ≥10 mg/day of prednisone or equivalent. Lifileucel is generated from resected tumor in a centralized GMP process. The regimen includes nonmyeloablative lymphodepletion, lifileucel infusion, and a short course of high-dose IL-2. The primary endpoint is feasibility, defined as ≥7/10 patients who undergo tumor harvest receiving lifileucel infusion. Secondary endpoints are safety, feasibility of manufacturing lifileucel in patients with MBM, and brain metastasis response rate (BMRR) per mRECIST 1.1. Exploratory endpoints include overall objective response rate by mRECIST 1.1, best extracranial response rate, intracranial progression-free survival (PFS), overall PFS, OS. Extensive correlative analyses of peripheral blood mononuclear cells, plasma, tumors, and cerebrospinal fluid are planned to better understand MBM growth, treatment resistance, and response of the central nervous system to lifileucel. Clinical trial information: NCT05640193 .

A phase 3 study (TILVANCE-301) to assess the efficacy and safety of lifileucel, an autologous tumor-infiltrating lymphocyte cell therapy, in combination with pembrolizumab compared with pembrolizumab alone in patients with untreated unresectable or metastatic melanoma.

TPS9607 Background: Most patients (pts) with advanced (unresectable or metastatic) melanoma receiving front-line immune checkpoint inhibitor (ICI) therapy progress within a year (Robert Lancet Oncol 2019; Larkin NEJM 2019; Tawbi NEJM 2022). Early-line therapies are needed to improve the rate of deep and durable responses and increase the proportion of pts with long-term benefit. Lifileucel demonstrated an ORR of 31.4% and median DOR not reached (median 36.5 mo follow-up) in pts with post-ICI advanced melanoma (Sarnaik SITC 2022). Earlier-line treatment with lifileucel plus pembrolizumab (pembro) in pts with ICI-naïve advanced melanoma demonstrated an ORR of 67%, including a CR rate of 25% (Iovance Press Release, April 5, 2022; O’Malley JITC 2021). TILVANCE-301 will evaluate the efficacy and safety of lifileucel plus pembro compared with pembro alone in pts with untreated advanced melanoma. Methods: TILVANCE-301 (NCT05727904) is a phase 3, multicenter, randomized, open-label, parallel group, treatment study that will randomize ~670 pts (1:1) to either Arm A: lifileucel plus pembro (study intervention includes tumor tissue resection, pembro, nonmyeloablative lymphodepletion [NMA-LD], lifileucel infusion, an abbreviated course of high-dose IL-2, and thereafter, continued pembro) or Arm B: pembro alone. Pts in Arm B who receive pembro and experience confirmed progressive disease verified by blinded independent review committee (BIRC) have the option to receive lifileucel as the immediate next line of treatment. Eligible adults have histologically confirmed advanced melanoma (Stage IIIC, IIID, or IV); ECOG PS of 0 or 1; estimated life expectancy > 6 mo; ≥1 resectable lesion ~1.5 cm in diameter postresection to generate lifileucel and ≥1 measurable lesion (RECIST v1.1); and adequate hematologic parameters and organ function. Neoadjuvant or adjuvant treatment including ICI meeting protocol-specified criteria may be allowed. Exclusion criteria include prior therapy for metastatic disease; symptomatic untreated brain metastases; organ allograft or prior cell transfer therapy; uveal/ocular melanoma; chronic systemic steroid therapy; active systemic infections; cardiovascular, respiratory, or immune system illnesses; primary/acquired immunodeficiency; or other primary malignancy in the last 3 y. The dual primary efficacy endpoints are BIRC-assessed (RECIST v1.1) ORR and PFS. Key secondary efficacy endpoint is OS. Additional secondary efficacy endpoints include BIRC-assessed CR rate, DOR, and EFS; investigator-assessed ORR, PFS, CR rate, DOR, EFS, and PFS2; and safety as characterized by severity and seriousness of TEAEs, and relationship to study drug. The study will enroll globally. Clinical trial information: NCT05727904 .

Rethinking Oncologic Treatment Strategies with Interleukin-2

High-dose recombinant human IL-2 (rhIL-2, aldesleukin) emerged as an important treatment option for selected patients with metastatic melanoma and metastatic renal cell carcinoma, producing durable and long-lasting antitumor responses in a small fraction of patients and heralding the potential of cancer immunotherapy. However, the adoption of high-dose rhIL-2 has been restricted by its severe treatment-related adverse event (TRAE) profile, which necessitates highly experienced clinical providers familiar with rhIL-2 administration and readily accessible critical care medicine support. Given the comparatively wide-ranging successes of immune checkpoint inhibitors and chimeric antigen receptor T cell therapies, there have been concerted efforts to significantly improve the efficacy and toxicities of IL-2-based immunotherapeutic approaches. In this review, we highlight novel drug development strategies, including biochemical modifications and engineered IL-2 variants, to expand the narrow therapeutic window of IL-2 by leveraging downstream activation of the IL-2 receptor to selectively expand anti-tumor CD8-positive T cells and natural killer cells. These modified IL-2 cytokines improve single-agent activity in solid tumor malignancies beyond the established United States Food and Drug Administration (FDA) indications of metastatic melanoma and renal cell carcinoma, and may also be safer in rational combinations with established treatment modalities, including anti-PD-(L)1 and anti-CTLA-4 immunotherapy, chemotherapies, and targeted therapy approaches.

Systemic Cancer Immunostimulatory Therapies and Acute Viral Infection Induce Corticosteroid-Mediated Thymic Involution and Naïve T cell Output in Mice and Humans

Abstract Thymic involution (TI) occurs with aging and during stress responses, decreasing naïve T cell output. Thymic progenitor apoptosis can occur directly by corticosteroids (CS) leading to transient involution followed by recovery. The effects of systemic immunotherapies (IT) used in cancer or immune stimulation via acute viral infection on the thymus has not been well-characterized and was the primary goal of this study. IT treatment of mice with high dose IL-2 (HD IL-2) or models of acute viral infection (mouse cytomegalovirus, MCMV) all resulted in rapid TI due to apoptosis of CD4/CD8 double-positive progenitors followed with a reduction in naïve T cell content. Interestingly, cessation of therapy or resolution of viral infection resulted in greater thymic size compared to untreated recipients, indicating a rebound effect. In both models, increased CS levels in the serum of mice preceded TI. Clinically, patients receiving HD IL-2 for cancer had significantly reduced T cell receptor excision circles, a marker for naïve T cell output, also correlating with increased cortisol levels. Using a model of sub-lethal MCMV infection that did not increase systemic pro-inflammatory cytokines, we observed a similar correlation of TI and increased CS levels. To further analyze the effects of CS on TI, mice were adrenalectomized and then given HD IL-2, where significantly reduced CS levels correlated with partial protection from TI. These results indicate that strong systemic immunostimulation by either IT or acute viral infections markedly induce transient CS-mediated TI, leading to a decrease in naïve T cells. Therefore, targeting CS responses in preventing TI and, especially with the aged, allow for naïve T cell populations to be maintained.

Abstract CT133: Trial in progress: a multicenter phase 1/1b dose escalation study of WTX-124 as a monotherapy and in combination with pembrolizumab in patients with selected advanced or metastatic solid tumors

Abstract Background: High-dose recombinant human IL-2 (HD IL-2) is an FDA-approved therapy that produces durable complete remissions in a subset of patients with advanced or metastatic cutaneous melanoma and renal cell carcinoma. However, the clinical benefit of HD IL-2 is counterbalanced by life-threatening toxicities such as capillary leak syndrome. WTX-124 is an engineered cytokine prodrug composed of a wild-type IL-2 fused via proprietary cleavable linkers to an inactivation domain and to a half-life extension domain. In the circulation and in normal tissues, WTX-124 is designed to remain inactive and not bind to high- (αβγ) and intermediate-affinity (βγ) IL-2 receptors. In contrast, proteolytic activation of WTX-124 in the tumor microenvironment (TME) is expected to liberate the IL-2 cytokine to stimulate antitumor immune responses. Preclinical data show that WTX-124 is activated by a range of dissociated human tumors in vitro but not by normal human cells or by patient serum. Moreover, WTX-124 demonstrates potent antitumor activity in multiple murine syngeneic tumor models with substantial expansion and activation of tumor-infiltrating lymphocytes. In the MC38 model, WTX-124 has a large therapeutic window of >19-fold compared to ~3-fold for recombinant human IL-2. Methods: This first-in-human, open-label phase 1/1b trial is investigating the safety, tolerability, pharmacokinetics, pharmacodynamics, immunogenicity, and antitumor activity of WTX-124 administered as a monotherapy or in combination with the anti-PD-1 antibody pembrolizumab to patients with advanced or metastatic solid tumors (NCT05479812). All patients must have ≥ 1 measurable lesion per RECIST 1.1 and a tumor type for which immune checkpoint inhibitor (ICI) therapy is indicated. Dose escalation uses a modified toxicity probability interval-2 study design with WTX-124 administered intravenously (IV) every two weeks in 28-day cycles. If the safety of WTX-124 as a monotherapy is established in the first three cohorts, the study will then initiate dose escalation of WTX-124 in combination with pembrolizumab 400mg IV every six weeks. Dose expansion is anticipated to include four arms that will enroll patients with either advanced or metastatic cutaneous melanoma or renal cell carcinoma to receive WTX-124 monotherapy or combination therapy (N=20 patients per arm). Patients with melanoma assigned to the WTX-124/pembrolizumab combination arm may be naïve to all prior systemic therapy for advanced disease, but all other patients in expansion must have previously received a standard of care ICI regimen. Pre- and on-treatment tumor biopsies, archival tumor tissue, and blood samples will be used to investigate multiple exploratory biomarkers. As of January 12, 2023, WTX-124 monotherapy dose escalation is proceeding. Citation Format: Ildefonso Ismael Rodriguez-Rivera, Justin C. Moser, Mateusz Opyrchal, Brendan Curti, Mehmet A. Bilen, Saero Park, Marissa Bruno, Paul Windt, Kulandayan K. Subramanian, Sameer S. Chopra, Randi Isaacs. Trial in progress: a multicenter phase 1/1b dose escalation study of WTX-124 as a monotherapy and in combination with pembrolizumab in patients with selected advanced or metastatic solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT133.

Abstract LB093: Engineering tumor infiltrating lymphocytes from sarcoma and colorectal tumors with membrane bound IL-15 for IL-2 independent expansion and enhanced cytotoxicity against autologous tumor cell lines

Abstract Tumor infiltrating lymphocytes (TIL) have shown promising efficacy in immunologically “hot” solid tumors that have a high level of T cell infiltration, such as melanoma. However, current treatment regimens require high dose IL-2 administration to support TIL survival, which limits their clinical applications due to IL-2 related toxicity. Obsidian Therapeutics is engineering TIL with membrane bound IL-15 (mbIL15) to eliminate the dependence of TIL on exogenous IL-2, potentially enhancing the tolerability of TIL therapies. Because there is a high unmet medical need in patients with tumors colder than melanoma, we evaluated mbIL15-engineered TIL expansion and functionality from colorectal cancer (CRC) and sarcoma biopsies. Using an IL2-independent, proprietary rapid expansion process (REP) we successfully expanded mbIL15-engineered TIL from both CRC and sarcoma, despite significantly lower T-cell numbers in the tumor tissues (average of 12-15% CD45+ TIL versus 64% in melanoma). mbIL15-engineered TIL showed an enrichment for CD8+ T cells throughout the REP and high T cell receptor variable beta chain (TCR Vbeta) diversity. mbIL15-engineered TIL also exhibited lower immune checkpoint expression (LAG3/PD-1) and higher activation marker expression (CD25/CD27/CD28) when compared with unengineered TIL expanded from the same tumors using a conventional REP with IL-2. mbIL15-engineered TIL were polyfunctional, as defined by expression of more than one effector molecules (CD107a, perforin, interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and granzyme b) in response to CD3/CD28 stimulation. To examine the cytotoxic function of mbIL15-engineered TIL, we developed autologous cell lines from the same tumors that were used to generate the TIL. Using whole exome and RNA sequencing we found that the autologous tumor cell lines maintained expression of conserved tumor antigens and HLA-expression when compared with the primary tumor. When co-cultured with the autologous tumor cell lines, mbIL15-engineered TIL secreted higher levels of IFN-γ and induced higher cytotoxicity as compared to unengineered TIL cultured with IL-2. Taken together, these data demonstrate that mbIL15-engineered TIL can successfully be expanded from comparatively “cold” tumors with low T-cell infiltration, such as CRC and sarcoma, while maintaining high TCR diversity and polyfunctionality and demonstrating higher cytokine production and cytotoxic activity against autologous tumor lines, compared to conventional TIL with IL2. Citation Format: Zheng Ao, Carmela Passaro, Bulent A. Aksoy, Balazs Koscso, Rachel Burga, Kyle Pedro, Natasha Ly, Nirzari Shah, Alonso V. Ocando, Gauri Kulkarni, Trisha Timpug, Seth M. Pollack, Jan ter Meulen, Michelle L. Ols. Engineering tumor infiltrating lymphocytes from sarcoma and colorectal tumors with membrane bound IL-15 for IL-2 independent expansion and enhanced cytotoxicity against autologous tumor cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB093.

Abstract 4055: CD40L stimulates melanoma infiltrating B cells and enhances ex vivo TIL expansion

Abstract Adoptive transfer of tumor infiltrating lymphocytes (TIL) is a feasible and effective therapy for melanoma and lung cancer[1,2]. Multiple factors may determine the quality of the TIL product including components of the tumor microenvironment. In this work, we analyzed the role of melanoma infiltrating B cells in the context of TIL expansion based on their documented association with response to other types of immunotherapies[3]. We stimulated melanoma infiltrating B cells using human recombinant CD40L on the first day of ex-vivo TIL expansion. Samples were expanded from cryopreserved melanoma tumor single cell suspensions, in high dose IL-2 alone (standard protocol), or in high dose IL-2 plus CD40L. After 48h, analysis of activation markers on the CD40-expressing cells by flow cytometry was performed. For further investigation of the changes induced by CD40L stimulation, TIL expansion cultures (+/- CD40L) were analyzed using scRNA-seq (10X Genomics Chromium NextGEM Single Cell 5’ v2 and V(D)J Reagent kits; Illumina NovaSeq 6000 instrument with S4 sequencing flow cell) at 48h of culture (n=7 patients). The TIL expansion success rate was 68% with the CD40L treatment condition compared to 36% with the standard protocol. TILs cultured in the presence of CD40L expanded to on average three times more than with the standard protocol (P ≤ 0.01). Treatment with CD40L increased the percentage of CD39- CD69- T cells (P ≤ 0.05). Within the tumor digests, a higher percentage of B cells, including switched memory B cells (CD27+ IgD− ), was associated with successful TIL expansion (P=0.04). scRNA-seq analysis demonstrated different clustering patterns within the B cell compartment based on culture conditions. No clear partition was observed for other cell types, including the myeloid compartment. B cells displayed 126 DEGs associated to CD40L addition, CCL22, CD83, EBI3 and CD58 were among the upregulated genes in the CD40L-treated B cells. Other cell types experienced minimal to no change in transcriptomic profiles. B cell clusters were sub-classified based on CD27 and IgD expression[4], showing a predominance of naïve and switched memory B cells. Our results show that higher presence of B cells within tumors is associated with better TIL expansion, suggesting an interplay between T and B cells, and providing rationale for the design of improved TIL expansion protocols based on B cell stimulation with CD40L. This work has been supported in part by the Flow Cytometry, Genomics and Biostatistics and Bioinformatics Core Facilities at Moffitt Cancer Center, an NCI designated Comprehensive Cancer Center (P30-CA076292). We acknowledge Moffitt’s Melanoma Center of Excellence and the Mark Foundation for the financial support. [1] Sarnaik, A.A. et al. JCO 39, 2656-2666 (2021). [2] Creelan, B. et al. Nat Med 27, 1410-1418 (2021). [3] Cabrita, R. et al. Nature 577, 561-565 (2020). [4] Sanz, I. et al. Front Immunol 10, 2458 (2019). Citation Format: Renata Ariza Marques Rossetti, Leticia Tordesillas, Matthew Beatty, Dongliang Du, Yian Ann Chen, Amod Sarnaik, Shari Pilon-Thomas, Daniel Abate-Daga. CD40L stimulates melanoma infiltrating B cells and enhances ex vivo TIL expansion. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4055.

A Sampling of Highlights from the Literature

The timing of immune interactions modulates antitumor responses (from LetsgomusicStyle via Wikimedia Commons)Immune cell interactions are critical to mounting effective antitumor responses. Wang et al. show that antitumor responses are coordinated by dendritic cell (DC)–CD8+ T-cell interactions and that expression of key circadian rhythm genes governs immune function, which is also affected by the time of day (day vs. night). Cervantes-Silva et al. reveal that DC antigen presentation is also dependent on expression of circadian rhythm genes, which regulate DC mitochondrial morphology and metabolism, and thus, affect T-cell priming. These studies highlight that induction of antitumor responses is a coordinated, rhythmic process, and a deeper understanding of immune rhythmicity can improve therapeutic strategies.Wang C, …, Scheiermann C. Nature 2022 December 5. DOI:10.1038/s41586-022-05605-0.Cervantes-Silva MP, …, Curtis AM. Nat Commun 2022 December 5;13:7217.IL2 is less active in the acidic tumor microenvironment (from BQUB17-ONadal via Wikimedia Commons)High-dose IL2 is approved for treating melanoma and renal cell carcinoma but is used infrequently because of limited efficacy and systemic on-target toxicity. Gaggero et al. find that the acidic pH of the tumor microenvironment (TME), created as a result of tumor production of lactic acid, reduces IL2 signaling in tumor-infiltrating CD8+ T cells, impairing the efficacy of high-dose IL2 therapy in preclinical models. A mutated IL2 variant (Switch-2) that binds IL2Rα with higher affinity at acidic pH than neutral pH shows enhanced antitumor activity with reduced on-target toxicity in mice. The data provide new insight into physicochemical properties of the TME that can impact immunotherapy efficacy and suggest ways to overcome this.Gaggero S, …, Mitra S. Sci Immuol 2022 December 2;7:eade5686.Eosinophils contrib-ute to the efficacy of ICB (from Blausen Medical via Wikimedia Commons)Understanding the mechanisms behind why immune checkpoint blockade (ICB) is not effective in many patients is key to improving treatment. Blomberg et al. find that eosinophils are enriched in ICB-responding patients with breast cancer. This observation was confirmed in a mouse model, and CD4+ T cells, IL5, and IL33 were identified as the driving factors for eosinophil enrichment. Increased eosinophil infiltration boosts eosinophil-dependent activation of CD8+ T cells, which in turn enhances efficacy of ICB. The data emphasize how immune cells other than T cells are affected by ICB and how they can be critical determinants of treatment efficacy.Blomberg OS, …, Kok M. Cancer Cell 2022 December 15;41:106–123.E10.Antitumor T-cell responses require two steps (from DENI070688 via Wikimedia Commons)Understanding T-cell differentiation can reveal insights into the mechanisms behind effective antitumor responses. Prokhnevska et al. demonstrate that T cells are activated via two key steps: first, initial priming and differentiation into a TCF-1+ stemlike phenotype in tumor-draining lymph nodes (TDLN); and second, acquisition of an effector program after infiltrating the tumor microenvironment (TME). Effector programming of CD8+ T cells is dependent on co-stimulation from antigen-presenting cells in the TME, as opposed to in the TDLNs. This process differs from canonical priming/activation of CD8+ T cells in other contexts and adds to our understanding of how T cells mount responses to cancer.Prokhnevska N, …, Kissick H. Immunity 2022 December 28;56:107–124.Eosinophils contribute to the efficacy of ICB (from Boghog2 via Wikimedia Commons)IL22 is known to facilitate tumorigenesis. Two new studies identify mechanisms by which IL22 promotes tumor metastasis and suggest the cytokine as a therapeutic target for preventing metastasis. Giannou et al. find that mice lacking IL22 and mice treated with an IL22-specific blocking antibody are protected from liver metastasis in a colorectal cancer model. Mechanistically, IL2 from liver-resident iNKT17 cells acts on endothelial cells, promoting cancer cell extravasation into the liver. In mouse models of lung and breast cancer, Briukhovetska et al. show that T helper–cell production of IL22 induces overexpression of CD155 on tumor cells. Binding of CD155 to CD226 on natural killer (NK) cells causes CD266 internalization and impaired NK-cell function, promoting metastasis.Giannou AD, …, Huber S. Immunity 2023 January 10;56:125–142.E12.Briukhovetska D, …, Kobold S. Immunity 2023 January 10;56:143–161.E11.Engineered live cancer cells can promote effective antitumor immunity (from Rita Elena Serda via NIH Flickr)The use of inactivated tumor cells as a therapeutic vaccine has been shown to provide limited clinical benefit. Chen et al. show that engineering live cancer cells to secrete IFNβ and GM-CSF, to be resistant to IFNβ, and to have a double kill switch (HSV1 thymidine kinase and rapamycin-activated caspase 9) yields therapeutic cancer cell–based vaccines that induce long-term antitumor immunity and improve survival in several preclinical models. The translatability of this approach is highlighted by the observation that similar engineering of an established human glioblastoma cell line yields a vaccine that reduces tumor growth and improves survival in a humanized model.Chen K-S, …, Shah K. Sci Trans Med 2023 January 4;14:eabo4778.

179P Early proof of concept of safety and clinical activity of clonal neoantigen-reactive T cells (cNeT)

cNeT therapy addresses limitations of TIL therapies including the requirement for high dose lymphodepletion and IL-2 which restricts application to fitter patients, and limited understanding of the active component that limits product optimisation and potency assay development. We have initiated trials evaluating cNeT in advanced, heavily pre-treated patients with NSCLC (CHIRON, NCT04032847) or melanoma (THETIS, NCT03997474) utilising lower dose lymphodepletion and IL-2 (1M IU/m2 sc x10d).

35MO Number of IL-2 doses and clinical outcomes of tumor-infiltrating lymphocyte (TIL) cell therapy: Post hoc analysis of the C-144-01 trial of lifileucel in patients with advanced melanoma

Aldesleukin (IL-2), at high doses in repeat cycles, is approved as monotherapy for patients (pts) with metastatic melanoma. Its limited efficacy is mediated through endogenous T cell activation, which also contributes to considerable toxicity (Rosenberg NEJM 1988). Lifileucel, a one-time investigational TIL cell therapy, showed a 31% IRC-assessed objective response rate (ORR) in 153 pts with advanced melanoma who progressed after immune checkpoint inhibitors and targeted therapy (if BRAF mutation positive) in the phase 2 C-144-01 trial (Sarnaik SITC 2022) using abbreviated high-dose IL-2 (≤6 doses) with 79% lower potential maximum cumulative exposure than monotherapy.

IL-2 Flexible Loops Might Play a Role in IL-2 Interaction with the High-Affinity IL-2 Receptor: A Molecular Dynamics (MD) Study

The clinical use of high-dose IL-2 in cancer immunotherapy faces several drawbacks such as toxicity and unfavorable pharmacokinetic profile. These drawbacks can be avoided by inhibiting IL-2 interaction with the CD25 subunit, which is a component of the high-affinity IL-2 receptor (IL-2Rαβγ). Several studies showed mutations of potential IL-2 residues such as R38, F42, Y45, and Y72 would produce IL-2 that is CD25-independent. In essence, structural comparison between wild-type (WT) IL-2 and CD25-independent IL-2 can be very insightful to assess the role of IL-2 flexibility and conformation in the IL-2 receptor interactions. Here, we investigated the flexibility loops and conformation of IL-2m (F24A, Y45A, and L72G), which is known to be CD25-independent, and IL-2m2 (F42Y and L72R) mutants along with WT IL-2 using MD simulations. Despite residue mutations, both IL-2m and IL-2m2 showed comparable conformational compactness and better stability than WT IL-2. Interestingly, IL-2m and IL-2m2 mutants showed rigid BC and CD loops in comparison to WT IL-2 . Also, the AB loop conformation of IL-2m was a bent structure compared to the WT IL-2 and IL-2m2. Principal component analysis (PCA) and free-energy landscape results suggested IL-2m and IL-2m2 have stable conformations compared to the WT IL-2. Therefore, these mutation sites of IL-2 produced stable and rigid loops that might prevent IL-2 from binding to the CD25 subunit. Our results can help to assess IL-2 flexibility loops to design new CD25-independent IL-2 mutants without compromising the IL-2 structure.

AVALIAÇÃO FUNCIONAL DE LINFÓCITOS T EM PACIENTES PORTADORES DE MIELOMA MÚLTIPLO PARA REPROGRAMAÇÃO GÊNICA E INSERÇÃO DE RECEPTORES DO TIPO CAR

T lymphocytes are able to recognize tumor antigens and control neoplasms. However, a series of functional alterations, such as immuno-senescence and exhaustion may affect an effective antitumor response. Multiple myeloma (MM) is a neoplasia characterized by an infiltration of neoplastic B cells in the bone marrow. The prevalent median age group in MM patients is 65 years old; therefore, patients are mainly experiencing an immunosenescent process that may potentially affect the functional status of T lymphocytes. Considering that autologous CAR-T cells (Chimeric Antigen Receptors) treatment for MM showed an overall rate of response of about 30%, we hypothesize that patients’ derived T lymphocytes may potentially present a dysfunctional profiling and generating inefficient CAR-T products. Thus, we aim to investigate the molecular landscape of T lymphocytes from patients with MM using multiparametric flow cytometry and functional assays. Healthy donors (HD) (n = 20) and MM patients’ (n = 30) peripheral blood will be collected with written consentient agreement. T lymphocytes from both groups will be obtained by ficoll gradient and further evaluated according to distinct functional assays. Cells will be submitted to distinct protocols of activation such as: anti-CD3/CD28 microbeads; variable concentrations of IL-2 and IL-7 cytokines; and PMA/ionomicin stimulation. After stimulated, we will investigate surface molecules associated to exhaustion and senescence: CTLA-4 CD160, CD244, PD1, CD62L, TIM3and LAG3; transcription factors: GATA-3, FOXP3 and T-bet; and intracellular cytokine/factors production: perforin, granzyme-B, TNF-alpha and IFN-gamma using flow cytometry. In addition, migration assays will be performed by cultivating T cells in trans well system in the presence of CXCL9 and CXCL10 chemokines. Moreover, we will evaluate differences in the activity of enzymes related to cell metabolism: EC2.7.1.1, EC 1.1.1.49, EC 2.7, and EC 2.7.1.40. Results: Preliminary set-up experiments revealed that T cells from HD individuals respond to the distinct stimulators by up-regulating all markers of activation. In addition, T cells cultivated in the presence high dose of IL-2 for five days showed an important rate of mortality. Experiments using T lymphocytes from MM patients are ongoing and will delineate the molecular and functional status of these cells in the course of the disease.

761P Tumor infiltrating lymphocytes expressing membrane-bound IL-2 and IL-12 exhibit enhanced proliferation, function, and persistence without requiring exogenous IL-2 support

Tumor Infiltrating Lymphocyte (TIL) therapies have shown significant solid tumor activity in patients, but many current TIL regimens require lymphodepletion and high-dose IL-2 after cell infusion to support TIL persistence and clinical activity. Removing these requirements that cause systemic toxicity while maintaining or improving TIL product functionality by ex vivo engineering of the TIL product with transient expression of mRNA could dramatically improve the patient experience, expand the eligible patient population, and potentially allow repeat dosing.

883TiP A phase I/II open-label study (IOV-GM1-201) of TALEN-mediated PD-1 inactivated autologous tumor-infiltrating lymphocytes (TIL; IOV-4001) in patients with advanced melanoma and NSCLC

Adoptive cell therapy with TIL has demonstrated efficacy in patients (pts) with advanced solid tumors, including melanoma (Sarnaik JCO 2021) and NSCLC (Schoenfeld SITC 2021). IOV-4001 is a TALEN®-mediated PDCD-1 knockout autologous TIL cell therapy product. Preclinical studies suggest that PD-1 inactivation by PDCD-1 gene knockout may enhance TIL cell therapy efficacy, with similar quality attributes and phenotypes to those of non-edited TIL (Natarajan AACR 2022). This first-in-human phase 1/2, open-label, nonrandomized, multicenter study (NCT05361174; open to enrollment) will enroll ∼53 adult pts. During the phase 1 portion, enrollment and dose level decisions will be based on emerging safety and tolerability data in a 28-day dose-limiting toxicity (DLT) observation period. Cohort 1 will include pts with unresectable/metastatic melanoma that has progressed during/within 12 wks of last anti–PD-1/PD-L1 dose (pts must have also received a BRAF ± MEK inhibitor if BRAF V600 mutation-positive). Cohort 2 will include pts with advanced NSCLC who have received ≤3 prior therapies and whose disease progressed either: (1) during/within 12 wks after last anti–PD-1/PD-L1 dose (pts without oncogene-driven mutations) or (2) during/after ≥1 targeted therapy and either platinum doublet chemotherapy or during/within 12 wks after last anti–PD-1/PD-L1 dose (pts with oncogene-driven tumors). Pts must have ECOG PS ≤1, ≥1 resectable lesion(s) (≥1.5 cm), ≥1 remaining RECIST-measurable lesion(s) and recovered from prior surgery/anticancer treatment-related AEs (grade ≤1). IOV-4001 is generated from resected tumor in a centralized GMP process. The regimen includes nonmyeloablative lymphodepletion, IOV-4001 infusion, and a short course of high-dose IL-2. The primary endpoints of phases 1 and 2 are safety (DLTs and AEs) and objective response rate per RECIST v1.1, respectively. Secondary endpoints include complete response rate, duration of response, disease control rate, progression-free survival, overall survival, feasibility, and additional safety. NCT05361174. Medical writing support was provided by Second City Science (Vaniam Group, LLC) and funded by Iovance Biotheraputics, Inc. Iovance Biotherapeutics, Inc.

Distinct Predictive Immunogenomic Profiles of Response to Immune Checkpoint Inhibitors and IL2: A Real-world Evidence Study of Patients with Advanced Renal Cancer.

Next-generation IL2 agents, designed for improved tolerability over traditional HD-IL2 (aldesleukin), are in clinical development. Retrospective molecular tumor profiling of patients treated with HD-IL2 or anti-PD-1 therapy provides insights into genomic characteristics of therapy response. This study revealed common and distinct immune-related predictive response markers for IL2 and anti-PD-1 therapy which may play a role in therapy guidance, and rational combination strategies for these agents.

ARTISTRY-7: A phase 3, multicenter study of nemvaleukin alfa, a novel engineered cytokine, in combination with pembrolizumab versus chemotherapy in patients with platinum-resistant epithelial ovarian, fallopian tube, or primary peritoneal cancer (326)

Objectives: ARTISTRY-7 will evaluate the novel engineered cytokine nemvaleukin alfa (nemvaleukin, ALKS 4230) in patients (pts) with gynecologic cancers. Nemvaleukin was designed to selectively bind to the intermediate-affinity IL-2 receptor, preferentially activating and expanding antitumor CD8+ T and NK cells with minimal expansion of Tregs. This selectivity may enhance tumor killing and improve safety/tolerability compared with high-dose IL-2. In clinical studies, nemvaleukin monotherapy and combination with pembrolizumab have shown evidence of clinical benefit in multiple tumor types. In ARTISTRY-1, four responses are observed in pts with ovarian cancer (OC), including one complete response in a pt with platinum-resistant OC and five prior lines of therapy who remains on treatment. Epithelial OC is the 7th most common cause of cancer mortality in women. The standard frontline treatment is platinum-based chemotherapy, to which many pts become resistant or are refractory. There is a high unmet medical need for platinum-resistant OC. Methods: ARTISTRY-7 is a phase III, multicenter, open-label randomized study of nemvaleukin and/or pembrolizumab versus chemotherapy (ClinicalTrials.gov registration forthcoming). Eligible pts included females (>18 y) with histologically confirmed epithelial OC (high-grade serous, endometrioid, clear cell), fallopian tube cancer, or primary peritoneal cancer. Pts must have received >1 prior line of systemic anticancer therapy in the platinum-sensitive setting, ≤5 prior lines in the platinum-resistant setting, and prior bevacizumab, with evidence of radiographic progression on most recent therapy. Primary platinum-refractory disease (progression during first-line platinum-based therapy) or primary platinum resistance (progression <3 months after completion of first-line platinum-based therapy) is exclusionary. Pts must have an ECOG performance status of 0 or 1, an estimated life expectancy of >3 months, and adequate hematologic reserve and hepatic and renal function. Approximately 376 pts will be randomized (3:1:1:3) to receive intravenous nemvaleukin (6 μg/kg) on days 1-5 and pembrolizumab (200 mg) on day 1 of each 21-day cycle, pembrolizumab monotherapy, nemvaleukin monotherapy, or investigator’s choice chemotherapy (pegylated liposomal doxorubicin, paclitaxel, topotecan, or gemcitabine) and stratified according to PD-L1 status, histologic subtype (high-grade vs non-high-grade serous), and chemotherapy (paclitaxel vs other). Pts will continue treatment in the absence of disease progression or intolerable toxicity (maximum 35 cycles for pembrolizumab; nemvaleukin can be continued). Investigator-assessed progression-free survival (RECIST v1.1) in pts treated with nemvaleukin/pembroli- zumab versus chemotherapy (primary endpoint), characterization of antitumor activity of nemvaleukin and pembrolizumab in combination and as monotherapy, safety, HRQOL, and pharmacokinetic/ pharmacodynamic effects will be collected. Objectives: ARTISTRY-7 will evaluate the novel engineered cytokine nemvaleukin alfa (nemvaleukin, ALKS 4230) in patients (pts) with gynecologic cancers. Nemvaleukin was designed to selectively bind to the intermediate-affinity IL-2 receptor, preferentially activating and expanding antitumor CD8+ T and NK cells with minimal expansion of Tregs. This selectivity may enhance tumor killing and improve safety/tolerability compared with high-dose IL-2. In clinical studies, nemvaleukin monotherapy and combination with pembrolizumab have shown evidence of clinical benefit in multiple tumor types. In ARTISTRY-1, four responses are observed in pts with ovarian cancer (OC), including one complete response in a pt with platinum-resistant OC and five prior lines of therapy who remains on treatment. Epithelial OC is the 7th most common cause of cancer mortality in women. The standard frontline treatment is platinum-based chemotherapy, to which many pts become resistant or are refractory. There is a high unmet medical need for platinum-resistant OC. Methods: ARTISTRY-7 is a phase III, multicenter, open-label randomized study of nemvaleukin and/or pembrolizumab versus chemotherapy (ClinicalTrials.gov registration forthcoming). Eligible pts included females (>18 y) with histologically confirmed epithelial OC (high-grade serous, endometrioid, clear cell), fallopian tube cancer, or primary peritoneal cancer. Pts must have received >1 prior line of systemic anticancer therapy in the platinum-sensitive setting, ≤5 prior lines in the platinum-resistant setting, and prior bevacizumab, with evidence of radiographic progression on most recent therapy. Primary platinum-refractory disease (progression during first-line platinum-based therapy) or primary platinum resistance (progression <3 months after completion of first-line platinum-based therapy) is exclusionary. Pts must have an ECOG performance status of 0 or 1, an estimated life expectancy of >3 months, and adequate hematologic reserve and hepatic and renal function. Approximately 376 pts will be randomized (3:1:1:3) to receive intravenous nemvaleukin (6 μg/kg) on days 1-5 and pembrolizumab (200 mg) on day 1 of each 21-day cycle, pembrolizumab monotherapy, nemvaleukin monotherapy, or investigator’s choice chemotherapy (pegylated liposomal doxorubicin, paclitaxel, topotecan, or gemcitabine) and stratified according to PD-L1 status, histologic subtype (high-grade vs non-high-grade serous), and chemotherapy (paclitaxel vs other). Pts will continue treatment in the absence of disease progression or intolerable toxicity (maximum 35 cycles for pembrolizumab; nemvaleukin can be continued). Investigator-assessed progression-free survival (RECIST v1.1) in pts treated with nemvaleukin/pembroli- zumab versus chemotherapy (primary endpoint), characterization of antitumor activity of nemvaleukin and pembrolizumab in combination and as monotherapy, safety, HRQOL, and pharmacokinetic/ pharmacodynamic effects will be collected.

Abstract 575: Ex vivo expansion of tumor infiltrating lymphocytes (TIL) from human renal cell carcinoma (RCC) tumors

Abstract In this study, we evaluated the feasibility of expanding tumor infiltrating lymphocytes (TIL) from surgically resected renal cell carcinoma (RCC) tumors. Tumors were collected from 43 patients undergoing surgery to remove primary kidney tumors. Tumor types included clear cell RCC (86.0%), papillary RCC (11.6%) as well as chromophobe RCC (2.3%). Tumors were minced into fragments, placed in individual wells of a 24-well plate containing media with high dose IL-2 (6000 IU/mL), and cultured for four weeks. Successful expansion was considered when at least one fragment expanded to a minimum of 2 wells. Successfully expanded TIL were then evaluated for T-cell phenotypes as well as reactivity to the autologous tumor. Of all RCC tumors collected, TIL were successfully grown for 79% of the samples (34/43). The T-cell phenotype of these TIL was highly variable across samples, although there was a higher percentage of CD4+ T-cells relative to CD8+ T-cells. The reactivity of expanded TIL was then assessed by IFNγ release. We found that TIL secreted IFNγ in response to autologous tumor in 71% of the samples. Together, these results demonstrated the feasibility of expanding functional tumor reactive TIL from RCC. Given that hypoxia plays an important role in the development of RCC tumors and is associated with poor prognosis, we then evaluated the ability to expand TIL under hypoxic conditions. To this end, primary TIL were cultured at various O2 levels (20% O2, 5% O2 or 1% O2) and the cell yield as well as the T-cell memory phenotypes were evaluated. We found that the yield of TIL grown in hypoxic conditions was lower when compared to normoxic conditions (20% O2), suggesting a reduction of TIL proliferation in hypoxia. TIL grown in hypoxic conditions displayed increased percentage of central memory T-cells compared to TIL cultured at 20% O2, as evaluated by CD45RA and CCR7 expression (CD45RA-CCR7+). Primary TIL were then expanded to larger numbers using a rapid expansion protocol (REP) by stimulation with anti-CD3 antibody in the presence of allogenic feeder cells. REP of TIL was carried out at either atmospheric O2 (20% O2) or at a hypoxic O2 level (5% O2), and the T-cell phenotype as well as reactivity to the autologous tumor were assessed. Unlike pre-REP TIL, TIL that underwent REP were able to expand in hypoxic conditions (5% O2) and displayed increased percentage of tissue resident memory T-cells (CD69+CD103+; 19%) when compared to the starting TIL population (0.11%) or TIL expanded at atmospheric O2 (0.17%). When co-cultured with the autologous tumor, hypoxic TILs displayed increased IFNγ, TNFα as well as increased Granzyme B release (p&amp;lt;0.001) when compared to TIL expanded in normoxia (20% O2). Collectively, these results support the advantage of adapting TIL to hypoxic conditions in the production of tumor-reactive TIL. Citation Format: Mohammed Alkhouli, Veronica Martinez-Brockhus, Matthew S. Beatty, Robert J. Gillies, Shari Pilon-Thomas, Jad Chahoud. Ex vivo expansion of tumor infiltrating lymphocytes (TIL) from human renal cell carcinoma (RCC) tumors [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 575.

Abstract 586: Orthogonal IL-2/IL-2RB signaling in adoptively transferred T cells controls tumor growth without the need for lymphodepletion in a B16 tumor model

Abstract Multiple adoptive T-cell therapy modalities (ACT) have delivered promising clinical responses in cancer patients. However, challenges including poor T cell effector function, lack of proliferation, and limited persistence have prevented ACTs from reaching their full curative potential. In addition, ACTs typically require lymphodepletion to aid cell engraftment. Lymphodepletion has been shown to improve persistence and efficacy of ACTs by elevating T-cell common gamma-chain cytokines like IL-7 and IL-15. However, lymphodepletion regimens have been identified as a risk factor for cytokine release syndrome (CRS) and infectious complications from opportunistic pathogens. IL-2, another common gamma-chain cytokine, is a potent stimulator of T cells, making it an attractive cytokine to support ACT and potentially bypass the need for lymphodepletion. However, therapeutic use of IL-2 is limited by systemic toxicity due its promiscuous activation of immune cells. To facilitate selective delivery of an IL-2 signal to engineered T cells and avoid signaling in bystander T cells and NK cells, we developed a mouse orthogonal receptor/ligand system consisting of a mutated IL-2 Receptor Beta (moRβ) and a pegylated, IL-2 mutein (moIL-2) that does not significantly activate the wild type IL-2β receptor but does activate moRβ. T cells from pmel-1 T cell receptor-transgenic mice, recognizing gp100 on B16 melanoma cells were transduced with moRβ (orthoPmel). A highly active moIL-2 was continuously dosed for four weeks in mice. Thy1.1+ orthoPmel T cells were tracked by FACS and IHC systemically and in the tumor. During orthoPmel manufacturing, moIL-2 specifically enriched orthoPmel compared to mouse WT IL-2. OrthoPmel in combination with moIL-2 controlled tumor growth in lymphoreplete mice bearing established B16 tumors while neither component alone inhibited tumor growth. moIL-2 significantly expanded orthoPmel systemically and intratumorally, with orthoPmel ultimately accounting for greater than 80% or 40% of all peripheral and intratumoral T cells, respectively. Systemic orthoPmel maintained a consistent central memory and effector memory mix throughout the four-week moIL-2 treatment course. moIL-2 also induced the expression of activation markers, CD25 and Granzyme B, in intratumoral orthoPmel. These findings validate than an orthogonal IL-2/IL-2Rβ platform can enhance efficacy of ACTs without peripheral expansion or activation of NK cells or non-tumor specific T cells and the toxicities typically associated with high dose IL-2 therapy. Importantly, these results demonstrate the potential of this platform overcome the requirement of lymphodepletion in adoptive cell therapies. Citation Format: Christina Kochel, Meng Sun, Navneet Ratti, Sandro Vivona, Mahalaksmi Ramadass, Marie Semana, Michele Bauer, Mohammed Ali, Jan Emmerich, Rob Kastelein, Patrick J. Lupardus, Paul-Joseph Aspuria, Martin Oft. Orthogonal IL-2/IL-2RB signaling in adoptively transferred T cells controls tumor growth without the need for lymphodepletion in a B16 tumor model [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 586.

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.