Novel engineered IL-2 Nemvaleukin alfa combined with PD1 checkpoint blockade enhances the systemic anti-tumor responses of radiation therapy.

BACKGROUND: Combining interleukin-2 (IL-2) with radiotherapy (RT) and immune checkpoint blockade (ICB) has emerged as a promising approach to address ICB resistance. However, conventional IL-2 cytokine therapy faces constraints owing to its brief half-life and adverse effects. RDB 1462, the mouse ortholog of Nemvaleukin alfa, is an engineered IL-2 with an intermediate affinity that selectively stimulates antitumor CD8 T and NK cells while limiting regulatory T cell expansion. This study aimed to evaluate the antitumor activity and mechanism of action of the combination of RDB 1462, RT, and anti-PD1 in mouse tumor models. METHODS: Two bilateral lung adenocarcinoma murine models were established using 344SQ-Parental and 344SQ anti-PD1-resistant cell lines. Primary tumors were treated with RT, and secondary tumors were observed for evidence of abscopal effects. We performed immune phenotyping by flow cytometry, analyzed 770 immune-related genes using NanoString, and performed T cell receptor (TCR) repertoire analysis. Serum pro-inflammatory cytokine markers were analyzed by 23-plex kit. RESULTS: Compared to native IL-2 (RDB 1475), RDB 1462 demonstrated superior systemic antitumoral responses, attributable, at least in part, to augmented levels of CD4 and CD8 T cells with the latter. Our findings reveal substantial reductions in primary and secondary tumor volumes compared to monotherapy controls, with some variability observed among different dosing schedules of RDB 1462 combined with RT. Blood and tumor tissue-based flow cytometric phenotyping reveals an increase in effector memory CD8 and CD4 T cells and a decrease in immunosuppressive cells accompanied by a significant increase in IL-2, IFN-γ, and GM-CSF levels in the combination group. Transcriptomic profiling and TCR sequencing reveal favorable gene expression and T cell repertoire patterns with the dual combination. Furthermore, integrating anti-PD1 therapy with RT and RDB 1462 further reduced primary and secondary tumor volumes, prolonged survival, and decreased lung metastasis. Observations of immune cell profiles indicated that RT with escalating doses of RDB 1462 significantly reduced tumor growth and increased tumor-specific immune cell populations. CONCLUSION: The addition of Nemvaleukin therapy may enhance responses to RT alone and in combination with anti-PD1.

IL-2 based cancer immunotherapies: an evolving paradigm.

Discovered over 4 decades ago in the supernatants of activated T cells, interleukin-2 (IL-2) is a potent pleiotropic cytokine involved in the regulation of immune responses. It is required for effector T cell expansion and differentiation as well as for peripheral tolerance induced by regulatory T cells. High-dose IL-2 treatment was the first FDA-approved immunotherapy for renal cell carcinoma and melanoma, achieving single agent complete and durable responses, albeit only in a small proportion of patients. The therapeutic potential of wild type IL-2 is clinically limited by its short half-life and severe vascular toxicity. Moreover, the activation of regulatory T cells and the terminal differentiation of effector T cells on IL-2 pose additional restrictions. To overcome the toxicity of IL-2 in order to realize its full potential for patients, several novel engineering strategies are being developed and IL-2 based immunotherapy for cancer has emerged as a burgeoning field of clinical and experimental research. In addition, combination of IL-2 with PD-1/L1 pathway blockade shows vastly improved anti-tumor efficacy over either monotherapy in preclinical tumor models. In this review we discuss the biological characteristics of IL-2 and its receptors, as well as its efficacy and treatment limiting toxicities in cancer patients. We also explore the efforts aimed at developing novel and safer IL-2 therapies to harness the full therapeutic potential of this cytokine.

Establishment of a protocol for rapidly expanding Epstein-Barr-virus-specific cytotoxic T cells with enhanced cytotoxicity.

BACKGROUND: Lytic Epstein-Barr virus (EBV) infection plays a major role in the pathogenesis of nasopharyngeal carcinoma (NPC). For patients with recurrent or metastatic NPC and resistant to conventional therapies, adoptive cell therapy using EBV-specific cytotoxic T cells (EBV-CTLs) is a promising option. However, the long production period (around 3 to 4 weeks) and low EBV-CTL purity (approximately 40% of total CD8 T cells) in the cell product limits the application of EBV-CTLs in clinics. Thus, this study aimed to establish a protocol for the rapid production of EBV-CTLs. METHODS: By culturing peripheral blood mononuclear cells (PBMCs) from EBV-seropositive donors with EBV-specific peptides and interleukin (IL)-2, IL-15, and interferon α (IFN-α) for 9 days, we identified that IL-15 can enhance IL-2-mediated CTL activation and significantly increase the yield of CTLs. RESULTS: When IFN-α was used in IL-2/IL-15-mediated CTL production from days 0 to 6, the productivity of EBV-CTLs and EBV-specific cytotoxicity significantly were reinforced relative to EBV-CTLs from IL-2/IL-15 treatment. Additionally, IFN-α-induced production improvement of virus-specific CTLs was not only the case for EBV-CTLs but also for cytomegalovirus-specific CTLs. CONCLUSION: We established a novel protocol to rapidly expand highly pure EBV-CTLs from PBMCs, which can produce EBV-CTLs in 9 days and does not require feeder cells during cultivation.

IL-2-armored peptide-major histocompatibility class I bispecific antibodies redirect antiviral effector memory CD8+ T cells to induce potent anti-cancer cytotoxic activity with limited cytokine release.

T cell engagers (TCEs) are becoming an integral class of biological therapeutic owing to their highly potent ability to eradicate cancer cells. Nevertheless, the widespread utility of classical CD3-targeted TCEs has been limited by narrow therapeutic index (TI) linked to systemic CD4+ T cell activation and aberrant cytokine release. One attractive approach to circumvent the systemic activation of pan CD3+ T cells and reduce the risk of cytokine release syndrome is to redirect specific subsets of T cells. A promising strategy is the use of peptide-major histocompatibility class I bispecific antibodies (pMHC-IgGs), which have emerged as an intriguing modality of TCE, based on their ability to selectively redirect highly reactive viral-specific effector memory cytotoxic CD8+ T cells to eliminate cancer cells. However, the relatively low frequency of these effector memory cells in human peripheral blood mononuclear cells (PBMCs) may hamper their redirection as effector cells for clinical applications. To mitigate this potential limitation, we report here the generation of a pMHC-IgG derivative known as guided-pMHC-staging (GPS) carrying a covalent fusion of a monovalent interleukin-2 (IL-2) mutein (H16A, F42A). Using an anti-epidermal growth factor receptor (EGFR) arm as a proof-of-concept, tumor-associated antigen paired with a single-chain HLA-A *02:01/CMVpp65 pMHC fusion moiety, we demonstrate in vitro that the IL-2-armored GPS modality robustly expands CMVpp65-specific CD8+ effector memory T cells and induces potent cytotoxic activity against target cancer cells. Similar to GPS, IL-2-armored GPS molecules induce modulated T cell activation and reduced cytokine release profile compared to an analogous CD3-targeted TCE. In vivo we show that IL-2-armored GPS, but not the corresponding GPS, effectively expands grafted CMVpp65 CD8+ T cells from unstimulated human PBMCs in an NSG mouse model. Lastly, we demonstrate that the IL-2-armored GPS modality exhibits a favorable developability profile and monoclonal antibody-like pharmacokinetic properties in human neonatal Fc receptor transgenic mice. Overall, IL-2-armored GPS represents an attractive approach for treating cancer with the potential for inducing vaccine-like antiviral T cell expansion, immune cell redirection as a TCE, and significantly widened TI due to reduced cytokine release.

IL-2 and TCR stimulation induce expression and secretion of IL-32ß by human T cells.

IL-32 expression is important for pathogen clearance but detrimental in chronic inflammation, autoimmunity, and cancer. T cells are major IL-32 producers in these diseases and key mediators of pathogen and tumor elimination but also autoimmune destruction. However, their contribution to IL-32 biology during immune responses is hardly understood due to several isoforms with divergent inflammatory properties. Here, we identified IL-32ß as the predominant isoform in various T cell subsets of healthy individuals and breast cancer patients with the highest levels detected in intratumoral regulatory T cells. We show that IL-32ß is induced by IL-2 but IL-32ß release requires T Cell Receptor rather than IL2R stimulation. Using inhibitors of protein secretion pathways and serial (ultra)centrifugation of T cell supernatants, we demonstrate that T cells actively secrete IL-32ß unconventionally, as a free protein and, to a minor degree, through exosomes. Thus, our data identify activated T cells as major IL-32ß secretors in health and cancer.

Dissecting the mediating role of inflammatory factors in the interaction between metabolites and sepsis: insights from bidirectional Mendelian randomization.

Sepsis, a life-threatening condition, involves complex interactions among metabolic alterations, inflammatory mediators, and host responses. This study utilized a bidirectional Mendelian randomization approach to investigate the causal relationships between 1400 metabolites and sepsis, and the mediating role of inflammatory factors. We identified 36 metabolites significantly associated with sepsis (p < 0.05), with AXIN1, FGF-19, FGF-23, IL-4, and OSM showing an inverse association, suggesting a protective role, while IL-2 exhibited a positive correlation, indicating a potential risk factor. Among these metabolites, Piperine and 9-Hydroxystearate demonstrated particularly interesting protective effects against sepsis. Piperine's protective effect was mediated through its interaction with AXIN1, contributing to a 16.296% reduction in sepsis risk. This suggests a potential pathway where Piperine influences sepsis outcomes by modulating AXIN1 levels. 9-Hydroxystearate also exhibited a protective role against sepsis, mediated through its positive association with FGF-19 and negative association with IL-2, contributing 9.436% and 12.565%, respectively, to its protective effect. Experimental validation confirmed significantly elevated IL-2 levels and reduced FGF-19, AXIN1, piperine, and 9-hydroxyoctadecanoic acid levels in sepsis patients compared to healthy controls. Piperine levels positively correlated with AXIN1, while 9-hydroxyoctadecanoic acid levels negatively correlated with IL-2 and positively correlated with FGF-19, supporting the Mendelian randomization findings. Our findings provide insights into the molecular mechanisms of sepsis, highlighting the unique roles and contributions of specific metabolites and their interactions with inflammatory mediators. This study enhances our understanding of sepsis pathophysiology and opens avenues for targeted therapeutic interventions and biomarker development for sepsis management. However, further research is essential to validate these pathways across diverse populations and fully explore the roles of these metabolites in sepsis.

Effect of pseudorabies virus infection on NMDA receptor expression in mice and its role in immunosuppression.

Pseudorabies virus (PRV), an α-herpesvirus, induces immunosuppression and can lead to severe neurological diseases. N-methyl-D-aspartate receptor (NMDAR), an important excitatory nerve receptor in the central nervous system, is linked to various nervous system pathologies. The link between NMDAR and PRV-induced neurological diseases has not been studied. In vivo studies revealed that PRV infection triggers a reduction in hippocampal NMDAR expression, mediated by inflammatory processes. Extensive hippocampal neuronal degeneration was found in mice on the 6th day by hematoxylin-eosin staining, which was strongly correlated with increased NMDAR protein expression. In vitro studies utilizing the CCK-8 assay demonstrated that treatment with an NMDAR antagonist significantly heightened the cytotoxic effects of PRV on T lymphocytes. Notably, NMDAR inhibition did not affect the replication ability of PRV. However, it facilitated the accumulation of pro-inflammatory cytokines in PRV-infected T cells and enhanced the transcription of the CD25 gene through the secretion of interleukin-2 (IL-2), consequently exacerbating immunosuppression. In this study, we found that NMDAR has functional activity in T lymphocytes and is crucial for the inflammatory and immune responses triggered by PRV infection. These discoveries highlight the significant role of NMDAR in PRV-induced neurological disease pathogenesis.

Tyrosine phosphorylation of both STAT5A and STAT5B is necessary for maximal IL-2 signaling and T cell proliferation.

Cytokine-mediated STAT5 protein activation is vital for lymphocyte development and function. In vitro tyrosine phosphorylation of a C-terminal tyrosine is critical for activation of STAT5A and STAT5B; however, the importance of STAT5 tyrosine phosphorylation in vivo has not been assessed. Here we generate Stat5a and Stat5b tyrosine-to-phenylalanine mutant knockin mice and find they have greatly reduced CD8+ T-cell numbers and profoundly diminished IL-2-induced proliferation of these cells, and this correlates with reduced induction of Myc, pRB, a range of cyclins and CDKs, and a partial G1→S phase-transition block. These mutant CD8+ T cells also exhibit decreased IL-2-mediated activation of pERK and pAKT, which we attribute in part to diminished expression of IL-2Rß and IL-2Rγ. Our findings thus demonstrate that tyrosine phosphorylation of both STAT5A and STAT5B is essential for maximal IL-2 signaling. Moreover, our transcriptomic and proteomic analyses elucidate the molecular basis of the IL-2-induced proliferation of CD8+ T cells.

Lymphotoxin limits Foxp3+ regulatory T cell development from Foxp3lo precursors via IL-4 signaling.

Regulatory T cells (Treg) are critical players of immune tolerance that develop in the thymus via two distinct developmental pathways involving CD25+Foxp3- and CD25-Foxp3lo precursors. However, the mechanisms regulating the recently identified Foxp3lo precursor pathway remain unclear. Here, we find that the membrane-bound lymphotoxin α1ß2 (LTα1ß2) heterocomplex is upregulated during Treg development upon TCR/CD28 and IL-2 stimulation. We show that Lta expression limits the maturational development of Treg from Foxp3lo precursors by regulating their proliferation, survival, and metabolic profile. Transgenic reporter mice and transcriptomic analyses further reveal that medullary thymic epithelial cells (mTEC) constitute an unexpected source of IL-4. We demonstrate that LTα1ß2-lymphotoxin ß receptor-mediated interactions with mTEC limit Treg development by down-regulating IL-4 expression in mTEC. Collectively, our findings identify the lymphotoxin axis as the first inhibitory checkpoint of thymic Treg development that fine-tunes the Foxp3lo Treg precursor pathway by limiting IL-4 availability.

Nanoparticles loaded with IL-2 and TGF-ß promote transplantation tolerance to alloantigen.

We have previously reported that nanoparticles (NPs) loaded with IL-2 and TGF-ß and targeted to T cells induced polyclonal T regulatory cells (Tregs) that protected mice from graft-versus-host disease (GvHD). Here, we evaluated whether administration of these NPs during alloantigen immunization could prevent allograft rejection by converting immunogenic responses to tolerogenic ones. Using C57BL/6 mice and BALB/c mice as either donors or recipients of allogeneic splenocytes, we found that treatment with the tolerogenic NPs in both strains of mice resulted in a marked inhibition of mixed lymphocyte reaction (MLR) to donor cell alloantigen but not to third-party control mouse cells after transfer of the allogeneic cells. The decreased alloreactivity associated with a four- to fivefold increase in the number of CD4+ and CD8+ T regulatory cells (Tregs) and the acquisition of a tolerogenic phenotype by recipient dendritic cells (DCs) in NP-treated mice. As allogeneic cells persisted in NP-treated mice, these findings suggest that tolerogenic NPs can induce alloantigen-specific Tregs and tolerogenic DCs promoting tolerogenic responses to alloantigen. By inhibiting reactivity to allotransplant, this approach could help reduce the need for immune suppression for the maintenance of allografts.

Phase I Study of Simlukafusp Alfa (FAP-IL2v) with or without Atezolizumab in Japanese Patients with Advanced Solid Tumors.

PURPOSE: The aim of the study was to evaluate the safety/tolerability and pharmacokinetics of simlukafusp alfa (FAP-IL2v), an immunocytokine containing an anti-fibroblast activation protein-α (FAP) antibody and an IL2 variant, administered alone or with the PDL1 inhibitor atezolizumab, in Japanese patients with advanced solid tumors. PATIENTS AND METHODS: In this phase 1, open-label, dose-escalation study, patients received i.v. FAP-IL2v at 10 or 15/20 mg alone or 10 mg when combined with i.v. atezolizumab. The primary objectives were identification of dose-limiting toxicities (DLT), recommended dose, and maximum tolerated dose, and evaluation of the safety/tolerability and pharmacokinetics of FAP-IL2v alone and combined with atezolizumab. RESULTS: All 11 patients experienced adverse events (AE) during FAP-IL2v treatment. Although most AEs were of mild severity, four treatment-related AEs led to study treatment discontinuation in two patients: one with infusion-related reaction, hypotension, and capillary leak syndrome, and the other with increased aspartate aminotransferase. No AE-related deaths occurred. One DLT (grade 3 hypotension) occurred in a patient receiving FAP-IL2v 15/20 mg alone. The recommended dose and maximum tolerated dose could not be determined. The pharmacokinetics of FAP-IL2v remained similar with or without atezolizumab. The study was terminated early as FAP-IL2v development was discontinued because of portfolio prioritization (not for efficacy/safety reasons). CONCLUSIONS: This study describes the safety/tolerability of FAP-IL2v 10 mg alone and in combination with atezolizumab in Japanese patients with advanced solid tumors; one DLT (hypotension) occurred with FAP-IL2v 15/20 mg. However, dose escalation of FAP-IL2v was not conducted because of early study termination. SIGNIFICANCE: This phase I study assessed the safety/tolerability and PK of simlukafusp alfa alone or combined with atezolizumab in Japanese patients with advanced solid tumors. No notable differences in PK were noted with the combination versus simlukafusp alfa alone; however, high-dose simlukafusp alfa treatment was associated with recombinant IL2-related toxicity, despite the drug's FAP targeting and IL2Rßγ-biased IL2 variant design.

Development of a highly sensitive platform for protein-protein interaction detection and regulation of T cell function.

We developed a highly sensitive assay for detecting protein-protein interaction using chimeric receptors comprising two molecules of interest in the extracellular domain and interferon alpha and beta receptor subunit 1 or 2 (IFNAR1/2) in the intracellular domain. This intracellular IFNAR1/2 reconstitution system (IFNARRS) proved markedly more sensitive than the NanoBiT system, currently considered one of the best detection systems for protein interaction. Employing chimeric receptors with extracellular domains from the IFNγ or IL-2 receptor and the intracellular domains of IFNAR1/2, the IFNARRS system effectively identifies low IFNγ or IL-2 levels. Cells stably expressing these chimeric receptors responded to IFNγ secreted by activated T cells following various stimuli, including a specific peptide-antigen. The activation signals were further enhanced by the expression of relevant genes, such as costimulators, via IFN-stimulated response elements in the promoters. Besides IFNγ or IL-2, the IFNARRS system demonstrated the capability to detect other cytokines by using the corresponding extracellular domains from these target cytokine receptors.

A Critical Role of Culture Medium Selection in Maximizing the Purity and Expansion of Natural Killer Cells.

Natural killer (NK) cells hold promise in cancer treatment due to their ability to spontaneously lyse cancer cells. For clinical use, high quantities of pure, functional NK cells are necessary. Combining adherence-based isolation with specialized media showed the unreliability of the isolation method, but demonstrated the superiority of the NK MACS® medium, particularly in suboptimal conditions. Neither human pooled serum, fetal calf serum (FCS), human platelet lysate, nor chemically defined serum replacement could substitute human AB serum. Interleukin (IL-)2, IL-15, IL-21, and combined CD2/NKp46 stimulation were assessed. IL-21 and CD2/NKp46 stimulation increased cytotoxicity, but reduced NK cell proliferation. IL-15 stimulation alone achieved the highest proliferation, but the more affordable IL-2 performed similarly. The RosetteSep™ human NK cell enrichment kit was effective for isolation, but the presence of peripheral blood mononuclear cells (PBMCs) in the culture enhanced NK cell proliferation, despite similar expression levels of CD16, NKp46, NKG2D, and ICAM-1. In line with this, purified NK cells cultured in NK MACS® medium with human AB serum and IL-2 demonstrated high cytotoxicity against primary glioblastoma stem cells.

Retrospective analysis of the clinical utility of multi-cytokine profiles in smear-negative pulmonary tuberculosis.

OBJECTIVES: To evaluate cytokine profiles and interferon-gamma release assay (IGRA) for their diagnostic capabilities in the differentiation of tuberculosis (TB) from non-TB conditions, as well as smear-negative pulmonary tuberculosis (SNPT) from smear-positive pulmonary tuberculosis (SPPT). METHODS: A total of 125 participants were included, 77 of whom had TB and 48 who didn't, and demographic, clinical, and laboratory data were collected, including cytokine levels and IGRA results. The TB patients were further divided into 2 subgroups: SNPT (n=42) and SPPT (n=35). RESULTS: Compared to non-TB, the TB group had lower BMI, higher WBC, neutrophils, monocytes, ESR and CRP (p<0.05). TB patients showed higher IL-2, IL-6, IFN-γ, IL-8 (p<0.001) and higher IGRA positivity (88.3% versus [vs.] 29.2%, p<0.001). Between SNPT and SPPT, moderate effect sizes were observed for IFN-α, IL-2, IL-10, IL-8 (Cohen's d 0.59-0.76), with lower IGRA positivity in SNPT (81.0% vs. 97.1%, p=0.015). ROC analysis indicated IFN-α, IL-2, IL-10, IL-8 had moderate accuracy for SNPT diagnosis (AUCs 0.668-0.734), and combining these improved accuracy (AUC 0.759, 80% sensitivity, 64.2% specificity). CONCLUSION: A multi-biomarker approach combining these cytokines demonstrates enhanced diagnostic accuracy for tuberculosis.

Older age is associated with a distinct and marked reduction of functionality of both alloreactive CD4+ and CD8+ T cells.

Introduction: Older recipient age is associated with a significant decreased risk for rejection after kidney transplantation which is incompletely understood. Methods: In a longitudinal study, circulating alloreactive T cells were assessed of young (≤45 years) and older (≥55 years) stable kidney transplant recipients. Alloreactive T-cells were identified by CD137-expression and phenotype, cytokine producing and proliferative capacity, were evaluated using multiparameter flowcytometry. Results: The results show that before transplantation frequencies of alloreactive CD4+ and CD8+ T-cells in older KT-recipients are significantly higher and shifted towards an effector memory-phenotype. However, the frequency of polyfunctional (≥2 pro-inflammatory cytokines) CD4+ T-cells was significantly lower and less IL2 was produced. The frequency of polyfunctional alloreactive CD4+ T-cells and proliferation of alloreactive T-cells donor-specifically declined after transplantation reaching a nadir at 12 months after transplantation, irrespective of age. A striking difference was observed for the proliferative response of alloreactive CD8+ T-cells. This was not only lower in older compared to younger recipients but could also not be restored by exogenous IL2 or IL15 in the majority of older recipients while the response to polyclonal stimulation was unaffected. Conclusion: In conclusion, older age is associated with a distinct and marked reduction of functionality of both alloreactive CD4+ and CD8+ T-cells.

Targeted Bias: The Next Swing at IL2 Therapy.

Despite its long history of toxicity and limited efficacy, IL2 has re-entered the clinic as a companion to the recently FDA-approved tumor infiltrating lymphocyte therapy. In back-to-back articles, Moynihan and Kaptein introduce a new fusion protein that delivers a biased IL2 mutein to CD8 T cells. See related article by Moynihan et al., p. 1206 (6). See related article by Kaptein et al., p. 1226 (7).

Effect of Short Peptides of Pregnancy-Specific ß1-Glycoprotein on Differentiation of Human Regulatory T Cells In Vitro and on Their Cytokine Profile.

Pregnancy-specific ß1-glycoprotein (PSG), one of the most important proteins of pregnancy, has a pronounced immunosuppressive effect. Short peptides of PSG, the so-called SLiMs (short linear motifs), are promising molecules for mild immunosuppression. We studied in vitro effect of short PSG peptides (YACS, YQCE, YVCS, and YECE) on differentiation and cytokine profile of human T-regulatory lymphocytes (Treg). T helpers isolated from the peripheral blood and polarized into the Treg phenotype with a T-cell activator (anti-CD2/3/28) and the cytokines IL-2 and transforming grown factor ß (TGFß) were used. PSG peptides were shown to have no direct modulatory effect on Treg differentiation in a culture of CD4+ cells polarized to the Treg phenotype. At the same time, PSG peptides had no effect on the viability and number of CD4+ cells in the in vitro culture. PSG peptides also had no effect on the levels of TNFα, IL-8, IL-2, macrophage inflammatory protein 1ß, IL-17, IL-10, IL-6, granulocyte-macrophage CSF, monocyte chemoattractant protein 1, IL-13, IL-5, IL-7, IL-12(p70), IL-1ß, granulocyte CSF, IL-4, but decreased IFNγ levels. The observed ability of the YQCE peptide to reduce the production of this proinflammatory Th1 cytokine by T helper cells can be interpreted as a positive effect. Our findings can be used for further development of safe peptide drugs based on SLiMs sequences.