Supplemental Figure S5. IL-18BP blockade alters the immune infiltrate composition of E0771 tumors.

Supplemental Figure S4. Anti-IL-18BP Ab induced immunogenic memory response in E0771 tumor model.

Supplemental Figure S3. Antitumor activity of anti-mouse-IL-18BP Ab in mice studies.

Supplemental Figure S1. IL-18BP is a potential soluble immune checkpoint in cancer.

Supplemental Figure S2. COM503 releases IL-18 activity in biochemical and functional assays.

Abstract Recombinant cytokines have limited anticancer efficacy mostly due to a narrow therapeutic window and systemic adverse effects. IL18 is an inflammasome-induced proinflammatory cytokine, which enhances T- and NK-cell activity and stimulates IFNγ production. The activity of IL18 is naturally blocked by a high-affinity endogenous binding protein (IL18BP). IL18BP is induced in the tumor microenvironment (TME) in response to IFNγ upregulation in a negative feedback mechanism. In this study, we found that IL18 is upregulated in the TME compared with the periphery across multiple human tumors and most of it is bound to IL18BP. Bound IL18 levels were largely above the amount required for T-cell activation in vitro, implying that releasing IL18 in the TME could lead to potent T-cell activation. To restore the activity of endogenous IL18, we generated COM503, a high-affinity anti-IL18BP that blocks the IL18BP:IL18 interaction and displaces precomplexed IL18, thereby enhancing T- and NK-cell activation. In vivo, administration of a surrogate anti-IL18BP, either alone or in combination with anti-PD-L1, resulted in significant tumor growth inhibition and increased survival across multiple mouse tumor models. Moreover, the anti-IL18BP induced pronounced TME-localized immune modulation including an increase in polyfunctional nonexhausted T- and NK-cell numbers and activation. In contrast, no increase in inflammatory cytokines and lymphocyte numbers or activation state was observed in serum and spleen. Taken together, blocking IL18BP using an Ab is a promising approach to harness cytokine biology for the treatment of cancer.

Abstract IL-18 is an inflammasome induced proinflammatory cytokine that augments T and NK cell activity and stimulates IFNγ production. The activity of IL-18 is naturally blocked by a high affinity endogenous binding protein (IL-18BP). IL-18BP is induced in the tumor microenvironment (TME) in response to IFNγ upregulation in a negative feedback mechanism. By evaluating 88 human tumor specimens and serum samples we were able to show that IL-18 is upregulated in the TME (median 11.2ng/gr) compared to serum samples (median 0.3ng/ml). Moreover, we showed that most of the IL-18 is bound by IL-18BP. IL-18BP-bound IL-18 levels were largely above the amount required for T cell activation in vitro (1.2ng/gr), implying that blocking IL-18BP has the potential to release IL-18 in tumors above the minimum range required for immune system stimulation. Next, to assess whether tumor endogenous IL-18 levels released by IL-18BP blockade are sufficient to provoke anti-tumor responses, COM503, a high affinity (<1pM) anti-IL-18BP Ab, was generated and examined in T and NK cell-based assays. In a co-culture assay of tumor cells with ex-vivo stimulated human tumor infiltrating CD8+ lymphocytes, COM503 was able to displace IL-18 from a pre-formed complex and enhance IFNγ (197%, p<0.01) and TNFα (84% p<0.01) secretion. Additionally, COM503 induced human NK cell activation as reflected by increased IFNγ secretion (26-fold, p<0.001). Finally, in human ex vivo dissociated tumor cells assay, COM503 mediated an increase in Granzyme B (25%), IFNγ (38%), TNFα (58%) and IL-12 (50%) production. In vivo, administration of an anti-mouse IL-18BP Ab resulted in potent anti-tumor responses and increased survival across multiple mouse tumor models. In orthotopic E0771 tumor model, anti-IL-18BP Ab induced significant tumor growth inhibition (91% TGI, p<0.0001), as well as pronounced TME-localized immune modulation. This modulation included an increase in CD8+ T cells expansion (108.5%, p=0.015) and activation, specifically in polyfunctional effector IFNγ+GrB+CD8+ T cells (259%, p=0.02) and IFNγ+TNFα+NK cells (77%, p=0.001). Similarly, anti-tumor effects were shown in MC38OVAdim model (58% TGI, p<0.001), accompanied by a robust TME-localized immune modulation including increased CD8+ T cells expansion (85%, p=0.009) and IFNγ secretion (76%, p=0.052). In contrast to immune modulation in the TME, no increase in inflammatory cytokines and lymphocyte numbers or activation state was observed in serum and spleen. Taken together, our data suggest that IL-18 is upregulated in the TME, mostly bound by IL-18BP, and could be exploited to induce pronounced TME-localized immune modulation. Anti-IL-18BP Ab approach has a leading edge in inhibiting tumor growth while avoiding peripheral toxicity associated with administration of a cytokine. COM503 is currently undergoing IND-enabling studies. Citation Format: Assaf Menachem, Zoya Alteber, Gady Cojocaru, Tal Fridman Kfir, Dan Blat, Olga Leiderman, Moran Galperin, Lital Sever, Nadav Cohen, Keren Cohen, Roy Zvi Granit, Sandra Vols, Masha Frenkel, Lior Faigenbloom, Liron Soffer, Karin Meyer, Keren Menachem, Hadas Galon Tilleman, Dina Morein, Itamar Borukhov, Amir Toporik, Michal Perpinial Shahor, Evgeny Tatirovsky, Pierre Ferre, Eran Ophir. Unleashing natural IL-18 activity using an anti-IL-18BP blocker antibody induces potent immune stimulation and anti-tumor effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4072.

List of 145 differentially-expressed genes in PVRIG-knockout vs wild-type CD8 TILs (day 18 post-implantation).

Supplementary Figures and Figure Legends

Background IL-18 is an inflammasome-induced proinflammatory cytokine that activates T and NK cells and stimulates IFNg production.1 2 The activity of IL-18 is naturally blocked by a high affinity endogenous binding-protein (IL-18BP)1 induced in response to IFNg upregulation as a negative feedback mechanism.3 Methods By assessing total and free IL-18 we examined whether bound-IL-18 levels in the tumor are above the level required for in-vitro human T-cell activation. To unleash endogenous bound IL-18 activity, COM503, an anti-IL-18BP blocker Ab, was generated and examined in T cell-based assays. In-vivo, IL-18BP blockade was evaluated in multiple mouse tumor models. Tumor microenvironment (TME) modulation was assessed by flow cytometry, scRNA sequencing and cytokine profiling. Results IL-18 levels were clearly elevated across 75 evaluated tumors compared to serum samples (figure 1A-B). Results show that most of tumor IL-18 was bound to IL-18BP, and its levels were above the amount required for T-cell activation in-vitro, implying that releasing tumor IL-18 locally could lead to T cell activation (figure 1C-D). By displacing IL-18 from IL-18:IL-18BP complex, COM503 was shown to enhance T-cell activation in an ex-vivo stimulated human CD8+ tumor infiltrating lymphocytes-tumor cells co-culture assay and in human dissociated tumor cells assay (figure 2). In addition, tumor growth inhibition was observed by anti-mouse IL-18BP Ab in multiple tumor models either alone or in combination with anti-PD-L1 (figure 3). Furthermore, in E0771 model, IL-18BP blockade induced significant increase in functional immune-cells and striking changes in clusters of lymphocytes, including a decrease in naïve T-cells and an increase in effector T-cells and T cell clonal expansion (figure 4). IL-18BP blockade also increased pro-inflammatory cytokine secretion and skewed cell populations of myeloid lineage to favor proinflammatory macrophages (figure 5). In MC38OVAdim model both TME and periphery were evaluated. Anti-mouse IL-18BP Ab induced potent TME immune-modulation, including increased CD8+ T-cell infiltration and IFNg secretion, while no increase of IFNg secretion, lymphocytes number or activation state was evident in the periphery (figure 6). Conclusions IL-18 is upregulated in human tumors and is mostly bound by IL-18BP. COM503, a high-affinity anti-IL-18BP Ab, induces human T-cell responses in-vitro and ex-vivo. An anti-mouse IL-18BP Ab induces potent anti-tumor responses and pronounced TME-constrained immune modulation, this in contrast to systemically administered therapeutic cytokines, which can generate systemic inflammatory responses (figure 7).4 Taken together, blocking IL-18BP is a promising novel approach to harness cytokine potency for the treatment of cancer. COM503 is currently undergoing IND-enabling studies. References Dinarello CA, Novick D, Kim S, Kaplanski G. Interleukin-18 and IL-18 binding protein. Front Immunol. 2013 Oct 8;4:289. Swain SL. Interleukin 18: Tipping the Balance towards a T Helper Cell 1 Response. J Exp Med. 2001 Aug 6;194(3):F11–4. Paulukat J, Bosmann M, Nold M, Garkisch S, Kämpfer H, Frank S, Raedle J, Zeuzem S, Pfeilschifter J, Mühl H. Expression and release of IL-18 binding protein in response to IFN-gamma. J Immunol Baltim Md 1950. 2001 Dec 15;167(12):7038–43. Propper DJ, Balkwill FR. Harnessing cytokines and chemokines for cancer therapy. Nat Rev Clin Oncol. 2022 Apr;19(4):237–53. Ethics Approval Mouse studies : Studies were approved by the Animal Care and Use Committee at Tel Aviv University; approval number 01–22-001 Human biopsies: This study was approved by Israel Institutional Review Board (IBR) ; approval number 0827–20RMC, 0711–15RMC, 0333–17-RMB-GN

AbstractA limitation to antitumor immunity is the dysfunction of T cells in the tumor microenvironment, in part due to upregulation of coinhibitory receptors such as PD-1. Here, we describe that poliovirus receptor–related immunoglobulin domain protein (PVRIG) acts as a coinhibitory receptor in mice. Murine PVRIG interacted weakly with poliovirus receptor (PVR) but bound poliovirus receptor–like 2 (PVRL2) strongly, making the latter its principal ligand. As in humans, murine NK and NKT cells constitutively expressed PVRIG. However, when compared with humans, less PVRIG transcript and surface protein was detected in murine CD8+ T cells ex vivo. However, activated CD8+ T cells upregulated PVRIG expression. In the mouse tumor microenvironment, infiltrating CD8+ T cells expressed PVRIG whereas its ligand, PVRL2, was detected predominantly on myeloid cells and tumor cells, mirroring the expression pattern in human tumors. PVRIG-deficient mouse CD8+ T cells mounted a stronger antigen-specific effector response compared with wild-type CD8+ T cells during acute Listeria monocytogenes infection. Furthermore, enhanced CD8+ T-cell effector function inhibited tumor growth in PVRIG−/− mice compared with wild-type mice and PD-L1 blockade conferred a synergistic antitumor response in PVRIG−/− mice. Therapeutic intervention with antagonistic anti-PVRIG in combination with anti–PD-L1 reduced tumor growth. Taken together, our results suggest PVRIG is an inducible checkpoint receptor and that targeting PVRIG–PVRL2 interactions results in increased CD8+ T-cell function and reduced tumor growth.See related article on p. 257

Purpose of review The mammalian mucosal surfaces are densely inhabited by a diverse microbial ecosystem termed the microbiota. Among these highly heterogeneous populations, the largest and richest is the gut microbiota, recently suggested to affect various physiological traits and susceptibility to disease. Novel metagenomic and metabolomic approaches, which have been developed in the past decade, have enabled the elucidation of the contribution of the microbiota to metabolic, immunologic, neurologic and endocrine homeostasis. Recent findings Dysbiosis, the alteration in the gut microbiota composition and function, has been lately associated with the pathogenesis of multifactorial diseases such as obesity, diabetes and cardiovascular disorders. Recent studies have also suggested associations between dysbiosis and essential hypertension, a common chronic medical condition affecting 20% or more of the adult population worldwide, which is considered a major causative factor for heart disease, stroke, chronic renal failure, blindness and dementia. Summary In this review, we discuss the accumulating research pointing to possible interplays between the gut microbiome and hypertension and highlight future prospects by which utilization of microbiome-related techniques may be incorporated into the diagnosis and therapeutic arsenal of hypertension management.

Purpose of review The mammalian mucosal surfaces are densely inhabited by a diverse microbial ecosystem termed the microbiota. Among these highly heterogeneous populations, the largest and richest is the gut microbiota, recently suggested to affect various physiological traits and susceptibility to disease. Novel metagenomic and metabolomic approaches, which have been developed in the past decade, have enabled the elucidation of the contribution of the microbiota to metabolic, immunologic, neurologic and endocrine homeostasis.

The intestinal microbiota undergoes diurnal compositional and functional oscillations that affect metabolic homeostasis, but the mechanisms by which the rhythmic microbiota influences host circadian activity remain elusive. Using integrated multi-omics and imaging approaches, we demonstrate that the gut microbiota features oscillating biogeographical localization and metabolome patterns that determine the rhythmic exposure of the intestinal epithelium to different bacterial species and their metabolites over the course of a day. This diurnal microbial behavior drives, in turn, the global programming of the host circadian transcriptional, epigenetic, and metabolite oscillations. Surprisingly, disruption of homeostatic microbiome rhythmicity not only abrogates normal chromatin and transcriptional oscillations of the host, but also incites genome-wide de novo oscillations in both intestine and liver, thereby impacting diurnal fluctuations of host physiology and disease susceptibility. As such, the rhythmic biogeography and metabolome of the intestinal microbiota regulates the temporal organization and functional outcome of host transcriptional and epigenetic programs.