Background Oral fingolimod, a sphingosine-1-phosphate–receptor modulator that prevents the egress of lymphocytes from lymph nodes, significantly improved relapse rates and end points measured on magnetic resonance imaging (MRI), as compared with either placebo or intramuscular interferon beta-1a, in phase 2 and 3 studies of multiple sclerosis. Methods In our 24-month, double-blind, randomized study, we enrolled patients who had relapsing–remitting multiple sclerosis, were 18 to 55 years of age, had a score of 0 to 5.5 on the Expanded Disability Status Scale (which ranges from 0 to 10, with higher scores indicating greater disability), and had had one or more relapses in the previous year or two or more in the previous 2 years. Patients received oral fingolimod at a dose of 0.5 mg or 1.25 mg daily or placebo. End points included the annualized relapse rate (the primary end point) and the time to disability progression (a secondary end point). Results A total of 1033 of the 1272 patients (81.2%) completed the study. The annualized relapse rate was 0.18 with 0.5 mg of fingolimod, 0.16 with 1.25 mg of fingolimod, and 0.40 with placebo (P<0.001 for either dose vs. placebo). Fingolimod at doses of 0.5 mg and 1.25 mg significantly reduced the risk of disability progression over the 24-month period (hazard ratio, 0.70 and 0.68, respectively; P = 0.02 vs. placebo, for both comparisons). The cumulative probability of disability progression (confirmed after 3 months) was 17.7% with 0.5 mg of fingolimod, 16.6% with 1.25 mg of fingolimod, and 24.1% with placebo. Both fingolimod doses were superior to placebo with regard to MRIrelated measures (number of new or enlarged lesions on T2 -weighted images, gadolinium-enhancing lesions, and brain-volume loss; P<0.001 for all comparisons at 24 months). Causes of study discontinuation and adverse events related to fingolimod included bradycardia and atrioventricular conduction block at the time of fingolimod initiation, macular edema, elevated liver-enzyme levels, and mild hypertension. Conclusions As compared with placebo, both doses of oral fingolimod improved the relapse rate, the risk of disability progression, and end points on MRI. These benefits will need to be weighed against possible long-term risks. (ClinicalTrials.gov number, NCT00289978.)

/pdf/a-placebo-controlled-trial-of-oral-fingolimod-in-relapsing-5bkxc6z0wg.pdf

A Placebo-Controlled Trial of Oral Fingolimod in Relapsing Multiple Sclerosis

BACKGROUND: Fingolimod (FTY720), a sphingosine-1-phosphate-receptor modulator that prevents lymphocyte egress from lymph nodes, showed clinical efficacy and improvement on imaging in a phase 2 study involving patients with multiple sclerosis. METHODS: In this 12-month, double-blind, double-dummy study, we randomly assigned 1292 patients with relapsing-remitting multiple sclerosis who had a recent history of at least one relapse to receive either oral fingolimod at a daily dose of either 1.25 or 0.5 mg or intramuscular interferon beta-1a (an established therapy for multiple sclerosis) at a weekly dose of 30 microg. The primary end point was the annualized relapse rate. Key secondary end points were the number of new or enlarged lesions on T(2)-weighted magnetic resonance imaging (MRI) scans at 12 months and progression of disability that was sustained for at least 3 months. RESULTS: A total of 1153 patients (89%) completed the study. The annualized relapse rate was significantly lower in both groups receiving fingolimod--0.20 (95% confidence interval [CI], 0.16 to 0.26) in the 1.25-mg group and 0.16 (95% CI, 0.12 to 0.21) in the 0.5-mg group--than in the interferon group (0.33; 95% CI, 0.26 to 0.42; P<0.001 for both comparisons). MRI findings supported the primary results. No significant differences were seen among the study groups with respect to progression of disability. Two fatal infections occurred in the group that received the 1.25-mg dose of fingolimod: disseminated primary varicella zoster and herpes simplex encephalitis. Other adverse events among patients receiving fingolimod were nonfatal herpesvirus infections, bradycardia and atrioventricular block, hypertension, macular edema, skin cancer, and elevated liver-enzyme levels. CONCLUSIONS: This trial showed the superior efficacy of oral fingolimod with respect to relapse rates and MRI outcomes in patients with multiple sclerosis, as compared with intramuscular interferon beta-1a. Longer studies are needed to assess the safety and efficacy of treatment beyond 1 year. (ClinicalTrials.gov number, NCT00340834.)

/pdf/oral-fingolimod-or-intramuscular-interferon-for-relapsing-6qpjeda41x.pdf

Oral fingolimod or intramuscular interferon for relapsing multiple sclerosis.

The spleen combines the innate and adaptive immune system in a uniquely organized way. The structure of the spleen enables it to remove older erythrocytes from the circulation and leads to the efficient removal of blood-borne microorganisms and cellular debris. This function, in combination with a highly organized lymphoid compartment, makes the spleen the most important organ for antibacterial and antifungal immune reactivity. A better understanding of the function of this complex organ has been gained from recent studies, as outlined in this Review article.

Structure and function of the spleen.

The Janus kinases (JAKs) and signal transducer and activator of transcription (STAT) proteins, particularly STAT3, are among the most promising new targets for cancer therapy. In addition to interleukin-6 (IL-6) and its family members, multiple pathways, including G-protein-coupled receptors (GPCRs), Toll-like receptors (TLRs) and microRNAs were recently identified to regulate JAK-STAT signalling in cancer. Well known for its role in tumour cell proliferation, survival, invasion and immunosuppression, JAK-STAT3 signalling also promotes cancer through inflammation, obesity, stem cells and the pre-metastatic niche. In addition to its established role as a transcription factor in cancer, STAT3 regulates mitochondrion functions, as well as gene expression through epigenetic mechanisms. Newly identified regulators and functions of JAK-STAT3 in tumours are important targets for potential therapeutic strategies in the treatment of cancer.

Revisiting STAT3 signalling in cancer: new and unexpected biological functions

The burgeoning field of leukocyte trafficking has created new and exciting opportunities in the clinic. Trafficking signals are being defined that finely control the movement of distinct subsets of immune cells into and out of specific tissues. Because the accumulation of leukocytes in tissues contributes to a wide variety of diseases, these 'molecular codes' have provided new targets for inhibiting tissue-specific inflammation, which have been confirmed in the clinic. However, immune cell migration is also critically important for the delivery of protective immune responses to tissues. Thus, the challenge for the future will be to identify the trafficking molecules that will most specifically inhibit the key subsets of cells that drive disease processes without affecting the migration and function of leukocytes required for protective immunity.

/pdf/immune-cell-migration-in-inflammation-present-and-future-3pllfoofal.pdf

Immune cell migration in inflammation: present and future therapeutic targets

Adaptive immunity depends on T-cell exit from the thymus and T and B cells travelling between secondary lymphoid organs to survey for antigens. After activation in lymphoid organs, T cells must again return to circulation to reach sites of infection; however, the mechanisms regulating lymphoid organ exit are unknown. An immunosuppressant drug, FTY720, inhibits lymphocyte emigration from lymphoid organs, and phosphorylated FTY720 binds and activates four of the five known sphingosine-1-phosphate (S1P) receptors1,2,3,4. However, the role of S1P receptors in normal immune cell trafficking is unclear. Here we show that in mice whose haematopoietic cells lack a single S1P receptor (S1P1; also known as Edg1) there are no T cells in the periphery because mature T cells are unable to exit the thymus. Although B cells are present in peripheral lymphoid organs, they are severely deficient in blood and lymph. Adoptive cell transfer experiments establish an intrinsic requirement for S1P1 in T and B cells for lymphoid organ egress. Furthermore, S1P1-dependent chemotactic responsiveness is strongly upregulated in T-cell development before exit from the thymus, whereas S1P1 is downregulated during peripheral lymphocyte activation, and this is associated with retention in lymphoid organs. We find that FTY720 treatment downregulates S1P1, creating a temporary pharmacological S1P1-null state in lymphocytes, providing an explanation for the mechanism of FTY720-induced lymphocyte sequestration. These findings establish that S1P1 is essential for lymphocyte recirculation and that it regulates egress from both thymus and peripheral lymphoid organs.

Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1

The splenic marginal zone is a site of blood flow, and the specialized B cell population that inhabits this compartment has been linked to the capture and follicular delivery of blood-borne antigens. However, the mechanism of this antigen transport has remained unknown. Here we show that marginal zone B cells were not confined to the marginal zone but continuously shuttled between the marginal zone and follicular areas, such that many of the cells visited a follicle every few hours. Migration to the follicle required the chemokine receptor CXCR5, whereas return to the marginal zone was promoted by the sphingosine 1-phosphate receptors S1P1 and S1P3. Treatment with an S1P1 antagonist caused displacement of marginal zone B cells from the marginal zone. Marginal zone-follicle shuttling of marginal zone B cells provides an efficient mechanism for systemic antigen capture and delivery to follicular dendritic cells.

/pdf/follicular-shuttling-of-marginal-zone-b-cells-facilitates-48qpti9kji.pdf

Follicular shuttling of marginal zone B cells facilitates antigen transport.

The factors directing marginal zone B cells to the splenic marginal zone are not well understood. Here we report that FTY720, a drug that targets sphingosine 1-phosphate (S1P) receptors, induced marginal zone B cell migration into follicles. Marginal zone B cells expressed S1P receptors 1 and 3 (S1P(1) and S1P(3), respectively). Using gene-targeted mice, we show that S1P(1) but not S1P(3) was required for localization in the marginal zone. In mice lacking the chemokine CXCL13, S1P(1)-deficient marginal zone B cells reacquired a marginal zone distribution. Exposure to lipopolysaccharide or antigen caused marginal zone B cells to downregulate S1P(1) and S1P(3) and to migrate into the splenic white pulp. These data suggest that marginal zone B cell localization to the marginal zone depends on responsiveness to the blood lysophospholipid S1P, with S1P(1) signaling overcoming the recruiting activity of CXCL13.

Sphingosine 1-phosphate receptor 1 promotes B cell localization in the splenic marginal zone

Abstract The poliovirus receptor (PVR, CD155) represents a resistance mechanism to approved immune checkpoint inhibitors (ICIs). It is a key regulator of immune activation, that modifies immune function through multiple mechanisms. Increased levels of PVR expression on tumor cells have been associated with resistance to anti-PD-(L)1 therapy in clinical settings, while loss of PVR led to reduced tumor growth in multiple pre-clinical models. Targeting PVR using blocking mAbs offers an attractive therapeutic approach for patients with advanced cancer. NTX-1088 is a first-in-class, potent, anti-PVR mAb being developed for the treatment of solid tumors. The antibody binds to PVR with high affinity, blocks its interactions with TIGIT and CD96, and thus interrupt their immunosuppressive signaling. However, NTX-1088 forte is manifested through its ability to block the critical interaction between PVR and the costimulatory receptor DNAM1 (CD226). This blockade prevents internalization of DNAM1, restores its expression on the surface of immune cells and results in a robust antitumor activation. NTX-1088 was tested using several tumor and immune cell co-culture systems. Various cancer cell lines were co-incubated with relevant immune effector cells from healthy human donors, in the presence of NTX-1088, as a single agent and in combination with anti-PD-1 mAb (pembrolizumab). NTX-1088 significantly increased immune cell activation, as measured by IFNg release from activated polyclonal CD8+ T cells, induction of CD137 and killing of tumor cells. When tested in combination with pembrolizumab, NTX-1088 further increased all measured activation parameters, suggesting a potential synergistic effect. When compared to anti-TIGIT mAb (tiragolumab), NTX-1088 demonstrated clear superiority in its ability to activate T and NK cells. Furthermore, NTX-1088 in combination with pembrolizumab was significantly superior to the combination of pembrolizumab with anti-TIGIT mAb. Interestingly, NTX-1088 as a single agent showed a comparable effect to that of the combined blockade of TIGIT and CD112R, and further synergized with anti-CD112R for maximal activity. NTX-1088 was the only intervention that significantly restored DNAM1 levels, whereas blockade of DNAM1 reduced the activity of NTX-1088 to levels comparable to that of anti-TIGIT mAb. Humanized murine models confirmed the above observations; NTX-1088 exhibited strong efficacy, inducing a robust tumor growth inhibition, accompanied by significantly higher prevalence of CD137+, DNAM1+, CD8+ tumor infiltrating cells, compared to control treated mice. This is the first report of drug-induced DNAM1 restoration and immune activation. NTX-1088 shows, for the first time, exclusive triple mechanism of action, whereby simultaneous and effective blockade of TIGIT and CD96 is complemented by the efficient restoration of DNAM1. This is a step change in antitumor immune activation, which will soon be tested in the clinic. Citation Format: Pini Tsukerman, Anas Atieh, Akram Obeidat, Keren Paz, Guy Cinamon, Tihana Lenac Rovis, Paola Kucan Brlic, Lea Hirsl, Stipan Jonjić, Ofer Mandelboim. NTX-1088, a potent first-in-class, anti-PVR mAb, restores expression and function of DNAM1 for optimal DNAM1-mediated antitumor immunity [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P075. 

Abstract P075: NTX-1088, a potent first-in-class, anti-PVR mAb, restores expression and function of DNAM1 for optimal DNAM1-mediated antitumor immunity

Background Poliovirus receptor (PVR, CD155) is a novel oncology target, differentially overexpressed in a wide range of solid tumors, which recently emerged as a main resistance mechanism to approved immune checkpoint inhibitors (ICIs). It is a key regulator of immune activation, that modifies function through multiple mechanisms, through interaction with the immune receptors DNAM1 (CD226), TIGIT and CD96. Increased PVR expression levels on tumor cells have been associated with resistance to anti-PD1 and PDL1 therapy in patients, while loss of PVR led to reduced tumor growth. Targeting PVR offers an attractive therapeutic approach for patients with advanced cancers, who are not responding to other ICIs. NTX1088 is a first-in-class, humanized, anti-PVR mAb, currently investigated in a Phase I. By binding PVR with high affinity, NTX1088 has a multi-faceted immune-stimulating role. It blocks the interaction between PVR and its receptors, leading to the restoration of DNAM1 expression and its immune activation function, while simultaneously neutralizing TIGIT and CD96 inhibitory signals in immune cells. DNAM1 downmodulation by PVR was recently identified as a key effector of immune surveillance, and its distinctive restoration by NTX1088 was never seen before by other therapies. Methods In vitro mechanistic studies demonstrated that NTX1088, as a monotherapy, significantly increased immune cell activation, and was superior to TIGIT, CD112R, and PD1 antibody blockade, leading to superior immune-mediated tumor cell killing, IFNg release, and CD137 induction. Importantly, only NTX1088 was able to restore DNAM1 to the surface of immune cells in all settings. Synergy was observed when NTX1088 was combined with PD1 blockers or with the anti-CD112R mAb, NTX2R13, in line with the restoration of DNAM1 expression. Results Numerous humanized murine xenograft models were investigated. NTX1088 as a monotherapy exhibited robust tumor growth inhibition of the PDAC cell line, HPAFII, coengrafted with human PBMC in NOD/SCID mice. The effect was significantly improved when NTX1088 was combined with a PD1 inhibitor. Furthermore, tumor growth inhibition by NTX1088 was observed in humanized A549 xenograft in which TIGIT and PD1 blockers failed to impact tumor growth. Tumor-infiltrating lymphocytes, harvested from NTX1088-treated mice, demonstrated a significantly higher prevalence of CD137+, DNAM1+, CD8+ T cells compared to all other interventions. Conclusions These promising preclinical findings, together with a clean safety profile in cynomolgus monkeys, paved the way to a Ph1 clinical study in which NTX1088 is tested as a monotherapy and in combination with pembrolizumab, in patients with locally advanced and metastatic solid tumors (NCT05378425).

/pdf/474-first-in-class-anti-pvr-mab-ntx1088-restores-expression-36hu2w70.pdf

Guy Cinamon

Papers

Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1

Follicular shuttling of marginal zone B cells facilitates antigen transport.

Sphingosine 1-phosphate receptor 1 promotes B cell localization in the splenic marginal zone

Abstract P075: NTX-1088, a potent first-in-class, anti-PVR mAb, restores expression and function of DNAM1 for optimal DNAM1-mediated antitumor immunity

474 First-in-class anti-PVR mAb NTX1088 restores expression of DNAM1 and augments antitumor immunity