TransCon IL-2 ß/γ: a novel long-acting prodrug with sustained release of an IL-2Rß/γ-selective IL-2 variant with improved pharmacokinetics and potent activation of cytotoxic immune cells for the treatment of cancer.

BACKGROUND: Recombinant interleukin-2 (IL-2, aldesleukin) is an approved cancer immunotherapy but causes severe toxicities including cytokine storm and vascular leak syndrome (VLS). IL-2 promotes antitumor function of IL-2Rß/γ+ natural killer (NK) cells and CD8+, CD4+ and gamma delta (γδ) T cells. However, IL-2 also potently activates immunosuppressive IL-2Rα+ regulatory T cells (Tregs) and IL-2Rα+ eosinophils and endothelial cells, which may promote VLS. Aldesleukin is rapidly cleared requiring frequent dosing, resulting in high Cmax likely potentiating toxicity. Thus, IL-2 cancer immunotherapy has two critical drawbacks: potent activation of undesired IL-2Rα+ cells and suboptimal pharmacokinetics with high Cmax and short half-life. METHODS: TransCon IL-2 ß/γ was designed to optimally address these drawbacks. To abolish IL-2Rα binding yet retain strong IL-2Rß/γ activity, IL-2 ß/γ was created by permanently attaching a small methoxy polyethylene glycol (mPEG) moiety in the IL-2Rα binding site. To improve pharmacokinetics, IL-2 ß/γ was transiently attached to a 40 kDa mPEG carrier via a TransCon (transient conjugation) linker creating a prodrug, TransCon IL-2 ß/γ, with sustained release of IL-2 ß/γ. IL-2 ß/γ was characterized in binding and primary cell assays while TransCon IL-2 ß/γ was studied in tumor-bearing mice and cynomolgus monkeys. RESULTS: IL-2 ß/γ demonstrated selective and potent human IL-2Rß/γ binding and activation without IL-2Rα interactions. TransCon IL-2 ß/γ showed slow-release pharmacokinetics with a low Cmax and a long (>30 hours) effective half-life for IL-2 ß/γ in monkeys. In mouse tumor models, TransCon IL-2 ß/γ promoted CD8+ T cell and NK cell activation and antitumor activity. In monkeys, TransCon IL-2 ß/γ induced robust activation and expansion of CD8+ T cells, NK cells and γδ T cells, relative to CD4+ T cells, Tregs and eosinophils, with no evidence of cytokine storm or VLS. Similarly, IL-2 ß/γ enhanced proliferation and cytotoxicity of primary human CD8+ T cells, NK cells and γδ T cells. SUMMARY: TransCon IL-2 ß/γ is a novel long-acting prodrug with sustained release of an IL-2Rß/γ-selective IL-2. It has remarkable and durable pharmacodynamic effects in monkeys and potential for improved clinical efficacy and tolerability compared with aldesleukin. TransCon IL-2 ß/γ is currently being evaluated in a Phase 1/2 clinical trial (NCT05081609).

The CTLA-4 x OX40 bispecific antibody ATOR-1015 induces anti-tumor effects through tumor-directed immune activation.

BACKGROUND: The CTLA-4 blocking antibody ipilimumab has demonstrated substantial and durable effects in patients with melanoma. While CTLA-4 therapy, both as monotherapy and in combination with PD-1 targeting therapies, has great potential in many indications, the toxicities of the current treatment regimens may limit their use. Thus, there is a medical need for new CTLA-4 targeting therapies with improved benefit-risk profile. METHODS: ATOR-1015 is a human CTLA-4 x OX40 targeting IgG1 bispecific antibody generated by linking an optimized version of the Ig-like V-type domain of human CD86, a natural CTLA-4 ligand, to an agonistic OX40 antibody. In vitro evaluation of T-cell activation and T regulatory cell (Treg) depletion was performed using purified cells from healthy human donors or cell lines. In vivo anti-tumor responses were studied using human OX40 transgenic (knock-in) mice with established syngeneic tumors. Tumors and spleens from treated mice were analyzed for CD8+ T cell and Treg frequencies, T-cell activation markers and tumor localization using flow cytometry. RESULTS: ATOR-1015 induces T-cell activation and Treg depletion in vitro. Treatment with ATOR-1015 reduces tumor growth and improves survival in several syngeneic tumor models, including bladder, colon and pancreas cancer models. It is further demonstrated that ATOR-1015 induces tumor-specific and long-term immunological memory and enhances the response to PD-1 inhibition. Moreover, ATOR-1015 localizes to the tumor area where it reduces the frequency of Tregs and increases the number and activation of CD8+ T cells. CONCLUSIONS: By targeting CTLA-4 and OX40 simultaneously, ATOR-1015 is directed to the tumor area where it induces enhanced immune activation, and thus has the potential to be a next generation CTLA-4 targeting therapy with improved clinical efficacy and reduced toxicity. ATOR-1015 is also expected to act synergistically with anti-PD-1/PD-L1 therapy. The pre-clinical data support clinical development of ATOR-1015, and a first-in-human trial has started (NCT03782467).

Functional effects of Toll-like receptor (TLR)3, 7, 9, RIG-I and MDA-5 stimulation in nasal epithelial cells.

BACKGROUND: The human nasal epithelium is an important physical barrier, and a part of the innate immune defense that protect against pathogens. The epithelial cells recognize microbial components by pattern-recognition receptors (PRRs), and thereby trigger an immune response. Even though TLR3, TLR7, TLR9, RIG-I and MDA-5 are all known to respond to viral stimulation, their potential role in chronic airway inflammation triggered by local cytokine release remains to be established. METHODS: mRNA and corresponding protein expression of TLR3, TLR7, TLR9, RIG-I and MDA-5 were analyzed in nasal biopsies and various upper airway epithelial cell lines using real-time reverse transcription PCR, immunohistochemistry and flow cytometry. Ligand induced, cytokine release, was evaluated with ELISA. RESULTS: Nasal biopsies were found to express TLR3, TLR7, TLR9, RIG-I and MDA-5, with the most abundant expression in the surface epithelium. These receptors were verified in primary human nasal epithelial cell (HNEC) as well as in the airway epithelial cell lines Detroit-562 and FaDu. Poly(I:C) (TLR3) and R-837 (TLR7) stimulation increased secretion of IL-6 and GM-CSF from the nasal mucosa and the epithelial cell lines. CpG (TLR9) stimulation caused release of IL-8 in the nasal mucosa and in FaDu. Poly(I:C)/LyoVec (RIG-I/MDA-5) stimulation activated the secretion of IFN-ß in the nasal mucosa. A corresponding release was also detected from HNEC and Detroit-562. CONCLUSION: The nasal epithelium has the ability to recognize viral intrusion through TLR and RLR receptors, and the subsequent response might have a role in exacerbation of inflammatory diseases like allergic rhinitis and chronic rhinosinusitis.

LEAP-2, LL-37 and RNase7 in tonsillar tissue: downregulated expression in seasonal allergic rhinitis.

In the upper airway, the production of antimicrobial peptides (AMPs) protects against bacteria, viruses and fungi. Previous investigations have revealed downregulated expression of AMPs in different manifestations of allergic disease. In this study, we examined the expression of LL-37, Ribonuclease7 (RNase7) and Liver-expressed antimicrobial peptide 2 (LEAP-2) in tonsillar tissue and studied a possible relation to seasonal allergic rhinitis (SAR). Tonsils, obtained from patients with SAR and nonallergic controls, were examined for the occurrence of LL-37, RNase7 and LEAP-2 with real-time RT-PCR and immunohistochemistry. Tonsillar mononuclear cells were cultured in presence or absence of LEAP-2 or LL-37 and analyzed for cytokine levels using ELISA. mRNA and protein for LL-37, RNase 7 and LEAP-2 were found in all tonsils. Immunohistochemistry revealed prominent staining for LL-37 and RNase7 in the tonsillar epithelium, whereas a moderate staining was seen with LEAP-2. Real-time RT-PCR showed a downregulation of RNase7 and LEAP-2 in the allergic as compared to the nonallergic group. Mononuclear cells cultured in presence of LEAP-2 or LL-37 demonstrated reduced levels of IL-10. The present study demonstrates the presence and function of LEAP-2, LL-37 and RNase7 in tonsils. Moreover, a downregulation of LEAP-2 and RNase7 is seen in SAR patients, indicating that allergic individuals may be more susceptible to respiratory tract infections due to an impaired antimicrobial defense.

Nod-like receptors in head and neck squamous cell carcinoma.

CONCLUSION: The capability of Nod1 to recognize bacteria along with its altered expression and ability to cause an immunological response in head and neck cancer suggest a novel pathway for bacteria to interfere with ongoing cancer inflammation. OBJECTIVE: Nucleotide oligomerization domain (Nod)-like receptors (NLRs) comprise a recently discovered family of pattern-recognition receptors. In addition to their protective function against infections, accumulating evidence suggests a role for these receptors in various diseases, including cancer. The present study was designed to explore the presence of NLRs in head and neck squamous cell carcinoma, and to determine if these cells have the ability to respond immunologically to ligand stimulation. METHODS: The pharyngeal squamous cell carcinoma cell lines Detroit-562 and FaDu were used as a model for head and neck cancer, and compared to healthy primary human nasal epithelial cells. Analyses were performed using immunohistochemistry, real-time RT-PCR, Luminex Multiplex Immunoassay, ELISA, and flow cytometry. RESULTS: The expression profile of NLRs in head and neck cancer cells differed from that seen in healthy epithelial cells. Further, Nod1 stimulation induced an immunological response in tumor cells that differed from the response in normal epithelial cells, especially regarding the expression of ß-defensin 2, granulocyte monocyte colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), intercellular adhesion molecule-1 (ICAM-1), and cell survival.

Upregulated levels of human ß-defensins in patients with seasonal allergic rhinitis after allergen-specific immunotherapy treatment.

BACKGROUND: Antimicrobial peptides (AMPs) are important actors in the innate immune system. One class of AMPs is the human ß-defensins (HBDs), a group of small peptides with a broad spectrum of antimicrobial activities. Expression of HBDs is downregulated in different manifestations of allergic disease. In this study, we examine whether allergen-specific immunotherapy (ASIT) affects the nasal levels of HBDs in patients with seasonal allergic rhinitis (SAR). METHODS: Nasal biopsies were examined for the occurrence of HBD1-3 with real-time reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. Nasal lavage (NAL) fluids from healthy individuals, untreated SAR patients and SAR patients before and after ASIT were analyzed for levels of HBD1-3 using enzyme-linked immunosorbent assay (ELISA). RESULTS: Examination of nasal biopsies revealed HBD1-3 expression at gene level as well as at protein level in all samples tested. HBD1 and HBD3 messenger RNA (mRNA) levels were downregulated in SAR patients compared to healthy individuals. All HBDs were found in NAL fluids. SAR patients having completed 3 years of ASIT displayed higher levels of HBD1 and HBD2 than before treatment, whereas levels of HBD3 were unaffected. CONCLUSION: The present study demonstrates an upregulation of HBD1 and HBD2 in SAR patients after completion of ASIT. This may reflect the importance of an intact innate immune response as part of our defense against infections among allergic individuals.

Reduced tonsillar expression of human ß-defensin 1, 2 and 3 in allergic rhinitis.

Airway infections are known to cause exacerbations of allergy and asthma. Tonsils constitute a primary site for microbial recognition and triggering of the immune system in the airways. Human ß-defensins (HBDs) are antimicrobial peptides with an important role in this defense. Our aim was to investigate HBD1-3 in tonsillar tissue and their potential role in allergic rhinitis (AR). Tonsils, obtained from patients with AR and non-allergic controls, and isolated tonsillar CD4(+), CD8(+) and CD19(+) lymphocytes were analyzed for HBD1-3 expression using real-time RT-PCR and/or immunohistochemistry. Tonsillar tissue, mixed tonsillar lymphocytes and airway epithelial cells (AECs) were cultured with or without IL-4, IL-5, IL-13 or histamine followed by measurements of HBD1-3 release using ELISA. HBD1-3 were present in tonsillar tissue, including epithelial, CD4(+), CD8(+) and CD19(+) cells. The expression was reduced in allergic compared to healthy tonsils. Stimulation of AECs with IL-4, IL-5 and histamine down-regulated the HBD release, whereas no effects were seen in cultured tonsils or lymphocytes. This study demonstrates presence of HBD1-3 in tonsils and that the levels are reduced in patients with AR. Together with the down-regulation of HBDs in epithelial cells in the presence of allergic mediators suggest that AR patients have an impaired antimicrobial defense that might make them more susceptible to respiratory tract infections.

Pattern-recognition receptors in human eosinophils.

The pattern-recognition receptor (PRR) family includes Toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD) -like receptors (NLRs), RIG-I-like receptors (RLRs), C-type lectin receptors (CLRs) and the receptor for advanced glycation end products (RAGE). They recognize various microbial signatures or host-derived danger signals and trigger an immune response. Eosinophils are multifunctional leucocytes involved in the pathogenesis of several inflammatory processes, including parasitic helminth infection, allergic diseases, tissue injury and tumour immunity. Human eosinophils express several PRRs, including TLR1-5, TLR7, TLR9, NOD1, NOD2, Dectin-1 and RAGE. Receptor stimulation induces survival, oxidative burst, activation of the adhesion system and release of cytokines (interleukin-1ß, interleukin-6, tumour necrosis factor-α and granulocyte-macrophage colony-stimulating factor), chemokines (interleukin-8 and growth-related oncogene-α) and cytotoxic granule proteins (eosinophil cationic protein, eosinophil-derived neurotoxin, eosinophil peroxidase and major basic protein). It is also evident that eosinophils play an immunomodulatory role by interacting with surrounding cells. The presence of a broad range of PRRs in eosinophils indicates that they are not only involved in defence against parasitic helminths, but also against bacteria, viruses and fungi. From a clinical perspective, eosinophilic PRRs seem to be involved in both allergic and malignant diseases by causing exacerbations and affecting tumour growth, respectively.

Diminished levels of nasal S100A7 (psoriasin) in seasonal allergic rhinitis: an effect mediated by Th2 cytokines.

BACKGROUND: S100A7 is an antimicrobial peptide involved in several inflammatory diseases. The aim of the present study was to explore the expression and regulation of S100A7 in seasonal allergic rhinitis (SAR). METHODS: Nasal lavage (NAL) fluid was obtained from healthy controls before and after lipopolysaccharide (LPS) provocation, from SAR patients before and after allergen challenge, and from SAR patients having completed allergen-specific immunotherapy (ASIT). Nasal biopsies, nasal epithelial cells and blood were acquired from healthy donors. The airway epithelial cell line FaDu was used for in vitro experiments. Real-time RT-PCR and immunohistochemistry were used to determine S100A7 expression in nasal tissue and cells. Release of S100A7 in NAL and culture supernatants was measured by ELISA. The function of recombinant S100A7 was explored in epithelial cells, neutrophils and peripheral blood mononuclear cells (PBMC). RESULTS: Nasal administration of LPS induced S100A7 release in healthy non-allergic subjects. The level of S100A7 was lower in NAL from SAR patients than from healthy controls, and it was further reduced in the SAR group 6 h post allergen provocation. In contrast, ASIT patients displayed higher levels after completed treatment. S100A7 was expressed in the nasal epithelium and in glands, and it was secreted by cultured epithelial cells. Stimulation with IL-4 and histamine repressed the epithelial S100A7 release. Further, recombinant S100A7 induced activation of neutrophils and PBMC. CONCLUSIONS: The present study shows an epithelial expression and excretion of S100A7 in the nose after microbial stimulation. The levels are diminished in rhinitis patients and in the presence of an allergic cytokine milieu, suggesting that the antimicrobial defense is compromised in patients with SAR.

Retinoic acid-inducible gene 1-like receptors in the upper respiratory tract.

BACKGROUND: Retinoic acid-inducible gene 1-like receptors (RLRs) are a novel family of pattern recognition receptors that include retinoic acid-inducible gene 1 (RIG-1), melanoma differentiation-associated gene 5 (MDA-5), and laboratory of genomics and physiology 2 (LGP-2). The knowledge of RLRs and their function in the human airway is limited. This study explores the role of RLRs in the upper respiratory tract. METHODS: Tonsils, adenoids, nasal polyps, and biopsy specimens from the nasal mucosa were examined for the occurrence of the RIG-1, MDA-5, and LGP-2 using real-time reverse-transcription polymerase chain reaction and immunohistochemistry. The nasopharyngeal epithelial cell line FaDu was cultured with the RIG-1/MDA-5 ligand poly(I:C)/LyoVec (Invivogen, San Diego, CA) and analyzed for cytokine release using ELISA. RESULTS: RIG-1, MDA-5, and LGP-2 mRNA were found in all tissues tested. The airway epithelium appeared to be their most prominent location. The RIG-1 and MDA-5 mRNA levels were higher in nasal polyps than in normal nasal mucosa, a state that seemed to be reversed by local steroid treatment. Culture of FaDu with poly(I:C)/LyoVec resulted in IL-6 and IL-8 release. No alteration in RLR expression in tonsils was seen on infection. CONCLUSION: This study shows the presence and functional activity of RLRs in the human upper airways. It also suggests a role for RLRs in nasal polyposis.

NOD-like receptors and RIG-I-like receptors in human eosinophils: activation by NOD1 and NOD2 agonists.

NOD-like receptors (NLRs) and RIG-I-like receptors (RLRs) are newly discovered pattern-recognition receptors. They detect substructures of bacterial peptidoglycan and viral RNA, respectively, thereby initiating an immune response. However, their role in eosinophil activation remains to be explored. The aim of this study was to characterize the expression of a range of NLRs and RLRs in purified human eosinophils and assess their functional importance. Expression of NOD1, NOD2, NLRP3, RIG-I and MDA-5 was investigated using real-time reverse transcription PCR, flow cytometry and immunohistochemistry. The effects of the corresponding agonists iE-DAP (NOD1), MDP (NOD2), alum (NLRP3) and poly(I:C)/LyoVec (RIG-I/MDA-5) were studied in terms of cytokine secretion, degranulation, survival, expression of adhesion molecules and activation markers, and chemotactic migration. Eosinophils expressed NOD1 and NOD2 mRNA and protein. Low levels of RIG-I and MDA-5 were found, whereas expression of NLRP3 was completely absent. In accordance, stimulation with iE-DAP and MDP was found to induce secretion of interleukin-8, up-regulate expression of CD11b, conversely down-regulate CD62 ligand, increase expression of CD69 and induce migration. The MDP also promoted release of eosinophil-derived neurotoxin, whereas iE-DAP failed to do so. No effects were seen upon stimulation with alum or poly(I:C)/LyoVec. Moreover, the NOD1-induced and NOD2-induced activation was mediated via the nuclear factor-κB signalling pathway and augmented by interleukin-5 and granulocyte-macrophage colony-stimulating factor, but not interferon-γ. Taken together, the NLR system represents a novel pathway for eosinophil activation. The responses are enhanced in the presence of cytokines that regulate T helper type 2 immunity, suggesting that the NLRs constitute a link between respiratory infections and exacerbations of allergic disease.