Targeting Interleukin-2 Research Articles

A novel nanobody-based immunocytokine of a mutant interleukin-2 as a potential cancer therapeutic

The immunotherapeutic application of interleukin-2 (IL-2) in cancer treatment is limited by its off-target effects on different cell populations and insufficient activation of anti-tumor effector cells at the site of the tumor upon tolerated doses. Targeting IL-2 to the tumor microenvironment by generating antibody-cytokine fusion proteins (immunocytokine) would be a promising approach to increase efficacy without associated toxicity. In this study, a novel nanobody-based immunocytokine is developed by the fusion of a mutant (m) IL-2 with a decreased affinity toward CD25 to an anti-vascular endothelial growth factor receptor-2 (VEGFR2) specific nanobody, denoted as VGRmIL2-IC. The antigen binding, cell proliferation, IFN-γ-secretion, and cytotoxicity of this new immunocytokine are evaluated and compared to mIL-2 alone. Furthermore, the pharmacokinetic properties are analyzed. Flow cytometry analysis shows that the VGRmIL2-IC molecule can selectively target VEGFR2-positive cells. The results reveal that the immunocytokine is comparable to mIL-2 alone in the stimulation of Primary Peripheral Blood Mononuclear Cells (PBMCs) and cytotoxicity in in vitro conditions. In vivo studies demonstrate improved pharmacokinetic properties of VGRmIL2-IC in comparison to the wild or mutant IL-2 proteins. The results presented here suggest VGRmIL2-IC could be considered a candidate for the treatment of VEGFR2-positive tumors.

Site-directed Fragnomics and MD Simulations Approaches to Identify Interleukin-2 Inhibitors.

The aberrant expression of Interleukin-2 (IL2), the chief regulator of immunity, is associated with many auto-immune diseases. At present, there is no FDA approved drug targeting IL2, which puts forth the need for small molecular inhibitors to block IL2 and its receptor interaction. Herein, we used the contemporary fragnomics approach to design novel drug-like inhibitors targeting IL2. Briefly, the RECAP (Retrosynthetic Combinatorial Analysis Procedure) package implemented in MOE (Molecular Operating Environment check) software suite was utilised to obtain fragments fulfilling the 'rule of three' criteria for fragments. The binding site of IL2 was divided into three smaller grooves, and the fragments were docked to screen their affinity for a particular site, followed by site-directed RECAP synthesis. A focused library of 10,000 compounds was prepared by re-combining the fragments according to their affinity for a particular site as observed in docking. Docking and subsequent analysis of newly synthesised compounds identified 40 privileged leads, presenting hydrogen bonding with basic residues of the pocket. A QSAR model was implied to predict the IC50 of the compounds and to analyse the electrostatic and hydrophobic contour maps. The resulting hits were found to be modest IL2 inhibitors with predicted inhibitory activity in the range of 5.17-4.40 nM. Further Dynamic simulation studies were carried out to determine the stability of the inhibitor-IL2 complex. Our findings underline the potential of the novel compounds as valuable pharmacological agents in diseases characterised by IL2 overexpression.

Clinical-grade N-(4-[18F]fluorobenzoyl)-interleukin-2 for PET imaging of activated T-cells in humans

BackgroundMolecular imaging of immune cells might be a potential tool for response prediction, treatment evaluation and patient selection in inflammatory diseases as well as oncology. Targeting interleukin-2 (IL2) receptors on activated T-cells using positron emission tomography (PET) with N-(4-[18F]fluorobenzoyl)-interleukin-2 ([18F]FB-IL2) could be such a strategy. This paper describes the challenging translation of the partly manual labeling of [18F]FB-IL2 for preclinical studies into an automated procedure following Good Manufacturing Practices (GMP), resulting in a radiopharmaceutical suitable for clinical use.MethodsThe preclinical synthesis of [18F]FB-IL2 was the starting point for translation to a clinical production method. To overcome several challenges, major adaptations in the production process were executed. The final analytical methods and production method were validated and documented. All data with regards to the quality and safety of the final drug product were documented in an investigational medicinal product dossier.ResultsRestrictions in the [18F]FB-IL2 production were imposed by hardware configuration of the automated synthesis equipment and by use of disposable cassettes. Critical steps in the [18F]FB-IL2 production comprised the purification method, stability of recombinant human IL2 and the final formulation. With the GMP compliant production method, [18F]FB-IL2 could reliably be produced with consistent quality complying to all specifications.ConclusionsTo enable the use of [18F]FB-IL2 in clinical studies, a fully automated GMP compliant production process was developed. [18F]FB-IL2 is now produced consistently for use in clinical studies.

Abstract 3811: Targeted Interleukin 2 and synergy with immune check-point inhibitors

Abstract Systemic high-dose Interleukin 2 (IL2) is able to elicit complete responses in patients suffering from metastatic melanoma. However, the majority of patients cannot receive the whole treatment dose due to severe toxicities. Other approved drugs for the treatment of metastatic melanoma include chemotherapeutics (e.g. Dacarbazine), a protein kinase B-raf inhibitor (Vemurafenib) and an immune check-point inhibitor (Ipilimumab, an anti CTLA-4 antibody). These drugs provide a clear benefit to patients, but cancer cures are still rare. Targeting IL2 to the site of disease represents a promising strategy to circumvent the toxicity and maximize the efficacy of this immunomodulatory payload. Various formats of targeted IL2 fusion proteins are currently in clinical development. Furthermore, recent successful approaches in the field of immunotherapy focus on the inhibition of check-point pathways, aiming to release the immunosuppressive brakes of the immune system. To explore the possibility of improving therapeutic efficacy of the single agents, a targeted version of IL2 (namely F8-IL2, where the F8 antibody recognizes the alternatively-spliced extra-domain A of fibronectin) was investigated in combination with an anti PD-1, anti PD-L1, or anti CTLA-4 antibody in an immunocompetent mouse model of CT26 colon carcinoma. In a first study, the combination of F8-IL2 and CTLA-4 blockade led to significant tumor growth retardation (curing 3/ 5 mice). Increasing the dose of F8-IL2 from 30 to 45 µg led to a 100 % cure rate with no signs of toxicity (body weight loss <5 %). Moreover, all cured mice acquired protective long-term immunity, which prevented tumor growth upon subsequent rechallenge with CT26 colon carcinoma cells. Furthermore, high dose F8-IL2 combined with the anti PD-1 antibody showed better therapeutic results than the respective monotherapies. In contrast, combination of F8-IL2 with the anti PD-L1 antibody did not improve the therapeutic outcome. Taken together, these data provide a strong rationale for the clinical use of targeted IL2 in combination with Ipilimumab. Citation Format: Cornelia Deborah Hutmacher-Berndt, Dario Neri. Targeted Interleukin 2 and synergy with immune check-point inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3811.

Targeting Interleukin-2 to the Bone Marrow Stroma for Therapy of Acute Myeloid Leukemia Relapsing after Allogeneic Hematopoietic Stem Cell Transplantation

The tumor-directed delivery of therapeutics using monoclonal antibodies specific to a tumor-associated antigen promises to accumulate large doses of the delivered payload at the tumor site while sparing healthy organs. The antibody-based delivery of interleukin-2 (IL-2) to extracellular targets expressed in the easily accessible tumor vasculature has shown promising results in animal models of solid tumors and hematological malignancies. In xenograft and immunocompetent murine models of acute myeloid leukemia (AML), IL-2-based vascular targeting antibody fusions have recently demonstrated potent anti-leukemic activity, especially when used in combination with cytarabine. Here, we report our experiences in four patients with relapsed AML after allogeneic hematopoietic stem cell transplantation (allo-HSCT), who were treated with the immunocytokine F16-IL2, consisting of a human monoclonal antibody specific to spliced large isoforms of tenascin-C fused to human IL-2, in combination with very low dose cytarabine (5 mg subcutaneously twice daily for 10 days). Clinical evidence of anti-leukemia efficacy was shown in all patients. One patient with rapidly progressing disseminated extramedullary AML lesions achieved a complete metabolic response in PET/CT, which lasted three months. Two out of three patients with bone marrow relapse achieved a blast reduction with transient molecular negativity (NPM1). One of the two enjoyed a short complete remission before AML relapse occurred two months after the first infusion of F16-IL2. The other patient did not regenerate neutrophil and thrombocyte counts and showed progressive disease after completion of the first cycle. In line with a site-directed delivery of the cytokine, F16-IL2 led to an extensive infiltration of immune effector cells (natural killer cells, CD8+ T cells, γδ T cells) in the bone marrow. Grade 2 fevers were the only non-hematological side effects in two patients. Grade 3 cytokine-release syndrome developed in the other two patients, required hospitalization, but was manageable in both cases with systemic glucocorticoids. No non-hematological grade 4 toxicities were observed. The concept of specifically targeting IL-2 to the leukemia-associated stroma using armed antibodies deserves further evaluation in clinical trials, especially in patients who relapse after allo-HSCT. DisclosuresOff Label Use: In this report, the antibody-cytokine fusion protein F16-IL2 has been used in a compassionate use setting in individual patients presenting with AML relapse after allogeneic stem cell transplantation. F16-IL2 is currently being evaluated in phase I/II studies in patients with solid cancer.. Neri:Philogen SpA: Employment, Equity Ownership.

Targeting Interleukin-2-Inducible T-cell Kinase (ITK) in T-Cell Related Diseases.

IL2-inducible T-cell kinase (ITK), a member of the Tec family tyrosine kinases, is the predominant Tec kinase in T cells and natural killer (NK) cells mediating T cell receptor (TCR) and Fc receptor (Fc R) initiated signal transduction. ITK deficiency results in impaired T and NK cell functions, leading to various disorders including malignancies, inflammation, and autoimmune diseases. In this mini-review, the role of ITK in T cell signaling and the development of small molecule inhibitors of ITK for the treatment of T-cell related disorders is examined.

Targeting Interleukin-2 to the Neovasculature Potentiates Rituximab‘s Activity Against Mantle Cell Lymphoma in Mice

AbstractAbstract 3716 Introduction:Antibody-based tumor targeting strategies promise to specifically target anti-neoplastic agents (e.g. radionuclides, drugs, cytokines) to the tumor site while sparing healthy tissues. In this context, the selective delivery of bioactive molecules to the vasculature seems particularly attractive, considering the fact that antigens expressed in blood vessels are the most accessible structures within a tumor mass and that a vigorous neo-vasculature development is a common feature of virtually all solid and hematologic malignancies. Vascular targeting antibodies functionalized with different payloads are currently being investigated in patients with solid tumors and are increasingly being considered also for the treatment of hematological malignancies. In the present work, we evaluated the efficacy of the fusion protein L19-IL2, comprising the human monoclonal antibody fragment scFv(L19) linked to the cytokine Interleukin-2 (IL-2), against Mantle Cell Lymphoma (MCL), a disease for which existing therapeutic approaches still remain highly unsatisfactory. Methods:Expression of the extra-domain B of fibronectin (EDB-Fn), a well-characterized marker of angiogenic blood vessels, was analyzed in Granta-519 and HBL-2 MCL xenografts and human MCL specimens using immunohistochemistry and dual-color immunofluorescence. The in vivo targeting performance of the EDB-Fn targeting immunocytokine L19-IL2 was investigated using ex vivo immunofluorescence analyses. SCID mice xenografted subcutaneously with Granta-519 or intravenously with HBL-2 MCL cells were used for mono- and combination therapy experiments, featuring i.v. injections of L19-IL2 (30 μg, corresponding to 9.9 μg or 177000 IU rIL-2 equivalents), unconjugated rIL-2 (9.9 μg), the anti-CD20 antibody rituximab (200 μg), rituximab plus IL-2, rituximab plus L19-IL2 or saline. Results:In vivo, L19-IL2 selectively homed to EDB-Fn expressing angiogenic blood vessels, thereby mediating the accumulation of IL-2 in MCL lesions. In mono-therapy experiments, L19-IL2 showed a superior anti-lymphoma activity compared to equimolar amounts of non-targeted recombinant IL-2. In combination with rituximab, L19-IL2 induced complete remissions (CR) of established subcutaneous Granta-519 tumors in 75 % (6/8) of the cases, whereas rituximab alone or in combination with free IL-2 only delayed lymphoma progression. Mice that experienced lymphoma relapse after having achieved a CR responded a second time to L19-IL2 and rituximab treatment. In the systemic HBL-2 MCL model, the combination of L19-IL2 and rituximab almost doubled survival of mice as compared to IL-2 and rituximab (median survival 63 vs. 33 days). Conclusions:In summary, we show that the vascular targeting immunocytokine L19-IL2 promotes a potent anti-lymphoma response in combination with anti-CD20 immunotherapy. The clinical evaluation of this combination is facilitated by the fact that L19-IL2 is entirely human and already being studied in Phase II clinical trials in patients with solid tumors. Disclosures:Menssen:Philogen: Employment. Neri:Philogen: Dario Neri is a co-founder and shareholder of Philogen, the biotech company which has licensed the L19 antibody from the ETH Zurich Other, Equity Ownership.

Intratumoral interleukin-2/agonist CD40 antibody drives CD4+ -independent resolution of treated-tumors and CD4+ -dependent systemic and memory responses.

Targeting interleukin-2 (IL-2) and/or agonist anti-CD40 antibody (Ab) into tumors represents an effective vaccination strategy that avoids systemic toxicity and resolves treated-site tumors. Here, we examined IL-2 and/or anti-CD40 Ab-driven local versus systemic T cell function and the installation of T cell memory. Single tumor studies showed that IL-2 induced a potent CD4+ and CD8+ T cell response that was limited to the draining lymph node and treated-site tumor, and lymph node tumor-specific CD8+ T cells did not upregulate CD44. A two-tumor model showed that while IL-2-treated-site tumors resolved, distal tumors continued to grow, implying limited systemic immunity. In contrast, anti-CD40 Ab treatment with or without IL-2 expanded the systemic T cell response to non-draining lymph nodes, and distal tumors resolved. Tumor-specific T cells in lymph nodes of anti-CD40 Ab ± IL-2-treated mice upregulated CD44, demonstrating activation and transition to effector/memory migratory cells. While CD40-activated CD4+ T cells were not required for eradicating treated-site tumors, they, plus CD8+ T cells, were crucial for removing distal tumors. Rechallenge/depletion experiments showed that the effector/memory phase required the presence of previously CD40/IL-2-activated CD4+ and CD8+ T cells to prevent recurrence. These novel findings show that different T cell effector mechanisms can operate for the eradication of local treated-site tumors versus untreated distal tumors and that signaling through CD40 generates a whole of body, effector/memory CD4+ and CD8+ T cell response that is amplified and prolonged via IL-2. Thus, successful immunotherapy needs to generate collaborating CD4+ and CD8+ T cells for a complete long-term protective cure.

Targeting IL-2 to the endoplasmic reticulum confines autocrine growth stimulation to NK-92 cells

Anti-tumor effects mediated by adoptively transferred natural killer (NK) cells are dependent on the presence of interleukin-2 (IL-2). IL-2 is considered to be a survival factor for NK cells and an enhancer of their cytotoxic potential. However, systemic administration of IL-2 is frequently impeded by undesirable side effects, such as high toxicity and nonlocalized administration. Genetic modification of NK cells expressing IL-2 in a localized and controlled manner could be a powerful tool for overcoming these obstacles. Consequently, we have cloned the IL-2 gene using PCR and designed constructs that target IL-2 to specific subcellular compartments. The IL-2-dependent NK-92 cell line was used to verify the functionality of the subcellularly targeted IL-2 constructs. IL-2 targeted specifically to the endoplasmic reticulum (ER) was sufficient to support growth of NK-92 cells. In such cell lines, IL-2 was verified to be localized to the ER. IL-2 was not detected in the supernatant and growth of non-IL-2-modified NK-92 cells was not supported during coculturing experiments. IL-2-transduced NK-92 cell lines showed comparable functional activity and cytotoxicity to parental NK-92 cells. We demonstrate the ability of ER-retained IL-2 to provide autocrine growth stimulation to NK-92 cells, without secretion of the cytokine to the extracellular compartment. Therapy with IL-2 gene-modified autoactivating NK cells may avoid side effects imposed by exogenously administered IL-2.