ABSTRACT
PURPOSE: Simlukafusp alfa [fibroblast activation protein α-targeted IL2 variant (FAP-IL2v)], a tumor-targeted immunocytokine, comprising an IL2 variant moiety with abolished CD25 binding fused to human IgG1, is directed against fibroblast activation protein α. This phase I, open-label, multicenter, dose-escalation, and extension study (NCT02627274) evaluated the safety, pharmacokinetics, pharmacodynamics, and antitumor activity of FAP-IL2v in patients with advanced/metastatic solid tumors. PATIENTS AND METHODS: Participants received FAP-IL2v intravenously once weekly. Dose escalation started at 5 mg; flat dosing (≤25 mg) and intraparticipant uptitration regimens (15/20, 20/25, 20/20/35, and 20/35/35 mg) were evaluated. Primary objectives were dose-limiting toxicities, maximum tolerated dose, recommended expansion dose, and pharmacokinetics. RESULTS: Sixty-one participants were enrolled. Dose-limiting toxicities included fatigue (flat dose 20 mg: n = 1), asthenia (25 mg: n = 1), drug-induced liver injury (uptitration regimen 20/25 mg: n = 1), transaminase increase (20/25 mg: n = 1), and pneumonia (20/35/35 mg: n = 1). The uptitration regimen 15/20 mg was determined as the maximum tolerated dose and was selected as the recommended expansion dose. Increases in peripheral blood absolute immune cell counts were seen for all tested doses [NK cells, 13-fold; CD4+ T cells (including regulatory T cells), 2-fold; CD8+ T cells, 3.5-fold] but without any percentage change in regulatory T cells. Clinical activity was observed from 5 mg [objective response rate, 5.1% (n = 3); disease control rate, 27.1% (n = 16)]. Responses were durable [n = 3, 2.8 (censored), 6.3, and 43.4 months]. CONCLUSIONS: FAP-IL2v had a manageable safety profile and showed initial signs of antitumor activity in advanced/metastatic solid tumors.
Subject(s)
Maximum Tolerated Dose , Neoplasms , Humans , Female , Male , Neoplasms/drug therapy , Neoplasms/pathology , Neoplasms/genetics , Middle Aged , Aged , Adult , Interleukin-2/administration & dosage , Interleukin-2/adverse effects , Interleukin-2/pharmacokinetics , Interleukin-2/genetics , Neoplasm Metastasis , Recombinant Fusion Proteins/pharmacokinetics , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/adverse effects , Recombinant Fusion Proteins/therapeutic use , Treatment Outcome , Endopeptidases/administration & dosage , Membrane ProteinsABSTRACT
Regulatory T cells (Tregs) are essential for maintaining immune homeostasis by serving as negative regulators of adaptive immune system effector cell responses. Reduced production or function of Tregs has been implicated in several human autoimmune diseases. The cytokine interleukin 2 plays a central role in promoting Treg differentiation, survival, and function in vivo and may therefore have therapeutic benefits for autoimmune diseases. mRNA-6231 is an investigational, lipid nanoparticle-encapsulated, mRNA-based therapy that encodes a modified human interleukin 2 mutein fused to human serum albumin (HSA-IL2m). Herein, we report the development of a semi-mechanistic kinetic-pharmacodynamic model to quantify the relationship between subcutaneous dose(s) of mRNA-6231, HSA-IL2m protein expression, and Treg expansion in nonhuman primates. The nonclinical kinetic-pharmacodynamic model was extrapolated to humans using allometric scaling principles and the physiological basis of pharmacological mechanisms to predict the clinical response to therapy a priori. Model-based simulations were used to inform the dose selection and design of the first-in-human clinical study (NCT04916431). The modeling approach used to predict human responses was validated when data became available from the phase I clinical study. This validation indicates that the approach is valuable in informing clinical decision-making.
Subject(s)
Interleukin-2 , RNA, Messenger , Humans , Interleukin-2/pharmacokinetics , Interleukin-2/genetics , Interleukin-2/pharmacology , Interleukin-2/administration & dosage , Animals , RNA, Messenger/genetics , T-Lymphocytes, Regulatory/drug effects , Nanoparticles , Models, Biological , Male , LiposomesABSTRACT
Direct injection of therapies into tumors has emerged as an administration route capable of achieving high local drug exposure and strong anti-tumor response. A diverse array of immune agonists ranging in size and target are under development as local immunotherapies. However, due to the relatively recent adoption of intratumoral administration, the pharmacokinetics of locally-injected biologics remains poorly defined, limiting rational design of tumor-localized immunotherapies. Here we define a pharmacokinetic framework for biologics injected intratumorally that can predict tumor exposure and effectiveness. We find empirically and computationally that extending the tumor exposure of locally-injected interleukin-2 by increasing molecular size and/or improving matrix-targeting affinity improves therapeutic efficacy in mice. By tracking the distribution of intratumorally-injected proteins using positron emission tomography, we observe size-dependent enhancement in tumor exposure occurs by slowing the rate of diffusive escape from the tumor and by increasing partitioning to an apparent viscous region of the tumor. In elucidating how molecular weight and matrix binding interplay to determine tumor exposure, our model can aid in the design of intratumoral therapies to exert maximal therapeutic effect.
Subject(s)
Collagen/genetics , Immunotherapy/methods , Interleukin-2/pharmacology , Melanoma, Experimental/therapy , Receptors, Immunologic/genetics , Skin Neoplasms/therapy , Allografts , Animals , Carrier Proteins/genetics , Carrier Proteins/immunology , Cell Line, Tumor , Collagen/immunology , Female , Gene Library , Injections, Intralesional , Interleukin-2/genetics , Interleukin-2/immunology , Interleukin-2/pharmacokinetics , Melanoma, Experimental/diagnostic imaging , Melanoma, Experimental/genetics , Melanoma, Experimental/mortality , Mice , Mice, Inbred C57BL , Peptides/genetics , Peptides/immunology , Positron-Emission Tomography , Protein Binding , Protein Engineering/methods , Receptors, Immunologic/immunology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Serum Albumin/genetics , Serum Albumin/immunology , Skin Neoplasms/diagnostic imaging , Skin Neoplasms/genetics , Skin Neoplasms/mortality , Survival Analysis , Tumor Burden/drug effectsABSTRACT
Protein drugs hold great promise as therapeutics for a wide range of diseases. Unfortunately, one of the greatest challenges to be addressed during clinical development of protein therapeutics is their short circulation half-life. Several protein conjugation strategies have been developed for half-life extension. However, these strategies have limitations and there remains room for improvement. Here, we report a novel nature-inspired strategy for enhancing the in vivo half-life of proteins. Our strategy involves conjugating proteins to a hydrophilic small molecule that binds reversibly to the plasma protein, transthyretin. We show here that our strategy is effective in enhancing the pharmacokinetic and pharmacodynamic properties of human interleukin 2 in rats, potentially opening the door for more effective and safer cancer immunotherapies. To our knowledge, this is the first example of successful use of a small-molecule that not only extends the half-life but also maintains the smaller size, binding potency, and hydrophilicity of proteins.
Subject(s)
Interleukin-2/pharmacokinetics , Prealbumin/metabolism , Small Molecule Libraries/metabolism , Amino Acid Sequence , Animals , Cell Line , Half-Life , Humans , Interleukin-2/chemistry , Interleukin-2/metabolism , Ligands , Male , Rats , Rats, Sprague-Dawley , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacokinetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tandem Mass SpectrometryABSTRACT
Nemvaleukin alfa (nemvaleukin, ALKS 4230) is a novel cytokine created by the fusion of circularly permuted interleukin-2 (IL-2) to the IL-2Rα subunit of the IL-2 receptor (IL-2R) complex that confers selectivity for the intermediate-affinity IL-2R expressed on CD8+ T cells and natural killer (NK) cells. The pharmacokinetics and selective pharmacodynamic properties of nemvaleukin have been demonstrated using in vitro and in vivo mouse models. The pharmacokinetic/pharmacodynamic effects of nemvaleukin on immune cell subtypes were evaluated in cynomolgus monkeys after intravenous and subcutaneous administration to inform dose selection and predict pharmacodynamic effects in humans. Male drug-naïve cynomolgus monkeys (N = 15) were administered either single-dose (0.3 mg/kg i.v.; 0.3 mg/kg or 1.0 mg/kg s.c.) or repeated doses (0.1 mg/kg i.v. on days 1-5 or 0.5 mg/kg s.c. on days 1 and 4) of nemvaleukin. Serial blood samples were collected for pharmacokinetic assessment, immunophenotyping by flow cytometry, and profiling of serum cytokines. Repeat-dose subcutaneous administration of nemvaleukin with less frequent dosing resulted in total systemic exposure and trough serum concentrations comparable to those seen with intravenous administration, with lower peak serum concentrations. Transient elevation of interferon-γ and IL-6 peaked at 2 and 8 hours after intravenous and subcutaneous administration, respectively. Selective expansion of immunoprotective central memory, effector memory, terminal effector CD8+ T cells, and CD56+ NK cells, and minimal expansion of immunosuppressive CD4+CD25+FoxP3+ regulatory T cells was observed after both intravenous and subcutaneous administration. These data support the ongoing clinical evaluation of intravenous and subcutaneous nemvaleukin. SIGNIFICANCE STATEMENT: Administration of the novel interleukin-2 receptor agonist nemvaleukin alfa to cynomolgus monkeys resulted in selective expansion of immune effector cells, including CD8+ T and natural killer cells with minimal effects on immunosuppressive CD4+ regulatory T cells, confirming the design of nemvaleukin and highlighting its potential as a cancer immunotherapy. Subcutaneous administration of nemvaleukin achieved systemic exposure and immunostimulatory effects similar to those observed after more frequent intravenous dosing and may represent a practical alternative in a clinical setting.
Subject(s)
Interleukin-2 Receptor alpha Subunit/metabolism , Interleukin-2/pharmacokinetics , Receptors, Interleukin-2/agonists , Receptors, Interleukin-2/metabolism , Administration, Intravenous , Animals , Dose-Response Relationship, Drug , Humans , Injections, Subcutaneous , Interleukin-2/administration & dosage , Lymphocytes/drug effects , Lymphocytes/metabolism , Macaca fascicularis , MaleABSTRACT
E7777 is a recombinant cytotoxic fusion protein composed of the diphtheria toxin fragments A and B and human interleukin-2. It shares an amino acid sequence with denileukin diftitox, but has improved purity and an increased percentage of active monomer. We undertook a multicenter, single-arm phase II study of E7777 in patients with relapsed or refractory peripheral T-cell lymphoma (PTCL) and cutaneous T-cell lymphoma (CTCL) to evaluate its efficacy, safety, pharmacokinetics, and immunogenicity. A total of 37 patients were enrolled, of which 17 and 19 patients had PTCL and CTCL, respectively, and one patient with another type of lymphoma (extranodal natural killer/T-cell lymphoma, nasal type), diagnosed by the Central Pathological Diagnosis Committee. Among the 36 patients with PTCL and CTCL, objective response rate based on the independent review was 36% (41% and 31%, respectively). The median progression-free survival was 3.1 months (2.1 months in PTCL and 4.2 months in CTCL). The common adverse events (AEs) observed were increased aspartate aminotransferase (AST) / alanine aminotransferase (ALT), hypoalbuminemia, lymphopenia, and pyrexia. Our results indicated that a 9 µg/kg/d dose of E7777 shows efficacy and a manageable safety profile in Japanese patients with relapsed or refractory PTCL and CTCL, with clinical activity observed across the range of CD25 expression. The common AEs were manageable, but increase in ALT / AST, hypoalbuminemia, and capillary leak syndrome should be carefully managed during the treatment.
Subject(s)
Interleukin-2/administration & dosage , Lymphoma, T-Cell, Cutaneous/drug therapy , Lymphoma, T-Cell, Peripheral/drug therapy , Neoplasm Recurrence, Local/drug therapy , Recombinant Fusion Proteins/administration & dosage , Administration, Intravenous , Binding Sites , Diphtheria Toxin/administration & dosage , Diphtheria Toxin/adverse effects , Diphtheria Toxin/chemistry , Diphtheria Toxin/genetics , Diphtheria Toxin/pharmacokinetics , Drug Administration Schedule , Female , Humans , Interleukin-2/adverse effects , Interleukin-2/chemistry , Interleukin-2/genetics , Interleukin-2/pharmacokinetics , Japan , Lymphoma, T-Cell, Cutaneous/blood , Lymphoma, T-Cell, Peripheral/blood , Male , Neoplasm Recurrence, Local/blood , Recombinant Fusion Proteins/adverse effects , Recombinant Fusion Proteins/pharmacokinetics , Survival Analysis , Treatment OutcomeABSTRACT
IL-2 is the master-regulator cytokine for T cell dependent responses and is crucial for proliferation and survival of T cells. However, IL-2-based treatments remained marginal, in part due to short half-life. Thus, we aimed to extend IL-2 half-life by flanking the IL-2 core with sequences derived from the extensively glycosylated hinge region of the NCR2 receptor. We termed this modified IL-2: "S2A". Importantly, S2A blood half-life was extended 14-fold compared to the clinical grade IL-2, Proleukin. Low doses inoculation of S2A significantly enhanced induction of Tregs (CD4+ Regulatory T cells) in vivo, as compared to Proleukin, while both S2A and Proleukin induced low levels of CD8+ T cells. In a B16 metastatic melanoma model, S2A treatment was unable to reduce the metastatic capacity of B16 melanoma, while enhancing induction and recruitment of Tregs, compared to Proleukin. Conversely, in two autoimmune models, rheumatoid arthritis and DSS-induced colitis, S2A treatment significantly reduced the progression of disease compared to Proleukin. Our results suggest new avenues for generating long-acting IL-2 for long-standing treatment and a new technique for manipulating short-life proteins for clinical and research uses.
Subject(s)
Autoimmunity/drug effects , Interleukin-2/analogs & derivatives , Natural Cytotoxicity Triggering Receptor 2/chemistry , T-Lymphocytes, Regulatory/drug effects , Animals , Arthritis, Rheumatoid/prevention & control , Delayed-Action Preparations , Drug Evaluation, Preclinical , Glycosylation , Half-Life , Interleukin-2/administration & dosage , Interleukin-2/pharmacokinetics , Mice, Inbred C57BL , Mice, KnockoutABSTRACT
Human Interleukin 2 (IL-2) has already achieved impressive results as a therapeutic agent for cancer and autoimmune diseases. However, one of the limitations associated with the clinical application of IL-2 is its short half-life owing to rapid clearance by the kidneys. Modification with fatty acids, as an albumin noncovalent ligand with the advantage of deep penetration into tissues and high activity-to-mass ratio, is a commonly used approach to improve the half-life of native peptides and proteins. In this investigation, we attempted to extend the half-life of IL-2 through conjugation with a fatty acid using sortase A (srtA). We initially designed and optimized three IL-2 analogues with different peptide linkers between the C-terminus of IL-2 and srtA recognition sequence (LPETG). Among these, analogue A3 was validated as the optimal IL-2 analogue for further modification. Next, six fatty acid moieties with the same fatty acid and different hydrophilic spacers were conjugated to A3 through srtA. The six bioconjugates generated were screened for in vitro biological activity, among which bioconjugate B6 was identified as near-optimal to IL-2. Additionally, B6 could effectively bind albumin through the conjugated fatty acid, which contributed to a significant improvement in its pharmacokinetic properties in vivo. In summary, we have developed a novel IL-2 bioconjugate, B6, modified with fatty acids using srtA, which may effectively serve as a new-generation long-acting IL-2 immunotherapeutic agent.
Subject(s)
Aminoacyltransferases/chemistry , Bacterial Proteins/chemistry , Cysteine Endopeptidases/chemistry , Fatty Acids/chemistry , Interleukin-2/pharmacology , Amino Acid Sequence , Half-Life , Humans , Interleukin-2/chemistry , Interleukin-2/pharmacokineticsABSTRACT
AIMS/HYPOTHESIS: IL-2 injections are a promising therapy for autoimmune type 1 diabetes but the short half-life of this cytokine in vivo limits effective tissue exposure and necessitates frequent injections. Here we have investigated whether an injectable hydrogel could be used to promote prolonged IL-2 release in vivo. METHODS: Capitalising on the IL-2-binding capabilities of heparin, an injectable hydrogel incorporating clinical-grade heparin, collagen and hyaluronan polymers was used to deliver IL-2. The IL-2-release kinetics and in vivo stability of this material were examined. The ability of soluble IL-2 vs hydrogel-mediated IL-2 injections to prevent autoimmune diabetes in the NOD mouse model of type 1 diabetes were compared. RESULTS: We observed in vitro that the hydrogel released IL-2 over a 12-day time frame and that injected hydrogel likewise persisted 12 days in vivo. Notably, heparin binding potentiates the activity of IL-2 and enhances IL-2- and TGFß-mediated expansion of forkhead box P3-positive regulatory T cells (FOXP3+ Tregs). Finally, weekly administration of IL-2-containing hydrogel partially prevented autoimmune diabetes while injections of soluble IL-2 did not. CONCLUSIONS/INTERPRETATION: Hydrogel delivery may reduce the number of injections required in IL-2 treatment protocols for autoimmune diabetes. Graphical abstract.
Subject(s)
Autoimmune Diseases/prevention & control , Diabetes Mellitus, Type 1/immunology , Diabetes Mellitus, Type 1/prevention & control , Hydrogels/administration & dosage , Interleukin-2/administration & dosage , Animals , Heparin/administration & dosage , Injections , Insulin-Secreting Cells/immunology , Interleukin-2/pharmacokinetics , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Solubility , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/physiologyABSTRACT
Recently, N-(4-18F-fluorobenzoyl)-interleukin-2 (18F-FB-IL2) was introduced as a PET tracer for T cell imaging. However, production is complex and time-consuming. Therefore, we developed 2 radiolabeled IL2 variants, namely aluminum 18F-fluoride-(restrained complexing agent)-IL2 (18F-AlF-RESCA-IL2) and 68Ga-gallium-(1,4,7-triazacyclononane-4,7-diacetic acid-1-glutaric acid)-IL2 (68Ga-Ga-NODAGA-IL2), and compared their in vitro and in vivo characteristics with 18F-FB-IL2. Methods: Radiolabeling of 18F-AlF-RESCA-IL2 and 68Ga-Ga-NODAGA-IL2 was optimized, and stability was evaluated in human serum. Receptor binding was studied with activated human peripheral blood mononuclear cells (hPBMCs). Ex vivo tracer biodistribution in immunocompetent BALB/cOlaHsd (BALB/c) mice was performed at 15, 60, and 90 min after tracer injection. In vivo binding characteristics were studied in severe combined immunodeficient (SCID) mice inoculated with activated hPBMCs in Matrigel. Tracer was injected 15 min after hPBMC inoculation, and a 60-min dynamic PET scan was acquired, followed by ex vivo biodistribution studies. Specific uptake was determined by coinjection of tracer with unlabeled IL2 and by evaluating uptake in a control group inoculated with Matrigel only. Results:68Ga-Ga-NODAGA-IL2 and 18F-AlF-RESCA-IL2 were produced with radiochemical purity of more than 95% and radiochemical yield of 13.1% ± 4.7% and 2.4% ± 1.6% within 60 and 90 min, respectively. Both tracers were stable in serum, with more than 90% being intact tracer after 1 h. In vitro, both tracers displayed preferential binding to activated hPBMCs. Ex vivo biodistribution studies on BALB/c mice showed higher uptake of 18F-AlF-RESCA-IL2 than of 18F-FB-IL2 in liver, kidney, spleen, bone, and bone marrow. 68Ga-Ga-NODAGA-IL2 uptake in liver and kidney was higher than 18F-FB-IL2 uptake. In vivo, all tracers revealed uptake in activated hPBMCs in SCID mice. Low uptake was seen after a blocking dose of IL2 and in the Matrigel control group. In addition, 18F-AlF-RESCA-IL2 yielded the highest-contrast PET images of target lymph nodes. Conclusion: Production of 18F-AlF-RESCA-IL2 and 68Ga-Ga-NODAGA-IL2 is simpler and faster than that of 18F-FB-IL2. Both tracers showed good in vitro and in vivo characteristics, with high uptake in lymphoid tissue and hPBMC xenografts.
Subject(s)
Interleukin-2/chemistry , Positron-Emission Tomography/methods , T-Lymphocytes/metabolism , Animals , BALB 3T3 Cells , Drug Discovery , Fluorine Radioisotopes/chemistry , Gallium Radioisotopes/chemistry , Humans , Interleukin-2/pharmacokinetics , Mice , Radioactive Tracers , Tissue DistributionABSTRACT
NKTR-214 (bempegaldesleukin) is a novel IL2 pathway agonist, designed to provide sustained signaling through heterodimeric IL2 receptor ßγ to drive increased proliferation and activation of CD8+ T and natural killer cells without unwanted expansion of T regulatory cells (Treg) in the tumor microenvironment. In this first-in-human multicenter phase I study, NKTR-214 administered as an outpatient regimen was well tolerated and showed clinical activity including tumor shrinkage and durable disease stabilization in heavily pretreated patients. Immune activation and increased numbers of immune cells were observed in the periphery across all doses and cycles with no loss of NKTR-214 activity with repeated administration. On-treatment tumor biopsies demonstrated that NKTR-214 promoted immune cell increase with limited increase of Tregs. Transcriptional analysis of tumor biopsies showed that NKTR-214 engaged the IL2 receptor pathway and significantly increased genes associated with an effector phenotype. Based on safety and pharmacodynamic markers, the recommended phase II dose was determined to be 0.006 mg/kg every three weeks. SIGNIFICANCE: We believe that IL2- and IL2 pathway-targeted agents such as NKTR-214 are key components to an optimal immunotherapy treatment algorithm. Based on its biological activity and tolerability, NKTR-214 is being studied with approved immuno-oncology agents including checkpoint inhibitors.See related commentary by Sullivan, p. 694.This article is highlighted in the In This Issue feature, p. 681.
Subject(s)
Interleukin-2 Receptor alpha Subunit/metabolism , Interleukin-2/analogs & derivatives , Interleukin-2/metabolism , Neoplasms/drug therapy , Neoplasms/metabolism , Polyethylene Glycols/therapeutic use , Signal Transduction/drug effects , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Biomarkers , CD8-Positive T-Lymphocytes , Cell Line, Tumor , Humans , Interleukin-2/administration & dosage , Interleukin-2/adverse effects , Interleukin-2/pharmacokinetics , Interleukin-2/therapeutic use , Neoplasms/etiology , Neoplasms/pathology , Polyethylene Glycols/administration & dosage , Polyethylene Glycols/adverse effects , Polyethylene Glycols/pharmacokinetics , Treatment Outcome , Tumor Microenvironment/drug effectsABSTRACT
Purpose: Optimal dosing is critical for immunocytokine-based cancer immunotherapy to maximize efficacy and minimize toxicity. Cergutuzumab amunaleukin (CEA-IL2v) is a novel CEA-targeted immunocytokine. We set out to develop a mathematical model to predict intratumoral CEA-IL2v concentrations following various systemic dosing intensities.Experimental Design: Sequential measurements of CEA-IL2v plasma concentrations in 74 patients with solid tumors were applied in a series of differential equations to devise a model that also incorporates the peripheral concentrations of IL2 receptor-positive cell populations (i.e., CD8+, CD4+, NK, and B cells), which affect tumor bioavailability of CEA-IL2v. Imaging data from a subset of 14 patients were subsequently utilized to additionally predict antibody uptake in tumor tissues.Results: We created a pharmacokinetic/pharmacodynamic mathematical model that incorporates the expansion of IL2R-positive target cells at multiple dose levels and different schedules of CEA-IL2v. Model-based prediction of drug levels correlated with the concentration of IL2R-positive cells in the peripheral blood of patients. The pharmacokinetic model was further refined and extended by adding a model of antibody uptake, which is based on drug dose and the biological properties of the tumor. In silico predictions of our model correlated with imaging data and demonstrated that a dose-dense schedule comprising escalating doses and shortened intervals of drug administration can improve intratumoral drug uptake and overcome consumption of CEA-IL2v by the expanding population of IL2R-positive cells.Conclusions: The model presented here allows simulation of individualized treatment plans for optimal dosing and scheduling of immunocytokines for anticancer immunotherapy. Clin Cancer Res; 24(14); 3325-33. ©2018 AACRSee related commentary by Ruiz-Cerdá et al., p. 3236.
Subject(s)
Cytokines/administration & dosage , Immunologic Factors/administration & dosage , Models, Theoretical , Neoplasms/drug therapy , Neoplasms/metabolism , Antineoplastic Agents, Immunological/administration & dosage , Antineoplastic Agents, Immunological/adverse effects , Antineoplastic Agents, Immunological/pharmacokinetics , Biomarkers , Cytokines/adverse effects , Cytokines/pharmacokinetics , Humans , Immunologic Factors/adverse effects , Immunologic Factors/pharmacokinetics , Immunotherapy , Interleukin-2/administration & dosage , Interleukin-2/adverse effects , Interleukin-2/pharmacokinetics , Models, Biological , Molecular Imaging , Neoplasms/diagnosis , Neoplasms/mortality , Prognosis , Receptors, Interleukin-2/metabolism , Treatment OutcomeABSTRACT
A biomimetic nanogel with tumor microenvironment responsive property is developed for the combinatorial antitumor effects of chemotherapy and immunotherapy. Nanogels are formulated with hydroxypropyl-ß-cyclodextrin acrylate and two opposite charged chitosan derivatives for entrapping anticancer drug paclitaxel and precisely controlling the pH responsive capability, respectively. The nanogel supported erythrocyte membrane can achieve "nanosponge" property for delivering immunotherapeutic agent interleukin-2 without reducing the bioactivity. By responsively releasing drugs in tumor microenvironment, the nanogels significantly enhanced antitumor activity with improved drug penetration, induction of calreticulin exposure, and increased antitumor immunity. The tumor microenvironment is remodeled by the combination of these drugs in low dosage, as evidenced by the promoted infiltration of immune effector cells and reduction of immunosuppressive factors.
Subject(s)
Antineoplastic Agents, Phytogenic/administration & dosage , Chitosan/analogs & derivatives , Gels/chemistry , Interleukin-2/administration & dosage , Neoplasms/therapy , Paclitaxel/administration & dosage , Tumor Microenvironment/drug effects , 2-Hydroxypropyl-beta-cyclodextrin/chemistry , Animals , Antineoplastic Agents, Phytogenic/pharmacokinetics , Antineoplastic Agents, Phytogenic/therapeutic use , Drug Carriers/chemistry , Drug Delivery Systems/methods , Humans , Immunotherapy/methods , Interleukin-2/pharmacokinetics , Interleukin-2/therapeutic use , Mice, Inbred C57BL , Neoplasms/drug therapy , Neoplasms/pathology , Paclitaxel/pharmacokinetics , Paclitaxel/therapeutic use , Rats, Sprague-DawleyABSTRACT
Cytokines are potent immune modulating agents but are not ideal medicines in their natural form due to their short half-life and pleiotropic systemic effects. NKTR-214 is a clinical-stage biologic that comprises interleukin-2 (IL2) protein bound by multiple releasable polyethylene glycol (PEG) chains. In this highly PEG-bound form, the IL2 is inactive; therefore, NKTR-214 is a biologic prodrug. When administered in vivo, the PEG chains slowly release, creating a cascade of increasingly active IL2 protein conjugates bound by fewer PEG chains. The 1-PEG-IL2 and 2-PEG-IL2 species derived from NKTR-214 are the most active conjugated-IL2 species. Free-IL2 protein is undetectable in vivo as it is eliminated faster than formed. The PEG chains on NKTR-214 are located at the region of IL2 that contacts the alpha (α) subunit of the heterotrimeric IL2 receptor complex, IL2Rαßγ, reducing its ability to bind and activate the heterotrimer. The IL2Rαßγ complex is constitutively expressed on regulatory T cells (Tregs). Therefore, without the use of mutations, PEGylation reduces the affinity for IL2Rαßγ to a greater extent than for IL2Rßγ, the receptor complex predominant on CD8 T cells. NKTR-214 treatment in vivo favors activation of CD8 T cells over Tregs in the tumor microenvironment to provide anti-tumor efficacy in multiple syngeneic models. Mechanistic modeling based on in vitro and in vivo kinetic data provides insight into the mechanism of NKTR-214 pharmacology. The model reveals that conjugated-IL2 protein derived from NKTR-214 occupy IL-2Rßγ to a greater extent compared to free-IL2 protein. The model accurately describes the sustained in vivo signaling observed after a single dose of NKTR-214 and explains how the properties of NKTR-214 impart a unique kinetically-controlled immunological mechanism of action.
Subject(s)
Immunotherapy/methods , Interleukin-2/analogs & derivatives , Neoplasms/therapy , Polyethylene Glycols/pharmacology , Receptors, Interleukin-2/agonists , Algorithms , Animals , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Line, Tumor , Drug Liberation , Female , Interleukin Receptor Common gamma Subunit/agonists , Interleukin Receptor Common gamma Subunit/metabolism , Interleukin-2/pharmacokinetics , Interleukin-2/pharmacology , Interleukin-2 Receptor alpha Subunit/agonists , Interleukin-2 Receptor alpha Subunit/metabolism , Interleukin-2 Receptor beta Subunit/agonists , Interleukin-2 Receptor beta Subunit/metabolism , Kinetics , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred C57BL , Models, Theoretical , Neoplasms/immunology , Neoplasms/metabolism , Phosphorylation/drug effects , Polyethylene Glycols/pharmacokinetics , Prodrugs/pharmacokinetics , Prodrugs/pharmacology , Receptors, Interleukin-2/metabolism , STAT5 Transcription Factor/metabolism , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Transplantation, Homologous , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunologyABSTRACT
The aim of this study was to develop and characterize rh- IL-2 loaded chitosan-based nanogels for the healing of wound incision in rats. Nanogels were prepared using chitosan and bovine serum albumin (BSA) by ionic gelation method and high temperature application, respectively. Particle size, zeta potential, and polydispersity index were measured for characterization of nanogels. The morphology of nanogels was examined by using SEM and AFM. The IL-2 loading capacity of nanogels was determined using ELISA method. In vitro release of IL-2 from nanogels was performed using Franz diffusion cells. Artificial neural network (ANN) models were developed using selected input parameters (stirring rate, chitosan%, BSA%, TPP%) where particle size was an output parameter for IL-2 free nanogels. Wound healing effect of IL-2 loaded chitosan-TPP nanogel was evaluated by determining the malondialdehyde (MDA) and glutathione (GSH) levels of wound tissues in rats. The particle size of IL-2 loaded chitosan-TPP nanogels was found to be larger than that of IL-2 loaded BSA-based chitosan nanogels. Drug loading capacity of nanogels was found 100% ± 0.010 for both nanogels. IL-2 was released slowly after the initial burst effect. According to SEM and AFM imaging, BSA-chitosan nanogel particles were of nanometer size and presented a swelling tendency, and chitosan-TPP nanogel particles were found to be spherical and homogenously dispersed. IL-2 loaded chitosan-TPP nanogel was found suitable for improving wound healing because it decreased the MDA levels and increased the GSH levels wound tissues comparing to control group.
Subject(s)
Chitosan , Drug Delivery Systems , Interleukin-2 , Polyethylene Glycols/pharmacology , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacokinetics , Biocompatible Materials/chemistry , Biocompatible Materials/pharmacology , Chitosan/chemistry , Chitosan/pharmacology , Diffusion , Gels , Interleukin-2/administration & dosage , Interleukin-2/pharmacokinetics , Nanoparticles , Nerve Net/drug effects , Particle Size , Rats , Solvents/pharmacology , Wound Healing/drug effectsABSTRACT
Ch14.18 manufactured in Chinese hamster ovary (CHO) cells is currently being evaluated in clinical trials. Short-term infusion (STI) (8-20 h/day; 4-5 days) of 100 mg/m2 ch14.18/CHO (dinutiximab ß) per cycle in combination with cytokines is standard treatment of neuroblastoma (NB) patients. As pain is a limiting factor, we investigated a novel delivery method by continuous long-term infusion (LTI) of 100 mg/m2 over 10 days. 53 NB patients were treated with 5-6 cycles of 6 × 106 IU/m2 subcutaneous interleukin-2 (d 1-5, 8-12), LTI of 100 mg/m2 ch14.18/CHO (d 8-18) and 160 mg/m2 oral 13-cis-retinoic acid (d 22-35). Human anti-chimeric antibody (HACA), antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity were determined. With LTI, we observed a maximum concentration of ch14.18/CHO (Cmax) of 12.56 ± 0.68 µg/ml and a terminal half-life time (t1/2 ß) of 32.7 ± 16.2 d. The clearance values for LTI and STI of 0.54 ± 0.13 and 0.41 ± 0.29 L/d m2 and area under the serum concentration-time curve (AUC) values of 189.6 ± 41.4 and 284.8 ± 156.8 µg×d/ml, respectively, were not significantly different. Importantly, we detected ch14.18/CHO trough concentration of ≥ 1 µg/ml at time points preceding subsequent antibody infusions after cycle 1, allowing a persistent activation of antibody effector mechanisms over the entire treatment period of 6 months. HACA responses were observed in 10/53 (19%) patients, similar to STI (21%), indicating LTI had no effect on the immunogenicity of ch14.18/CHO. In conclusion, LTI of ch14.18/CHO induced effector mechanisms over the entire treatment period, and may therefore emerge as the preferred delivery method of anti-GD2 immunotherapy to NB patients.
Subject(s)
Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/pharmacokinetics , Interleukin-2/administration & dosage , Interleukin-2/pharmacokinetics , Neuroblastoma , Adolescent , Adult , Animals , CHO Cells , Child , Child, Preschool , Cricetinae , Cricetulus , Female , Humans , Infant , Isotretinoin/administration & dosage , Isotretinoin/pharmacokinetics , Male , Neuroblastoma/drug therapy , Neuroblastoma/metabolismABSTRACT
Cancer immunotherapies under development have generally focused on either stimulating T cell immunity or driving antibody-directed effector functions of the innate immune system such as antibody-dependent cell-mediated cytotoxicity (ADCC). We find that a combination of an anti-tumor antigen antibody and an untargeted IL-2 fusion protein with delayed systemic clearance induces significant tumor control in aggressive isogenic tumor models via a concerted innate and adaptive response involving neutrophils, NK cells, macrophages, and CD8(+) T cells. This combination therapy induces an intratumoral "cytokine storm" and extensive lymphocyte infiltration. Adoptive transfer of anti-tumor T cells together with this combination therapy leads to robust cures of established tumors and development of immunological memory.
Subject(s)
Neoplasms/therapy , Adaptive Immunity , Animals , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , Drug Synergism , Half-Life , Immunity, Innate , Immunotherapy , Interleukin-2/metabolism , Interleukin-2/pharmacokinetics , Interleukin-2/pharmacology , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Mice , Mice, Inbred C57BL , Neoplasms/immunologyABSTRACT
Cytokine therapy can activate potent, sustained antitumor responses, but collateral toxicity often limits dosages. Although antibody-cytokine fusions (immunocytokines) have been designed with the intent to localize cytokine activity, systemic dose-limiting side effects are not fully ameliorated by attempted tumor targeting. Using the s.c. B16F10 melanoma model, we found that a nontoxic dose of IL-2 immunocytokine synergized with tumor-specific antibody to significantly enhance therapeutic outcomes compared with immunocytokine monotherapy, concomitant with increased tumor saturation and intratumoral cytokine responses. Examination of cell subset biodistribution showed that the immunocytokine associated mainly with IL-2R-expressing innate immune cells, with more bound immunocytokine present in systemic organs than the tumor microenvironment. More surprisingly, immunocytokine antigen specificity and Fcγ receptor interactions did not seem necessary for therapeutic efficacy or biodistribution patterns because immunocytokines with irrelevant specificity and/or inactive mutant Fc domains behaved similarly to tumor-specific immunocytokine. IL-2-IL-2R interactions, rather than antibody-antigen targeting, dictated immunocytokine localization; however, the lack of tumor targeting did not preclude successful antibody combination therapy. Mathematical modeling revealed immunocytokine size as another driver of antigen targeting efficiency. This work presents a safe, straightforward strategy for augmenting immunocytokine efficacy by supplementary antibody dosing and explores underappreciated factors that can subvert efforts to purposefully alter cytokine biodistribution.
Subject(s)
Epitopes/immunology , Interleukin-2/pharmacokinetics , Interleukin-2/therapeutic use , Recombinant Fusion Proteins/pharmacokinetics , Recombinant Fusion Proteins/therapeutic use , Animals , Antibodies, Neoplasm/immunology , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Immunity, Innate/drug effects , Melanoma, Experimental/drug therapy , Melanoma, Experimental/immunology , Mice, Inbred C57BL , Models, Immunological , Receptors, IgG/metabolism , Tissue Distribution , Treatment OutcomeABSTRACT
Antibody-cytokine fusion proteins ("immunocytokines") represent a promising class of armed antibody products, which allow the selective delivery of potent pro-inflammatory payloads at the tumor site. The antibody-based selective delivery of interleukin-2 (IL2) is particularly attractive for the treatment of metastatic melanoma, an indication for which this cytokine received marketing approval from the US Food and drug administration. We used the K1735M2 immunocompetent syngeneic model of murine melanoma to study the therapeutic activity of F8-IL2, an immunocytokine based on the F8 antibody in diabody format, fused to human IL2. F8-IL2 was shown to selectively localize at the tumor site in vivo, following intravenous administration, and to mediate tumor growth retardation, which was potentiated by the combination with paclitaxel or dacarbazine. Combination treatment led to a substantially more effective tumor growth inhibition, compared to the cytotoxic drugs used as single agents, without additional toxicity. Analysis of the immune infiltrate revealed a significant accumulation of CD4(+) T cells 24 h after the administration of the combination. The fusion proteins F8-IL2 and L19-IL2, specific to the alternatively spliced extra domain A and extra domain B of fibronectin respectively, were also studied in combination with tumor necrosis factor (TNF)-based immunocytokines. The combination treatment was superior to the action of the individual immunocytokines and was able to eradicate neoplastic lesions after a single intratumoral injection, a procedure that is being clinically used for the treatment of Stage IIIC melanoma. Collectively, these data reinforce the rationale for the use of IL2-based immunocytokines in combination with cytotoxic agents or TNF-based immunotherapy for the treatment of melanoma patients.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Immunotherapy/methods , Interleukin-2/pharmacology , Melanoma, Experimental/therapy , Recombinant Fusion Proteins/pharmacology , Tumor Necrosis Factor-alpha/pharmacology , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacokinetics , Antibodies, Monoclonal, Humanized , Cell Line, Tumor , Combined Modality Therapy , Dacarbazine/administration & dosage , Drug Synergism , Female , Interleukin-2/administration & dosage , Interleukin-2/immunology , Interleukin-2/pharmacokinetics , Melanoma, Experimental/drug therapy , Melanoma, Experimental/immunology , Melanoma, Experimental/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Paclitaxel/administration & dosage , Recombinant Fusion Proteins/administration & dosage , Tissue Distribution , Tumor Necrosis Factor-alpha/immunologyABSTRACT
The present study reports the preparation and physicochemical characterization of surface-modified poly(lactide-co-glycolide) (PLGA) microparticles containing interleukin-2 (rhIL-2) for pulmonary delivery. The surface of the microparticles was modified with mucoadhesive polymers such as chitosan and Carbopol 971P. The feasibility of this surface modification was confirmed by measuring the zeta potential. Chitosan-modified PLGA microparticles showed a positive zeta potential, while Carbopol-modified PLGA microparticles were negatively charged. The mucin binding efficiency values have shown that the positively charged chitosan coated microparticles showed a higher adhesive percent to the mucin than the negatively charged un-coated or Carbopol 971P coated microparticles. Furthermore, surface modification of microparticles with chitosan and Carbopol 971P has yielded a slight decrease in the amount of protein initially released. These findings suggest the suitability of surface-modified PLGA microparticles as an efficient carrier system for delivery peptides and proteins to the respiratory tract.