Therapeutic Evaluation of Antibody-Based Targeted Delivery of Interleukin 9 in Experimental Pulmonary Hypertension.

BACKGROUND AND AIMS: Pulmonary hypertension (PH) is a heterogeneous disorder associated with poor prognosis. For the majority of patients, only limited therapeutic options are available. Thus, there is great interest to develop novel treatment strategies focusing on pulmonary vascular and right ventricular remodeling. Interleukin 9 (IL9) is a pleiotropic cytokine with pro- and anti-inflammatory functions. The aim of this study was to evaluate the therapeutic activity of F8IL9F8 consisting of IL9 fused to the F8 antibody, specific to the alternatively-spliced EDA domain of fibronectin, which is abundantly expressed in pulmonary vasculature and right ventricular myocardium in PH. METHODS: The efficacy of F8IL9F8 in attenuating PH progression in the monocrotaline mouse model was evaluated in comparison to an endothelin receptor antagonist (ERA) or an IL9 based immunocytokine with irrelevant antibody specificity (KSFIL9KSF). Treatment effects were assessed by right heart catheterization, echocardiography as well as histological and immunohistochemical tissue analyses. RESULTS: Compared to controls, systolic right ventricular pressure (RVPsys) was significantly elevated and a variety of right ventricular echocardiographic parameters were significantly impaired in all MCT-induced PH groups except for the F8IL9F8 group. Both, F8IL9F8 and ERA treatments lead to a significant reduction in RVPsys and an improvement of echocardiographic parameters when compared to the MCT group not observable for the KSFIL9KSF group. Only F8IL9F8 significantly reduced lung tissue damage and displayed a significant decrease of leukocyte and macrophage accumulation in the lungs and right ventricles. CONCLUSIONS: Our study provides first pre-clinical evidence for the use of F8IL9F8 as a new therapeutic agent for PH in terms of a disease-modifying concept addressing cardiovascular remodeling.

Antibody-based delivery of interleukin-9 to neovascular structures: Therapeutic evaluation in cancer and arthritis.

Interleukin-9 is a cytokine with multiple functions, including the ability to activate group 2 innate lymphoid cells, which has been postulated to be therapeutically active in mouse models of arthritis. Similarly, interleukin-9 has been suggested to play an important role in tumor immunity. Here, we describe the cloning, expression, and characterization of three fusion proteins based on murine interleukin-9 and the F8 antibody, specific to the alternatively spliced EDA domain of fibronectin. EDA is strongly expressed in cancer and in various arthritic conditions, while being undetectable in the majority of healthy organs. Interleukin-9-based fusion proteins with an irrelevant antibody specific to hen egg lysozyme served as negative control in our study. The fusion proteins were characterized by quantitative biodistribution analysis in tumor-bearing mice using radioiodinated protein preparations. The highest tumor uptake and best tumor:organ ratios were observed for a format, in which the interleukin-9 moiety was flanked by two units of the F8 antibody in single-chain Fv format. Biological activity of interleukin-9 was retained when the payload was fused to antibodies. However, the targeted delivery of interleukin-9 to the disease site resulted in a modest anti-tumor activity in three different murine models of cancer (K1735M2, CT26, and F9), while no therapeutic benefit was observed in a collagen induced model of arthritis. Collectively, these results confirm the possibility to deliver interleukin-9 to the site of disease but cast doubts about the alleged therapeutic activity of this cytokine in cancer and arthritis, which has been postulated in previous publications.

A novel format for recombinant antibody-interleukin-2 fusion proteins exhibits superior tumor-targeting properties in vivo.

The targeted delivery of interleukin-2 to the tumor is gaining attention as an avenue to potentiate the action of T and NK cells at the site of disease. We have previously described the fusion of the L19 antibody, specific to the EDB domain of fibronectin, with human interleukin-2, using a non-covalent homodimeric diabody format. Here, we describe four novel formats for the L19-IL2 fusion, featuring different arrangements of antibody and IL2. A comparative quantitative biodistribution analysis in tumor-bearing mice using radioiodinated proteins revealed that the novel format (L19L19-IL2, with the antibody in single-chain diabody format) exhibited the best biodistribution results. In vitro assays on peripheral blood mononuclear cells showed a decrease activation of regulatory T cells when single IL2 domain was used. In vivo, both L19-IL2 and L19L19-IL2 inhibited tumor growth in immunocompetent mouse models of cancer. T-cell analysis revealed similar levels of CD4+ and FoxP3+ cells, with an expansion of the CD8+ T cell in mice treated with L19-IL2 and L19L19-IL2. The percentage of CD4+ regulatory T cells was markedly decreased with L19L19-IL2 combined with a mouse-specific PD-1 blocker. Collectively, these data indicate that the new L19L19-IL2 format exhibits favorable tumor-homing properties and mediates a potent anti-cancer activity in vivo.

Sortase-Mediated Site-Specific Modification of Interleukin-2 for the Generation of a Tumor-Targeting Acetazolamide-Cytokine Conjugate.

Small ligands specific to tumor-associated antigens can be used as alternatives to antibodies for the delivery of small payloads such as radionuclides, cytotoxic drugs, and fluorophores. Their use as a delivery moiety of bioactive proteins such as cytokines remains largely unexplored. Here, we describe the preparation and in vivo characterization of the first small molecule-cytokine conjugate targeting carbonic anhydrase IX (CAIX), a marker of renal cell carcinoma and hypoxia. Site-specific conjugation between interleukin-2 and acetazolamide was obtained by sortase A-mediated transpeptidation. Binding of the conjugate to the cognate CAIX antigen was confirmed by surface plasmon resonance. The in vivo targeting of structures expressing carbonic anhydrase IX was assessed by biodistribution experiments in tumor-bearing mice. Optimization of manufacturability and tumor-targeting performance of acetazolamide-cytokine products will be required in order to enable industrial applications.

The antibody-based delivery of interleukin-12 to solid tumors boosts NK and CD8+ T cell activity and synergizes with immune checkpoint inhibitors.

We describe the cloning and characterization of a novel fusion protein (termed L19-mIL12), consisting of murine interleukin-12 in single-chain format, sequentially fused to the L19 antibody in tandem diabody format. The fusion protein bound avidly to the cognate antigen (the alternatively spliced EDB domain of fibronectin), retained the activity of the parental cytokine and was able to selectively localize to murine tumors in vivo, as shown by quantitative biodistribution analysis. L19-mIL12 exhibited a potent antitumor activity in immunocompetent mice bearing CT26 carcinomas and WEHI-164 sarcomas, which could be boosted by combination with checkpoint blockade, leading to durable cancer eradication. L19-mIL12 also inhibited tumor growth in mice with Lewis lung carcinoma (LLC), but in this case, cancer cures could not be obtained, both in monotherapy and in combination. A microscopic analysis and a depletion experiment of tumor-infiltrating leukocytes illustrated the contribution of NK cells and CD8+ T cells for the anticancer activity observed in both tumor models. Upon L19-mIL12 treatment, the density of regulatory T cells (Tregs) was strongly increased in LLC, but not in CT26 tumors. A FACS analysis also revealed that the majority of CD8+ T cells in CT26 tumors were specific to the retroviral AH1 antigen.

Targeted enhancement of the therapeutic window of L19-TNF by transient and selective inhibition of RIPK1-signaling cascade.

INTRODUCTION: Cytokine-based products are gaining importance for cancer immunotherapy. L19-TNF is a clinical-stage antibody-cytokine fusion protein that selectively accumulates to tumors and displays potent anticancer activity in preclinical models. Here, we describe an innovative approach to transiently inhibit off-target toxicity of L19-TNF, while maintaining antitumor activity. METHODS: GSK'963, a potent small molecule inhibitor of RIPK1, was tested in tumor-bearing mice for its ability to reduce acute toxicity associated with TNF signaling. The biological effects of L19-TNF on tumor cells, lymphocytes and tumor vessels were investigated with the aim to enable the administration of TNF doses, which would otherwise be lethal. RESULTS: Transient inhibition of RIPK1 allowed to increase the maximal tolerated dose of L19-TNF. The protective effect of GSK'963 did not affect the selective localization of the immunocytokine to tumors as evidenced by quantitative biodistribution analysis and allowed to reach high local TNF concentrations around tumor blood vessels, causing diffused vascular shutdown and hemorrhagic necrosis within the neoplastic mass. CONCLUSIONS: The selective inhibition of RIPK1 with small molecule inhibitors can be used as a pharmaceutical tool to transiently mask TNF activity and improve the therapeutic window of TNF-based biopharmaceuticals. Similar approaches may be applicable to other pro-inflammatory cytokines.

A Novel Fully-Human Potency-Matched Dual Cytokine-Antibody Fusion Protein Targets Carbonic Anhydrase IX in Renal Cell Carcinomas.

Certain cytokines synergize in activating anti-cancer immunity at the site of disease and it may be desirable to generate biopharmaceutical agents, capable of simultaneous delivery of cytokine pairs to the tumor. In this article, we have described the cloning, expression and characterization of IL2-XE114-TNFmut, a dual-cytokine biopharmaceutical featuring the sequential fusion of interleukin-2 (IL2) with the XE114 antibody in scFv format and a tumor necrosis factor mutant (TNFmut). The fusion protein recognized the cognate antigen (carbonic anhydrase IX, a marker of hypoxia and of renal cell carcinoma) with high affinity and specificity. IL2-XE114-TNFmut formed a stable non-covalent homotrimeric structure, displayed cytokine activity in in vitro tests and preferentially localized to solid tumors in vivo. The product exhibited a partial growth inhibition of murine CT26 tumors transfected for carbonic anhydrase IX. When administered to Cynomolgus monkey as intravenous injection, IL2-XE114-TNFmut showed the expected plasma concentration of ~1,500 ng/ml at early time points, indicating the absence of any in vivo trapping events, and a half-life of ~2 h. IL2-XE114-TNFmut may thus be considered as a promising biopharmaceutical for the treatment of metastatic clear-cell renal cell carcinoma, since these tumors are known to be sensitive to IL2 and to TNF.

A novel anti-cancer L19-interleukin-12 fusion protein with an optimized peptide linker efficiently localizes in vivo at the site of tumors.

Antibody-cytokine fusion proteins are a class of biopharmaceuticals, with the potential to modulate the activity of the immune system at the site of disease. The molecular format used to connect antibody moiety and cytokine payload can have a profound influence on biological activity and pharmacokinetic properties. The optimization of fusion protein format is particularly challenging for heterodimeric cytokines, since various molecular arrangements can be considered. In this article, we have explored the role of linker in a tumor-targeting IL12 fusion protein, based on the L19 antibody, specific to the extra-domain B of fibronectin. In biodistribution studies performed in tumor-bearing mice using radioiodinated protein preparations, fusion of human IL12 at the N-terminus of the L19 antibody in tandem-diabody format led to higher tumor uptake and improved tumor-to-organ ratios, compared to a similar fusion protein featuring L19 in IgG1 format. Moreover, optimization of the amino acid composition in eight variants of the linker connecting the IL12 moiety to the tandem-diabody revealed that a 15-amino acid linker (GSADGGSSAGGSDAG) displayed the best tumor targeting characteristics, with a long residence time at the tumor site and a rapid clearance from blood and normal organs. The product is being developed for industrial and clinical applications.