An antibody-based enzymatic therapy for cancer treatment: The selective localization of D-amino acid oxidase to EDA fibronectin.

In order to generate an antibody directed enzyme prodrug therapy, here we designed a chimeric protein by fusing the F8 antibody that recognizes the EDA of fibronectin (expressed on the tumor neovasculature) and an evolved variant of the ROS-generating enzyme D-amino acid oxidase (DAAO). The F8(scFv)-DAAO-Q144R recombinant protein is expressed by both CHO-S and E. coli cells. The F8(scFv)-DAAO-Q144R from E. coli cells is fully soluble, shows a high specific activity, is more thermostable in blood than the native DAAO, possesses a binding affinity for EDA well suited for efficient tumor accumulation, and localizes in tumor tissues. Notably, the F8(scFv)-DAAO-Q144R conjugate generates a stronger cytotoxicity to tumor cells than the native enzyme, especially when an inhibitor of heme oxygenase-1 (HO-1) is used, making it a promising candidate for a selective antitumor oxidative therapy controlled by the substrate addition, in the so called "activity on demand", thus sparing normal tissue from damage.

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.

Antibody-Based Delivery of Cytokine Payloads to Carbonic Anhydrase IX Leads to Cancer Cures in Immunocompetent Tumor-Bearing Mice.

Antibody-cytokine fusion proteins can have the potential to increase the density and activity of subsets of leukocytes within the tumor mass. Here, we describe the design, production, and characterization of four novel antibody-cytokine fusion proteins directed against human carbonic anhydrase IX, a highly validated marker of hypoxia that is overexpressed in clear cell renal cell carcinoma and other malignancies. As immunomodulatory payloads we used TNF, IL2, IFNα2 (corresponding to products that are in clinical use), and IL12 (as this cytokine potently activates T cells and NK cells). Therapy experiments were performed in BALB/c mice, bearing CT26 tumors transfected with human carbonic anhydrase IX, in order to assess the performance of the fusion proteins in an immunocompetent setting. The biopharmaceuticals featuring TNF, IL2, or IL12 as payloads cured all mice in their therapy groups, whereas only a subset of mice was cured by the antibody-based delivery of IFNα2. Although the antibody fusion with TNF mediated a rapid hemorrhagic necrosis of the tumor mass, a slower regression of the neoplastic lesions (which continued after the last injection) was observed with the other fusion proteins, and treated mice acquired protective anticancer immunity. A high proportion of tumor-infiltrating CD8+ T cells was specific to the retroviral antigen AH1; however, the LGPGREYRAL peptide derived from human carbonic anhydrase IX was also present on tumor cells. The results described herein provide a rationale for the clinical use of fully human antibody-cytokine fusions specific to carbonic anhydrase IX.

Targeted delivery of calreticulin to ED-A fibronectin leads to tumor-growth retardation.

We report the design and characterization of novel fusion proteins, consisting of the F8 antibody and of murine calreticulin (Calr). The F8 antibody recognizes the alternatively-spliced ED-A domain of fibronectin, an extracellular matrix component found in most tumor types, while calreticulin has previously been described as an "eat-me" signal for dendritic cells and phagocytes. Four fusion proteins, differing in antibody formats and peptide linkers, were produced in mammalian cells, purified to homogeneity and tested in vitro and in vivo. A quantitative biodistribution in F9 tumor-bearing mice revealed that the homobivalent F8-F8-Calr format, featuring a tandem diabody structure, had the best tumor-homing properties and, for this reason, this protein was studied in therapy experiments in CT26 tumor-bearing mice. Intravenous administration of F8-F8-Calr led to a tumor growth retardation, which could be further improved by combination with anti-PD1 antibody treatment. Immunohistochemical analysis revealed an increased density of CD8+ T cells, CD11c+ dendritic cells and F4/80+ macrophages in tumor tissue. Even though F8-F8-Calr did not lead to cancer cures at the doses tested, the excellent tolerability profile and the ability to favor a leukocyte infiltration into the neoplastic mass suggests that the targeted delivery of calreticulin may be considered for combination therapy approaches.

Enhanced Therapeutic Activity of Non-Internalizing Small-Molecule-Drug Conjugates Targeting Carbonic Anhydrase IX in Combination with Targeted Interleukin-2.

Purpose: Antibody-drug conjugates and small-molecule-drug conjugates have been proposed as alternatives to conventional anticancer cytotoxic agents, with the potential to deliver bioactive payloads to the site of disease, helping spare normal tissues.Experimental Design: Here, we describe a novel small-molecule-drug conjugate, based on a high-affinity ligand specific to carbonic anhydrase IX. The product featured a peptidic linker, suitable for cleavage in the tumor extracellular environment, and monomethyl auristatin E as cytotoxic payload.Results: A potent anticancer activity was observed in nude mice bearing SKRC-52 renal cell carcinoma xenografts, but no durable complete responses could be observed in this model. However, when the product was administered together with L19-IL2 (a clinical-stage fusion protein capable of delivering IL2 to the tumor neovasculature), all treated mice in the combination group could be rendered tumor free, in a process that favored the influx of natural killer cells into the tumor mass. The combination of L19-IL2 and the new small-molecule-drug conjugate also eradicated cancer in 100% of immunocompetent mice, bearing subcutaneously grafted CT26 colorectal cancer cells, which stably expressed carbonic anhydrase IX.Conclusions: These findings may be of clinical significance, because carbonic anhydrase IX is overexpressed in the majority of clear cell renal cell carcinomas and in approximately 30% of colorectal cancers. The targeted delivery of IL2 helps potentiate the action of targeted cytotoxics, leading to cancer eradication in models that cannot be cured by conventional chemotherapy. Clin Cancer Res; 24(15); 3656-67. ©2018 AACR.

Intratumoral administration of IL2- and TNF-based fusion proteins cures cancer without establishing protective immunity.

AIM: The combination of tumor-targeting IL2- and TNF-based antibody-cytokine fusions has exhibited encouraging results in mouse and men. Here, we studied their combination to assess efficacy and mechanism of action in four different immunocompetent mouse models of cancer. METHODS: Mice receiving a single intratumoral injection of F8-IL2, F8-TNF or the combination were investigated for tumor-infiltrating leukocytes and rechallenged when cured. RESULTS: In three models, a proportion of treated animals could be cured, most probably by infiltrating NK and CD8+ T cells. Most of the cured mice did not acquire protective immunity when rechallenged with the same tumor cell line. CONCLUSION: Immunocompetent mouse tumor models may not be adequate enough to predict the search for more efficacious therapy regimens.

Antibody-Based Targeted Delivery of Interleukin-22 Promotes Rapid Clinical Recovery in Mice With DSS-Induced Colitis.

BACKGROUND: We have recently described the potential of the alternatively spliced extradomain A of fibronectin as a target for antibody-based pharmacodelivery applications in ulcerative colitis. Here, we report on the cloning and therapeutic properties of novel antibody-based fusion proteins, comprising the F8 antibody specific to extradomain A and murine interleukin (IL)-22, a globular cytokine belonging to the IL10 family. A protective function for IL22 in colitis has previously been described, as this cytokine induces antimicrobial, proliferative, and antiapoptotic pathways, preventing tissue damage and promoting epithelial repair. METHODS: Two fusion proteins comprising IL22, fused at the N- or at the C-terminus of the F8 antibody in diabody format, were expressed in mammalian cells. The ability of radiolabeled preparations of the 2 fusion proteins to localize at sites of disease was assessed by autoradiography in a murine model of dextran sodium sulfate-induced colitis and by quantitative biodistribution analysis in a syngeneic mouse teratocarcinoma model. Therapeutic activity was assessed in mice with dextran sodium sulfate-induced colitis, which received intravenous injections of antibody-cytokine fusion proteins. RESULTS: Both fusion proteins were able to selectively accumulate at the site of disease. The fusion protein with the cytokine moiety at the N-terminal extremity (IL22-F8) exhibited better results than the C-terminal fusion, both in terms of targeting selectivity and therapeutic efficacy. Mice treated with IL22-F8 showed a more rapid recovery from clinical symptoms compared with controls and improved macroscopic and microscopic morphology of the colon. CONCLUSIONS: IL22-F8 is a promising biopharmaceutical drug candidate for the treatment of ulcerative colitis.