Cloning and characterization of novel tumor-targeting immunocytokines based on murine IL7

Pasche, Nadine; Woytschak, Janine; Wulhfard, Sarah; Villa, Alessandra; Frey, Karl; Neri, Dario

doi:10.1016/j.jbiotec.2011.04.003

Cited by 58 publications

(75 citation statements)

References 46 publications

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“…The gene encoding for the F8 antibody, respectively the KSF antibody and the gene encoding murine IL4 were PCR amplified and PCR assembled using standard procedures previously described by our lab. 18 The product was ligated into the mammalian expression vector pcDNA3.1(1) (Invitrogen) by a HindIII=NotI restriction site (for sequences of F8-IL4 and KSF-IL4 see Supporting Information S1).…”

Section: Cloning Expression and Protein In Vitro Characterizationmentioning

confidence: 99%

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The antibody‐based targeted delivery of interleukin‐4 and 12 to the tumor neovasculature eradicates tumors in three mouse models of cancer

Hemmerle

Neri

2013

Intl Journal of Cancer

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Preclinical studies with recombinant murine interleukin 4 (IL4) in models of cancer have shown potent tumor growth inhibition. However, systemic administration of human IL4 to cancer patients exhibited modest antitumor activity and considerable toxicities. To improve the therapeutic index and reduce side effects of this cytokine, we developed of a novel "immunocytokine" based on sequential fusion of murine IL4 with the antibody fragment F8 (specific to the alternatively spliced extra-domain A of fibronectin, a marker for tumor-angiogenesis) in diabody format. The resulting fusion protein, termed F8-IL4, retained full antigen-binding activity and cytokine bioactivity and was able to selectively localize on solid tumors in vivo. When used as single agent, F8-IL4 inhibited tumor growth in three different immunocompetent murine cancer models (F9 teratocarcinoma, CT26 colon carcinoma and A20 lymphoma). Furthermore, F8-IL4 showed synergistic effects when coadministered with immunocytokines based on IL2 and IL12. Indeed, combination therapy with an IL12-based immunocytokine yielded complete tumor eradication, in spite of the fact that IL4 and IL12 display opposite immunological mechanisms of action in terms of their polarization of T-cell based responses. No weight loss or any signs of toxicity were observed in treated mice, both in monotherapy and in combination, indicating a good tolerability of the immunocytokine treatment. Interestingly, mice cured from CT26 tumors acquired a durable protective antitumor immunity. Depletion experiments indicated that the antitumor activity was mediated by CD81 T cells and by NK cells.Cytokines are potent modulators of the activity of the immune system, which can be used for therapeutic purposes. A number of cytokines (e.g., IL2, interferon-alpha and TNF) are frequently used for the treatment of cancer patients, but these agents can be toxic even at low doses (i.e., few milligrams), preventing the escalation to therapeutically active regimens.1 The antibody-based targeted delivery of cytokines ("immunocytokines") to the site of disease represents a promising avenue for increasing the therapeutic index of cytokine products, increasing activity and sparing normal organs.2 Proinflammatory immunocytokines (e.g., those based on IL2, IL12, IL15 and TNF) often display a potent antitumoral effect in mouse models of cancer.3-7 By contrast, antiinflammatory immunocytokines (e.g., those based on IL10) confer a therapeutic benefit in mouse models of chronic inflammatory conditions (rheumatoid arthritis and endometriosis 8,9 ) but have no impact on tumor growth (Trachsel and Neri, unpublished results).We have mainly used antibodies specific to splice-isoforms of fibronectin and of tenascin-C as vehicles for pharmacodelivery applications, as these antigens are virtually undetectable in the normal healthy adult (exception made for placenta, endometrium and some vessels in the ovaries) while being strongly expressed in the majority of solid tumors and lymphomas, with a prominent vascular staining p...

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Section: Cloning Expression and Protein In Vitro Characterizationmentioning

confidence: 99%

“…into the lateral tail vein of tumorbearing mice (for labeling protocol see Ref. 18). Mice were sacrificed 24 hr after injection, organs were excised, weighed and radioactivity was measured using a Packard Cobra g counter.…”

Section: Biodistribution Studiesmentioning

confidence: 99%

The antibody‐based targeted delivery of interleukin‐4 and 12 to the tumor neovasculature eradicates tumors in three mouse models of cancer

Hemmerle

Neri

2013

Intl Journal of Cancer

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“…For the cloning of F8-based immunocytokines, the gene coding for the F8 diabody was PCR amplified from F8-mIL7 (39). The IL12 hp40 and hp35 genes were PCR amplified from the vectors used for F8-mp35/mp40-F8 production, previously reported by our group (32).…”

Section: Cloning Of Fusion Proteinsmentioning

confidence: 99%

“…The corresponding murine mp40 and mp35 genes were PCR amplified from the L19-mIL12 clone (27). All immunocytokine genes were PCR assembled and cloned into the mammalian cell expression vector pcDNA3.1(þ) (Invitrogen) using a strategy similar to the one used by our laboratory for the preparation of IL7-based immunocytokines (39). More detailed information can be found in the Supplementary Material section (S1.2).…”

Section: Cloning Of Fusion Proteinsmentioning

confidence: 99%

The Antibody-Based Delivery of Interleukin-12 to the Tumor Neovasculature Eradicates Murine Models of Cancer in Combination with Paclitaxel

Pasche

Wulhfard

Pretto

et al. 2012

Clinical Cancer Research

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104

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Purpose: Interleukin-12 (IL12) is a potent proinflammatory cytokine with antitumor activity. Its heterodimeric nature makes it compatible with a large variety of different immunocytokine formats. Here we report the design, production, and characterization of a novel immunocytokine, based on the fusion of the F8 antibody (specific to the alternatively spliced EDA domain of fibronectin, a marker of tumor neovasculature) with IL12 (termed IL12-F8-F8).Experimental Design: We developed a novel immunocytokine based on the sequential fusion of interleukin-12 as a single polypeptide with two F8 antibodies in single-chain Fv (scFv) format. The fusion protein was characterized in vitro, and its targeting performance was assessed in vivo. The immunocytokine antitumor activity was studied as monotherapy as well as in combination therapies in three different murine tumor models. Moreover, depletion experiments and tumor analysis revealed a dominant role of natural killer cells for the mechanism of action.Results: IL12-F8-F8 can be produced in mammalian cells, yielding a product of good pharmaceutical quality, capable of selective localization on the tumor neovasculature in vivo, as judged by quantitative biodistribution analysis with radioiodinated protein preparations. The protein potently inhibited tumor growth in three different immunocompetent syngeneic models of cancer. The treatment was generally well tolerated. Moreover, the IL12-F8-F8 fusion protein could be produced both with murine IL12 (mIL12) and with human IL12 (hIL12).Conclusions: The potent antitumor activity of mIL12-F8-F8, studied alone or in combination with paclitaxel in different tumor models, paves the way to the clinical development of the fully human immunocytokine. Clin Cancer Res; 18(15); 4092-103. Ó2012 AACR.

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“…Other cytokines have shown limitations either in tumor targeting or in therapy experiments (e.g., IL7, IL17, IL15, and IL18; refs. [21][22][23], indicating that the immunocytokine format and the choice of payload have to be evaluated from case to case. At one extreme, anti-inflammatory cytokines (such as IL10) can be used as fusion partners with disease-homing antibodies with no inhibition of tumor growth (E. Trachsel and D. Neri; unpublished data) but with a substantial inhibition of the autoimmune and/or inflammatory conditions (9,24).…”

Section: Introductionmentioning

confidence: 99%

The Dose-Dependent Tumor Targeting of Antibody–IFNγ Fusion Proteins Reveals an Unexpected Receptor-Trapping Mechanism In Vivo

Hemmerle

Neri

2014

Cancer Immunology Research

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Cytokines often display substantial toxicities at low concentrations, preventing their escalation for therapeutic treatment of cancer. Fusion proteins comprising cytokines and recombinant antibodies may improve the anticancer activity of proinflammatory cytokines. Murine IFNg was appended in the diabody format at the C-terminus of the F8 antibody, generating the F8-IFNg fusion protein. The F8 antibody is specific for the extra-domain A (EDA) of fibronectin, a tumor-associated antigen that is expressed in the vasculature and stroma of almost all tumor types. Tumor-targeting properties were measured in vivo using a radioiodinated preparation of the fusion protein. Therapy experiments were performed in three syngeneic murine models of cancer [F9 teratocarcinoma, WEHI-164 fibrosarcoma, and Lewis lung carcinoma (LLC)]. F8-IFNg retained the biologic activity of both the antibody and the cytokine moiety in vitro, but, unlike the parental F8 antibody, it did not preferentially localize to the tumors in vivo. However, when unlabeled F8-IFNg was administered before radioiodinated F8-IFNg, a selective accumulation at the tumor site was observed. F8-IFNg showed dose-dependent anticancer activity with a clear superiority over untargeted recombinant IFNg. The anticancer activity was potentiated by combining with F8-IL4 without additional toxicities, whereas combination of F8-IFNg with F8-TNF was lethal in all mice. Unlike other antibodycytokine fusions, the use of IFNg as payload for anticancer therapy is associated with a receptor-trapping mechanism, which can be overcome by the administration of a sufficiently large amount of the fusion protein without any detectable toxicity at the doses used. Cancer Immunol Res; 2(6); 559-67. Ó2014 AACR.

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Cloning and characterization of novel tumor-targeting immunocytokines based on murine IL7

Cited by 58 publications

References 46 publications

The antibody‐based targeted delivery of interleukin‐4 and 12 to the tumor neovasculature eradicates tumors in three mouse models of cancer

The antibody‐based targeted delivery of interleukin‐4 and 12 to the tumor neovasculature eradicates tumors in three mouse models of cancer

The Antibody-Based Delivery of Interleukin-12 to the Tumor Neovasculature Eradicates Murine Models of Cancer in Combination with Paclitaxel

The Dose-Dependent Tumor Targeting of Antibody–IFNγ Fusion Proteins Reveals an Unexpected Receptor-Trapping Mechanism In Vivo

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