Community Research and Development Information Service - CORDIS


ZAUBERKUGEL Report Summary

Project ID: 670603
Funded under: H2020-EU.1.1.

Periodic Reporting for period 1 - ZAUBERKUGEL (Fulfilling Paul Ehrlich’s Dream: therapeutics with activity on demand)

Reporting period: 2015-09-01 to 2017-02-28

Summary of the context and overall objectives of the project

The main objective of this Research Proposal is the development of a novel class of pharmaceutical agents, with the potential to yield unprecedented levels of activity and in vivo selectivity. The novel class of therapeutic agents should gain biological activity once they have selectively localized at the site of disease, thus fulfilling Paul Ehrlich’s dream of “magic bullets” (Zauberkugeln), which target pathological structures while sparing healthy tissues.

While the main focus of the planned development activities are in the oncology field, the novel therapeutic concepts should be applicable for other indications.

The targeted pharmaceuticals studied in the ZAUBERKUGEL Project can be grouped into two categories:

a) Cytokines (i.e., proteins capable of modulating the activity of the immune system) fused to suitable antibody molecules, serving as “delivery vehicles”

b) Cytotoxic agents (i.e., anti-cancer chemotherapeutic agents) coupled to antibodies or to small organic molecules, serving as “delivery vehicles”

The main challenge for many types of pharmacotherapy consists in the inability to achieve a selective killing or growth inhibition of disease-associated cells in vivo, while sparing normal tissues. This problem is particularly acute for the pharmacotherapy of cancer. Indeed, it is becoming increasingly clear that conventional therapeutics based on small molecules (e.g., cytotoxic agents) or on therapeutic proteins (e.g., cytokines) do not selectively localize at the site of disease, causing considerable toxicity to normal organs and preventing dose escalation to therapeutically active regimens.

If successful, the therapeutic strategies investigated in the frame of the ZAUBERKUGEL Project will generate new products, suitable for industrial development activities and for the initiation of novel clinical trials.

The strategies outlined in this Project should be readily translatable to other disease areas (e.g., chronic inflammation), for which the use of immunocytokines and targeted small molecule drugs (e.g., targeted corticosteroids) has been proposed.

During the first 18 months of the projects, many goals have already been achieved, including the following ones:

We have generated and tested in tumor-bearing mice new antibody-cytokine fusion proteins, which have exhibited an extremely potent therapeutic activity. The most impressive anti-cancer activity has probably been achieved with a novel class of biopharmaceuticals, called “potency-matched dual cytokine fusions), featuring interleukin-2 and tumor necrosis factor as payloads. In addition, we have implemented a novel therapeutic strategy (termed “split cytokine fusions”), based on the sequential administration of antibody-based fusion proteins, which reassemble into a functional unit at the site of disease.

Considering the fact that conventional anti-cancer therapeutics do not efficiently localize at the tumor site, we have studied ligand-based drug delivery strategies, in order to generate products that are more active and selective against cancer. In particular, we have developed certain antibody-drug conjugates (ADCs), which localize on the tumor extracellular matrix and liberate highly cytotoxic payloads, leading to cures in mouse models of the disease. Similarly, we have developed and tested in mice novel small molecule drug conjugates (SMDCs), which have exhibited a potent therapeutic activity in mouse models of kidney cancer.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

In this section, I have copied the Workpackage (WP) description from the original application and I have indicates some of the main results achieved. As you can see, for all eight Workpackages we have made considerable progress, which is documented in articles which have been published or in manuscripts which have been submitted for publication.

WP1: Design, implementation and testing (in vitro and in vivo) of modular antibody-cytokine fusion proteins, capable of re-assembly at the tumor site. Initially, the focus will be on immunocytokines, based on the p40 and p35 subunits of murine interleukin-12, but the work will expand to include cytokine-masking strategies and allosteric regulation.

We have generated and tested in vivo a novel class of antibody-cytokine products (termed “split cytokine fusions”), which can be administered sequentially and can reconstitute a therapeutic activity at the site of disease, thus helping spare normal tissues from undesired toxicity. We have initially focused on split cytokine fusions based on the cysteine->serine mutants of the p40 and p35 subunits of murine interleukin-12, showing how these products can reassemble in vitro and how they perform in vivo (both in biodistribution studies and in terms of their biological activity).
The results of these studies have now been published [Venetz et al. (2016) J. Biol. Chem., 291, 18139].

WP2: Investigation of the synergistic therapeutic activity of immunocytokine pairs, first by the co-administration of immunocytokine products and, if successful, by the simultaneous engineering of two cytokine payloads into the same antibody-based fusion protein.

We have investigated the combination of two antibody-cytokine fusions (e.g., those based on IL2 and TNF, or IL4 and IL12) in various models of tumor-bearing mice, in order to discover which product pairs are most suitable for future clinical applications. In particular, we have successfully cloned, expressed and characterized a novel class of therapeutic antibodies (termed “potency-matched dual cytokine fusions), based on interleukin-2 and tumor necrosis factor as payloads, which has demonstrated the ability to cure various types of tumor-bearing mice, when used as single agent. Importantly, the cancer models tested did not respond to conventional anti-cancer cytotoxic agents or to anti-PD-1 antibodies.
The results of the study on potency-matched dual cytokine fusions have now been submitted for publication [De Luca et al., manuscript submitted].

WP3: Investigation of the interplay between administration modality (i.e., rate of infusion), tolerability and therapeutic activity of two IL2-based and TNF-based immunocytokines in mouse models of cancer.

We have initially focused on the combination of two immunocytokines (F8-IL2 and F8-TNF), administered to four different immunocompetent syngeneic mouse models of cancer (WEHI-164 sarcoma, Lewis Lung Carcinoma, F9 teratocarcinoma and TIB-49 myeloma) as intratumoral injection. We have observed that the activity of the therapeutic intervention is largely dependent on the tumor size at the beginning of therapy. We could achieve cancer cures in all models, except TIB-49.
The results of this study have been written in a manuscript, which is almost ready for submission [Ziffels et al., manuscript in preparation].

WP4: Study of ADCC potentiation by the co-administration of therapeutic antibodies in IgG format and IL2- based immunocytokines. Some of these studies may be performed in nude mice bearing subcutaneously-grafted tumors, as these immunodeficient mice still contain functional NK cells.

We have started to investigate whether an intact murine monoclonal antibody (TA99) in IgG format, specific to a melanoma surface antigens (melanosome gp75 antigen), can selectively localize to B16 melanomas in vivo and can display a measurable anticancer activity. Quantitative biodistribution studies, obtained with radioiodinated antibody preparations, evidenced a rather modest tumor targeting performance for this product, in spite of the fact that it has been used in many prominent publications by the group of Jeff Ravetch and of other authors. An ex vivo a analysis of tumor targeting, performed using fluorescence microscopy methodologies, confirmed that the TA99 antibody was able to reach melanoma cells in vivo. In therapy studies performed with solid tumors of different size, the TA99 antibody exhibited only a modest tumor-growth inhibition activity. Studies on the inhibition of metastasis formation are currently in progress.
Based on the results of this set of experiments, we will decide whether to go ahead with combination studies, using IL2-based immunocytokines.
In parallel, we have collaborated with Prof. Wolfgang Berdel and PD Dr. Christoph Schliemann in Münster (Germany) and reported a synergistic activity between L19-IL2 and rituximab in a mouse model of Mantle Cell Lymphoma [Börschel et al. (2015) Leukemia Res., 39, 739]. In addition, our collaborative efforts have documented a mechanistic role associated with the targeting of IL2 to the bone marrow stroma for therapy of acute myeloid leukemia relapsing after allogeneic hematopoietic stem cell transplantation [Schliemann et al. (2015) Cancer Immunol. Res., 3, 547].

WP5: Design and experimental implementation of “release on demand” strategies for non-internalizing antibody-drug conjugates, based on the use of reducing agents, tetrazines or other chemical methodologies for the cleavage of antibody-drug linkers.

We have focused on the generation and characterization of antibody-drug conjugates (ADCs), based on the human monoclonal antibody F16 (specific to the alternatively-spliced A1 domain of tenascin-C) and on various cytotoxic payloads (including monomethyl auristatin E and the highly-potent antracycline derivative PNU-159682. The antibody was used either in Small Immune Protein (SIP) format or in IgG format. In both cases, the products were engineered in order to have a single cysteine residue available for site-specific chemical modification with a linker-drug payload. We have observed that the ADC products could lead to cancer cures, even though the antibody was chosen as a non-internalizing ligand to a very abundant extracellular matrix component in the tumor environment. IgG-based products performed better in vivo and we have studies these derivatives in most detail. We also investigated various linkers, in order to identify the ones which were most suitable for therapeutic intervention. We discovered that an excellent selectivity can be achieved, exploiting the fact that the tumor environment is rich in proteases (probably as a result of tumor cell death events). By contrast, attempts to trigger drug release with exogenous agents were less successful and we decided to focus on the design of ADCs, which would be activated by tumor-intrinsic factors.
The results of these studies have already generated a number of publications, including the following ones:

Antibody Format and Drug Release Rate Determine the Therapeutic Activity of Noninternalizing Antibody-Drug Conjugates.
Gébleux R, Wulhfard S, Casi G, Neri D.
Mol Cancer Ther. 2015 Nov;14(11):2606-12.

Non-internalizing antibody-drug conjugates display potent anti-cancer activity upon proteolytic release of monomethyl auristatin E in the subendothelial extracellular matrix.
Gébleux R, Stringhini M, Casanova R, Soltermann A, Neri D.
Int J Cancer. 2017 Apr 1;140(7):1670-1679.

WP6: Design and experimental implementation of “release on demand” strategies for non-internalizing small molecule-drug conjugates, based on the use of reducing agents, tetrazines or other chemical methodologies for the cleavage of antibody-drug linkers.

We have mainly focused on non-internalizing small molecule-drug conjugates (SMDCs), specific to carbonic anhydrase IX (CAIX). This membrane protein is expressed in the majority of renal cell carcinomas, but also in other malignancies (e.g., colorectal tumors). In normal tissues, expression is confined to stomach, gallbladder and duodenum.
We have generated and characterized in vivo (both using quantitative biodistribution studies with radiolabeled material and near-infrared fluorescence imaging) various CAIX ligands and identified two molecules, which were more promising for subsequent SMDC studies.
As for the ADC experience described in WP5, the results obtained with exogenous drug release triggering agents were disappointing, whereas enzymatic activities present in the tumor environment (e.g., certain proteases) allowed an efficient and selective mechanism of SMDC activation.
We have extensively studied in vitro and in vivo SMDC products, differing in terms of CAIX ligands, linkers and payloads. Potent therapeutic activity was observed in nude mice, bearing human SKRC52 kidney cancer, but cures were rarely seen. However, the combination of SMDCs with an IL2-based immunocytokine (L19-IL2) yielded 100% of cancer cures in the same model (which is otherwise insensitive to standard therapeutics, such as sunitinb and sorafenib). The mechanism of action relies on NK cells, which are potentiated by L19-IL2 and by the tumor cell damage, caused by the SMDC product.
The results of these studies have already generated a number of publications, including the following ones:

Acetazolamide Serves as Selective Delivery Vehicle for Dipeptide-Linked Drugs to Renal Cell Carcinoma.
Cazzamalli S, Dal Corso A, Neri D.
Mol Cancer Ther. 2016 Dec;15(12):2926-2935.

Linker stability influences the anti-tumor activity of acetazolamide-drug conjugates for the therapy of renal cell carcinoma.
Cazzamalli S, Dal Corso A, Neri D.
J Control Release. 2017 Jan 28;246:39-45.

WP7: Experimental determination of the MHC-I peptidome analysis of murine tumor cell lines, which are used in preclinical therapy studies in the lab, in fully immunocompetent syngeneic settings.

We have extablished methodologies for the characterization of the MHC class I and class II peptidome, both on murine and human material.
This work has already led to various publications, including the following ones:

High-resolution analysis of the murine MHC class II immunopeptidome.
Sofron A, Ritz D, Neri D, Fugmann T.
Eur J Immunol. 2016 Feb;46(2):319-28.

High-sensitivity HLA class I peptidome analysis enables a precise definition of peptide motifs and the identification of peptides from cell lines and patients' sera.
Ritz D, Gloger A, Weide B, Garbe C, Neri D, Fugmann T.
Proteomics. 2016 May;16(10):1570-80.

Mass spectrometric analysis of the HLA class I peptidome of melanoma cell lines as a promising tool for the identification of putative tumor-associated HLA epitopes.
Gloger A, Ritz D, Fugmann T, Neri D.
Cancer Immunol Immunother. 2016 Nov;65(11):1377-1393.

Purification of soluble HLA class I complexes from human serum or plasma deliver high quality immuno peptidomes required for biomarker discovery.
Ritz D, Gloger A, Neri D, Fugmann T.
Proteomics. 2017, in press.

The MHC Class II Immunopeptidome of Lymph Nodes in Health and in Chemically Induced Colitis.
Fugmann T, Sofron A, Ritz D, Bootz F, Neri D.
J Immunol. 2017 Feb 1;198(3):1357-1364.

In addition, we have used the methodology in order to perform detailed studies on the mechanism of tumor rejection of immunocompetent mice with soft-tissue sarcoma, cured as a result of therapeutic intervention with doxorubicin and a TNF-based immunocytokine [F8-TNF; Hemmerle et al. (2013) Br. J. Cancer, 109, 1206]. We have observed that BALB/c mice, cured from WEHI-164 sarcomas, could reject other BALB/c tumors (e.g., CT26 and C51 colorectal cancers), in a process dependent on CD8+ T cells. Exome sequencing of the WEHI-164 cell line revealed the presence of over 1700 non-synonymous mutations compared to wild-type BALB/c. However, a mass-spectrometry based peptidome analysis could not identify neo-epitopes on MHC class I on the tumor cells. By contrast, all three tumor cell lines presented the AH1 peptide, corresponding to a gene of retroviral origin. The functional implications of these findings are described in the next WP (see below). The results have been written in a manuscript, which has now been revised twice for Cancer Research and which will hopefully be published soon.

WP8: Generation of multiplex tetramers based on the MHC-I peptidomes, identified in WP7, for the FACS- based analysis of T-cell specificities before and after therapeutic intervention.

For the description of the experimental setting investigated in this WP, see WP7.
Flow cytometry and tetramer-based analysis of spleen sections showed an increase of AH1 peptide specific CD8+ T cells in mice after F8-TNF plus doxorubicin treatment, suggesting that cognate CD8+ T cells contribute to the rejection process. Additionally, the cytotoxic potential of AH1-specific cells was shown in an in vitro cytotoxicity assay. Sequence analysis of T cell receptors of CD8+ T cells revealed the presence of H-2Ld/AH1-specific T cells and an expansion of sequence diversity in treated mice. Overall, these findings provide evidence that retroviral genes contribute to the immune surveillance of tumors, in a process, which can be boosted by the F8-TNF fusion protein. The combination of doxorubicin with antibody-TNF fusions is currently being investigated in clinical trials in sarcoma patients.
The results of this study have been written in a manuscript, which has now been revised twice for Cancer Research and which will hopefully be published soon [Probst et al., manuscript submitted].

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Thanks to the use of complementary and interdisciplinary methodologies, the Proposal aims at the generation and in vivo testing of novel biopharmaceutical agents, with the potential to display unprecedented levels of activity and selectivity.

Immunocytokines and targeted cytotoxics (ADC and SMDC products) are already being actively investigated in clinical trials. The ZAUBERKUGEL Project aims at pushing research in these fields to the next level, by the implementation of “activity on demand” strategies, which trigger therapeutic activity at the site of disease, while sparing normal organs.

While the Proposal is initially focused on cancer therapy, the strategies outlined in this Project should be readily translatable to other disease areas (e.g., chronic inflammation), for which the use of immunocytokines and targeted small molecule drugs (e.g., targeted corticosteroids) has been proposed.
Follow us on: RSS Facebook Twitter YouTube Managed by the EU Publications Office Top