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Fulfilling Paul Ehrlich’s Dream: therapeutics with activity on demand

Periodic Reporting for period 3 - ZAUBERKUGEL (Fulfilling Paul Ehrlich’s Dream: therapeutics with activity on demand)

Reporting period: 2018-09-01 to 2020-02-29

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.
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 rath
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.