Immunocytokines with activity-on-demand by combination with small molecule inhibitors.

Dose-limiting systemic toxicity constitutes a major impediment to the application of cytokines in cancer therapy. To enhance the therapeutic index, tumor-directed antibody-cytokine fusion proteins, i.e.

Mechanosensory feedback loops during chronic inflammation.

Epithelial tissues are crucial to maintaining healthy organization and compartmentalization in various organs and act as a first line of defense against infection in barrier organs such as the skin, lungs and intestine. Disruption or injury to these barriers can lead to infiltration of resident or foreign microbes, initiating local inflammation. One often overlooked aspect of this response is local changes in tissue mechanics during inflammation.

Targeting Co-Stimulatory Receptors of the TNF Superfamily for Cancer Immunotherapy.

The clinical approval of immune checkpoint inhibitors is an important advancement in the field of cancer immunotherapy. However, the percentage of beneficiaries is still limited and it is becoming clear that combination therapies are required to further enhance the treatment efficacy. The potential of strategies targeting the immunoregulatory network by "hitting the gas pedal" as opposed to "blocking the brakes" is being recognized and intensively investigated.

Influence of antigen density and immunosuppressive factors on tumor-targeted costimulation with antibody-fusion proteins and bispecific antibody-mediated T cell response.

Target expression heterogeneity and the presence of an immunosuppressive microenvironment can hamper severely the efficiency of immunotherapeutic approaches. We have analyzed the potential to encounter and overcome such conditions by a combinatory two-target approach involving a bispecific antibody retargeting T cells to tumor cells and tumor-directed antibody-fusion proteins with costimulatory members of the B7 and TNF superfamily. Targeting the tumor-associated antigens EpCAM and EGFR with the bispecific antibody and costimulatory fusion proteins, respectively, we analyzed the impact of target expression and the influence of the immunosuppressive factors IDO, IL-10, TGF-β, PD-1 and CTLA-4 on the targeting-mediated stimulation of T cells.

IL15-Based Trifunctional Antibody-Fusion Proteins with Costimulatory TNF-Superfamily Ligands in the Single-Chain Format for Cancer Immunotherapy.

IL15 and costimulatory receptors of the tumor necrosis superfamily (TNFRSF) have shown great potential to support and drive an antitumor immune response. However, their efficacy as monotherapy is limited. Here, we present the development of a novel format for a trifunctional antibody-fusion protein that combines and focuses the activity of IL15/TNFSF-ligand in a targeting-mediated manner to the tumor site.

Duokines: a novel class of dual-acting co-stimulatory molecules acting in cis or trans.

Co-stimulatory signals induced by ligands of the tumor necrosis factor superfamily (TNFSF) play a central role in T cell activation and have emerged as a promising strategy in cancer immunotherapy. Here, we established a novel class of bifunctional co-stimulatory fusion proteins with the aim to boost T cell activation at the level of T cell - antigen-presenting cell (APC) interaction. These novel dual-acting cytokine fusion proteins were created by connecting two different homotrimeric TNFSF ligands to form homotrimeric bifunctional molecules (Duokines) or by connecting single-chain derivatives of two different homotrimeric TNFSF with a single, flexible linker (single-chain Duokines, scDuokines).

Production, Purification, and Characterization of Antibody-TNF Superfamily Ligand Fusion Proteins.

Antibody-fusion proteins with ligands, e.g., of the TNF superfamily (TNFSF) can be adequately produced in mammalian expression systems.

Tumor-targeted costimulation with antibody-fusion proteins improves bispecific antibody-mediated immune response in presence of immunosuppressive factors.

Therapeutic strategies aiming for the induction of an effective immune response at the tumor site can be severely hampered by the encounter of an immunosuppressive microenvironment. We investigated here the potential of concerted costimulation by tumor-directed antibody-fusion proteins with B7.1, 4-1BBL and OX40L to enforce bispecific antibody-induced T cell stimulation in presence of recognized immunosuppressive factors including IL-10, TGF-β, indoleamine 2,3-dioxygenase (IDO), PD-L1 and regulatory T cells.

Advancing targeted co-stimulation with antibody-fusion proteins by introducing TNF superfamily members in a single-chain format.

Co-stimulation via receptors of the tumor necrosis factor superfamily (TNFSF) emerges as promising strategy to support antitumor immune responses. Targeted strategies with antibody-fusion proteins composed of a tumor-directed antibody part and the extracellular domain of a co-stimulatory ligand of the TNFSF constitute an attractive option to focus the co-stimulatory activity to the tumor site. Since TNFSF members intrinsically form functional units of non-covalently linked homotrimers, the protein engineering of suitable antibody-fusion proteins is challenging.

Antibody fusions with immunomodulatory proteins for cancer therapy.

The potential of immunomodulatory proteins, in particular cytokines, for cancer therapy is well recognized, but hampered by the toxicity associated with their systemic application. In order to address this problem, targeted delivery by antibody fusion proteins has been early proposed and their development intensively pursued over the last decade. Here, factors influencing the selection and modification of cytokines and antibody formats for this approach are being discussed, indicating current developments and translational advances in the field.

miR149 functions as a tumor suppressor by controlling breast epithelial cell migration and invasion.

Deregulated molecular signaling pathways are responsible for the altered adhesive, migratory, and invasive properties of cancer cells. The different breast cancer subtypes are characterized by the expression of distinct miRNAs, short non-coding RNAs that posttranscriptionally modulate the expression of entire gene networks. Profiling studies have revealed downregulation of miR149 in basal breast cancer.

Combining antibody-directed presentation of IL-15 and 4-1BBL in a trifunctional fusion protein for cancer immunotherapy.

Influencing the cytokine receptor network that modulates the immune response holds great potential for cancer immunotherapy. Although encouraging results have been obtained by focusing on individual members of the common γ-chain (γc) receptor family and TNF receptor superfamily so far, combination strategies might be required to further improve the effectiveness of the antitumor response. Here, we propose the combination of interleukin (IL)-15 and 4-1BBL in a single, tumor-directed molecule.

Antibody-cytokine fusion proteins for cancer immunotherapy: an update on recent developments.

Treatment with cytokines holds great potential for cancer immunotherapy, but is generally restricted by systemic toxicity. Tumor-directed targeting in the form of antibody fusion proteins appears to be an attractive strategy to overcome this problem. In the last twenty years, continuous efforts in developing appropriate molecules have retrieved a variety of antibody fusion proteins that reveal promising therapeutic effects in preclinical studies.

Evaluating combinations of costimulatory antibody-ligand fusion proteins for targeted cancer immunotherapy.

Combinatory strategies are becoming of increasing interest in cancer immunotherapy. Costimulation by individual members of the immunoglobulin-like (Ig)- and TNF superfamily have already shown promising antitumor potential, thus prompting the exploration of their synergistic abilities in combinatorial approaches. Here, we pursued a targeted strategy with antibody-fusion proteins composed of a tumor-directed antibody and the extracellular domain of the costimulatory ligand B7.

Targeted cancer immunotherapy: Mimicking physiological trans-presentation of IL-15.

Under physiological conditions, the trans-presentation of interleukin-15 (IL-15) by the IL-15 receptor α on the cell surface allows to confine and tune the IL-15-mediated immune responses. Therefore, targeting strategies that mimic this situation at the tumor sites appear especially promising for anticancer immunotherapy.

Combination of a bispecific antibody and costimulatory antibody-ligand fusion proteins for targeted cancer immunotherapy.

Initiation of a tumor-directed immune response and appropriate modulation of its progress are key issues in cancer immunotherapy. Combinatorial strategies addressing both aspects might therefore be especially suitable. Here, we report a targeted approach combining a bispecific antibody with 2 costimulatory antibody-ligand fusion proteins.

An antibody fusion protein for cancer immunotherapy mimicking IL-15 trans-presentation at the tumor site.

Cytokines driving the immune response are powerful tools for cancer immunotherapy, but their application is generally limited by severe systemic toxicity. Targeted approaches by means of antibody-cytokine fusion proteins might enable focus on the cytokine activity to the tumor site, thereby reducing unwanted side effects. Here, we investigated the possibility to improve the efficiency of interleukin (IL)-15 presentation in a targeted approach by the incorporation of an IL-15Rα chain fragment, mimicking physiologic trans-presentation.

Tumor-specific crosslinking of GITR as costimulation for immunotherapy.

Activation of murine glucocorticoid-induced tumor necrosis factor-related receptor (mGITR) by its natural ligand (GITRL) or antiGITR agonist mAb enhances T-cell responses, inhibits regulatory T-cell (Treg)-mediated suppression and induces tumor immunity in a variety of murine tumor models. However, systemic administration of these costimulatory agents can lead to global T-cell activation and autoimmunity. To specifically manipulate the T-cell compartment in the tumor microenvironment we propose to target the tumor infiltrating T cells with a bispecific mGITRL fusion protein.

The effects of affinity and valency of an albumin-binding domain (ABD) on the half-life of a single-chain diabody-ABD fusion protein.

Fusion of small recombinant antibody fragments to an albumin-binding domain (ABD) from streptococcal protein G strongly extends their plasma half-life. This ABD binds with nanomolar affinity to human (HSA) and mouse serum albumin (MSA). It was speculated that an increase in albumin-binding affinity should lead to a further increase in half-life.

Humanization of a mouse monoclonal antibody directed against a cell surface-exposed epitope of membrane-associated heat shock protein 70 (Hsp70).

The translocation of heat shock protein 70 (mHsp70) into the plasma membrane has been found to be associated with various cancers including breast cancer, head-and-neck cancer, and acute myeloid leukemia. Parts of the C-terminal substrate-binding domain (SBD) of mHsp70 are accessible to binding by monoclonal antibodies (mAb). One of these mAbs, cmHsp70.

Bispecific antibodies for cancer immunotherapy: Current perspectives.

The concept of using bispecific antibodies to retarget immune effector cells for cancer therapy was conceived more than 20 years ago. However, initial clinical studies were rather disappointing mainly due to low efficacy, severe adverse effects and immunogenicity of the bispecific antibodies. A deeper understanding of effector cell biology and especially developments in the field of antibody engineering has led to the generation of new classes of bispecific antibodies capable of circumventing many of these obstacles.

Targeted delivery of SiRNA to CD33-positive tumor cells with liposomal carrier systems.

SiRNA molecules represent promising therapeutic molecules, e.g. for cancer therapy.

Biodistribution of a bispecific single-chain diabody and its half-life extended derivatives.

Small recombinant antibody molecules such as bispecific single-chain diabodies (scDb) possessing a molecular mass of approximately 55 kDa are rapidly cleared from circulation. We have recently extended the plasma half-life of scDb applying various strategies including PEGylation, N-glycosylation and fusion to an albumin-binding domain (ABD) from streptococcal protein G. Here, we further analyzed the influence of these modifications on the biodistribution of a scDb directed against carcinoembryonic antigen (CEA) and CD3 capable of retargeting T cells to CEA-expressing tumor cells.

Targeting of epidermal growth factor receptor (EGFR)-expressing tumor cells with sterically stabilized affibody liposomes (SAL).

Affibody molecules are small and stable antigen-binding molecules derived from the B domain of protein A. We applied a bivalent, high-affinity epidermal growth factor receptor (EGFR)-specific affibody molecule for the generation of targeted PEGylated liposomes. These sterically stabilized affibody liposomes (SAL) were produced by chemical coupling of the cysteine-modified affibody molecule to maleimide-PEG(2000)-DSPE and subsequent insertion into PEGylated liposomes.