High-Affinity Superantigen-Based Trifunctional Immune Cell Engager Synergizes NK and T Cell Activation for Tumor Suppression.

The development of immune cell engagers (ICEs) can be limited by logistical and functional restrictions associated with fusion protein designs, thus limiting immune cell recruitment to solid tumors. Herein, a high affinity superantigen-based multivalent ICE is developed for simultaneous activation and recruitment of NK and T cells for tumor treatment. Yeast library-based directed evolution is adopted to identify superantigen variants possessing enhanced binding affinity to immunoreceptors expressed on human T cells and NK cells. High-affinity superantigens exhibiting improved immune-stimulatory activities are then incorporated into a superantigen-based tri-functional yeast-display-enhanced multivalent immune cell engager (STYMIE), which is functionalized with a nanobody, a Neo-2/15 cytokine, and an Fc domain for tumor targeting, immune stimulation, and prolonged circulation, respectively. Intravenous administration of STYMIE enhances NK and T cell recruitment into solid tumors, leading to enhanced inhibition in multiple tumor models. The study offers design principles for multifunctional ICEs.

Overcoming the nutritional immunity by engineering iron-scavenging bacteria for cancer therapy.

Certain bacteria demonstrate the ability to target and colonize the tumor microenvironment, a characteristic that positions them as innovative carriers for delivering various therapeutic agents in cancer therapy. Nevertheless, our understanding of how bacteria adapt their physiological condition to the tumor microenvironment remains elusive. In this work, we employed liquid chromatography-tandem mass spectrometry to examine the proteome of E. coli colonized in murine tumors. Compared to E. coli cultivated in the rich medium, we found that E. coli colonized in tumors notably upregulated the processes related to ferric ions, including the enterobactin biosynthesis and iron homeostasis. This finding indicated that the tumor is an iron-deficient environment to E. coli. We also found that the colonization of E. coli in the tumor led to an increased expression of lipocalin 2 (LCN2), a host protein that can sequester the enterobactin. We therefore engineered E. coli in order to evade the nutritional immunity provided by LCN2. By introducing the IroA cluster, the E. coli synthesizes the glycosylated enterobactin, which creates steric hindrance to avoid the LCN2 sequestration. The IroA-E. coli showed enhanced resistance to LCN2 and significantly improved the anti-tumor activity in mice. Moreover, the mice cured by the IroA-E. coli treatment became resistant to the tumor re-challenge, indicating the establishment of immunological memory. Overall, our study underscores the crucial role of bacteria's ability to acquire ferric ions within the tumor microenvironment for effective cancer therapy.

Development of a TNF-α-mediated Trojan Horse for bacteria-based cancer therapy.

Tumor necrosis factor α (TNF-α) is upregulated in a chronic inflammatory environment, including tumors, and has been recognized as a pro-tumor factor in many cancers. Applying the traditional TNF-α antibodies that neutralize TNF-α activity, however, only exerts modest anti-tumor efficacy in clinical studies. Here, we develop an innovative approach to target TNF-α that is distinct from the neutralization mechanism. We employed phage display and yeast display to select non-neutralizing antibodies that can piggyback on TNF-α and co-internalize into cells through receptor ligation. When conjugating with toxins, the antibody exhibited cytotoxicity to cancer cells in a TNF-α-dependent manner. We further implemented the immunotoxin to an E. coli vehicle specially engineered for a high secretion level. In a syngeneic murine melanoma model, the bacteria stimulated TNF-α expression that synergized with the secreted immunotoxin and greatly inhibited tumor growth. The treatment also dramatically remodeled the tumor microenvironment in favor of several anti-tumor immune cells, including N1 neutrophils, M1 macrophages, and activated CD4+ and CD8+ lymphocytes. We anticipate that our new piggyback strategy is generalizable to targeting other soluble ligands and/or conjugates with different drugs for managing a diverse set of diseases.

Development of a Novel Cytokine Vehicle Using Filamentous Phage Display for Colorectal Cancer Treatment.

Due to its highly immunogenic nature and the great engineerability, filamentous phage has shown promising antitumor activities in preclinical studies. Previous designs of antitumor phage mainly focused on tumor targeting using a cancer-specific moiety displayed on the minor capsid protein, pIII. In this work, we developed a new therapeutic platform of filamentous phage, in which the major capsid protein pVIII was utilized for displaying an antitumor cytokine. We showcased that a 16.1-kD cytokine GM-CSF could be efficiently presented on the M13 phage particle using the 8 + 8 type display system through a highly tolerable pVIII variant P8(1a). We verified that the GM-CSF phage was a potent activator for STAT5 signaling in murine macrophage. The GM-CSF phage significantly reduced the tumor size by more than 50% as compared to the unmodified phage in a murine colorectal cancer model. Immunological profiling of the tumor-infiltrating leukocytes revealed that an increase of CD4+ lymphocytes in the GM-CSF phage treatment group. Furthermore, the combined therapy of the GM-CSF phage and radiation greatly improved the therapeutic potency with a 100% survival rate and a 25% complete remission rate. We observed that the IFN-γ expression was dramatically up-regulated by the combined therapy in multiple types of tumor-infiltrating immune cells. Overall, we created a novel vehicle for cytokine therapy using the pVIII filamentous phage display. This new platform can be multiplexed with other phage engineering approaches, such as displaying targeting ligands on pIII or encapsulating therapeutic genes inside phage capsids, to create multifunctional nanoparticles for cancer therapy.

Development of a Sensitive Enzyme-Linked Immunosorbent Assay and Rapid Gold Nanoparticle Immunochromatographic Strip for Detecting Citrinin in Monascus Fermented Food.

Antibodies against citrinin (CTN) were generated from rabbits, which were injected with CTN-keyhole limpet hemocyanin (KLH). This work involved the development of a sensitive competitive direct enzyme-linked immunosorbent assay (cdELISA) and a rapid gold nanoparticle immunochromatographic strip (immunostrip) method for analyzing CTN in Monascus-fermented food. CTN at a concentration of 5.0 ng/mL caused 50% inhibition (IC50) of CTN-horseradish peroxidase (CTN-HRP) binding to the antibodies in the cdELISA. The capable on-site detection of CTN was accomplished by a rapid antibody-gold nanoparticle immunostrip with a detection limit of 20 ng/mL and that was completed within 15 min. A close inspection of 19 Monascus-fermented foods by cdELISA confirmed that 14 were contaminated with citrinin at levels from 28.6⁻9454 ng/g. Further analysis with the immunostrip is consistent with those results obtained using cdELISA. Both means are sensitive enough for the rapid examination of CTN in Monascus-fermented food products.