TNF- was initially put on the TNFR1-ECD cells accompanied by the antibody incubation. piggyback strategy is usually generalizable to targeting other soluble ligands and/or conjugates with different drugs for managing a diverse set of diseases. Keywords:TNF-, antibody, immunotoxin, bacterial therapy, melanoma, phage display, yeast surface display, immunotherapy, tumor-infiltrating immune cells == Graphical abstract == A bacteria-delivered immunotoxin is designed to piggyback on TNF- and co-internalize into tumor cells for cancer therapy. == Introduction == Tumor necrosis factor (TNF-) is usually a pleiotropic cytokine that is tightly regulated at a very low level in the body but greatly increased at inflammation sites.1TNF- exacerbates the inflammation mainly through its ligation to the ubiquitously expressed receptor TNFR1. It is one of the most prominent drug targets for autoimmune diseases, with five neutralizing antibodies/biologics approved by the US Food and Drug Administration (FDA).2In oncology, TNF- was originally named because of its ability to induce tumor necrosis at a high concentration.3However, recent studies showed that a moderate upregulation of TNF-, which is often found in the tumor microenvironment owing to chronic inflammation, could act as HSPA1 a pro-tumor signal in various stages of cancer development. For example, the seminal work by Balkwill and co-workers revealed that TNF- is usually involved in tumor initiation, as TNF-/mice in four genetic backgrounds were resistant to skin tumors induced by chemical carcinogens.4TNF- promotes the immunosuppressive environment by inducing COP9 signalosome 5 (CSN5) expression, which stabilizes programmed cell death-ligand 1 (PD-L1) in cancer cells5as well as impairing the accumulation of CD8+tumor-infiltrating lymphocytes.6Cancer cells overexpressing TNF- resulted in increased vascularity and enlarged tumor sizes in mouse melanoma, lung cancer, and mammary cancer models.7In vitrostudies revealed that TNF- significantly increased melanoma cell migration and invasion through protease activation.8Angiotropism, an extravascular migratory metastasis process specific to melanoma, has also been shown to associate with TNF- stimulation.9Because of its pro-tumor activity, targeting TNF- using neutralizing biologics has shown promising outcomes in pre-clinical studies, such as reduced aggressivenessin vitro10and inhibited tumor growthin vivo.6However, early clinical trials showed that this administration of TNF- blockades only exerted modest efficacy in ovarian cancer11and renal cell carcinoma patients.12It is likely that this pro-tumor signaling by TNF- is redundant and can be easily replaced GSK2838232A by other pathways once TNF- is blocked.13 Bacterial therapy has emerged as a revitalizing modality for cancer treatment14,15,16,17,18,19,20,21since the pioneering works by William Coley in the late 19thcentury. Although the mechanism was elusive at the time, it is now acknowledged that Coleys approach was the very first immunotherapy against cancer in history.22Modern studies have demonstrated several advantages of bacterial therapy against cancer,23,24,25,26including (1) selective colonization in tumors,27(2) pathogen-associated molecular patterns to stimulate the immune system,28and (3) high engineerability, especially for model microorganisms such asS.typhimuriumandE.coli.29For example, the seminal works by Hoffman and co-workers demonstrated that engineered auxotrophs ofS.typhimuriumexhibited impressive tumor-targeting ability and anti-tumor activity in multiple moue GSK2838232A models.30,31,32,33An early work by Zhang et al. showed that theE.coli-overexpressing tumor GSK2838232A necrosis factor-related apoptosis-inducing ligand caused a dramatic reduction in tumor size in mouse models.34More recently, Danino and co-workers elegantly demonstrated that immune-checkpoint blockades can be locally delivered in tumors byE. coliwith a synthetic lysis circuit and greatly inhibited tumor growth.35,36Encouraged by these promising pre-clinical results, a phase 1 clinical trial for treating advanced solid tumors and lymphoma was recently launched using an engineeredE.colitargeting the STING pathway.37The treatment was well tolerated and achieved target engagement as well as durable disease stabilization in two patients. Here, we propose an unconventional strategy for TNF- targeting and couple its delivery using bacteria. Instead of using TNF- blockades widely utilized in GSK2838232A clinics, we designed non-neutralizing TNF- antibodies, which can piggyback on TNF- and co-internalize into cells upon TNF- ligating to its receptor. We showed that this piggyback antibody, when conjugated with chemical drugs or fused with protein toxins, exerted TNF–dependent cytotoxicity to melanoma cellsin vitro. We deliberately implemented this immunotoxin into anE.colivehicle for a synergistic effect, i.e., the bacteria stimulated the TNF- expression that facilitates the immunotoxin internalization. The immunotoxin-secreting bacteria effectively suppressed the tumor growth in a syngeneic mouse model of melanoma. Tumor-infiltrating leukocyte analysis revealed several beneficial immunomodulations by the treatment, including increased M1 macrophages, decreased M2 macrophages, and increased activated GSK2838232A CD4+and CD8+lymphocytes. Our approach resembles a Trojan Horse strategy, which exploits the pro-tumor signal and converts it into an anti-tumor drug for cancer therapy. == Results == == Rationale of designing a TNF–mediated Trojan Horse delivered by bacteria == All of.