ABSTRACT
T cells are critical mediators of antigen-specific immune responses and are common targets for immunotherapy. Biomaterial scaffolds have previously been used to stimulate antigen-presenting cells to elicit antigen-specific immune responses; however, structural and molecular features that directly stimulate and expand naïve, endogenous, tumor-specific T cells in vivo have not been defined. Here, an artificial lymph node (aLN) matrix is created, which consists of an extracellular matrix hydrogel conjugated with peptide-loaded-MHC complex (Signal 1), the co-stimulatory signal anti-CD28 (Signal 2), and a tethered IL-2 (Signal 3), that can bypass challenges faced by other approaches to activate T cells in situ such as vaccines. This dynamic immune-stimulating platform enables direct, in vivo antigen-specific CD8+ T cell stimulation, as well as recruitment and coordination of host immune cells, providing an immuno-stimulatory microenvironment for antigen-specific T cell activation and expansion. Co-injecting the aLN with naïve, wild-type CD8+ T cells results in robust activation and expansion of tumor-targeted T cells that kill target cells and slow tumor growth in several distal tumor models. The aLN platform induces potent in vivo antigen-specific CD8+ T cell stimulation without the need for ex vivo priming or expansion and enables in situ manipulation of antigen-specific responses for immunotherapies.
Subject(s)
CD8-Positive T-Lymphocytes , Lymph Nodes , Animals , Lymph Nodes/immunology , CD8-Positive T-Lymphocytes/immunology , Mice , Lymphocyte Activation , Hydrogels/chemistry , Immunotherapy/methods , Extracellular Matrix/metabolism , CD28 Antigens/immunology , CD28 Antigens/metabolism , Humans , Interleukin-2/metabolism , Peptides/chemistry , Cell Line, Tumor , Mice, Inbred C57BLABSTRACT
The clinical utility of human interleukin-2 (hIL-2) is limited by its short serum half-life, preferential activation of regulatory T (TReg) over immune effector cells, and dose-limiting toxicities. We previously engineered F10 immunocytokine (IC), an intramolecularly assembled cytokine/antibody fusion protein that linked hIL-2 to an anti-IL-2 antibody (denoted F10) that extended IL-2 half-life and augmented the immune effector to TReg ratio. Here, we leveraged molecular engineering to improve the anti-tumor therapeutic efficacy and tolerability of F10 IC by developing an iteration, denoted F10 IC-CBD (collagen binding domain), designed for intratumoral administration and in situ retention based on collagen affinity. F10 IC-CBD retained IL-2 bioactivity exclusively in the tumor and eliminated IL-2-associated toxicities. Furthermore, F10 IC exhibited potent single-agent therapeutic efficacy and synergy with systemic immune checkpoint blockade and elicited an abscopal response in mouse tumors models. This engineered fusion protein presents a prototype for the design of intratumoral therapies.
Subject(s)
Interleukin-2 , Neoplasms , Humans , Mice , Animals , Interleukin-2/genetics , Interleukin-2/pharmacology , Interleukin-2/therapeutic use , Biological Availability , CollagenABSTRACT
Helper (CD4+) T cells perform direct therapeutic functions and augment responses of cells such as cytotoxic (CD8+) T cells against a wide variety of diseases and pathogens. Nevertheless, inefficient synthetic technologies for expansion of antigen-specific CD4+ T cells hinders consistency and scalability of CD4+ T cell-based therapies, and complicates mechanistic studies. Here we describe a nanoparticle platform for ex vivo CD4+ T cell culture that mimics antigen presenting cells (APC) through display of major histocompatibility class II (MHC II) molecules. When combined with soluble co-stimulation signals, MHC II artificial APCs (aAPCs) expand cognate murine CD4+ T cells, including rare endogenous subsets, to induce potent effector functions in vitro and in vivo. Moreover, MHC II aAPCs provide help signals that enhance antitumor function of aAPC-activated CD8+ T cells in a mouse tumor model. Lastly, human leukocyte antigen class II-based aAPCs expand rare subsets of functional, antigen-specific human CD4+ T cells. Overall, MHC II aAPCs provide a promising approach for harnessing targeted CD4+ T cell responses.
Subject(s)
Immunotherapy, Adoptive , Nanoparticles , Animals , Antigen-Presenting Cells , CD4-Positive T-Lymphocytes , CD8-Positive T-Lymphocytes , HLA Antigens , Humans , MiceABSTRACT
Since the FDA approval of the first therapeutic antibody 35 years ago, antibody-based products have gained prominence in the pharmaceutical market. Building on the early successes of monoclonal antibodies, more recent efforts have capitalized on the exquisite specificity and/or favorable pharmacokinetic properties of antibodies by developing fusion proteins that enable targeted delivery of therapeutic payloads which are otherwise ineffective when administered systemically. This review focuses on recent engineering and translational advances for therapeutics that genetically fuse antibodies to disease-relevant payloads, including cytokines, toxins, enzymes, neuroprotective agents, and soluble factor traps. With numerous antibody fusion proteins in the clinic and other innovative molecules poised to follow suit, these potent, multifunctional drug candidates promise to be a major player in the therapeutic development landscape for years to come.
Subject(s)
Antibodies, Monoclonal , Cytokines , HumansABSTRACT
Streptococcus pyogenes (group A Streptococcus [GAS]), a major human-specific pathogen, relies on efficient nutrient acquisition for successful infection within its host. The phosphotransferase system (PTS) couples the import of carbohydrates with their phosphorylation prior to metabolism and has been linked to GAS pathogenesis. In a screen of an insertional mutant library of all 14 annotated PTS permease (EIIC) genes in MGAS5005, the annotated ß-glucoside PTS transporter (bglP) was found to be crucial for GAS growth and survival in human blood and was validated in another M1T1 GAS strain, 5448. In 5448, bglP was shown to be in an operon with a putative phospho-ß-glucosidase (bglB) downstream and a predicted antiterminator (licT) upstream. Using defined nonpolar mutants of the ß-glucoside permease (bglP) and ß-glucosidase enzyme (bglB) in 5448, we showed that bglB, not bglP, was important for growth in blood. Furthermore, transcription of the licT-blgPB operon was found to be repressed by glucose and induced by the ß-glucoside salicin as the sole carbon source. Investigation of the individual bglP and bglB mutants determined that they influence in vitro growth in the ß-glucoside salicin; however, only bglP was necessary for growth in other non-ß-glucoside PTS sugars, such as fructose and mannose. Additionally, loss of BglP and BglB suggests that they are important for the regulation of virulence-related genes that control biofilm formation, streptolysin S (SLS)-mediated hemolysis, and localized ulcerative lesion progression during subcutaneous infections in mice. Thus, our results indicate that the ß-glucoside PTS transports salicin and its metabolism can differentially influence GAS pathophysiology during soft tissue infection.
Subject(s)
Benzyl Alcohols/metabolism , Glucosides/metabolism , Phosphoenolpyruvate Sugar Phosphotransferase System/metabolism , Soft Tissue Infections/pathology , Streptococcal Infections/pathology , Streptococcus pyogenes/metabolism , Streptococcus pyogenes/pathogenicity , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biofilms/growth & development , Catabolite Repression , Gene Expression Regulation, Bacterial , Hemolysis/genetics , Humans , Mice , Microbial Viability/genetics , Mutation , Operon , Phosphoenolpyruvate Sugar Phosphotransferase System/genetics , Soft Tissue Infections/metabolism , Soft Tissue Infections/microbiology , Streptococcal Infections/metabolism , Streptococcal Infections/microbiology , Streptococcus pyogenes/genetics , Streptococcus pyogenes/growth & development , Sugars/metabolism , Virulence/geneticsABSTRACT
The transport and metabolism of glucose has been shown to have far reaching consequences in the transcriptional profile of many bacteria. As glucose is most often the preferred carbon source for bacteria, its presence in the environment leads to the repression of many alternate carbohydrate pathways, a condition known as carbon catabolite repression (CCR). Additionally, the expression of many virulence factors is also dependent on the presence of glucose. Despite its importance, little is known about the transport routes of glucose in the human pathogen Streptococcus pyogenes. Considering that Streptococcus pyogenes is an important human pathogen responsible for over 500,000 deaths every year, we characterized the routes of glucose transport in an effort to understand its importance in GAS pathogenesis. Using a deletion of glucokinase (ΔnagC) to block utilization of glucose imported by non-PTS pathways, we determined that of the two glucose transport pathways in GAS (PTS and non-PTS), the non-PTS pathway played a more significant role in glucose transport. However, the expression of both pathways is linked by a currently unknown mechanism, as blocking the non-PTS uptake of glucose reduces ptsI (EI) expression. Similar to the effects of the deletion of the PTS pathway, lack of the non-PTS pathway also leads to the early activity of Streptolysin S. However, this early activity did not adversely or favorably affect survival of ΔnagC in whole human blood. In a subcutaneous murine infection model, ΔnagC-infected mice showed increased lesion severity at the local site of infection; although, lesion size and dissemination from the site of infection was similar to wild type. Here, we show that glucose transport in GAS is primarily via a non-PTS pathway. The route of glucose transport differentially affects the survival of GAS in whole human blood, as well as the lesion size at the local site of infection in a murine skin infection model.