Cytokine/Antibody Fusion Protein Design and Evaluation.

Cytokines constitute a class of secreted proteins that activate transmembrane receptors to coordinate a vast array of physiological processes, particularly those related to immune activity. Due to their vital role in immune regulation, cytokines have garnered great interest as potential therapeutic agents. Unfortunately, the clinical success of cytokine drugs has been limited by their multifunctional activities, which hinder therapeutic performance and lead to harmful toxicities. In addition, the strikingly short circulation half-life of cytokines further hampers their efficacy as drugs. To overcome the translational challenges associated with natural cytokines, significant efforts have focused on engineering cytokines to target their activities and improve their pharmacological properties. One such strategy is the design of fusion proteins that tether a cytokine to an anti-cytokine antibody that selectively biases its functions and extends its serum half-life. These cytokine/antibody fusion proteins (termed immunocytokines) assemble intramolecularly to bias cytokine signaling behavior through multi-layered structural and molecular effects. Here, we present a detailed workflow for the design, production, and functional validation of intramolecularly assembled immunocytokines. In-depth procedures are presented for gene manipulation, mammalian cell-based expression and purification, binding analysis via bio-layer interferometry, and interrogation of cytokine signaling activity on human primary cells. In contrast with immunocytokines in which the tethered cytokine and antibody do not bind one another, intramolecularly assembled immunocytokines require special considerations with respect to their production to avoid oligomerization and/or aggregation. The protocol herein was developed based on experience with immunocytokines that incorporate interleukin-2 (IL-2); however, this modular approach can be extended to any cytokine of interest for a broad range of biomedical applications. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Design and generation of immunocytokine genes Basic Protocol 2: Immunocytokine expression and purification Basic Protocol 3: Validation of immunocytokine assembly and binding by bio-layer interferometry Basic Protocol 4: Analysis of immunocytokine signaling on human primary cells.

In Vivo Stimulation of Therapeutic Antigen-Specific T Cells in an Artificial Lymph Node Matrix.

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.

Engineered cytokine/antibody fusion proteins improve delivery of IL-2 to pro-inflammatory cells and promote antitumor activity.

Progress in cytokine engineering is driving therapeutic translation by overcoming the inherent limitations of these proteins as drugs. The interleukin-2 (IL-2) cytokine harbors great promise as an immune stimulant for cancer treatment. However, the cytokine's concurrent activation of both pro-inflammatory immune effector cells and anti-inflammatory regulatory T cells, its toxicity at high doses, and its short serum half-life have limited clinical application. One promising approach to improve the selectivity, safety, and longevity of IL-2 is complexation with anti-IL-2 antibodies that bias the cytokine towards the activation of immune effector cells (i.e., effector T cells and natural killer cells). Although this strategy shows therapeutic potential in preclinical cancer models, clinical translation of a cytokine/antibody complex is complicated by challenges in formulating a multi-protein drug and concerns about complex stability. Here, we introduce a versatile approach to designing intramolecularly assembled single-agent fusion proteins (immunocytokines, ICs) comprising IL-2 and a biasing anti-IL-2 antibody that directs the cytokine's activities towards immune effector cells. We establish the optimal IC construction and further engineer the cytokine/antibody affinity to improve immune biasing function. We demonstrate that our IC preferentially activates and expands immune effector cells, leading to superior antitumor activity compared to natural IL-2 without inducing toxicities associated with IL-2 administration. Collectively, this work presents a roadmap for the design and translation of immunomodulatory cytokine/antibody fusion proteins.

Supramolecular filaments for concurrent ACE2 docking and enzymatic activity silencing enable coronavirus capture and infection prevention.

Coronaviruses have historically precipitated global pandemics of severe acute respiratory syndrome (SARS) into devastating public health crises. Despite the virus's rapid rate of mutation, all SARS coronavirus 2 (SARS-CoV-2) variants are known to gain entry into host cells primarily through complexation with angiotensin-converting enzyme 2 (ACE2). Although ACE2 has potential as a druggable decoy to block viral entry, its clinical use is complicated by its essential biological role as a carboxypeptidase and hindered by its structural and chemical instability. Here we designed supramolecular filaments, called fACE2, that can silence ACE2's enzymatic activity and immobilize ACE2 to their surface through enzyme-substrate complexation. This docking strategy enables ACE2 to be effectively delivered in inhalable aerosols and improves its structural stability and functional preservation. fACE2 exhibits enhanced and prolonged inhibition of viral entry compared with ACE2 alone while mitigating lung injury in vivo.

Nanoparticle-based modulation of CD4+ T cell effector and helper functions enhances adoptive immunotherapy.

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.

Antibody-Invertase Fusion Protein Enables Quantitative Detection of SARS-CoV-2 Antibodies Using Widely Available Glucometers.

Rapid diagnostics that can accurately inform patients of disease risk and protection are critical to mitigating the spread of the current COVID-19 pandemic and future infectious disease outbreaks. To be effective, such diagnostics must rely on simple, cost-effective, and widely available equipment and should be compatible with existing telehealth infrastructure to facilitate data access and remote care. Commercial glucometers are an established detection technology that can overcome the cost, time, and trained personnel requirements of current benchtop-based antibody serology assays when paired with reporter molecules that catalyze glucose conversion. To this end, we developed an enzymatic reporter that, when bound to disease-specific patient antibodies, produces glucose in proportion to the level of antibodies present in the patient sample. Although a straightforward concept, the coupling of enzymatic reporters to secondary antibodies or antigens often results in low yields, indeterminant stoichiometry, reduced target binding, and poor catalytic efficiency. Our enzymatic reporter is a novel fusion protein that comprises an antihuman immunoglobulin G (IgG) antibody genetically fused to two invertase molecules. The resulting fusion protein retains the binding affinity and catalytic activity of the constituent proteins and serves as an accurate reporter for immunoassays. Using this fusion, we demonstrate quantitative glucometer-based measurement of anti-SARS-CoV-2 spike protein antibodies in blinded clinical sample training sets. Our results demonstrate the ability to detect SARS-CoV-2-specific IgGs in patient serum with precise agreement to benchmark commercial immunoassays. Because our fusion protein binds all human IgG isotypes, it represents a versatile tool for detection of disease-specific antibodies in a broad range of biomedical applications.

IgM anti-ACE2 autoantibodies in severe COVID-19 activate complement and perturb vascular endothelial function.

BackgroundSome clinical features of severe COVID-19 represent blood vessel damage induced by activation of host immune responses initiated by the coronavirus SARS-CoV-2. We hypothesized autoantibodies against angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 receptor expressed on vascular endothelium, are generated during COVID-19 and are of mechanistic importance.MethodsIn an opportunity sample of 118 COVID-19 inpatients, autoantibodies recognizing ACE2 were detected by ELISA. Binding properties of anti-ACE2 IgM were analyzed via biolayer interferometry. Effects of anti-ACE2 IgM on complement activation and endothelial function were demonstrated in a tissue-engineered pulmonary microvessel model.ResultsAnti-ACE2 IgM (not IgG) autoantibodies were associated with severe COVID-19 and found in 18/66 (27.2%) patients with severe disease compared with 2/52 (3.8%) of patients with moderate disease (OR 9.38, 95% CI 2.38-42.0; P = 0.0009). Anti-ACE2 IgM autoantibodies were rare (2/50) in non-COVID-19 ventilated patients with acute respiratory distress syndrome. Unexpectedly, ACE2-reactive IgM autoantibodies in COVID-19 did not undergo class-switching to IgG and had apparent KD values of 5.6-21.7 nM, indicating they are T cell independent. Anti-ACE2 IgMs activated complement and initiated complement-binding and functional changes in endothelial cells in microvessels, suggesting they contribute to the angiocentric pathology of COVID-19.ConclusionWe identify anti-ACE2 IgM as a mechanism-based biomarker strongly associated with severe clinical outcomes in SARS-CoV-2 infection, which has therapeutic implications.FUNDINGBill & Melinda Gates Foundation, Gates Philanthropy Partners, Donald B. and Dorothy L. Stabler Foundation, and Jerome L. Greene Foundation; NIH R01 AR073208, R01 AR069569, Institutional Research and Academic Career Development Award (5K12GM123914-03), National Heart, Lung, and Blood Institute R21HL145216, and Division of Intramural Research, National Institute of Allergy and Infectious Diseases; National Science Foundation Graduate Research Fellowship (DGE1746891).

Engineered antibody fusion proteins for targeted disease therapy.

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.

IgM autoantibodies recognizing ACE2 are associated with severe COVID-19.

SARS-CoV-2 infection induces severe disease in a subpopulation of patients, but the underlying mechanisms remain unclear. We demonstrate robust IgM autoantibodies that recognize angiotensin converting enzyme-2 (ACE2) in 18/66 (27%) patients with severe COVID-19, which are rare (2/52; 3.8%) in hospitalized patients who are not ventilated. The antibodies do not undergo class-switching to IgG, suggesting a T-independent antibody response. Purified IgM from anti-ACE2 patients activates complement. Pathological analysis of lung obtained at autopsy shows endothelial cell staining for IgM in blood vessels in some patients. We propose that vascular endothelial ACE2 expression focuses the pathogenic effects of these autoantibodies on blood vessels, and contributes to the angiocentric pathology observed in some severe COVID-19 patients. These findings may have predictive and therapeutic implications.

Weaponizing T-cell receptors through molecular engineering.

T-cell receptors (TCRs) recognize pathogens to ignite immune responses, making them attractive scaffolds for development as immunotherapeutics. However, manipulation of TCRs has been impeded by difficulties in their engineering and expression. Wagner and colleagues now establish new platforms to generate high-affinity TCR variants that potently activate T cells, and they also create soluble TCR fusion proteins that specifically recognize cognate peptides. This work provides specific tools to combat cytomegalovirus (CMV) infection and helps illuminate a general path to actuation of engineered TCR-based therapeutics.

Design and validation of a corneal bioreactor.

Mechanical strain is an important signal that influences the behavior and properties of cells in a wide variety of tissues. Physiologically similar mechanical strain can revert cultured cells to a more normal phenotype. Here, we have demonstrated that 3% equibiaxial (EB) and uniaxial strains confer favorable protein expression in cultured rabbit corneal fibroblasts (RCFs), with approximately 35% and 65% reduction in expression of α-smooth muscle actin (α-SMA), respectively. We have designed a novel bioreactor that is capable of imparting up to 7% EB strain and up to 6% EB strain using a cornea-shaped post. Additional features of the bioreactor include the application of shear stress to cells in culture and the ability to image cells using optical coherence microscopy (OCM) without being removed from the system.