Inhibiting membrane rupture with NINJ1 antibodies limits tissue injury.

Plasma membrane rupture (PMR) in dying cells undergoing pyroptosis or apoptosis requires the cell-surface protein NINJ11. PMR releases pro-inflammatory cytoplasmic molecules, collectively called damage-associated molecular patterns (DAMPs), that activate immune cells. Therefore, inhibiting NINJ1 and PMR may limit the inflammation that is associated with excessive cell death. Here we describe an anti-NINJ1 monoclonal antibody that specifically targets mouse NINJ1 and blocks oligomerization of NINJ1, preventing PMR. Electron microscopy studies showed that this antibody prevents NINJ1 from forming oligomeric filaments. In mice, inhibition of NINJ1 or Ninj1 deficiency ameliorated hepatocellular PMR induced with TNF plus D-galactosamine, concanavalin A, Jo2 anti-Fas agonist antibody or ischaemia-reperfusion injury. Accordingly, serum levels of lactate dehydrogenase, the liver enzymes alanine aminotransaminase and aspartate aminotransferase, and the DAMPs interleukin 18 and HMGB1 were reduced. Moreover, in the liver ischaemia-reperfusion injury model, there was an attendant reduction in neutrophil infiltration. These data indicate that NINJ1 mediates PMR and inflammation in diseases driven by aberrant hepatocellular death.

Diagnostic Pitfalls of Chylothorax After Dasatinib Treatment of Chronic Myeloid Leukemia.

BACKGROUND Chronic myeloid leukemia (CML) is a myeloproliferative malignancy generally treated with Dasatinib, a tyrosin-kinase inhibitor. Pleural effusions are a known adverse effect, but only 0.8% of patients develop pleural effusions after 6 years of use. Recent case reports have implicated Dasatinib as a rare cause of chylothorax. CASE REPORT We describe a woman in her 30's with a history of chronic myeloid leukemia, who had been taking Dasatinib for 10 years and presented to the Emergency Department after a chest X-ray revealed bilateral pleural effusions in the setting of worsening dyspnea on exertion for 6 months. She had previously received radiotherapy at age 11 prior to an allogenic bone marrow transplant nearly 30 years prior. Thoracentesis removed 900 cc of chylous fluid, and flow cytometry and cultures found no evidence of infection or malignancy. Dasatinib was discontinued, and she was treated with diuretics, steroids, and a low-fat diet. The effusions reaccumulated twice in the following month and required 2 additional thoracenteses and courses of steroids. Months later, the bilateral chylous effusions recurred, and MR lymphangiogram demonstrated 2 thoracic duct tears. CONCLUSIONS While previous reports have indicated that Dasatinib can rarely cause chylous pleural effusions, it is unlikely after 5 years of use, and other etiologies must be considered by clinicians. Initial misattribution to Dasatinib alone can delay further necessary investigations, including lymphangiography. In our patient, it is more likely that other factors contributed to her chylothorax, including her previous radiotherapy 30 years prior, given her recurrence of chylous effusions following cessation of the medication.

The Safety of Blood Flow Restriction Training as a Therapeutic Intervention for Patients With Musculoskeletal Disorders: A Systematic Review.

BACKGROUND: The effectiveness of blood flow restriction training (BFRT) as compared with other forms of training, such as resistance training, has been evaluated in the literature in clinical and nonclinical populations. However, the safety of this intervention has been summarized only in healthy populations and not in clinical populations with musculoskeletal disorders. PURPOSE: To evaluate the safety and adverse events associated with BFRT in patients with musculoskeletal disorders. STUDY DESIGN: Systematic review. METHODS: A literature search was conducted with 3 online databases (MEDLINE, CINAHL, and Embase). Eligibility criteria for selecting studies were as follows: (1) BFRT was used as a clinical intervention, (2) study participants had a disorder of the musculoskeletal system, (3) authors addressed adverse events, (4) studies were published in English, and (5) the intervention was performed with human participants. RESULTS: Nineteen studies met eligibility criteria, with a pooled sample size of 322. Diagnoses included various knee-related disorders, inclusion body myositis, polymyositis or dermatomyositis, thoracic outlet syndrome, Achilles tendon rupture, and bony fractures. Nine studies reported no adverse events, while 3 reported rare adverse events, including an upper extremity deep vein thrombosis and rhabdomyolysis. Three case studies reported common adverse events, including acute muscle pain and acute muscle fatigue. In the randomized controlled trials, individuals exposed to BFRT were not more likely to have an adverse event than individuals exposed to exercise alone. Of the 19 studies, the adverse events were as follows: overall, 14 of 322; rare overall, 3 of 322; rare BFRT, 3 of 168; rare control, 0 of 154; any adverse BFRT, 10 of 168; any adverse control, 4 of 154. A majority of studies were excluded because they did not address safety. CONCLUSION: BFRT appears to be a safe strengthening approach for knee-related musculoskeletal disorders, but further research is needed to make definitive conclusions and to evaluate the safety in other musculoskeletal conditions. Improved definitions of adverse events related to BFRT are needed to include clear criteria for differentiating among common, uncommon, and rare adverse events. Finally, further research is needed to effectively screen who might be at risk for rare adverse events.

An engineered antibody binds a distinct epitope and is a potent inhibitor of murine and human VISTA.

V-domain immunoglobulin (Ig) suppressor of T cell activation (VISTA) is an immune checkpoint that maintains peripheral T cell quiescence and inhibits anti-tumor immune responses. VISTA functions by dampening the interaction between myeloid cells and T cells, orthogonal to PD-1 and other checkpoints of the tumor-T cell signaling axis. Here, we report the use of yeast surface display to engineer an anti-VISTA antibody that binds with high affinity to mouse, human, and cynomolgus monkey VISTA. Our anti-VISTA antibody (SG7) inhibits VISTA function and blocks purported interactions with both PSGL-1 and VSIG3 proteins. SG7 binds a unique epitope on the surface of VISTA, which partially overlaps with other clinically relevant antibodies. As a monotherapy, and to a greater extent as a combination with anti-PD1, SG7 slows tumor growth in multiple syngeneic mouse models. SG7 is a promising clinical candidate that can be tested in fully immunocompetent mouse models and its binding epitope can be used for future campaigns to develop species cross-reactive inhibitors of VISTA.

Reduction of Nonspecificity Motifs in Synthetic Antibody Libraries.

Successful antibody development requires both functional binding and desirable biophysical characteristics. In the current study, we analyze the causes of one hurdle to clinical development, off-target reactivity, or nonspecificity. We used a high-throughput nonspecificity assay to isolate panels of nonspecific antibodies from two synthetic single-chain variable fragment libraries expressed on the surface of yeast, identifying both individual amino acids and motifs within the complementarity-determining regions which contribute to the phenotype. We find enrichment of glycine, valine, and arginine as both individual amino acids and as a part of motifs, and additionally enrichment of motifs containing tryptophan. Insertion of any of these motifs into the complementarity-determining region H3 of a "clean" antibody increased its nonspecificity, with greatest increases in antibodies containing Trp or Val motifs. We next applied these rules to the creation of a synthetic diversity library based on natural frameworks with significantly decreased incorporation of such motifs and demonstrated its ability to isolate binders to a wide panel of antigens. This work both provides a greater understanding of the drivers of nonspecificity and provides design rules to increase efficiency in the isolation of antibodies with drug-like properties.

A Raf-Competitive K-Ras Binder Can Fail to Functionally Antagonize Signaling.

Mutated in approximately 30% of human cancers, Ras GTPases are the most common drivers of oncogenesis and render tumors unresponsive to many standard therapies. Despite decades of research, no drugs directly targeting Ras are currently available. We have previously characterized a small protein antagonist of K-Ras, R11.1.6, and demonstrated its direct competition with Raf for Ras binding. Here we evaluate the effects of R11.1.6 on Ras signaling and cellular proliferation in a panel of human cancer cell lines. Through lentiviral transduction, we generated cell lines that constitutively or through induction with doxycycline express R11.1.6 or a control protein YW1 and show specific binding by R11.1.6 to endogenous Ras through microscopy and co-immunoprecipitation experiments. Genetically encoded intracellular expression of this high-affinity Ras antagonist, however, fails to measurably disrupt signaling through either the MAPK or PI3K pathway. Consistently, cellular proliferation was unaffected as well. To understand this lack of signaling inhibition, we quantified the number of molecules of R11.1.6 expressed by the inducible cell lines and developed a simple mathematical model describing the competitive binding of Ras by R11.1.6 and Raf. This model supports a potential mechanism for the lack of biological effects that we observed, suggesting stoichiometric and thermodynamic barriers that should be overcome in pharmacologic efforts to directly compete with downstream effector proteins localized to membranes at very high effective concentrations. Mol Cancer Ther; 17(8); 1773-80. ©2018 AACR.

Directed evolution of broadly crossreactive chemokine-blocking antibodies efficacious in arthritis.

Chemokine receptors typically have multiple ligands. Consequently, treatment with a blocking antibody against a single chemokine is expected to be insufficient for efficacy. Here we show single-chain antibodies can be engineered for broad crossreactivity toward multiple human and mouse proinflammatory ELR+ CXC chemokines. The engineered molecules recognize functional epitopes of ELR+ CXC chemokines and inhibit neutrophil activation ex vivo. Furthermore, an albumin fusion of the most crossreactive single-chain antibody prevents and reverses inflammation in the K/BxN mouse model of arthritis. Thus, we report an approach for the molecular evolution and selection of broadly crossreactive antibodies towards a family of structurally related, yet sequence-diverse protein targets, with general implications for the development of novel therapeutics.

Nonspecificity in a nonimmune human scFv repertoire.

Efforts to develop effective antibody therapeutics are frequently hampered by issues such as aggregation and nonspecificity, often only detected in late stages of the development process. In this study, we used a high throughput cross-reactivity assay to select nonspecific clones from a naïve human repertoire scFv library displayed on the surface of yeast. Most antibody families were de-enriched; however, the rarely expressed VH6 family was highly enriched among nonspecific clones, representing almost 90% of isolated clones. Mutational analysis of this family reveals a dominant role of CDRH2 in driving nonspecific binding. Homology modeling of a panel of VH6 antibodies shows a constrained ß-sheet structure in CDRH2 that is not present in other families, potentially contributing to nonspecificity of the family. These findings confirm the common decision to exclude VH6 from synthetic antibody libraries, and support VH6 polyreactivity as a possible important role for the family in early ontogeny and cause for its overabundance in cases of some forms of autoimmunity.

Chaperone proteins as single component reagents to assess antibody nonspecificity.

Early stage assays that evaluate monoclonal antibody drug-like properties serve as valuable tools for selection of lead candidates. One liability for clinical development, off-target reactivity, is often assessed by binding to a mixture or panel of noncognate proteins. While robust, these mixes are often ill-defined, and can suffer from issues such as lot-to-lot variability. In this study, we discovered in immunoprecipitation experiments that certain chaperones are present in one of these mixtures;we then explored the use of recombinant chaperone proteins as well-characterized agents to predict antibody nonspecificity. Antibody binding to the heat shock proteins HSP70, HSP90, or trigger factor all served as predictors of cross-interaction propensity, with HSP90 providing the greatest ability to predict antibody clearance rates in mouse. Individual chaperone binding correlates surprisingly closely with binding to complex cell extracts, with the exception of a few "false negatives" (assuming a complex cell extract as the "true" value). As defined reagents, these chaperone reagents present advantages for high throughput assays of nonspecificity.

A platform for constructing, evaluating, and screening bioconjugates on the yeast surface.

The combination of protein display technologies and noncanonical amino acids (ncAAs) offers unprecedented opportunities for the high throughput discovery and characterization of molecules suitable for addressing fundamental and applied problems in biological systems. Here we demonstrate that ncAA-compatible yeast display facilitates evaluations of conjugation chemistry and stability that would be challenging or impossible to perform with existing mRNA, phage, or E. coli platforms. Our approach enables site-specific introduction of ncAAs into displayed proteins, robust modification at azide-containing residues, and quantitative evaluation of conjugates directly on the yeast surface. Moreover, screening allows for the selective enrichment of chemically modified constructs while maintaining a genotype-phenotype linkage with encoded azide functionalities. Thus, this platform is suitable for the high throughput characterization and screening of libraries of chemically modified polypeptides for therapeutic lead discovery and other biological applications.

Target-independent variable region mediated effects on antibody clearance can be FcRn independent.

The importance of the neonatal Fc receptor (FcRn) in extending the serum half-life of monoclonal antibodies (mAbs) is well demonstrated, and has led to the development of multiple engineering approaches designed to alter Fc interactions with FcRn. Recent reports have additionally highlighted the effect of nonspecific interactions on antibody pharmacokinetics (PK), suggesting an FcRn-independent mechanism for mAb clearance. In this report we examine a case study of 2 anti-interleukin-12/23 antibodies, ustekinumab and briakinumab, which share the same target and Fc, but differ in variable region sequences. Ustekinumab displayed near baseline signal in a wide range of early stage developability assays for undesirable protein/protein interactions, while briakinumab showed significant propensity for self- and cross-interactions. This phenotypic difference correlates with faster clearance rates for briakinumab in both human FcRn transgenic and FcRn knockout mice. These findings support a dominant contribution for FcRn-independent clearance for antibodies with high nonspecificity, and highlight a key role for early stage developability screening to eliminate clones with such high nonspecific disposition PK.

Temporally Programmed CD8α+ DC Activation Enhances Combination Cancer Immunotherapy.

Numerous synergistic cancer immunotherapy combinations have been identified, but the effects of relative dose timing are rarely considered. In established syngeneic mouse tumor models, we found that staggering interferon-α (IFNα) administration after, rather than before or simultaneously with, serum-persistent interleukin-2 (IL-2) and tumor-specific antibody significantly increased long-term survival. Successful combination therapy required IFNα-induced activation of cross-presenting CD8α+ dendritic cells (DCs) following the release of antigenic tumor debris by the IL-2- and antibody-mediated immune response. Due to decreased phagocytic ability post-maturation, DCs activated too early captured less antigen and could not effectively prime CD8+ T cells. Temporally programming DC activation to occur after tumoricidal activity enhanced tumor control by multiple distinct combination immunotherapies, highlighting dose schedule as an underappreciated factor that can profoundly affect the success of multi-component immunotherapies.

Eradication of large established tumors in mice by combination immunotherapy that engages innate and adaptive immune responses.

Checkpoint blockade with antibodies specific for cytotoxic T lymphocyte-associated protein (CTLA)-4 or programmed cell death 1 (PDCD1; also known as PD-1) elicits durable tumor regression in metastatic cancer, but these dramatic responses are confined to a minority of patients. This suboptimal outcome is probably due in part to the complex network of immunosuppressive pathways present in advanced tumors, which are unlikely to be overcome by intervention at a single signaling checkpoint. Here we describe a combination immunotherapy that recruits a variety of innate and adaptive immune cells to eliminate large tumor burdens in syngeneic tumor models and a genetically engineered mouse model of melanoma; to our knowledge tumors of this size have not previously been curable by treatments relying on endogenous immunity. Maximal antitumor efficacy required four components: a tumor-antigen-targeting antibody, a recombinant interleukin-2 with an extended half-life, anti-PD-1 and a powerful T cell vaccine. Depletion experiments revealed that CD8+ T cells, cross-presenting dendritic cells and several other innate immune cell subsets were required for tumor regression. Effective treatment induced infiltration of immune cells and production of inflammatory cytokines in the tumor, enhanced antibody-mediated tumor antigen uptake and promoted antigen spreading. These results demonstrate the capacity of an elicited endogenous immune response to destroy large, established tumors and elucidate essential characteristics of combination immunotherapies that are capable of curing a majority of tumors in experimental settings typically viewed as intractable.

A switchable yeast display/secretion system.

Display technologies such as yeast and phage display offer powerful alternatives to traditional immunization-based antibody discovery, but require conversion of displayed proteins into soluble form prior to downstream characterization. Here we utilize amber suppression to implement a yeast-based switchable display/secretion system that enables the immediate production of soluble, antibody-like reagents at the end of screening efforts. Model selections in the switchable format remain efficient, and library screening in the switchable format yields renewable sources of affinity reagents exhibiting nanomolar binding affinities. These results confirm that this system provides a seamless link between display-based screening and the production and evaluation of soluble forms of candidate binding proteins. Switchable display/secretion libraries provide a cloning-free, accessible approach to affinity reagent generation.

High throughput cross-interaction measures for human IgG1 antibodies correlate with clearance rates in mice.

Although improvements in technology for the isolation of potential therapeutic antibodies have made the process increasingly predictable, the development of biologically active monoclonal antibodies (mAbs) into drugs can often be impeded by developability issues such as poor expression, solubility, and promiscuous cross-reactivity. Establishing early stage developability screening assays capable of predicting late stage behavior is therefore of high value to minimize development risks. Toward this goal, we selected a panel of 16 monoclonal antibodies (mAbs) representing different developability profiles, in terms of self- and cross-interaction propensity, and examined their downstream behavior from expression titer to accelerated stability and pharmacokinetics in mice. Clearance rates showed significant rank-order correlations to 2 cross-interaction related assays, with the closest correlation to a non-specificity assay on the surface of yeast. Additionally, 2 self-association assays correlated with each other but not to mouse clearance rate. This case study suggests that combining assays capable of high throughput screening of self- and cross-interaction early in the discovery stage could significantly lower downstream development risks.

A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

Chemokines play a key role in leukocyte recruitment during inflammation and are implicated in the pathogenesis of a number of autoimmune diseases. As such, inhibiting chemokine signaling has been of keen interest for the development of therapeutic agents. This endeavor, however, has been hampered due to complexities in the chemokine system. Many chemokines have been shown to signal through multiple receptors and, conversely, most chemokine receptors bind to more than one chemokine. One approach to overcoming this complexity is to develop a single therapeutic agent that binds and inactivates multiple chemokines, similar to an immune evasion strategy utilized by a number of viruses. Here, we describe the development and characterization of a novel therapeutic antibody that targets a subset of human CC chemokines, specifically CCL3, CCL4, and CCL5, involved in chronic inflammatory diseases. Using a sequential immunization approach, followed by humanization and phage display affinity maturation, a therapeutic antibody was developed that displays high binding affinity towards the three targeted chemokines. In vitro, this antibody potently inhibits chemotaxis and chemokine-mediated signaling through CCR1 and CCR5, primary chemokine receptors for the targeted chemokines. Furthermore, we have demonstrated in vivo efficacy of the antibody in a SCID-hu mouse model of skin leukocyte migration, thus confirming its potential as a novel therapeutic chemokine antagonist. We anticipate that this antibody will have broad therapeutic utility in the treatment of a number of autoimmune diseases due to its ability to simultaneously neutralize multiple chemokines implicated in disease pathogenesis.