Alcohol dependence activates ventral tegmental area projections to central amygdala in male mice and rats.

The neural adaptations that occur during the transition to alcohol dependence are not entirely understood but may include a gradual recruitment of brain stress circuitry by mesolimbic reward circuitry that is activated during early stages of alcohol use. Here, we focused on dopaminergic and nondopaminergic projections from the ventral tegmental area (VTA), important for mediating acute alcohol reinforcement, to the central nucleus of the amygdala (CeA), important for alcohol dependence-related negative affect and escalated alcohol drinking. The VTA projects directly to the CeA, but the functional relevance of this circuit is not fully established. Therefore, we combined retrograde and anterograde tracing, anatomical, and electrophysiological experiments in mice and rats to demonstrate that the CeA receives input from both dopaminergic and nondopaminergic projection neurons primarily from the lateral VTA. We then used slice electrophysiology and fos immunohistochemistry to test the effects of alcohol dependence on activity and activation profiles of CeA-projecting neurons in the VTA. Our data indicate that alcohol dependence activates midbrain projections to the central amygdala, suggesting that VTA projections may trigger plasticity in the CeA during the transition to alcohol dependence and that this circuit may be involved in mediating behavioral dysregulation associated with alcohol dependence.

Novel Anti-TM4SF1 Antibody-Drug Conjugates with Activity against Tumor Cells and Tumor Vasculature.

Antibody-drug conjugates (ADC) represent a promising therapeutic modality for managing cancer. Here, we report a novel humanized ADC that targets the tetraspanin-like protein TM4SF1. TM4SF1 is highly expressed on the plasma membranes of many human cancer cells and also on the endothelial cells lining tumor blood vessels. TM4SF1 is internalized upon interaction with antibodies. We hypothesized that an ADC against TM4SF1 would inhibit cancer growth directly by killing cancer cells and indirectly by attacking the tumor vasculature. We generated a humanized anti-human TM4SF1 monoclonal antibody, v1.10, and armed it with an auristatin cytotoxic agent LP2 (chemical name mc-3377). v1.10-LP2 selectively killed cultured human tumor cell lines and human endothelial cells that express TM4SF1. Acting as a single agent, v1.10-LP2 induced complete regression of several TM4SF1-expressing tumor xenografts in nude mice, including non-small cell lung cancer and pancreas, prostate, and colon cancers. As v1.10 did not react with mouse TM4SF1, it could not target the mouse tumor vasculature. Therefore, we generated a surrogate anti-mouse TM4SF1 antibody, 2A7A, and conjugated it to LP2. At 3 mpk, 2A7A-LP2 regressed several tumor xenografts without noticeable toxicity. Combination therapy with v1.10-LP2 and 2A7A-LP2 together was more effective than either ADC alone. These data provide proof-of-concept that TM4SF1-targeting ADCs have potential as anticancer agents with dual action against tumor cells and the tumor vasculature. Such agents could offer exceptional therapeutic value and warrant further investigation. Mol Cancer Ther; 14(8); 1868-76. ©2015 AACR.

Secretion from bacterial versus mammalian cells yields a recombinant scFv with variable folding properties.

Escherichia coli (E. coli) is the most commonly used organism for expressing antibody fragments such as single chain antibody Fvs (scFvs). Previously, we have utilized E. coli to express well-folded scFvs for characterization and engineering purposes with the goal of using these engineered proteins as building blocks for generating IgG-like bispecific antibodies (BsAbs). In the study, described here, we observed a significant difference in the secondary structure of an scFv produced in E. coli and the same scFv expressed and secreted from chinese hamster ovary (CHO) cells as part of a BsAb. We devised a proteolytic procedure to separate the CHO-derived scFv from its antibody-fusion partner and compared its properties with those of the E. coli-derived scFv. In comparison to the CHO-derived scFv, the E. coli-derived scFv was found trapped in a misfolded, but monomeric state that was stable for months at 4 °C. The misfolded state bound antigen in a heterogeneous fashion that included non-specific binding, which made functional characterization challenging. This odd incidence of obtaining a misfolded scFv from bacteria suggests careful characterization of the folded properties of bacterially expressed scFvs is warranted if anomalous issues with antigen-binding or non-specificity occur during an engineering campaign. Additionally, our proteolytic methodology for obtaining significant levels of intact scFvs from highly expressed IgG-like antibody proteins serves as a robust method for producing scFvs in CHO without the use of designed cleavage motifs.

A stable IgG-like bispecific antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor demonstrates superior anti-tumor activity.

The epidermal growth factor receptor (EGFR) and the type I insulin-like growth factor receptor (IGF-1R) are two cell surface receptor tyrosine kinases known to cooperate to promote tumor progression and drug resistance. Combined blockade of EGFR and IGF-1R has shown improved anti-tumor activity in preclinical models. Here, we report the characterization of a stable IgG-like bispecific antibody (BsAb) dual-targeting EGFR and IGF-1R that was developed for cancer therapy. The BsAb molecule (EI-04), constructed with a stability-engineered single chain variable fragment (scFv) against IGF-1R attached to the carboxyl-terminus of an IgG against EGFR, displays favorable biophysical properties for biopharmaceutical development. Biochemically, EI-04 bound to human EGFR and IGF-1R with sub nanomolar affinity, co-engaged the two receptors simultaneously, and blocked the binding of their respective ligands with similar potency compared to the parental monoclonal antibodies (mAbs). In tumor cells, EI-04 effectively inhibited EGFR and IGF-1R phosphorylation, and concurrently blocked downstream AKT and ERK activation, resulting in greater inhibition of tumor cell growth and cell cycle progression than the single mAbs. EI-04, likely due to its tetravalent bispecific format, exhibited high avidity binding to BxPC3 tumor cells co-expressing EGFR and IGF-1R, and consequently improved potency at inhibiting IGF-driven cell growth over the mAb combination. Importantly, EI-04 demonstrated enhanced in vivo anti-tumor efficacy over the parental mAbs in two xenograft models, and even over the mAb combination in the BxPC3 model. Our data support the clinical investigation of EI-04 as a superior cancer therapeutic in treating EGFR and IGF-1R pathway responsive tumors.

Stable IgG-like bispecific antibodies directed toward the type I insulin-like growth factor receptor demonstrate enhanced ligand blockade and anti-tumor activity.

Bispecific antibodies (BsAbs) target multiple epitopes on the same molecular target or different targets. Although interest in BsAbs has persisted for decades, production of stable and active BsAbs has hindered their clinical evaluation. Here, we describe the production and characterization of tetravalent IgG-like BsAbs that combine the activities of allosteric and competitive inhibitors of the type-I insulin-like growth factor receptor (IGF-1R). The BsAbs, which were engineered for thermal stability, express well, demonstrate favorable biophysical properties, and recognize both epitopes on IGF-1R. Only one BsAb with a unique geometry, denoted BIIB4-5scFv, was capable of engaging all four of its binding arms simultaneously. All the BsAbs (especially BIIB4-5scFv) demonstrated enhanced ligand blocking over the single monoclonal antibodies (mAbs), particularly at high ligand concentrations. The pharmacokinetic profiles of two IgG-like BsAbs were tested in nude mice and shown to be comparable with that of the parental mAbs. The BsAbs, especially BIIB4-5scFv, demonstrated an improved ability to reduce the growth of multiple tumor cell lines and to inhibit ligand-induced IGF-1R signaling in tumor cells over the parental mAbs. BIIB4-5scFv also led to superior tumor growth inhibition over its parental mAbs in vivo. In summary, BsAbs that bridge multiple inhibitory mechanisms against a single target may generally represent a more effective strategy for intervention in oncology or other indications compared with traditional mAb therapy.

Stability engineering of scFvs for the development of bispecific and multivalent antibodies.

Single-chain Fvs (scFvs) are commonly used building blocks for creating engineered diagnostic and therapeutic antibody molecules. Bispecific antibodies (BsAbs) hold particular interest due to their ability to simultaneously bind and engage two distinct targets. We describe a technology for producing stable, scalable IgG-like bispecific and multivalent antibodies based on methods for rapidly engineering thermally stable scFvs. Focused libraries of mutant scFvs were designed using a combination of sequence-based statistical analyses and structure-, and knowledge-based methods. Libraries encoding these designs were expressed in E. coli and culture supernatants-containing soluble scFvs screened in a high-throughput assay incorporating a thermal challenge prior to an antigen-binding assay. Thermally stable scFvs were identified that retain full antigen-binding affinity. Single mutations were found that increased the measured T(m) of either the V(H) or V(L) domain by as much as 14 degrees C relative to the wild-type scFv. Combinations of mutations further increased the T(m) by as much as an additional 12 degrees C. Introduction of a stability-engineered scFv as part of an IgG-like BsAb enabled scalable production and purification of BsAb with favorable biophysical properties.

Anti-tumor activity of stability-engineered IgG-like bispecific antibodies targeting TRAIL-R2 and LTbetaR.

Bispecific antibodies (BsAbs) represent an emerging class of biologics that achieve dual targeting with a single agent. Recombinant DNA technologies have facilitated a variety of creative bispecific designs with many promising therapeutic applications; however, practical methods for producing high quality BsAbs that have good product stability, long serum half-life, straightforward purification, and scalable production have largely been limiting. Here we describe a protein-engineering approach for producing stable, scalable tetravalent IgG-like BsAbs. The stability-engineered IgG-like BsAb was envisioned to target and crosslink two TNF family member receptors, TRAIL-R2 (TNF-Related Apoptosis Inducing Ligand Receptor-2) and LTbetaR (Lymphotoxin-beta Receptor), expressed on the surface of epithelial tumor cells with the goal of triggering an enhanced anti-tumor effect. Our IgG-like BsAbs consists of a stability-engineered anti-LTbetaR single chain Fv (scFv) genetically fused to either the N- or C-terminus of the heavy chain of a fulllength anti-TRAIL-R2 IgG1 monoclonal antibody. Both N- or C-terminal BsAbs were active in inhibiting tumor cell growth in vitro, and with some cell lines demonstrated enhanced activity relative to the combination of parental Abs. Pharmacokinetic studies in mice revealed long serum half-lives for the BsAbs. In murine tumor xenograft models, therapeutic treatment with the BsAbs resulted in reduction in tumor volume either comparable to or greater than the combination of parental antibodies, indicating that simultaneously targeting and cross-linking receptor pairs is an effective strategy for treating tumor cells. These studies support that stability-engineering is an enabling step for producing scalable IgG-like BsAbs with properties desirable for biopharmaceutical development.

Endosome-associated complex, ESCRT-II, recruits transport machinery for protein sorting at the multivesicular body.

Sorting of ubiquitinated endosomal membrane proteins into the MVB pathway is executed by the class E Vps protein complexes ESCRT-I, -II, and -III, and the AAA-type ATPase Vps4. This study characterizes ESCRT-II, a soluble approximately 155 kDa protein complex formed by the class E Vps proteins Vps22, Vps25, and Vps36. This protein complex transiently associates with the endosomal membrane and thereby initiates the formation of ESCRT-III, a membrane-associated protein complex that functions immediately downstream of ESCRT-II during sorting of MVB cargo. ESCRT-II in turn functions downstream of ESCRT-I, a protein complex that binds to ubiquitinated endosomal cargo. We propose that the ESCRT complexes perform a coordinated cascade of events to select and sort MVB cargoes for delivery to the lumen of the vacuole/lysosome.

Peroxisome remnants in pex3delta cells and the requirement of Pex3p for interactions between the peroxisomal docking and translocation subcomplexes.

During peroxisomal matrix protein import, the peroxisomal targeting signal receptors recognize cargo in the cytosol and interact with docking and translocation subcomplexes on the peroxisomal membrane. Using immunoprecipitations of multiple protein components, we show that in Pichia pastoris the docking subcomplex consists of the unique peroxins Pex13p, Pex14p and Pex17p, whereas the putative translocation subcomplex has all three RING-finger peroxins, Pex2p, Pex10p and Pex12p, as unique constituents. We identify Pex3p as a shared component of both subcomplexes. In pex3delta cells, the unique constituents of the docking subcomplex interact as they do in wild-type cells, but the assembly of the translocation subcomplex is impaired and its components are present at reduced levels. Furthermore, several interactions detected in wild-type cells between translocation and docking subcomplex components are undetectable in pex3delta cells. Contrary to previous reports, pex3delta cells have peroxisome remnants that pellet during high-speed centrifugation, associate with membranes on floatation gradients and can be visualized by deconvolution microscopy using antibodies to several peroxins which were not available earlier. We discuss roles for Pex3p in the assembly of specific peroxisomal membrane protein subcomplexes whose formation is necessary for matrix protein import.