Intratumoral aluminum hydroxide-anchored IL-12 drives potent antitumor activity by remodeling the tumor microenvironment.

IL-12 is a potent cytokine that can promote innate and adaptive anticancer immunity, but its clinical development has been limited by toxicity when delivered systemically. Intratumoral (i.t.) administration can expand the therapeutic window of IL-12 and other cytokines but is in turn limited by rapid drug clearance from the tumor, which reduces efficacy, necessitates frequent administration, and increases systemic accumulation. To address these limitations, we developed an anchored IL-12 designated ANK-101, composed of an engineered IL-12 variant that forms a stable complex with the FDA-approved vaccine adjuvant aluminum hydroxide (Alhydrogel). Following i.t. administration of murine ANK-101 (mANK-101) in early intervention syngeneic mouse tumors, the complex formed a depot that was locally retained for weeks as measured by IVIS or SPECT/CT imaging, while unanchored protein injected i.t. was cleared within hours. One or 2 i.t. injections of mANK-101 induced single-agent antitumor activity across a diverse range of syngeneic tumors, including models resistant to checkpoint blockade at doses where unanchored IL-12 had no efficacy. Local treatment with mANK-101 further induced regressions of noninjected lesions, especially when combined with systemic checkpoint blockade. Antitumor activity was associated with remodeling of the tumor microenvironment, including prolonged IFN-γ and chemokine expression, recruitment and activation of T and NK cells, M1 myeloid cell skewing, and increased antigen processing and presentation. Subcutaneous administration of ANK-101 in cynomolgus macaques was well tolerated. Together, these data demonstrate that ANK-101 has an enhanced efficacy and safety profile and warrants future clinical development.

Intratumorally anchored cytokine therapy.

INTRODUCTION: On-target, off-tumor toxicity severely limits systemic dosing of cytokines and agonist antibodies for cancer. Intratumoral administration is increasingly being explored to mitigate this problem. Full exploitation of this mode of administration must include a mechanism for sustained retention of the drug; otherwise, rapid diffusion out of the tumor eliminates any advantage. AREAS COVERED: We focus here on strategies for anchoring immune agonists in accessible formats. Such anchoring may utilize extracellular matrix components, cell surface receptor targets, or exogenously administered particulate materials. Promising alternative strategies not reviewed here include slow release from the interior of a material depot, expression following local transfection, and conditional proteolytic activation of masked molecules. EXPERT OPINION: An effective mechanism for tissue retention is a critical component of intratumorally anchored cytokine therapy, as leakage leads to decreased tumor drug exposure and increased systemic toxicity. Matching variable drug release kinetics with receptor-mediated cellular uptake is an intrinsic requirement for the alternative strategies mentioned above. Bioavailability of an anchored form of the administered drug is key to obviating this balancing act.

[Bempedoic Acid]. / Bempedoinsäure.

ATP-Citrate-Lyase is a key enzyme of cholesterol biosynthesis. Its liver-specific inhibition by the bempedoic acid opens new possibilities to effectively escalate a cholesterol-lowering therapy while avoiding muscle-related side effects. Herein, we present the properties of this new first-in-class pharmaceutical agent and discuss potential consequences for pharmacotherapy.

Differentiated agonistic antibody targeting CD137 eradicates large tumors without hepatotoxicity.

CD137 (4-1BB) is a member of the TNFR superfamily that represents a promising target for cancer immunotherapy. Recent insights into the function of TNFR agonist antibodies implicate epitope, affinity, and IgG subclass as critical features, and these observations help explain the limited activity and toxicity seen with clinically tested CD137 agonists. Here, we describe the preclinical characterization of CTX-471, a fully human IgG4 agonist of CD137 that engages a unique epitope that is shared by human, cynomolgus monkey, and mouse and is associated with a differentiated pharmacology and toxicology profile. In vitro, CTX-471 increased IFN-γ production by human T cells in an Fcγ receptor-dependent (FcγR-dependent) manner, displaying an intermediate level of activity between 2 clinical-stage anti-CD137 antibodies. In mice, CTX-471 exhibited curative monotherapy activity in various syngeneic tumor models and showed a unique ability to cure mice of very large (~500 mm3) tumors compared with validated antibodies against checkpoints and TNFR superfamily members. Extremely high doses of CTX-471 were well tolerated, with no signs of hepatic toxicity. Collectively, these data demonstrate that CTX-471 is a unique CD137 agonist that displays an excellent safety profile and an unprecedented level of monotherapy efficacy against very large tumors.

Crystal structure of an HSA/FcRn complex reveals recycling by competitive mimicry of HSA ligands at a pH-dependent hydrophobic interface.

The long circulating half-life of serum albumin, the most abundant protein in mammalian plasma, derives from pH-dependent endosomal salvage from degradation, mediated by the neonatal Fc receptor (FcRn). Using yeast display, we identified human serum albumin (HSA) variants with increased affinity for human FcRn at endosomal pH, enabling us to solve the crystal structure of a variant HSA/FcRn complex. We find an extensive, primarily hydrophobic interface stabilized by hydrogen-bonding networks involving protonated histidines internal to each protein. The interface features two key FcRn tryptophan side chains inserting into deep hydrophobic pockets on HSA that overlap albumin ligand binding sites. We find that fatty acids (FAs) compete with FcRn, revealing a clash between ligand binding and recycling, and that our high-affinity HSA variants have significantly increased circulating half-lives in mice and monkeys. These observations open the way for the creation of biotherapeutics with significantly improved pharmacokinetics.

Practical theoretic guidance for the design of tumor-targeting agents.

Theoretical analyses of targeting agent pharmacokinetics provides specific guidance with respect to desirable design objectives such as agent size, affinity, and target antigen. These analyses suggest that IgG-sized macromolecular constructs exhibit the most favorable balance between systemic clearance and vascular extravasation, resulting in maximal tumor uptake. Quantitative predictions of the effects of dose and binding affinity on tumor uptake and penetration are also provided. The single bolus dose required for saturation of xenografted tumors in mice can be predicted from knowledge of antigen expression level and metabolic half-life. The role of high binding affinity in tumor uptake can be summarized as: essential for small peptides, less important for antibodies, and negligible for nanoparticles.

A modeling analysis of the effects of molecular size and binding affinity on tumor targeting.

A diverse array of tumor targeting agents ranging in size from peptides to nanoparticles is currently under development for applications in cancer imaging and therapy. However, it remains largely unclear how size differences among these molecules influence their targeting properties. Here, we develop a simple, mechanistic model that can be used to understand and predict the complex interplay between molecular size, affinity, and tumor uptake. Empirical relationships between molecular radius and capillary permeability, interstitial diffusivity, available volume fraction, and plasma clearance were obtained using data in the literature. These relationships were incorporated into a compartmental model of tumor targeting using MATLAB to predict the magnitude, specificity, time dependence, and affinity dependence of tumor uptake for molecules across a broad size spectrum. In the typical size range for proteins, the model uncovers a complex trend in which intermediate-sized targeting agents (MW, approximately 25 kDa) have the lowest tumor uptake, whereas higher tumor uptake levels are achieved by smaller and larger agents. Small peptides accumulate rapidly in the tumor but require high affinity to be retained, whereas larger proteins can achieve similar retention with >100-fold weaker binding. For molecules in the size range of liposomes, the model predicts that antigen targeting will not significantly increase tumor uptake relative to untargeted molecules. All model predictions are shown to be consistent with experimental observations from published targeting studies. The results and techniques have implications for drug development, imaging, and therapeutic dosing.

Antibody tumor penetration: transport opposed by systemic and antigen-mediated clearance.

Antibodies have proven to be effective agents in cancer imaging and therapy. One of the major challenges still facing the field is the heterogeneous distribution of these agents in tumors when administered systemically. Large regions of untargeted cells can therefore escape therapy and potentially select for more resistant cells. We present here a summary of theoretical and experimental approaches to analyze and improve antibody penetration in tumor tissue.

Zebrafish brd2a and brd2b are paralogous members of the bromodomain-ET (BET) family of transcriptional coregulators that show structural and expression divergence.

BACKGROUND: Brd2 belongs to the bromodomain-extraterminal domain (BET) family of transcriptional co-regulators, and functions as a pivotal histone-directed recruitment scaffold in chromatin modification complexes affecting signal-dependent transcription. Brd2 facilitates expression of genes promoting proliferation and is implicated in apoptosis and in egg maturation and meiotic competence in mammals; it is also a susceptibility gene for juvenile myoclonic epilepsy (JME) in humans. The brd2 ortholog in Drosophila is a maternal effect, embryonic lethal gene that regulates several homeotic loci, including Ultrabithorax. Despite its importance, there are few systematic studies of Brd2 developmental expression in any organism. To help elucidate both conserved and novel gene functions, we cloned and characterized expression of brd2 cDNAs in zebrafish, a vertebrate system useful for genetic analysis of development and disease, and for study of the evolution of gene families and functional diversity in chordates. RESULTS: We identify cDNAs representing two paralogous brd2 loci in zebrafish, brd2a on chromosome 19 and brd2b on chromosome 16. By sequence similarity, syntenic and phylogenetic analyses, we present evidence for structural divergence of brd2 after gene duplication in fishes. brd2 paralogs show potential for modular domain combinations, and exhibit distinct RNA expression patterns throughout development. RNA in situ hybridizations in oocytes and embryos implicate brd2a and brd2b as maternal effect genes involved in egg polarity and egg to embryo transition, and as zygotic genes important for development of the vertebrate nervous system and for morphogenesis and differentiation of the digestive tract. Patterns of brd2 developmental expression in zebrafish are consistent with its proposed role in Homeobox gene regulation. CONCLUSION: Expression profiles of zebrafish brd2 paralogs support a role in vertebrate developmental patterning and morphogenesis. Our study uncovers both maternal and zygotic contributions of brd2, the analysis of which may provide insight into the earliest events in vertebrate development, and the etiology of some forms of epilepsy, for which zebrafish is an important model. Knockdowns of brd2 paralogs in zebrafish may now test proposed function and interaction with homeotic loci in vertebrates, and help reveal the extent to which functional novelty or partitioning has occurred after gene duplication.

Kinetics of anti-carcinoembryonic antigen antibody internalization: effects of affinity, bivalency, and stability.

Theoretical analyses suggest that the cellular internalization and catabolism of bound antibodies contribute significantly to poor penetration into tumors. Here we quantitatively assess the internalization of antibodies and antibody fragments against the commonly targeted antigen carcinoembryonic antigen (CEA). Although CEA is often referred to as a non-internalizing or shed antigen, anti-CEA antibodies and antibody fragments are shown to be slowly endocytosed by LS174T cells with a half-time of 10-16 h, a time scale consistent with the metabolic turnover rate of CEA in the absence of antibody. Anti-CEA single chain variable fragments (scFvs) with significant differences in affinity, stability against protease digestion, and valency exhibit similar uptake rates of bound antibody. In contrast, one anti-CEA IgG exhibits unique binding and trafficking properties with twice as many molecules bound per cell at saturation and significantly faster cellular internalization after binding. The internalization rates measured herein can be used in simple computational models to predict the microdistribution of these antibodies in tumor spheroids.

Monovalent, reduced-size quantum dots for imaging receptors on living cells.

We describe a method to generate monovalent quantum dots (QDs) using agarose gel electrophoresis. We passivated QDs with a carboxy-terminated polyethylene-glycol ligand, yielding particles with half the diameter of commercial QDs, which we conjugated to a single copy of a high-affinity targeting moiety (monovalent streptavidin or antibody to carcinoembryonic antigen) to label cell-surface proteins. The small size improved access of QD-labeled glutamate receptors to neuronal synapses, and monovalency prevented EphA3 tyrosine kinase activation.

A33 antigen displays persistent surface expression.

The A33 antigen is a cell surface glycoprotein of the small intestine and colonic epithelium with homology to tight junction-associated proteins of the immunoglobulin superfamily, including CAR and JAM. Its restricted tissue localization and high level of expression have led to its use as a target in colon cancer immunotherapy. Although the antigen is also present in normal intestine, radiolabeled antibodies against A33 are selectively retained by tumors in the gut as well as in metastatic lesions for as long as 6 weeks. Accordingly, we have studied the trafficking and kinetic properties of the antigen to determine its promise in two-step, pretargeted therapies. The localization, mobility, and persistence of the antigen were investigated, and this work has demonstrated that the antigen is both highly immobile and extremely persistent-retaining its surface localization for a turnover halflife of greater than 2 days. In order to explain these unusual properties, we explored the possibility that A33 is a component of the tight junction. The simple property of surface persistence, described here, may contribute to the prolonged retention of the clinically administered antibodies, and their uncommon ability to penetrate solid tumors.

Factors determining antibody distribution in tumors.

The development of antibody therapies for cancer is increasing rapidly, primarily owing to their specificity. Antibody distribution in tumors is often extremely uneven, however, leading to some malignant cells being exposed to saturating concentrations of antibody, whereas others are completely untargeted. This is detrimental because large regions of cells escape therapy, whereas other regions might be exposed to suboptimal concentrations that promote a selection of resistant mutants. The distribution of antibody depends on a variety of factors, including dose, affinity, antigens per cell and molecular size. Because these parameters are often known or easily estimated, a quick calculation based on simple modeling considerations can predict the uniformity of targeting within a tumor. Such analyses should enable experimental researchers to identify in a straightforward way the limitations in achieving evenly distributed antibody, and design and test improved antibody therapeutics more rationally.