Correlation of In Vitro Kinetic Stability to Preclinical In Vivo Pharmacokinetics for a Panel of Anti-PD-1 Monoclonal Antibody Interleukin 21 Mutein Immunocytokines.

The development of therapeutic fusion protein drugs is often impeded by the unintended consequences that occur from fusing together domains from independent naturally occurring proteins, consequences such as altered biodistribution, tissue uptake, or rapid clearance and potential immunogenicity. For therapeutic fusion proteins containing globular domains, we hypothesized that aberrant in vivo behavior could be related to low kinetic stability of these domains leading to local unfolding and susceptibility to partial proteolysis and/or salvage and uptake. Herein we describe an assay to measure kinetic stability of therapeutic fusion proteins by way of their sensitivity to the protease thermolysin. The results indicate that in vivo pharmacokinetics of a panel of anti-programmed cell death protein 1 monocolonal antibody:interleukin 21 immunocytokines in both mice and nonhuman primates are highly correlated with their in vitro susceptibility to thermolysin-mediated proteolysis. This assay can be used as a tool to quickly identify in vivo liabilities of globular domains of therapeutic proteins, thus aiding in the optimization and development of new multispecific drug candidates. SIGNIFICANCE STATEMENT: This work describes a novel assay utilizing protein kinetic stability to identify preclinical in vivo pharmacokinetic liabilities of multispecific therapeutic fusion proteins. This provides an efficient, inexpensive method to ascertain inherent protein stability in vitro before conducting in vivo studies, which can rapidly increase the speed of preclinical drug development.

The biodistribution of therapeutic proteins: Mechanism, implications for pharmacokinetics, and methods of evaluation.

Therapeutic proteins (TPs) are a diverse drug class that include monoclonal antibodies (mAbs), recombinantly expressed enzymes, hormones and growth factors, cytokines (e.g. chemokines, interleukins, interferons), as well as a wide range of engineered fusion scaffolds containing IgG1 Fc domain for half-life extension. As the pharmaceutical industry advances more potent and selective protein-based medicines through discovery and into the clinical stages of development, it has become widely appreciated that a comprehensive understanding of the mechanisms of TP biodistribution can aid this endeavor. This review aims to highlight the literature that has advanced our understanding of the determinants of TP biodistribution. A particular emphasis is placed on the multi-faceted role of the neonatal Fc receptor (FcRn) in mAb and Fc-fusion protein disposition. In addition, characterization of the TP-target interaction at the cell-level is discussed as an essential strategy to establish pharmacokinetic-pharmacodynamic (PK/PD) relationships that may lead to more informed human dose projections during clinical development. Methods for incorporation of tissue and cell-level parameters defining these characteristics into higher-order mechanistic and semi-mechanistic PK models will also be presented.

Targeting Phosphatidylserine with Calcium-Dependent Protein-Drug Conjugates for the Treatment of Cancer.

In response to cellular stress, phosphatidylserine is exposed on the outer membrane leaflet of tumor blood vessels and cancer cells, motivating the development of phosphatidylserine-specific therapies. The generation of drug-conjugated phosphatidylserine-targeting agents represents an unexplored therapeutic approach, for which antitumor effects are critically dependent on efficient internalization and lysosomal delivery of the cytotoxic drug. In the current study, we have generated phosphatidylserine-targeting agents by fusing phosphatidylserine-binding domains to a human IgG1-derived Fc fragment. The tumor localization and pharmacokinetics of several phosphatidylserine-specific Fc fusions have been analyzed in mice and demonstrate that Fc-Syt1, a fusion containing the synaptotagmin 1 C2A domain, effectively targets tumor tissue. Conjugation of Fc-Syt1 to the cytotoxic drug monomethyl auristatin E results in a protein-drug conjugate (PDC) that is internalized into target cells and, due to the Ca2+ dependence of phosphatidylserine binding, dissociates from phosphatidylserine in early endosomes. The released PDC is efficiently delivered to lysosomes and has potent antitumor effects in mouse xenograft tumor models. Interestingly, although an engineered, tetravalent Fc-Syt1 fusion shows increased binding to target cells, this higher avidity variant demonstrates reduced persistence and therapeutic effects compared with bivalent Fc-Syt1. Collectively, these studies show that finely tuned, Ca2+-switched phosphatidylserine-targeting agents can be therapeutically efficacious. Mol Cancer Ther; 17(1); 169-82. ©2017 AACR.

Myelin oligodendrocyte glycoprotein-specific antibodies from multiple sclerosis patients exacerbate disease in a humanized mouse model.

Myelin oligodendrocyte glycoprotein (MOG) is exposed on the outer surface of the myelin sheath, and as such, represents a possible target antigen for antibodies in multiple sclerosis (MS) and other demyelinating diseases. However, despite extensive analyses, whether MOG-specific antibodies contribute to pathogenesis in human MS remains an area of uncertainty. In the current study we demonstrate that antibodies derived from adult MS patients exacerbate experimental autoimmune encephalomyelitis (EAE) in 'humanized' mice that transgenically express human FcγRs (hFcγRs). Importantly, this exacerbation is dependent on MOG recognition by the human-derived antibodies. The use of mice that express hFcγRs has allowed us to also investigate the contribution of these receptors to disease in the absence of confounding effects of cross-species differences. Specifically, by engineering the Fc region of MOG-specific antibodies to modulate FcγR and complement (C1q) binding, we reveal that FcγRs but not complement activation contribute to EAE pathogenesis. Importantly, selective enhancement of the affinities of these antibodies for specific FcγRs reveals that FcγRIIA is more important than FcγRIIIA in mediating disease exacerbation. These studies not only provide definitive evidence for the contribution of MOG-specific antibodies to MS, but also reveal mechanistic insight that could lead to new therapeutic targets.

Engineered clearing agents for the selective depletion of antigen-specific antibodies.

Here we have designed a novel class of engineered antibody-based reagents ('Seldegs') that induce the selective degradation of antigen-specific antibodies. We demonstrate the rapid and specific clearance of antibodies recognizing the autoantigen, myelin oligodendrocyte glycoprotein and tumour target, HER2. Seldegs have considerable potential in multiple areas, including the treatment of antibody-mediated autoimmunity and diagnostic imaging.

Targeting FcRn for the modulation of antibody dynamics.

The MHC class I-related receptor, FcRn, is a multitasking protein that transports its IgG ligand within and across cells of diverse origins. The role of this receptor as a global regulator of IgG homeostasis and transport, combined with knowledge of the molecular details of FcRn-IgG interactions, has led to opportunities to modulate the in vivo dynamics of antibodies and their antigens through protein engineering. Consequently, the generation of half-life extended antibodies has shown a rapid expansion over the past decade. Further, FcRn itself can be targeted by inhibitors to induce decreased levels of circulating IgGs, which could have applications in multiple clinical settings. The engineering of antibody-antigen interactions to reduce antibody-mediated buffering of soluble ligand has also developed into an active area of investigation, leading to novel antibody platforms designed to result in more effective antigen clearance. Similarly, the target-mediated elimination of antibodies by internalizing, membrane bound antigens (receptors) can be decreased using novel engineering approaches. These strategies, combined with subcellular trafficking analyses of antibody/antigen/FcRn behavior in cells to predict in vivo behavior, have considerable promise for the production of next generation therapeutics and diagnostics.

The effect of pH dependence of antibody-antigen interactions on subcellular trafficking dynamics.

A drawback of targeting soluble antigens such as cytokines or toxins with long-lived antibodies is that such antibodies can prolong the half-life of the target antigen by a "buffering" effect. This has motivated the design of antibodies that bind to target with higher affinity at near neutral pH relative to acidic endosomal pH (~pH 6.0). Such antibodies are expected to release antigen within endosomes following uptake into cells, whereas antibody will be recycled and exocytosed in FcRn-expressing cells. To understand how the pH dependence of antibody-antigen interactions affects intracellular trafficking, we generated three antibodies that bind IL-6 with different pH dependencies in the range pH 6.0-7.4. The behavior of antigen in the presence of these antibodies has been characterized using a combination of fixed and live cell fluorescence microscopy. As the affinity of the antibody:IL-6 interaction at pH 6.0 decreases, an increasing amount of antigen dissociates from FcRn-bound antibody in early and late endosomes, and then enters lysosomes. Segregation of antibody and FcRn from endosomes in tubulovesicular transport carriers (TCs) into the recycling pathway can also be observed in live cells, and the extent of IL-6 association with TCs correlates with increasing affinity of the antibody:IL-6 interaction at acidic pH. These analyses result in an understanding, in spatiotemporal terms, of the effect of pH dependence of antibody-antigen interactions on subcellular trafficking and inform the design of antibodies with optimized binding properties for antigen elimination.

Prediction of IgE-binding epitopes by means of allergen surface comparison and correlation to cross-reactivity.

BACKGROUND: The experimental determination of conformational allergen epitopes recognized by IgE is a difficult task because they often involve discontinuous amino acid residues, being separated in the primary allergen sequence, and require the correct allergen fold. OBJECTIVE: We sought to develop a computational tool for the localization of conformational IgE epitopes by using a structure-based comparison of allergen surfaces and IgE cross-reactivity data. METHODS: Our approach involves the quantitative analysis of geometric and physicochemical surface parameters and the subsequent correlation of surface similarity scores to immunologic data. The software tool Surface comparison-based Prediction of Allergenic Discontinuous Epitopes (SPADE) is able to predict the IgE epitopes of an allergen given the availability of at least 2 structural models and IgE reactivity data. RESULTS: We report on the application of our tool to 3 allergen families: the lipocalins, the group 10 pathogenesis-related proteins, and the group 2/3 grass pollen allergens. First, we succeeded in the partial relocalization of IgE epitopes of bovine ß-lactoglobulin and grass pollen Phl p 2 as known from the x-ray structures of their antibody complexes. Second, we measured the relative binding of anti-Bet v 1 IgE to 10 homologous proteins and correlated these data to surface comparison results involving Bet v 1, 5 of the homologs, and 2 hypoallergenic Bet v 1 isoforms. Thereby we predicted IgE-reactive surface portions in agreement with IgE epitope-mapping studies. CONCLUSION: Our approach is the first for the prediction of IgE epitopes by combining structural and IgE cross-reactivity data. It should be useful for the development of point-mutated or structurally disrupted allergen derivatives for allergen-specific immunotherapy.

Biophysical characterization of recombinant HIV-1 subtype C virus infectivity factor.

HIV-1 virus infectivity factor (Vif) is one of the four accessory proteins that are characteristic of primate lentiviruses and critically required for the infection of host cells. Vif plays a key role in replication and transmission of the virus in non-permissive cells, such as primary T cells and macrophages. Using co-precipitation and co-fractionation techniques, evidence has been provided that Vif interacts with a variety of host proteins, such as the cytidine deaminases APOBEC3G and 3F, the Cullin5/EloBC ubiquitin-ligase complex, Fyn and Hck tyrosine kinases, as well as with viral components, such as the immature Gag precursor and viral RNA. We report on the expression, purification and molecular characterization of a folded recombinant subtype C Vif. Vif was expressed in E. coli with a C-terminal hexahistidine tag and purified by nickel affinity chromatography. We obtained approximately 5 mg protein per liter of bacterial culture, with a purity >95%. The expected molecular mass of 23.7 kDa was confirmed by mass spectrometry. Although dynamic light scattering and small angle X-ray scattering measurements revealed the presence of high molecular weight aggregates in the protein preparation, circular dichroism analysis showed that the protein contains mainly folded ß-sheet elements and exhibits remarkable thermal stability (T (m) > 95°C). Recombinant Vif may be used as a tool to study its biological functions and tertiary structure, as well as for the development of diagnostic, therapeutic and preventive strategies for HIV-1 infections.