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Therapeutic proteins provide interventions for some of the most complex and intractable diseases and are an essential part of modern medicine. Immunogenicity is the development of immune responses, usually measured by antibodies, to therapeutic proteins. These responses can adversely affect the safety and efficacy of the therapeutic agent and may have the following consequences: neutralization of a life-saving biotherapeutic, crossreactivity to non-redundant endogenous proteins, and hypersensitivity responses. These concerns have been underscored by the discontinuation of development of several drugs in recent years owing to immunogenicity issues. We review here recent progress in technological approaches that are useful for the clinical and non-clinical risk assessment of immunogenicity as well as mitigation strategies including deimmunizing protein molecules and inducing immune tolerance to the therapeutic protein. Highlights Immunogenicity compromises the safety and/or efficacy of therapeutic proteins, is a priority for regulatory agencies, and adds to the risks and costs associated with drug development. In the clinical setting, the risks and consequences of anti-drug antibodies translate into complex decision-making, and reliable immunogenicity testing strategies are therefore needed to inform clinical decisions. A multitude of non-clinical approaches (computational, in vitro , ex vivo , and animal models) are currently used to assess immunogenicity risk. Newly emerging technologies are expected to contribute to the rapid advancement of non-clinical assessments and have the potential to reduce the burden of immunogenicity. Novel strategies applied to protein therapeutic deimmunization or immune tolerance induction have the potential to reduce the incidence of immunogenicity and aid in better clinical management. [ABSTRACT FROM AUTHOR]

The yeast Pdr5 multidrug transporter is an important member of the ATP-binding cassette superfamily of proteins. We describe a novel mutation (S558Y) in transmembrane helix 2 of Pdr5 identified in a screen for suppressors that eliminated Pdr5-mediated cycloheximide hyper-resistance. Nucleotides as well as transport substrates bind to the mutant Pdr5 with an affinity comparable with that for wild-type Pdr5. Wild-type and mutant Pdr5s show ATPase activity with comparable Km(ATP) values. Nonetheless, drug sensitivity is equivalent in the mutant pdr5 and the pdr5 deletion. Finally, the transport substrate clotrimazole, which is a non-competitive inhibitor of Pdr5 ATPase activity, has a minimal effect on ATP hydrolysis by the S558Y mutant. These results suggest that the drug sensitivity of the mutant Pdr5 is attributable to the uncoupling of NTPase activity and transport. We screened for amino acid alterations in the nucleotide-binding domains that would reverse the phenotypic effect of the S558Y mutation. A second-site mutation, N242K, located between the Walker A and signature motifs of the N-terminal nucleotide-binding domain, restores significant function. This region of the nucleotide-binding domain interacts with the transmembrane domains via the intracellular loop-1 (which connects transmembrane helices 2 and 3) in the crystal structure of Sav1866, a bacterial ATP-binding cassette drug transporter. These structural studies are supported by biochemical and genetic evidence presented here that interactions between transmembrane helix 2 and the nucleotide-binding domain, via the intracellular loop-1, may define at least part of the translocation pathway for coupling ATP hydrolysis to drug transport. [ABSTRACT FROM AUTHOR]

The transport cycle of ABC transporters in general and P-glycoprotein in particular has been extensively studied, but the molecular mechanism remains controversial. We identify stable reaction intermediates in the progression of the P-glycoprotein-mediated ATPase reaction equivalent to the enzyme-substrate (E·S, P-glycoprotein·ATP) and enzyme-product (E·P, P-glycoprotein· ADP·Pi) reaction intermediates. These have been characterized using the photoaffinity analog 8-azido-[α-32P]ATP as well as under equilibrium conditions using [α-32P]ATP, in which a cross-linking step is not involved. Similar results were obtained when 8-azido-[α-32p]ATP or [α-32P]ATP was used. The reaction intermediates were characterized based on their kinetic properties and the nature (triphosphate/diphosphate) of the trapped nucleotide. Using this defined framework and the Walker B E556Q/E1201Q mutant that traps nucleotide in the absence of vanadate or beryllium fluoride, the high to low affinity switch in the transport substrate binding site can be attributed to the formation of the E·S reaction intermediate of the ATPase reaction. Importantly, the posthydrolysis E·P state continues to have low affinity for substrate, suggesting that conformational changes that form the E·S complex are coupled to the conformational change at the transport substrate site to do mechanical work. Thus, the formation of E·S reaction intermediate during a single turnover of the catalytic cycle appears to provide the initial power stroke for movement of drug substrate from inner leaflet to outer leaflet of lipid bilayer. This novel approach applies transition state theory to elucidate the mechanism of P-glycoprotein and other ABC transporters and has wider applications in testing cause-effect hypotheses in coupled systems. [ABSTRACT FROM AUTHOR]

Multidrug resistance protein 4 (MRP4/ABCC4), transports cyclic nucleoside monophosphates, nucleoside analog drugs, chemotherapeutic agents, and prostaglandins. In this study we characterize ATP hydrolysis by human MRP4 expressed in insect cells. MRP4 hydrolyzes ATP (Km, 0.62 mM), which is inhibited by orthovanadate and beryllium fluoride. However, unlike ATPase activity of P-glycoprotein, which is equally sensitive to both inhibitors, MRP4-ATPase is more sensitive to beryllium fluoride than to orthovanadate. 8-Azido[α-32p]ATP binds to MRP4 (concentration for half-maximal binding ∼3 μM) nnd is displaced by ATP or by its nonhydrolyzable analog AMPPNP (concentrations for half-maximal inhibition of 13.3 and 308 μM). MRP4 substrates, the prostaglandins E1 and E2, stimulate ATP hydrolysis 2- to 3-fold but do not affect the Km for ATP. Several other substrates, azidothymidine, 9-(2-phosphonylmethoxyethyl)adenine, and methotrexate do not stimulate ATP hydrolysis but inhibit prostaglandin E2-stimulated ATP hydrolysis. Although both post-hydrolysis transition states MRP4·8-azido[α-s2p]ADP·Vi and MRP4·8-azido[α-s2p]ADP·beryllium fluoride can be generated, nucleotide trapping is ∼4-fold higher with beryllium fluoride. The divalent cations Mg2+ and Mn2+ support comparable levels of nucleotide binding, hydrolysis, and trapping. However, Co2+ increases 8-azido[α-32p]ATP binding and beryllium fluoride-induced 8-azido[α-32p]ADP trapping but does not support steady-state ATP hydrolysis. ADP inhibits basal and prostaglandin E2-stimulated ATP hydrolysis (concentrations for half-maximal inhibition 0.19 and 0.25 mM, respectively) and beryllium fluoride-induced 8-azido[α-32p]ADP trapping, whereas Pi has no effect up to 20 mM. In aggregate, our results demonstrate that MRP4 exhibits substrate-stimulated ATP hydrolysis, and we propose a kinetic scheme suggesting that ADP release from the post-hydrolysis transition state may be the rate-limiting step during the catalytic cycle. [ABSTRACT FROM AUTHOR]

Intercellular transfer of proteins is a mode of communication between cells that is crucial for certain physiological processes. Chemotherapy is the treatment of choice for ∼50% of all cancers. However, multidrug resistance mediated by drug-efflux pumps such as P-glycoprotein (Pgp) minimizes the effectiveness of such therapy in a large number of patients. A new study demonstrates the functional intercellular transfer of Pgp. Non-genetic transfer of the multidrug resistance phenotype raises fascinating questions about the mechanism and regulation of cell-surface membrane-protein-mediated spread of traits. [Copyright &y& Elsevier]

Recombinant therapeutic proteins are a broad class of biological products used to replace dysfunctional human proteins in individuals with genetic defects (e.g., factor VIII for hemophilia) or, in the case of monoclonal antibodies, bind to disease targets involved in cancers, autoimmune disorders, or other conditions. Unfortunately, immunogenicity (immune response to the drug) remains a key impediment, potentially affecting the safety and efficacy of these therapeutics. Immunogenicity risk is routinely evaluated during the licensure of therapeutic proteins. However, despite eliciting anti-drug immune responses in at least some patients, most protein drugs are nevertheless licensed as they address unmet medical needs. The pre-licensure immunogenicity assessments of therapeutic proteins are the subject of numerous reviews and white papers. However, observation and clinical management of the immunogenicity of approved therapeutic proteins face additional challenges. We survey the immunogenicity of approved therapeutic proteins, discuss the clinical management of immunogenicity, and identify the challenges to establishing clinically relevant immunogenicity assays for use in routine clinical practice. Immunogenicity can compromise the safety and/or efficacy of therapeutic protein products, and is a priority issue for regulatory agencies. In addition to poor patient outcomes, the social and economic costs associated with neutralizing antibodies are considerable. Reliable assays for monitoring anti-drug antibodies (ADAs) and neutralizing antibodies post-licensure are lacking for most therapeutic proteins. [ABSTRACT FROM AUTHOR]

Inducing immune tolerance to proteins used in therapeutic applications is needed for these medications to meet their full potential. There has been a surge in new approaches to induce immune tolerance to therapeutic proteins; however, challenges remain in applying these to the clinic. Effective immune tolerance is necessary because many therapeutic proteins are no longer safe and/or effective due to immunogenicity. Immunogenicity affects the safety and efficacy of therapeutic proteins. This review is focused on approaches for inducing immunological tolerance to circumvent the immunogenicity of therapeutic proteins in the clinic. The few immune tolerance strategies that are used in the clinic tend to be inefficient and expensive and typically involve global immunosuppression, putting patients at risk of infections. The hallmark of a desirable immune tolerance regimen is the specific alleviation of immune responses to the therapeutic protein. In the past decade, proof-of-principle studies have demonstrated that emerging technologies, including nanoparticle-based delivery of immunomodulators, cellular targeting and depletion, cellular engineering, gene therapy, and gene editing, can be leveraged to promote tolerance to therapeutic proteins. We discuss the potential of these novel approaches and the barriers that need to be overcome for translation into the clinic. Immunogenicity affects the safety and efficacy of therapeutic proteins. This review is focused on approaches for inducing immunological tolerance to circumvent the immunogenicity of therapeutic proteins in the clinic. The few immune tolerance strategies that are used in the clinic tend to be inefficient and expensive and typically involve global immunosuppression, putting patients at risk of infections. The hallmark of a desirable immune tolerance regimen is the specific alleviation of immune responses to the therapeutic protein. In the last decade, proof-of-principle studies have demonstrated that emerging technologies, including nanoparticle-based delivery of immunomodulators, cellular targeting and depletion, cellular engineering, gene therapy, and gene editing, can be leveraged to promote tolerance to therapeutic proteins. We discuss the potential of these novel approaches and the barriers that need to be overcome for translation into the clinic. [ABSTRACT FROM AUTHOR]

Abstract: Members of the superfamily of ATP-binding cassette (ABC) transporters mediate the movement of a variety of substrates including simple ions, complex lipids and xenobiotics. At least 18 ABC transport proteins are associated with disease conditions. P-glycoprotein (Pgp, ABCB1) is the archetypical mammalian ABC transport protein and its mechanism of action has received considerable attention. There is strong biochemical evidence that Pgp moves molecular cargo against a concentration gradient using the energy of ATP hydrolysis. However, the molecular details of how the energy of ATP hydrolysis is coupled to transport remain in dispute and it has not been possible to reconcile the data from various laboratories into a single model. The functional unit of Pgp consists of two nucleotide binding domains (NBDs) and two trans-membrane domains which are involved in the transport of drug substrates. Considerable progress has been made in recent years in characterizing these functionally and spatially distinct domains of Pgp. In addition, our understanding of the domains has been augmented by the resolution of structures of several non-mammalian ABC proteins. This review considers: (i) the role of specific conserved amino acids in ATP hydrolysis mediated by Pgp; (ii) emerging insights into the dimensions of the drug binding pocket and the interactions between Pgp and the transport substrates and (iii) our current understanding of the mechanisms of coupling between energy derived from ATP binding and/or hydrolysis and efflux of drug substrates. [Copyright &y& Elsevier]

The platform technology of fragment crystallizable (Fc) fusion, in which the Fc region of an antibody is genetically linked to an active protein drug, is among the most successful of a new generation of bioengineering strategies. Immunogenicity is a critical safety concern in the development of any protein therapeutic. While the therapeutic goal of generating Fc-fusion proteins has been to extend half-life, there is a critical mass of literature from immunology indicating that appropriate design of the Fc component has the potential to engage the immune system for product-specific outcomes. In the context of Fc-fusion therapeutics, a review of progress in understanding Fc biology suggests the prospect of engineering products that have an extended half-life and are able to modulate the immune system. [ABSTRACT FROM AUTHOR]

Highlights: [•] Synonymous mutations, which do not result in a change in amino acid sequence, have been considered biologically silent. [•] Studies in molecular evolution show that synonymous codons are under selective pressure. [•] Synonymous mutation may affect the function of translated protein through diverse cellular mechanisms. [•] Multiple methods should be applied to investigate synonymous variants and decipher the exact mechanisms by which they impact biological function. [ABSTRACT FROM AUTHOR]

Highlights: [•] Most native proteins do not make for good drugs. [•] New generations of engineered therapeutic proteins are rapidly becoming the norm. [•] Advantages of engineered therapeutic proteins can be considerable. [•] Engineering proteins can also affect the safety and efficacy of drugs. [ABSTRACT FROM AUTHOR]

The protease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat) cleaves multimers of von Willebrand factor, thus regulating platelet aggregation. ADAMTS13 deficiency leads to the fatal disorder thrombotic thrombocytopenic purpura (TTP). It has been observed that cyclosporin A (CsA) treatment, particularly in transplant patients, may sometimes be linked to the development of TTP. Until now, the reason for such a link was unclear. Here we provide evidence demonstrating that cyclophilin B (CypB) activity plays an important role in the secretion of active ADAMTS13. We found that CsA, an inhibitor of CypB, reduces the secretion of ADAMTS13 and leads to conformational changes in the protein resulting in diminished ADAMTS13 proteolytic activity. A direct, functional interaction between CypB (which possesses peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone functions) and ADAMTS13 is demonstrated using immunoprecipitation and siRNA knockdown of CypB. Finally, CypB knockout mice were found to have reduced ADAMTS13 levels. Taken together, our findings indicate that cyclophilin-mediated activity is an important factor affecting secretion and activity of ADAMTS13. The large number of proline residues in ADAMTS13 is consistent with the important role of cis-trans isomerization in the proper folding of this protein. These results altogether provide a novel mechanistic explanation for CsA-induced TTP in transplant patients. [ABSTRACT FROM AUTHOR]

The arsRDABC operon of Escherichia coli plasmid R773 encodes the ArsAB pump that catalyzes extrusion of the metalloids As(III) and Sb(III), conferring metalloid resistance. The catalytic subunit, ArsA, is an ATPase with two homologous halves, A1 and A2, connected by a short linker. Each half contains a nucleotide binding domain. The overall rate of ATP hydrolysis is slow in the absence of metalloid and is accelerated by metalloid binding. The results of photolabeling of ArsA with the ATP analogue 8-azidoadenosine 5′-[α-32 P]-triphosphate at 4 °C indicate that metalloid stimulation correlates with a > 10-fold increase in affinity for nucleotide. To investigate the relative contributions of the two nucleotide binding domains to catalysis, a thrombin site was introduced in the linker. This allowed discrimination between incorporation of labeled nucleotides into the two halves of ArsA. The results indicate that both the A1 and A2 nucleotide binding domains bind and hydrolyze trinucleotide, even in the absence of metalloid. Sb(III) increases the affinity of the A1 nucleotide binding domain to a greater extent than the A2 nucleotide binding domain. The ATP analogue labeled with 32P at the γ position was used to measure hydrolysis of trinucleotide at 37°C. Under these catalytic conditions, both nucleotide binding domains hydrolyze ATP, but hydrolysis in A1 is stimulated to a greater degree by Sb(III) than A2. These results suggest that the two homologous halves of the ArsA may be functionally nonequivalent. [ABSTRACT FROM AUTHOR]