Impact of site-specific conjugation strategies on the pharmacokinetics of antibody conjugated radiotherapeutics.

Antibody radionuclide conjugates are an emerging modality for targeted imaging and potent therapy of disseminated disease. Coupling of radionuclides to monoclonal antibodies (mAbs) is typically achieved by applying non-site-specific labelling techniques. With the ambition of reducing variability, increasing labelling efficacy and stability, several site-specific conjugation strategies have been developed in recent years for toxin- and fluorophore-mAb conjugates. In this study, we studied two site-specific labelling strategies for the conjugation of the macrocyclic chelating agent, DOTA, to the anti-Leucine Rich Repeat Containing 15 (LRRC15) mAb DUNP19. Specifically, one approach utilized a DOTA-bearing peptide (FcIII) with a strong affinity for the fragment crystallizable (Fc) domain of the human IgG1 of DUNP19 (DUNP19LF-FcIII-DOTASS), while the other leveraged a chemo-enzymatic technique to substitute the N-linked bi-antennary oligosaccharides in the human IgG1 Fc domain with DOTA (DUNP19LF-gly-DOTASS). To assess if these methods impact the antibody's binding properties and targeting efficacy, comparative in vitro and in vivo studies of the generated DUNP19-conjugates were performed. While the LRRC15 binding of both radioimmunoconjugates remained intact, the conjugation methods had different impacts on their abilities to interact with FcRn and FcγRs. In vitro assessments of DUNP19LF-FcIII-DOTASS and DUNP19LF-gly-DOTASS demonstrated markedly decreased affinity for FcRn and FcγRIIIa (CD16), respectively. DUNP19LF-FcIII-DOTASS demonstrated increased blood and tissue kinetics in vivo, confirming loss of FcRn binding. While the ablated FcγR interaction of DUNP19LF-gly-DOTASS had no immediate impact on in vivo biodistribution, reduced immunotherapeutic effect can be expected in future studies as a result of reduced NK-cells interaction. In conclusion, our findings underscore the necessity for meticulous consideration and evaluation of mAb labelling strategies, extending beyond mere conjugation efficiency and radiolabeling yields. Notably, site-specific labelling methods were found to significantly influence the immunological impact of Fc interactions. Therefore, it is of paramount importance to consider the intended diagnostic or therapeutic application of the construct and to adopt conjugation strategies that ensure the preservation of critical pharmacological properties and functionality of the antibody in use.

A Nitroreductase-activatable Metabolic Reporter for Covalent Labeling of Pathological Hypoxic Cells in Tumorigenesis.

Aberrant hypoxic stress will initiate a cascade of pathological consequence observed prominently in tumorigenesis. Understanding of hypoxia's role in tumorigenesis is  highly essential for developing effective therapeutics, which necessitates reliable tools to specifically distinguish hypoxic tumor cells (or tissues) and correlate their dynamics with the status of disease in complex living settings for precise theranostics. So far, disparate hypoxia-responsive probe molecules and prodrugs were designed via chemical or enzymatic reactions, yet their capability in real-time reporting pathogenesis development is often compromised due to unrestricted diffusion and less selectivity towards the environmental responsiveness. Herein we present an oxygen-insensitive nitroreductase (NTR)-activatable glycan metabolic reporter (pNB-ManNAz) capable of covalently labeling hypoxic tumor cells and tissues. Under pathophysiological hypoxic environments, the caged non-metabolizable precursor pNB-ManNAz exhibited unique responsiveness to cellular NTR, culminating in structural self-immolation and the resultant ManNAz could incorporate onto cell surface glycoproteins, thereby facilitating fluorescence labeling via bioorthogonal chemistry. This NTR-responsive metabolic reporter demonstrated broad applicability for multicellular hypoxia labeling, particularly in the dynamic monitoring of orthotopic tumorigenesis and targeted tumor phototherapy in vivo. We anticipate that this approach holds promise for investigating hypoxia-related pathological progression, offering valuable insights for accurate diagnosis and treatment.

Reading and Writing the Human Glycocode.

The complex carbohydrate structures decorating human proteins and lipids, also called glycans, are abundantly present at cell surfaces and in the secretome. Glycosylation is vital for biological processes including cell-cell recognition, immune responses, and signaling pathways. Therefore, the structural and functional characterization of the human glycome is gaining more and more interest in basic biochemistry research and in the context of developing new therapies, diagnostic tools, and biotechnology applications. For glycomics to reach its full potential in these fields, it is critical to appreciate the specific factors defining the function of the human glycome. Here, we review the glycosyltransferases (the writers) that form the glycome and the glycan-binding proteins (the readers) with an essential role in decoding glycan functions. While abundantly present throughout different cells and tissues, the function of specific glycosylation features is highly dependent on their context. In this review, we highlight the relevance of studying the glycome in the context of specific carrier proteins, cell types, and subcellular locations. With this, we hope to contribute to a richer understanding of the glycome and a more systematic approach to identifying the roles of glycosylation in human physiology.

Efficient Expression of Functionally Active Aflibercept with Designed N-glycans.

Aflibercept is a therapeutic recombinant fusion protein comprising extracellular domains of human vascular endothelial growth factor receptors (VEGFRs) and IgG1-Fc. It is a highly glycosylated protein with five N-glycosylation sites that might impact it structurally and/or functionally. Aflibercept is produced in mammalian cells and exhibits large glycan heterogeneity, which hampers glycan-associated investigations. Here, we report the expression of aflibercept in a plant-based system with targeted N-glycosylation profiles. Nicotiana benthamiana-based glycoengineering resulted in the production of aflibercept variants carrying designed carbohydrates, namely, N-glycans with terminal GlcNAc and sialic acid residues, herein referred to as AFLIGnGn and AFLISia, respectively. Both variants were transiently expressed in unusually high amounts (2 g/kg fresh leaf material) in leaves and properly assembled to dimers. Mass spectrometric site-specific glycosylation analyses of purified aflibercept showed the presence of two to four glycoforms in a consistent manner. We also demonstrate incomplete occupancy of some glycosites. Both AFLIGnGn and AFLISia displayed similar binding potency to VEGF165, with a tendency of lower binding to variants with increased sialylation. Collectively, we show the expression of functionally active aflibercept in significant amounts with controlled glycosylation. The results provide the basis for further studies in order to generate optimized products in the best-case scenario.

Spin-labeling Insights into How Chemical Fixation Impacts Glycan Organization on Cells.

As new methods to interrogate glycan organization on cells develop, it is important to have a molecular level understanding of how chemical fixation can impact results and interpretations. Site-directed spin labeling technologies are well suited to study how the spin label mobility is impacted by local environmental conditions, such as those imposed by cross-linking effects of paraformaldehyde cell fixation methods. Here, we utilize three different azide-containing sugars for metabolic glycan engineering with HeLa cells to incorporate azido glycans that are modified with a DBCO-based nitroxide moiety via click reaction. Continuous wave X-band electron paramagnetic resonance spectroscopy is employed to characterize how the chronological sequence of chemical fixation and spin labeling impacts the local mobility and accessibility of the nitroxide-labeled glycans in the glycocalyx of HeLa cells. Results demonstrate that chemical fixation with paraformaldehyde can alter local glycan mobility and care should be taken in the analysis of data in any study where chemical fixation and cellular labeling occur.

A genome-edited N. benthamiana line for industrial-scale production of recombinant glycoproteins with targeted N-glycosylation.

Control over glycosylation is an important quality parameter in recombinant protein production. Here, we demonstrate the generation of a marker-free genome edited Nicotiana benthamiana N-glycosylation mutant (NbXF-KO) carrying inactivated ß1,2-xylosyltransferase and α1,3-fucosyltransferase genes. The knockout of seven genes and their stable inheritance was confirmed by DNA sequencing. Mass spectrometric analyses showed the synthesis of N-glycans devoid of plant-specific ß1,2-xylose and core α 1,3-fucose on endogenous proteins and a series of recombinantly expressed glycoproteins with different complexities. Further transient glycan engineering towards more diverse human-type N-glycans resulted in the production of recombinant proteins decorated with ß1,4-galactosylated and α2,6-sialylated structures, respectively. Notably, a monoclonal antibody expressed in the NbXF-KO displayed glycosylation-dependent activities. Collectively, the engineered plants grow normally and are well suited for upscaling, thereby meeting industrial and regulatory requirements for the production of high-quality therapeutic proteins.

Spin-labeling Insights into How Chemical Fixation Impacts Glycan Organization on Cells.

As new methods to interrogate glycan organization on cells develop, it is important to have a molecular level understanding of how chemical fixation can impact results and interpretations. Site-directed spin labeling technologies are well suited to study how the spin label mobility is impacted by local environmental conditions, such as those imposed by cross-linking effects of paraformaldehyde cell fixation methods. Here, we utilize three different azide-containing sugars for metabolic glycan engineering with HeLa cells to incorporate azido glycans that are modified with a DBCO-based nitroxide moiety via click reaction. Continuous wave X-band electron paramagnetic resonance spectroscopy is employed to characterize how the chronological sequence of chemical fixation and spin labeling impacts the local mobility and accessibility of the nitroxide-labeled glycans in the glycocalyx of HeLa cells. Results demonstrate that chemical fixation with paraformaldehyde can alter local glycan mobility and care should be taken in the analysis of data in any study where chemical fixation and cellular labeling occur.

Practical Antibody Recruiting by Metabolic Labeling with Caged Glycans.

We propose a de novo glycan display approach that combines metabolic labeling and a glycan-caging strategy as a facile editing method for cell-surface glycans. This method enables the introduction of antigen glycans onto cancer cells to induce immune responses through antibody recruiting. The caging strategy prevents the capture of α-rhamnose (an antigen glycan) by endogenous antibodies during the introduction of the glycan to the targeted cell surface, and subsequent uncaging successfully induces immune responses. Therefore, this study proposes a practical method for editing the cell-surface glycocalyx under promiscuous conditions, such as those in vivo, which paves the way for the development of glycan function analysis and regulation.

Glycoprotein In Vitro N-Glycan Processing Using Enzymes Expressed in E. coli.

Protein N-glycosylation is a common post-translational modification that plays significant roles on the structure, property, and function of glycoproteins. Due to N-glycan heterogeneity of naturally occurring glycoproteins, the functions of specific N-glycans on a particular glycoprotein are not always clear. Glycoprotein in vitro N-glycan engineering using purified recombinant enzymes is an attractive strategy to produce glycoproteins with homogeneous N-glycoforms to elucidate the specific functions of N-glycans and develop better glycoprotein therapeutics. Toward this goal, we have successfully expressed in E. coli glycoside hydrolases and glycosyltransferases from bacterial and human origins and developed a robust enzymatic platform for in vitro processing glycoprotein N-glycans from high-mannose-type to α2-6- or α2-3-disialylated biantennary complex type. The recombinant enzymes are highly efficient in step-wise or one-pot reactions. The platform can find broad applications in N-glycan engineering of therapeutic glycoproteins.

The Light-Controlled Release of 2-fluoro-l-fucose, an Inhibitor of the Root Cell Elongation, from a nitrobenzyl-caged Derivative.

Glycan metabolic engineering is a powerful tool for studying the glycosylation in living plant cells. The use of modified monosaccharides such as deoxy or fluorine-containing glycosides has been reported as a powerful pharmacological approach for studying the carbohydrate metabolism. 1,3,4-tri-O-acetyl-2-fluoro-l-fucose (2F-Fuc) is a potent inhibitor of the plant cell elongation. After feeding plant seedlings with 2F-Fuc, this monosaccharide derivative is deacetylated and converted by the endogenous metabolic machinery into the corresponding nucleotide-sugar, which then efficiently inhibits Golgi-localized fucosyltransferases. Among plant cell wall polymers, defects in the fucosylation of the pectic rhamnogalacturonan-II cause a decrease in RG-II dimerization, which in turn induce the arrest of the cell elongation. In order to perform the inhibition of the cell elongation process in a spatio-temporal manner, we synthesized a caged 3,4-di-O-acetyl-1-hydroxy-2-fluoro-l-fucose (1-OH-2F-Fuc) derivative carrying a photolabile ortho-nitrobenzyl alcohol function at the anomeric position: 3,4-di-O-acetyl-1-ortho-nitrobenzyl-2-fluoro-l-fucose (2F-Fuc-NB). The photorelease of the trapped 1-OH-2F-Fuc was performed under a 365 nm LED illumination. We demonstrated that the in planta elimination by photoexcitation of the photolabile group releases free 2F-Fuc in plant cells, which in turn inhibits in a dose-dependent manner and, reversibly, the root cell elongation.

An Augmented Method for Collecting PLGA Nanoparticles and the Fabrication of 1, 3, 4, 6-Tetra-O-acetyl-2-azido-2-deoxy-D-glucopyranose (Ac42AzGlc)-Loaded PLGA Nanoparticles for Efficient and Prospective in Vivo Metabolic Processing.

We investigated two ways for fabricating 1, 3, 4, 6-tetra-O-acetyl-2-azido-2-deoxy-D-glucopyranose (Ac42AzGlc)-loaded poly (lactic-co-glycolic acid) PLGA nanoparticles in this article : 1) single emulsion solvent evaporation and 2) the nanoprecipitation method. Among the available methods of collecting nanoparticles using an ultra-high-speed centrifuge, we improvised a less-known method for collecting synthesized nanoparticles without a high-speed centrifuge, based on molecular weight (MW)-dependent centrifugal filters. These nanoparticles were collected in a tabletop centrifuge at a meager centrifugal force in the range of 200-300 xg whereas the conventional high-speed centrifuge method for nanoparticle recovery results in a hard nanoparticle pellet with poor resuspendability which hampers the yield and outcomes of the product. The Ac42AzGlc-loaded PLGA nanoparticles were spherical in shape with consistent and reliable nanometric particle size. The polydispersity indices were well within the acceptable limits. The preliminary studies in RAW 264.7 cell and C57BL/6 mice advocated efficient engineering in the former; however, the latter needs further confirmatory investigations. Preliminary in vivo studies with un-encapsulated Ac42AzGlc showed poor engineering of cardiac glycoproteins, opening up avenues for Ac42AzGlc-loaded nanoparticles for improved bioavailability and efficient metabolic engineering.

Subtle Influence of ACE2 Glycan Processing on SARS-CoV-2 Recognition.

The severity of SARS-CoV-2 infection is highly variable and yet the molecular basis for this effect remains elusive. One potential contribution are differences in the glycosylation of target human cells, particularly as SARS-CoV-2 has the capacity to bind sialic acid which is a common, and highly variable, terminal modification of glycans. The viral spike glycoprotein (S) of SARS-CoV-2 and the human cellular receptor, angiotensin-converting enzyme 2 (ACE2) are both densely glycosylated. We therefore sought to investigate whether the glycosylation state of ACE2 impacts the interaction with SARS-CoV-2 viral spike. We generated a panel of engineered ACE2 glycoforms which were analyzed by mass spectrometry to reveal the site-specific glycan modifications. We then probed the impact of ACE2 glycosylation on S binding and revealed a subtle sensitivity with hypersialylated or oligomannose-type glycans slightly impeding the interaction. In contrast, deglycosylation of ACE2 did not influence SARS-CoV-2 binding. Overall, ACE2 glycosylation does not significantly influence viral spike binding. We suggest that any role of glycosylation in the pathobiology of SARS-CoV-2 will lie beyond its immediate impact of receptor glycosylation on virus binding.

Expression Profiling and Glycan Engineering of IgG Subclass 1-4 in Nicotiana benthamiana.

IgG, the main serum immunoglobulin isotype, exists in four subclasses which selectively appear with distinctive glycosylation profiles. However, very little is known about the biological consequences mainly due to the difficulties in the generation of distinct IgG subtypes with targeted glycosylation. Here, we show a comprehensive expression and glycan modulation profiling of IgG variants in planta that are identical in their antigen binding domain but differ in their subclass appearance. While IgG1, 2, and 4 exhibit similar expression levels and purification yields, IgG3 is generated only at low levels due to the in planta degradation of the heavy chain. All IgG subtypes are produced with four distinct N-glycosylation profiles, differing in sugar residues previously shown to impact IgG activities, i.e., galactosylation, sialylation and core fucosylation. Affinity purified IgG variants are shown to be fully assembled to heterodimers but display different biochemical/physical features. All subtypes are equally well amenable to targeted glycosylation, except sialylated IgG4 which frequently accumulates substantial fractions of unusual oligo-mannosidic structures. IgG variants show significant differences in aggregate formation and endotoxin contamination which are eliminated by additional polishing steps (size exclusion chromatography, endotoxin removal treatments). Collectively we demonstrate the generation of 16 IgG variants at high purity and large glycan homogeneity which constitute an excellent toolbox to further study the biological impact of the two main Fc features, subclass and glycosylation.

Off-target glycans encountered along the synthetic biology route toward humanized N-glycans in Pichia pastoris.

The glycosylation pathways of several eukaryotic protein expression hosts are being engineered to enable the production of therapeutic glycoproteins with humanized application-customized glycan structures. In several expression hosts, this has been quite successful, but one caveat is that the new N-glycan structures inadvertently might be substrates for one or more of the multitude of endogenous glycosyltransferases in such heterologous background. This then results in the formation of novel, undesired glycan structures, which often remain insufficiently characterized. When expressing mouse interleukin-22 in a Pichia pastoris (syn. Komagataella phaffii) GlycoSwitchM5 strain, which had been optimized to produce Man5 GlcNAc2 N-glycans, glycan profiling revealed two major species: Man5 GlcNAc2 and an unexpected, partially α-mannosidase-resistant structure. A detailed structural analysis using exoglycosidase sequencing, mass spectrometry, linkage analysis, and nuclear magnetic resonance revealed that this novel glycan was Man5 GlcNAc2 modified with a Glcα-1,2-Manß-1,2-Manß-1,3-Glcα-1,3-R tetrasaccharide. Expression of a Golgi-targeted GlcNAc transferase-I strongly inhibited the formation of this novel modification, resulting in more homogeneous modification with the targeted GlcNAcMan5 GlcNAc2 structure. Our findings reinforce accumulating evidence that robustly customizing the N-glycosylation pathway in P. pastoris to produce particular human-type structures is still an incompletely solved synthetic biology challenge, which will require further innovation to enable safe glycoprotein pharmaceutical production.

In Planta Glycan Engineering and Functional Activities of IgE Antibodies.

Human immunoglobulin E (IgE) is the most extensively glycosylated antibody isotype so glycans attached to the seven N-glycosites (NGS) in its Fab and Fc domains may modulate its functions. However, targeted modification of glycans in multiply glycosylated proteins remains a challenge. Here, we applied an in vivo approach that allows the manipulation of IgE N-glycans, using a trastuzumab equivalent IgE (HER2-IgE) as a model. Taking advantage of plant inherent features, i.e., synthesis of largely homogeneous complex N-glycans and susceptibility to glycan engineering, we generated targeted glycoforms of HER2-IgE largely resembling those found in serum IgE. Plant-derived HER2-IgE exhibited N-glycans terminating with GlcNAc, galactose or sialic acid, lacking, or carrying core fucose and xylose. We were able to not only modulate the five NGSs naturally decorated with complex N-glycans, but to also induce targeted glycosylation at the usually unoccupied NGS6, thus increasing the overall glycosylation content of HER2-IgE. Recombinant human cell-derived HER2-IgE exhibited large N-glycan heterogeneity. All HER2-IgE variants demonstrated glycosylation-independent binding to the target antigen and the high affinity receptor FcεRI, and subsequent similar capacity to trigger mast cell degranulation. In contrast, binding to the low affinity receptor CD23 (FcεRII) was modulated by the glycan profile, with increased binding to IgE variants with glycans terminating with GlcNAc residues. Here we offer an efficient in planta approach to generate defined glycoforms on multiply glycosylated IgE, allowing the precise exploration of glycosylation-dependent activities.

Differential inhibition of mucin-type O-glycosylation (MTOG) induced by peracetyl N-thioglycolyl-d-galactosamine (Ac5GalNTGc) in myeloid cells.

Investigations on the structure and functional roles of glycosylation - an intricate, complex, and dynamic post translational modification on proteins - in biological processes has been a challenging task. Glycan modifications vary depending on the specific cell type, its developmental stage, and resting or activated state. In the present study, we aim to understand the differences between the mucin-type O-glycosylation (MTOG) of two functionally divergent human cell lines, K562 (chronic myeloid leukemia) and U937 (histiocytic lymphoma), having myeloid origins. MTOG is initiated by the addition of N-acetyl-α-d-galactosamine (GalNAc) to Ser/Thr of glycoproteins. We exploited the metabolic glycan engineering (MGE) strategy using the peracetyl N-thioglycolyl-d-galactosamine (Ac5GalNTGc), a synthetic GalNAc analogue, to engineer the glycoconjugates. Ac5GalNTGc was metabolized and incorporated as N-thioglycolyl-d-galactosamine (GalNTGc) in cell surface glycoproteins in both the cell lines with varying degrees of efficiency. Notably, metabolic incorporation of GalNTGc resulted in differential inhibition of MTOG. It was observed that endogenous glycosylation machinery of K562 is relatively more stringent for selecting GalNTGc whereas U937 is flexible towards this selection. Additionally, we studied how the glycan modifications vary on a given CD antigen in these cell lines. Particularly, MTOG on CD43 was differentially inhibited in K562 and U937 as revealed by glycan-dependent and glycan-independent antibodies. It was observed that the effect of MGE on CD43 was similar to global effects on both cell lines. Consequences of MGE using GalNAc analogues depend on the expression and activity of various glycosyl transferases which determine global glycosylation on cell surface as well as on specific glycoproteins.

Advanced Plant-Based Glycan Engineering.

With respect to biomanufacturing, glycosylation is one of the most addressed post-translational modifications, since it is well-known that the attachment of sugar residues efficiently affects protein homogeneity and functionality. Much effort has been taken into engineering various expression systems to control glycosylation and to generate molecules with targeted sugar profiles. Nevertheless, engineering of N- and O-linked glycans on well-established expression systems remains challenging. On the one side the glycosylation machinery in mammalian cells is hard to control due to its complexity. Most bacteria, on the other side, completely lack such glycan formations, and in general exhibit fundamental differences in their glycosylation abilities. Beyond that, plants generate complex N-glycans typical of higher eukaryotes, but simpler than those produced by mammals. Paradoxically, it seems that the limited glycosylation capacity of plant cells is an advantage for specific glycan manipulations. This review focuses on recent achievements in plant glycan engineering and provides a short outlook on how new developments (in synthetic biology) might have a positive impact.

Genetic disruption of multiple α1,2-mannosidases generates mammalian cells producing recombinant proteins with high-mannose-type N-glycans.

Recombinant therapeutic proteins are becoming very important pharmaceutical agents for treating intractable diseases. Most biopharmaceutical proteins are produced in mammalian cells because this ensures correct folding and glycosylation for protein stability and function. However, protein production in mammalian cells has several drawbacks, including heterogeneity of glycans attached to the produced protein. In this study, we established cell lines with high-mannose-type N-linked, low-complexity glycans. We first knocked out two genes encoding Golgi mannosidases (MAN1A1 and MAN1A2) in HEK293 cells. Single knockout (KO) cells did not exhibit changes in N-glycan structures, whereas double KO cells displayed increased high-mannose-type and decreased complex-type glycans. In our effort to eliminate the remaining complex-type glycans, we found that knocking out a gene encoding the endoplasmic reticulum mannosidase I (MAN1B1) in the double KO cells reduced most of the complex-type glycans. In triple KO (MAN1A1, MAN1A2, and MAN1B1) cells, Man9GlcNAc2 and Man8GlcNAc2 were the major N-glycan structures. Therefore, we expressed two lysosomal enzymes, α-galactosidase-A and lysosomal acid lipase, in the triple KO cells and found that the glycans on these enzymes were sensitive to endoglycosidase H treatment. The N-glycan structures on recombinant proteins expressed in triple KO cells were simplified and changed from complex types to high-mannose types at the protein level. Our results indicate that the triple KO HEK293 cells are suitable for producing recombinant proteins, including lysosomal enzymes with high-mannose-type N-glycans.

Engineered Glycocalyx Regulates Stem Cell Proliferation in Murine Crypt Organoids.

At the base of the intestinal crypt, long-lived Lgr5+ stem cells are intercalated by Paneth cells that provide essential niche signals for stem cell maintenance. This unique epithelial anatomy makes the intestinal crypt one of the most accessible models for the study of adult stem cell biology. The glycosylation patterns of this compartment are poorly characterized, and the impact of glycans on stem cell differentiation remains largely unexplored. We find that Paneth cells, but not Lgr5+ stem cells, express abundant terminal N-acetyllactosamine (LacNAc). Employing an enzymatic method to edit glycans in cultured crypt organoids, we assess the functional role of LacNAc in the intestinal crypt. We discover that blocking access to LacNAc on Paneth cells leads to hyperproliferation of the neighboring Lgr5+ stem cells, which is accompanied by the downregulation of genes that are known as negative regulators of proliferation.