Human B-lymphocytes express alpha2-6-sialylated 6-sulfo-N-acetyllactosamine serving as a preferred ligand for CD22/Siglec-2

CD22/Siglec-2, an important inhibitory co-receptor on B-lymphocytes, is known to recognize alpha2-6-sialylated glycan as a specific ligand. Here we propose that the alpha2-6-sialylated and 6-GlcNAc-sulfated determinant serves as a preferred ligand for CD22 because the binding of a human B-cell line to CD22 was almost completely abrogated after incubating the cells with NaClO3, an inhibitor of cellular sulfate metabolism, and was also significantly inhibited by a newly generated monoclonal antibody specific to the alpha2-6-sialylated 6-sulfo-N-acetyllactosamine (LacNAc) determinant (KN343, murine IgM). The alpha2-6-sialylated 6-sulfo-LacNAc determinant defined by the antibody was significantly expressed on a majority of normal human peripheral B-lymphocytes as well as follicular B-lymphocytes in peripheral lymph nodes. The determinant was also expressed in endothelial cells of high endothelial venules of secondary lymphoid tissues, including lymph nodes, tonsils, and intestine-associated lymphoid tissues, more strongly than on B-lymphocytes, suggesting a role for CD22 in B-cell interaction with blood vessels and trafficking. These results indicate that the alpha2-6-sialylated 6-sulfo-LacNAc determinant serves as an endogenous ligand for human CD22 and suggest the possibility that 6-GlcNAc sulfation as well as alpha2-6-sialylation may regulate CD22/Siglec-2 functions in humans.     

Antibody-drug therapeutic conjugates: Potential of antibody-siRNAs in cancer therapy

Codelivery is a promising strategy of targeted delivery of cytotoxic drugs for eradicating tumor cells. This rapidly growing method of drug delivery uses a conjugate containing drug linked to a smart carrier. Both two parts usually have therapeutic properties on the tumor cells. Monoclonal antibodies and their derivatives, such as Fab, scFv, and bsAb due to targeting high potent have now been attractive candidates as drug targeting carrier systems. The success of some therapeutic agents like small interfering RNA (siRNA), a small noncoding RNAs, with having problems such as enzymatic degradation and rapid renal filtration need to an appropriate carrier. Therefore, the aim of this study is to review the recent enhancements in development of antibody drug conjugates (ADCs), especially antibody–siRNA conjugates (SRCs), its characterizations and mechanisms in innovative cancer therapy approaches.     

Fabrication of Langmuir-Blodgett film of a fullerene derivative with a cyclic peptide as an anchor

A novel cyclic octapeptide carrying a fullerene unit and poly(ethylene glycol) at the side chain (cyclo8-C 60 + PEG) was synthesized, and its monolayer formation at the air/water interface and on a substrate was studied. Surface pressure-area per molecule isotherms indicated that cyclo8-C 60 + PEG formed a stable monolayer at the air/water interface. The cyclo8-C 60 + PEG monolayers prepared from various spreading volumes (i.e., from various initial areas per molecule) overlapped nicely on a single curve, suggesting that the molecules were uniformly dispersed on the surface without aggregation of the fullerene units. The uniform dispersibility is due to the scaffold effect of the cyclic peptide unit to keep the fullerene units away from each other. The formed monolayer could be quantitatively transferred onto a solid substrate. UV-vis absorption spectroscopy of the Langmuir-Blodgett (LB) monolayer showed that the electronic structure of the fullerene unit was not affected by the formation of the monolayer. Cyclic voltammetry of the LB monolayer in an aqueous solution containing redox species indicated that the LB monolayer was densely packed. Furthermore, reversible redox peaks attributed to the one-electron reduction of the fullerene unit were observed, showing that the redox property of the fullerene unit was also retained in the monolayer. It is thus concluded that the cyclic peptide is a good candidate as a scaffold for stable monolayer formation at the air/water interface and for intact immobilization of the fullerene moiety onto a substrate.     

Engineering Antibodies as Drugs: Principles and Practice

Over the last forty years, recombinant antibodies have been transformed from an unproven experimental approach to a therapeutic modality with multiple success stories in the treatment of cancer, inflammation, infections and cardiometabolic diseases. Owing to their high affinity and selectivity for the target antigen, their multimodal tunable mode of action, their modular nature and long half-life, antibodies now hold prominent positions in the pipelines of major biopharmaceutical companies. In this brief report, I aim to highlight the themes that have shaped the therapeutic antibody engineering as it exists today and to offer a personal perspective on its future developments. Distinct antibody engineering history, developments and trends in Russian Federation will not be discussed since they are detailed elsewhere in this journal issue.     

Liposomes surface conjugated with human hemoglobin target delivery to macrophages

Current strategies to deliver therapeutic molecules to specific cell and tissue types rely on conjugation of antibodies and other targeting ligands directly to the therapeutic molecule itself or its carrier. This work describes a novel strategy to deliver therapeutic molecules into macrophages that takes advantage of the native hemoglobin (Hb) scavenging activity of plasma haptoglobin (Hp) and the subsequent uptake of the Hb-Hp complex into macrophages via CD163 receptor-mediated endocytosis. The drug delivery system described in this work consists of Hb decorated liposomes that can encapsulate any therapeutic molecule of interest, in this case the model fluorescent dye calcein was used in this study. The results of this study clearly demonstrate that this delivery system is specific towards macrophages and demonstrates the feasibility of using this approach in targeted drug delivery.     

The active site and substrates binding mode of malonyl-CoA synthetase determined by transferred nuclear Overhauser effect spectroscopy, site-directed mutagenesis, and comparative modeling studies

The active sites and substrate bindings of Rhizobium trifolii molonyl-CoA synthetase (MCS) catalyzing the malonyl-CoA formation from malonate and CoA have been determined based on NMR spectroscopy, site-directed mutagenesis, and comparative modeling methods. The MCS-bound conformation of malonyl-CoA was determined from two-dimensional-transferred nuclear Overhauser effect spectroscopy data. MCS protein folds into two structural domains and consists of 16 alpha-helices, 24 beta-strands, and several long loops. The core active site was determined as a wide cleft close to the end of the small C-terminal domain. The catalytic substrate malonate is placed between ATP and His206 in the MCS enzyme, supporting His206 in its catalytic role as it generates reaction intermediate, malonyl-AMP. These findings are strongly supported by previous biochemical data, as well as by the site-directed mutagenesis data reported here. This structure reveals the biochemical role as well as the substrate specificity that conservative residues of adenylate-forming enzymes have.     

中枢神经系统疾病治疗性抗体药物应用进展

单克隆抗体药物是一种新兴的治疗药物,具有高选择性,被用于多种疾病的治疗,如肿瘤、免疫疾病等,也可以用于中枢神经系统疾病,如阿尔茨海默病、帕金森病、中风和脑肿瘤等。然而,因为血脑屏障低通透性,限制了抗体药物在中枢神经系统疾病治疗中的应用,在很多神经系统疾病临床试验中,抗体药物并没有取得预期效果。如今,人们利用血脑屏障上内源性转运蛋白介导,设计了可以通过血脑屏障的抗体药物。对通过血脑屏障治疗性抗体药物研发进展及其应用前景进行了综述。     

Isolation of human B-cell subpopulations for pharmacological studies.

Three groups of human peripheral blood B-lymphocytes were separated from each other by countercurrent centrifugal elutriation and free-flow electrophoresis. They differed in their state of maturation and in their capability to produce antibodies in vitro. These B-cell subpopulations were used to study features of a drug such as BAY R 1005. BAY R 1005 is a synthetic glycolipid analogue (GLA), which is supposed to modulate antibody synthesis. Mature, immunoglobulin- (Ig-) secreting B-lymphocytes secreted equal quantities of antibodies in the presence and in the absence of the GLA. BAY R 1005 was found to be without mitogenic activity on resting B-cells and did not induce them to produce antibodies. However, it supported the antibody production of preactivated B-lymphocytes. The in vitro preactivated B-cells were affected via monocytes. Only in vivo preactivated B-lymphocytes increased their antibody production under the direct influence of BAY R 1005.     

Complexity generated by iteration of hierarchical modules in bryozoa

Growth in colonial organisms by iteration of modules inherently provides for an increase in available morpho-ecospace relative to their solitary relatives. Therefore, the interpretation of the functional or evolutionary significance of complexity within groups that exhibit modular growth may need to be considered under criteria modified from those used to interpret complexity in solitary organisms. Primary modules, corresponding to individuals, are the fundamental building blocks of a colonial organism. Groups of primary modules commonly form a second-order modular unit, such as a branch, which may then be iterated to form a more complex colony. Aspects of overall colony form, along with their implications for ecology and evolution, are reflected in second-order modular (structural) units to a far greater degree than by primary modular units (zooids). A colony generated by modular growth can be classified by identifying its second-order modular (structural) unit and then by characterizing the nature and relationships of these iterated units within the colony. This approach to classifying modular growth habits provides a standardized terminology and allows for direct comparison of a suite of functionally analogous character states among taxa with specific parameters of their ecology.     

Active-site residue,domain and module swaps in modular polyketide synthases

Sequence comparisons of multiple acyltransferase (AT) domains from modular polyketide synthases (PKSs) have highlighted a correlation between a short sequence motif and the nature of the extender unit selected. When this motif was specifically altered in the bimodular model PKS DEBS1-TE of Saccharopolyspora erythraea, the products included triketide lactones in which acetate extension units had been incorporated instead of propionate units at the predicted positions. We also describe a cassette system for convenient construction of hybrid modular PKSs based on the tylosin PKS in Streptomyces fradiae and demonstrate its use in domain and module swaps.     

Policing starter unit selection of the enterocin type II polyketide synthase by the type II thioesterase EncL

Enterocin is an atypical type II polyketide synthase (PKS) product from the marine actinomycete 'Streptomyces maritimus'. The enterocin biosynthesis gene cluster (enc) codes for proteins involved in the assembly and attachment of the rare benzoate primer that initiates polyketide assembly with the addition of seven malonate molecules and culminates in a Favorskii-like rearrangement of the linear poly-β-ketone to give its distinctive non-aromatic, caged core structure. Fundamental to enterocin biosynthesis, which utilizes a single acyl carrier protein (ACP), EncC, for both priming with benzoate and elongating with malonate, involves maintaining the correct balance of acyl-EncC substrates for efficient polyketide assembly. Here, we report the characterization of EncL as a type II thioesterase that functions to edit starter unit (mis)priming of EncC. We performed a series of in vivo mutational studies, heterologous expression experiments, in vitro reconstitution studies, and Fourier-transform mass spectrometry-monitored competitive enzyme assays that together support the proposed selective hydrolase activity of EncL toward misprimed acetyl-ACP over benzoyl-ACP to facilitate benzoyl priming of the enterocin PKS complex. While this system resembles the R1128 PKS that also utilizes an editing thioesterase (ZhuC) to purge acetate molecules from its initiation module ACP in favor of alkylacyl groups, the enterocin system is distinct in its usage of a single ACP for both priming and elongating reactions with different substrates.     

Alteration of the substrate specificity of a modular polyketide synthase acyltransferase domain through site-specific mutations.

Cassette replacement of acyltransferase (AT) domains in 6-deoxyerythronolide B synthase (DEBS) with heterologous AT domains with different substrate specificities usually yields the predicted polyketide analogues. As reported here, however, several AT replacements in module 4 of DEBS failed to produce detectable polyketide under standard conditions, suggesting that module 4 is sensitive to perturbation of the protein structure when the AT is replaced. Alignments between different modular polyketide synthase AT domains and the Escherichia coli fatty acid synthase transacylase crystal structure were used to select motifs within the AT domain of module 4 to re-engineer its substrate selectivity and minimize potential alterations to protein folding. Three distinct primary regions of AT4 believed to confer specificity for methylmalonyl-CoA were mutated into the sequence seen in malonyl-CoA-specific domains. Each individual mutation as well as the three in combination resulted in functional DEBSs that produced mixtures of the natural polyketide, 6-deoxyerythronolide B, and the desired novel analogue, 6-desmethyl-6-deoxyerythronolide B. Production of the latter compound indicates that the identified sequence motifs do contribute to AT specificity and that DEBS can process a polyketide chain incorporating a malonate unit at module 4. This is the first example in which the extender unit specificity of a PKS module has been altered by site-specific mutation and provides a useful alternate method for engineering AT specificity in the combinatorial biosynthesis of polyketides.     

Investigation of parameters influencing incorporation,retention and cellular cytotoxicity in liposomal formulations of poorly soluble camptothecin

Abstract

Improving tumor delivery of lipophilic drugs through identifying advanced drug carrier systems with efficient carrier potency is of high importance. We have performed an investigative approach to identify parameters that affect liposomes’ ability to effectively deliver lipophilic camptothecin (CPT) to target cells. CPT is a potent anticancer drug, but its undesired physiological properties are impairing its therapeutic use. In this study, we have identified parameters influencing incorporation and retention of lipophilic CPT in liposomes, evaluating the effect of lipid composition, lipid chemical structure (head and tail group variations, polymer inclusion), zeta potential and anisotropy. Polyethyleneglycol (PEG) surface decoration was included to avoid liposome fusing and increase the potential for prolonged in vivo circulation time. The in vitro effect of the different carrier formulations on cell cytotoxicity was compared and the effect of active targeting of one of the formulations was evaluated. We found that a combination of liposome surface charge, lipid headgroup and carbon chain unsaturation affect CPT incorporation. Retention in liposomes was highly dependent on the liposomal surroundings and liposome zeta potential. Inclusion of lipid tethered PEG provided stability and prevented liposome fusing. PEGylation negatively affected CPT incorporation while improving retention. In vitro cell culture testing demonstrated that all formulations increased CPT potency compared to free CPT, while cationic formulations proved significantly more toxic to cancer cells that healthy cells. Finally, antibody mediated targeting of one liposome formulation further enhanced the selectivity towards targeted cancer cells, rendering normal cells fully viable after 1 hour exposure to targeted liposomes.     

Multivariate modular metabolic engineering of Escherichia coli to produce resveratrol from l-tyrosine

Microbial fermentations and bioconversion promise to revolutionize the conventional extraction of resveratrol from natural plant sources. However, the development of efficient and feasible microbial processes remains challenging. Current fermentation strategies often require supplementation of expensive phenylpropanoic precursors and two separate fermentation protocols, which are significantly more difficult and expensive to undertake when migrating to large-scale fermentation processes. In this study, an Escherichia coli fermentation system, consisting of tyrosine ammonia lyase (TAL), 4-coumarate:CoA ligase (4CL), stilbene synthase (STS), malonate synthetase, and malonate carrier protein, was developed to produce resveratrol from l-tyrosine. Multivariate modular metabolic engineering, which redefined the overall pathway as a collection of distinct modules, was employed to assess and alleviate pathway bottlenecks. Using this strategy, the optimum strain was capable of producing 35.02 mg/L of resveratrol from l-tyrosine in a single medium. The strategy described here paves the way to the development of a simple and economical process for microbial production of resveratrol and other similar stilbene chemicals.     

Synthesis and evaluation of malonate-based inhibitors of phosphosugar-metabolizing enzymes: class II fructose-1,6-bis-phosphate aldolases, type I phosphomannose isomerase, and phosphoglucose isomerase

In the design of inhibitors of phosphosugar metabolizing enzymes and receptors with therapeutic interest, malonate has been reported in a number of cases as a good and hydrolytically-stable surrogate of the phosphate group, since both functions are dianionic at physiological pH and of comparable size. We have investigated a series of malonate-based mimics of the best known phosphate inhibitors of class II (zinc) fructose-1,6-bis-phosphate aldolases (FBAs) (e.g., from Mycobacterium tuberculosis), type I (zinc) phosphomannose isomerase (PMI) from Escherichia coli, and phosphoglucose isomerase (PGI) from yeast. In the case of FBAs, replacement of one phosphate by one malonate on a bis-phosphorylated inhibitor (1) led to a new compound (4) still showing a strong inhibition (K(i) in the nM range) and class II versus class I selectivity (up to 8×10(4)). Replacement of the other phosphate however strongly affected binding efficiency and selectivity. In the case of PGI and PMI, 5-deoxy-5-malonate-D-arabinonohydroxamic acid (8) yielded a strong decrease in binding affinities when compared to its phosphorylated parent compound 5-phospho-D-arabinonohydroxamic acid (2). Analysis of the deposited 3D structures of the kinetically evaluated enzymes complexed to the phosphate-based inhibitors indicate that malonate could be a good phosphate surrogate only if phosphate is not tightly bound at the enzyme active site, such as in position 7 of compound 1 for FBAs. These observations are of importance for further design of inhibitors of phosphorylated-compounds metabolizing enzymes with therapeutic interest.     

Targeting hepatitis B virus-infected cells with a T-cell receptor-like antibody

Virus-specific CD8 T cells are activated when their T-cell receptors (TCRs) recognize the specific viral peptide/major histocompatibility complex (MHC) class I (pMHC) complexes present on the surface of infected cells. Antibodies able to recognize the specific pMHC can mimic TCR specificity and both represent a valuable biological tool to visualize pMHC complexes on infected cells and serve as a delivery system for highly targeted therapies. To evaluate these possibilities, we created a monoclonal antibody able to specifically recognize a hepatitis B virus (HBV) envelope epitope (Env at positions 183 to 91 [Env183-91]) presented by the HLA-A201 molecule, and we tested its ability to recognize HBV-infected hepatocytes and to deliver a cargo to a specific target. We demonstrate that this antibody detects and visualizes the processed product of HBV proteins produced in naturally HBV-infected cells, is not inhibited by soluble HBV proteins present in patient sera, and mediates the intracellular delivery of a fluorescent molecule to target cells. Additionally, compared to CD8 T cells specific for the same HBV epitope, the TCR-like antibody has both a superior sensitivity and a specificity focused on distinct amino acids within the epitope. These data demonstrate that a T-cell receptor-like antibody can be used to determine the quantitative relationship between HBV replication and specific antigen presentation to CD8 T cells and serves as a novel therapeutic delivery platform for personalized health care for HBV-infected patients.     

Humoral response of cynomolgus macaques to human soluble CD4: antibody reactivity restricted to xeno-human determinants.

The CD4 cell surface glycoprotein which is expressed primarily by a subset of T lymphocytes plays a key role in normal immune responses. In the immunopathogenesis of AIDS, it serves as the high-affinity receptor for HIV, facilitating viral attachment and entry into CD4+ cells. As such, the truncated soluble form of this molecule (sT4) has been proposed as a therapeutic drug for the treatment of AIDS whereby it would act as decoy for viral entry into cells or facilitate elimination of soluble viral envelope glycoprotein. In a study designed to look at the effect of sT4 on immune function, sT4 was administrated to experimentally naive primates. In this report, we show that administration of sT4 to cynomolgus macaque monkeys over a period of up to 3 weeks results in antibody responses with specificities for human CD4 molecules. Antisera thus generated bound sT4 and cell surface CD4 expressed on human T lymphocytes but failed to bind to cynomolgus lymphocytes. These antibodies caused no apparent adverse effects on normal immune functions of the cynomolgus macaques. We conclude from these data that the antibody response to soluble CD4 in cynomolgus monkeys is directed at determinants present on human CD4 but absent on monkey CD4. The restricted xenogeneic specificity of the antibody response indicates that soluble CD4 may not be highly immunogenic in syngeneic hosts. The present study also shows that these antibodies can block HIV-induced syncytium formation indicating that the antibodies bind to regions on the CD4 molecule close to the HIV-env gp120 binding site. The gp120 binding site, which resides within the N-terminal V1 domain of CD4, encompasses a region which corresponds to the complementarity determining regions (CDRs) of immunoglobulins. The CDR-like regions of CD4-V1 manifest the greatest species divergence, are tolerant to experimental in vitro mutagenesis, and generate the predominant antibody response in mice immunized with human CD4 indicating that differences in the V1 sequence between human and other non-human primates may localize to this regions.     

Coiled coil peptides as universal linkers for the attachment of recombinant proteins to polymer therapeutics

We have designed, synthesized, and characterized peptides containing four repeats of the sequences VAALEKE (peptide E) or VAALKEK (peptide K). While the peptides alone adopt in aqueous solutions a random coil conformation, their equimolar mixture forms heterodimeric coiled coils as confirmed by CD spectroscopy. 5-Azidopentanoic acid was connected to the N-terminus of peptide E via a short poly(ethylene glycol) spacer. The terminal azide group enabled conjugation of the peptide with a synthetic drug carrier based on the N-(2-hydroxypropyl)methacrylamide copolymer containing propargyl groups using "click" chemistry. When incorporated into the polymer drug carrier, peptide E formed a stable noncovalent complex with peptide K belonging to a recombinant single-chain fragment (scFv) of the M75 antibody. The complex thereby mediates a noncovalent linkage between the polymer drug carrier and the protein. The recombinant scFv antibody fragment was selected as a targeting ligand against carbonic anhydrase IX-a marker overexpressed by tumor cells of various human carcinomas. The antigen binding affinity of the polymer-scFv complex was confirmed by ELISA. This approach offers a well-defined, specific, and nondestructive universal method for the preparation of protein (antibody)-targeted polymer drug and gene carriers designed for cell-specific delivery.     

Pre-Clinical Development of a Humanized Anti-CD47 Antibody with Anti-Cancer Therapeutic Potential

CD47 is a widely expressed cell surface protein that functions as a regulator of phagocytosis mediated by cells of the innate immune system, such as macrophages and dendritic cells. CD47 serves as the ligand for a receptor on these innate immune cells, SIRP-alpha, which in turn delivers an inhibitory signal for phagocytosis. We previously found increased expression of CD47 on primary human acute myeloid leukemia (AML) stem cells, and demonstrated that blocking monoclonal antibodies directed against CD47 enabled the phagocytosis and elimination of AML, non-Hodgkin’s lymphoma (NHL), and many solid tumors in xenograft models. Here, we report the development of a humanized anti-CD47 antibody with potent efficacy and favorable toxicokinetic properties as a candidate therapeutic. A novel monoclonal anti-human CD47 antibody, 5F9, was generated, and antibody humanization was carried out by grafting its complementarity determining regions (CDRs) onto a human IgG4 format. The resulting humanized 5F9 antibody (Hu5F9-G4) bound monomeric human CD47 with an 8 nM affinity. Hu5F9-G4 induced potent macrophage-mediated phagocytosis of primary human AML cells in vitro and completely eradicated human AML in vivo, leading to long-term disease-free survival of patient-derived xenografts. Moreover, Hu5F9-G4 synergized with rituximab to eliminate NHL engraftment and cure xenografted mice. Finally, toxicokinetic studies in non-human primates showed that Hu5F9-G4 could be safely administered intravenously at doses able to achieve potentially therapeutic serum levels. Thus, Hu5F9-G4 is actively being developed for and has been entered into clinical trials in patients with AML and solid tumors (ClinicalTrials.gov identifier: NCT02216409).     

Learning the selectivity of V2 and V4 neurons using non-linear multi-layer wavelet networks

We investigate a non-linear network with two processing stages optimized to reduce the statistical dependencies in natural images. This network serves as a model for the neural information processing in the higher visual areas of primates (visual cortices V2-V4). The resulting population is analyzed with regard to non-linear selectivity and invariance properties. We find units that are very selective with respect to the space spanned by all possible input signals and units that are invariant with respect to certain stimulus classes. In comparison to the measured distribution of selectivity in V2 neurons, the selectivity histogram of the network units shows an even more pronounced tendency towards higher selectivities. A special property of the system is the emergence of non-linear interactions between coefficients from different scales and orientations, which are necessary for the exploitation of higher-order statistical redundancies of natural images. We extend the concept to multi-layer systems and present some simulation results.