Characterization of Disulfide Bond Rebridged Fab–Drug Conjugates Prepared Using a Dual Maleimide Pyrrolobenzodiazepine Cytotoxic Payload

We describe the characterization of antigen binding fragments (Fab)–drug conjugates prepared using a dual maleimide pyrrolobenzodiazepine dimer cytotoxic payload (SG3710). Pyrrolobenzodiazepine dimers, which are DNA cross-linkers, are a class of payloads used in antibody–drug conjugates (ADCs). SG3710 was designed to rebridge two adjacent cysteines, such as those that form the canonical interchain disulfide bond between the light and heavy chain in Fab fragments. The rebridging generated homogenous Fab conjugates, with a drug-to-Fab ratio of one, as demonstrated by the preparation of rebridged Fabs derived from the anti-HER2 trastuzumab antibody and from a negative control antibody both prepared using recombinant expression and papain digestion. The resulting anti-HER2 trastuzumab Fab-rebridged conjugate retained antigen binding, was stable in rat serum, and demonstrated potent and antigen-dependent cancer cell-killing ability. Disulfide rebridging with SG3710 is a generic approach to prepare Fab–pyrrolobenzodiazepine dimer conjugates, which does not require the Fabs to be engineered for conjugation. Thus, SG3710 offers a flexible and straightforward platform for the controlled assembly of pyrrolobenzodiazepine dimer conjugates from any Fab for oncology applications.     

Efficient Site-Specific Antibody–Drug Conjugation by Engineering a Nature-Derived Recognition Tag for Microbial Transglutaminase

Microbial transglutaminase (mTG) has recently emerged as a powerful tool for antibody engineering. In nature, it catalyzes the formation of amide bonds between glutamine side chains and primary amines. Being applied to numerous research fields from material sciences to medicine, mTG enables efficient site-specific conjugation of molecular architectures that possess suitable recognition motifs. In monoclonal antibodies, the lack of native transamidation sites is bypassed by incorporating specific peptide recognition sequences. Herein, we report a rapid and efficient mTG-catalyzed bioconjugation that relies on a novel recognition motif derived from its native substrate Streptomyces papain inhibitor (SPI_P). Improved reaction kinetics compared to commonly applied sequences were demonstrated for model peptides and for biotinylation of Her2-targeting antibody trastuzumab variants. Moreover, an antibody–drug conjugate assembled from trastuzumab that was C-terminally tagged with the novel recognition sequence revealed a higher payload-antibody ratio than the reference antibody.     

Recent Chemical Approaches for Site-Specific Conjugation of Native Antibodies: Technologies toward Next-Generation Antibody–Drug Conjugates

Antibody–drug conjugates (ADCs), which consist of three components, antibody, linker, and payload, can function as “magic bullets”. These conjugates offer the ability to target drug delivery to specific cells, based on cell-specific recognition and the binding of an antigen by a monoclonal antibody (mAb). In particular, by delivering a cytotoxic payload to cancer cells, ADCs are expected to provide a breakthrough in oncology treatments by providing a way to increase efficacy and decrease toxicity, in comparison with traditional chemotherapeutic treatments. The development of ADC therapeutics has dramatically progressed in the past decade and two ADCs have been approved and used as anticancer drugs in the clinic. However, several critical issues regarding the performance of ADCs are still being discussed and investigated. Indeed, in the past few years, several groups have reported that, changing the number and position of the drug payloads in the ADCs, affects the pharmacokinetics, drug release rates, and biological activity. The use of conventional heterogeneous conjugation methods for ADC preparation results in the drug/antibody ratio and connecting position of the payload having stochastic distributions. Therefore, it is important to investigate how these potential problems can be circumvented through site-specific conjugation. Herein, various site-specific chemical conjugation strategies with native mAbs that are currently used for the production of ADCs, including residue-selective labeling for generating ADCs, disulfide rebridging, and affinity-peptide-mediated site-specific chemical conjugation technologies, are reviewed and described.     

Drug Conjugates Such as Antibody Drug Conjugates (ADCs), Immunotoxins and Immunoliposomes Challenge Daily Clinical Practice

Drug conjugates have been studied extensively in preclinical in vitro and in vivo models but to date only a few compounds have progressed to the clinical setting. This situation is now changing with the publication of studies demonstrating a significant impact on clinical practice and highlighting the potential of this new class of targeted therapies. This review summarizes the pharmacological and molecular background of the main drug conjugation systems, namely antibody drug conjugates (ADCs), immunotoxins and immunoliposomes. All these compounds combine the specific targeting moiety of an antibody or similar construct with the efficacy of a toxic drug. The aim of this strategy is to target tumor cells specifically while sparing normal tissue, thus resulting in high efficacy and low toxicity. Recently, several strategies have been investigated in phase I clinical trials and some have entered phase III clinical development. This review provides a detailed overview of various strategies and critically discusses the most relevant achievements. Examples of the most advanced compounds include T-DM1 and brentuximab vedotin. However, additional promising strategies such as immunotoxins and immunoliposmes are already in clinical development. In summary, targeted drug delivery by drug conjugates is a new emerging class of anti-cancer therapy that may play a major role in the future.     

Design,Synthesis, and in vitro Evaluation of Multivalent Drug Linkers for High‐Drug‐Load Antibody–Drug Conjugates

A series of novel multivalent drug linkers (MDLs) containing cytotoxic agents were synthesized and conjugated to antibodies to yield highly potent antibody–drug conjugates (ADCs) with drug/antibody ratios (DARs) higher than those typically reported in the literature (10 vs. ≈4). These MDLs contain two copies of a cytotoxic agent attached to biocompatible scaffolds composed of a branched peptide core and discrete polyethylene glycol (PEG) chains to enhance solubility and decrease aggregation. These drug linkers produced well‐defined ADCs, whose DARs could be accurately determined by LC–MS. Using this approach, ADCs with significantly lower aggregation and higher DAR than those of conventional drug linker design were obtained with highly hydrophobic cytotoxic agents such as monomethyldolastatin 10 (MMAD). The in vitro potencies of the MDL‐derived conjugates matched that of ADCs of similar DAR with conventional linkers, and the potency increased proportionally with drug loading. This approach may provide a means to prepare highly potent ADCs from a broader range of drugs, including those with lower cytotoxicity or poor solubility, which otherwise limits their use for antibody–drug conjugates. This may also provide a means to further improve the potency achievable with cytotoxins currently used in ADCs.     

Controlling Toxicity of Peptide–Drug Conjugates by Different Chemical Linker Structures

The side effects of chemotherapy can be overcome by linking toxic agents to tumor‐targeting peptides with cleavable linkers. Herein, this concept is demonstrated by addressing the human Y₁ receptor (hY₁R), overexpressed in breast tumors, with analogues of the hY₁R‐preferring [F⁷,P³⁴]NPY. First, carboxytetramethylrhodamine was connected to [F⁷,P³⁴]NPY by an amide, ester, disulfide, or enzymatic linkage. Live imaging revealed hY₁R‐mediated delivery and allowed visualization of time‐dependent intracellular release. Next, the fluorophore was replaced by the toxic agent methotrexate (MTX). In addition to linkage through the amide, ester, disulfide bond, or enzymatic cleavage site, a novel disulfide/ester linker was designed and coupled to [F⁷,P³⁴]NPY by solid‐phase peptide synthesis. Internalization studies showed hY₁R subtype selective uptake, and cell viability experiments demonstrated hY₁R‐mediated toxicity that was clearly dependent on the linkage type. Fast release profiles for fluorophore‐[F⁷,P³⁴]NPY analogues correlated with high toxicities of MTX conjugates carrying the same linker types and emphasize the relevance of new structures connecting the toxophore and the carrier.     

Synthesis of Lysozyme–Metallacarborane Conjugates and the Effect of Boron Cluster Modification on Protein Structure and Function

Two complementary methods, “in solution” and “in solid state”, for the synthesis of lysozyme modified with metallacarborane (cobalt bis(dicarbollide), Co(C₂B₉H₁₁)₂^2?) were developed. As metallacarborane donors, oxonium adducts of cobalt bis(dicarbollide) and 1,4‐dioxane or tetrahydropyran were used. The physicochemical and biochemical properties of the obtained lysozyme–metallacarborane conjugates were studied for changes in secondary and tertiary structure, aggregation behavior, and biological activity. Only minor changes in primary, secondary, and tertiary protein structure were observed, caused by the single substitution of metallacarborane on lysozyme. However, the modification produced significant changes in lysozyme enzymatic activity and a tendency toward time‐ and temperature‐dependent aggregation.     

PEG Linker Improves Antitumor Efficacy and Safety of Affibody-Based Drug Conjugates

Antibody drug conjugates (ADCs) have become an important modality of clinical cancer treatment. However, traditional ADCs have some limitations, such as reduced permeability in solid tumors due to the high molecular weight of monoclonal antibodies, difficulty in preparation and heterogeneity of products due to the high drug/antibody ratio (4–8 small molecules per antibody). Miniaturized ADCs may be a potential solution, although their short circulation half-life may lead to new problems. In this study, we propose a novel design strategy for miniaturized ADCs in which drug molecules and small ligand proteins are site-specifically coupled via a bifunctional poly(ethylene glycol) (PEG) chain. The results showed that the inserted PEG chains significantly prolonged the circulation half-life but also obviously reduced the cytotoxicity of the conjugates. Compared with the conjugate Z_HER2-SMCC-MMAE (HM), which has no PEG insertion, Z_HER2-PEG4K-MMAE (HP4KM) and Z_HER2-PEG10K-MMAE (HP10KM) with 4 or 10 kDa PEG insertions have 2.5- and 11.2-fold half-life extensions and 4.5- and 22-fold in vitro cytotoxicity reductions, respectively. The combined effect leads to HP10KM having the most ideal tumor therapeutic ability at the same dosages in the animal model, and its off-target toxicity was also reduced by more than 4 times compared with that of HM. These results may indicate that prolonging the half-life is very helpful in improving the therapeutic capacity of miniaturized ADCs. In the future, the design of better strategies that can prolong half-life without affecting cytotoxicity may be useful for further improving the therapeutic potential of these molecules.     

Development and Application of a Label-Free Fluorescence Method for Determining the Composition of Gold Nanoparticle–Protein Conjugates

A method was developed for determining the composition of the conjugates between gold nanoparticles and proteins based on the intrinsic fluorescence of unbound protein molecules. The fluorescence was evaluated after separation of the conjugates from the reaction mixture by centrifugation. Gold nanoparticles obtained using the citrate technique (average diameter 24 nm) were conjugated at pH 5.4 with the following four proteins: human immunoglobulin G (IgG), bovine serum albumin (BSA), recombinant streptococcal protein G (protein G), and Kunitz-type soybean trypsin inhibitor (STI). The compositions of these conjugates were determined using the developed method. The conjugate compositions were dependent on the concentration of the added protein, and in all cases reached saturation. The equilibrium dissociation constants of the gold nanoparticle conjugates with IgG, BSA, protein G, STI in the initial section of the concentration dependence curve were 4, 6, 10, and 15 nM, respectively. Close to saturation, the corresponding values were 25, 76, 175, and 100 nM, respectively. The maximal binding capacities of a single gold nanoparticle for IgG, BSA, Protein G, and STI were 52, 90, 500, and 550, respectively, which agrees well with the hypothesis of monolayer immobilization.     

Ribonuclease A modification with poly[N-(2-hydroxypropyl)methacrylamide] copolymers: new route of synthesis and purification

The cover image is based on the Original Article Ribonuclease A modification with poly[N-(2-hydroxypropyl)methacrylamide] copolymers: new route of synthesis and purification by Marco Rito-Palomares, Calef Sanchz-Trasviña, and Karla Mayolo-Deloisa., https://doi.org/10.1002/jctb.6367 .

     

Selective Delivery of Doxorubicin to EGFR+ Cancer Cells by Cetuximab–DNA Conjugates

Doxorubicin is a hydrophobic anticancer drug that has poor selectivity, due to the lack of active targeting capability. Here, learning lessons from the success of antibody–drug conjugates, we have designed a new doxorubicin delivery system without conjugating doxorubicin to antibody directly. In this setup, cetuximab, an antibody that targets the epidermal growth factor receptor (EGFR) in cancer cells, was conjugated to a single-stranded DNA with a carefully designed sequence in a site-selective manner by using the DNA-templated protein conjugation (DTPC) method. The DNA duplex in the conjugates serves as a carrier of doxorubicin through noncovalent intercalation, and cetuximab functions as the targeting agent; this could drastically decrease systemic toxicity and potentially avoid under- or overdosing. The size of conjugates loaded with doxorubicin was about 8.77 or 16.61 nm when characterized by dynamic light scattering and atomic force microscopy, respectively. In vitro cytotoxicity and selective cancer cell killing was investigated against two EGFR⁺ cell lines (KB and MDA-MB-231) and one EGFR⁻ cell line (NIH-3T3). Cytotoxicity and flow cytometry data showed that doxorubicin loaded in cetuximab–DNA conjugates was more potent in terms of cell cytotoxicity than free doxorubicin in EGFR-overexpressed cell lines, thus suggesting that the conjugates were more selectively and easily taken up into cells, followed by rapid release of doxorubicin from the system into the cytoplasm from endosomes.     

Breaking the Fourth Wall: Modulating Quaternary Associations for Protein Regulation and Drug Discovery

Protein–protein interactions (PPIs) are an effective means to orchestrate intricate biological processes required to sustain life. Approximately 650 000 PPIs underlie the human interactome; thus underscoring its complexity and the manifold signaling outputs altered in response to changes in specific PPIs. This minireview illustrates the growing arsenal of PPI assemblies and offers insights into how these varied PPI regulatory modalities are relevant to customized drug discovery, with a focus on cancer. First, known and emerging PPIs and PPI-targeted drugs of both natural and synthetic origin are categorized. Building on these discussions, the merits of PPI-guided therapeutics over traditional drug design are discussed. Finally, a compare-and-contrast section for different PPI blockers, with gain-of-function PPI interventions, such as PROTACS, is provided.     

2nd PSL Chemical Biology Symposium (2019): At the Crossroads of Chemistry and Biology

Chemical Biology is the science of designing chemical tools to dissect and manipulate biology at different scales. It provides the fertile ground from which to address important problems of our society, such as human health and environment.     

Lipid-Drug Conjugates and Nanoparticles for the Cutaneous Delivery of Cannabidiol

Lipid nanoparticles are currently used to deliver drugs to specific sites in the body, known as targeted therapy. Conjugates of lipids and drugs to produce drug-enriched phospholipid micelles have been proposed to increase the lipophilic character of drugs to overcome biological barriers. However, their applicability at the topical level is still minimal. Phospholipid micelles are amphiphilic colloidal systems of nanometric dimensions, composed of a lipophilic nucleus and a hydrophilic outer surface. They are currently used successfully as pharmaceutical vehicles for poorly water-soluble drugs. These micelles have high in vitro and in vivo stability and high biocompatibility. This review discusses the use of lipid-drug conjugates as biocompatible carriers for cutaneous application. This work provides a metadata analysis of publications concerning the conjugation of cannabidiol with lipids as a suitable approach and as a new delivery system for this drug.     

Antibody–Bactericidal Macrocyclic Peptide Conjugates To Target Gram‐Negative Bacteria

To combat antimicrobial infections, new active molecules are needed. Antimicrobial peptides, ever abundant in nature, are a fertile starting point to develop new antimicrobial agents but suffer from low stability, low specificity, and off‐target toxicity. These drawbacks have limited their development. To overcome some of these limitations, we developed antibody–bactericidal macrocyclic peptide conjugates (ABCs), in which the antibody directs the bioactive macrocyclic peptide to the targeted Gram‐negative bacteria. We used cysteine S_NAr chemistry to synthesize and systematically study a library of large (>30‐mer) macrocyclic antimicrobial peptides (mAMPs) to discover variants with extended proteolytic stability in human serum and low hemolytic activity while maintaining bioactivity. We then conjugated, by using sortase A, these bioactive variants onto an Escherichia coli targeted monoclonal antibody. We found that these ABCs had minimized hemolytic activity and were able to kill E. coli at nanomolar concentrations. Our findings suggest macrocyclic peptides if fused to antibodies may facilitate the discovery of new agents to treat bacterial infections.     

Derivatization of Antibody Fab Fragments: A Designer Enzyme for Native Protein Modification

Bioconjugates, such as antibody–drug conjugates, have gained recent attention because of their increasing use in therapeutic and diagnostic applications. Commonly used conjugation reactions based upon chemoselective reagents exhibit a number of drawbacks: most of these reactions lack regio‐ and stereospecificity, thus resulting in loss of protein functionality due to random modifications. Enzymes provide an obvious solution to this problem, but the intrinsic (natural) substrate specificities of existing enzymes pose severe limitations to the kind of modifications that can be introduced. Here we describe the application of the novel trypsin variant trypsiligase for site‐specific modification of the C terminus of a Fab antibody fragment via a stable peptide bond. The suitability of this designed biocatalyst was demonstrated by coupling the Her2‐specific Fab to artificial functionalities of either therapeutic (PEG) or diagnostic (fluorescein) relevance. In both cases we obtained homogeneously modified Fab products bearing the artificial functionality exclusively at the desired position.     

Pharmaceutical Functionalization of Monomeric Ionic Liquid for the Preparation of Ionic Graft Polymer Conjugates

Polymerizable choline-based ionic liquid (IL), i.e., [2-(methacryloyloxy)ethyl]-trimethylammonium (TMAMA/Cl¯), was functionalized by an ion exchange reaction with pharmaceutical anions, i.e., cloxacillin (CLX¯) and fusidate (FUS¯), as the antibacterial agents. The modified biocompatible IL monomers (TMAMA/CLX¯, TMAMA/FUS¯) were copolymerized with methyl methacrylate (MMA) to prepare the graft copolymers (19–50 mol% of TMAMA units) serving as the drug (co)delivery systems. The in vitro drug release, which was driven by the exchange reaction of the pharmaceutical anions to phosphate ones in PBS medium, was observed for 44% of CLX¯ (2.7 μg/mL) and 53% of FUS¯ (3.6 μg/mL) in the single systems. Similar amounts of released drugs were detected for the dual system, i.e., 41% of CLX¯ (2.2 μg/mL) and 33% of FUS¯ (2.0 μg/mL). The investigated drug ionic polymer conjugates were examined for their cytotoxicity by MTT test, showing a low toxic effect against human bronchial epithelial cells (BEAS-2B) and normal human dermal fibroblasts (NHDF) as the normal cell lines. The satisfactory drug contents and the release profiles attained for the well-defined graft polymers with ionically bonded pharmaceuticals in the side chains make them promising drug carriers in both separate and combined drug delivery systems.     

压裂液用抗温交联剂的合成

硼酸盐是水力压裂施工中植物胶压裂液中最常用的交联剂之一,但是其交联时间非常短,耐温性能差,为了合成具有缓交联性能并且抗温性较好的交联剂,在实验室里以硼酸为起始原料,经过与有机碱反应生成硼酸酯后,在催化剂作用下与有机配位体进行络合反应,最终制备了具有缓交联、抗温性好的交联剂.考察了以硼酸酯为原料合成抗温交联剂的反应条件,得到了合成交联剂的最佳工艺.反应温度60～65 ℃,合成反应时间是3h左右,催化剂用量是0.3%(质量比),络合配位剂用量是35%(质量比).     

Protein Modification via Nitrile Oxide−Dehydroalanine Cycloaddition: Formation of Isoxazoline Ring on the Protein Backbone

Here we describe a novel catalyst-free 1,3-dipolar cycloaddition bioconjugation approach for chemical modification of proteins. The dehydroalanine (Dha)-containing protein reacts with nitrile oxides generated in situ through 1,3-dipolar cycloaddition in fully aqueous-buffered systems. This leads to the formation of a new isoxazoline ring at a pre-defined site (Dha) of the protein. Furthermore, the 1-pyrene isoxazoline-installed annexin V acts as a fluorescent probe, which successfully labels the outer cellular membranes of human cholangiocarcinoma (HuCCA-1) cells for detection of apoptosis.