Methods for site-specific drug conjugation to antibodies

Antibody drug conjugates (ADCs) are an emerging class of targeted therapeutics with the potential to improve therapeutic index over traditional chemotherapy. Drugs and linkers have been the current focus of ADC development, in addition to antibody and target selection. Recently, however, the importance of conjugate homogeneity has been realized. The current methods for drug attachment lead to a heterogeneous mixture, and some populations of that mixture have poor in vivo performance. New methods for site-specific drug attachment lead to more homogeneous conjugates and allow control of the site of drug attachment. These subtle improvements can have profound effects on in vivo efficacy and therapeutic index. This review examines current methods for site-specific drug conjugation to antibodies, and compares in vivo results with their non-specifically conjugated counterparts. The apparent improvement in pharmacokinetics and the reduced off target toxicity warrant further development of this site-specific modification approach for future ADC development.     

Site-specific antibody drug conjugates for cancer therapy

Antibody therapeutics have revolutionized the treatment of cancer over the past two decades. Antibodies that specifically bind tumor surface antigens can be effective therapeutics; however, many unmodified antibodies lack therapeutic activity. These antibodies can instead be applied successfully as guided missiles to deliver potent cytotoxic drugs in the form of antibody drug conjugates (ADCs). The success of ADCs is dependent on four factors—target antigen, antibody, linker, and payload. The field has made great progress in these areas, marked by the recent approval by the US Food and Drug Administration of two ADCs, brentuximab vedotin (Adcetris^®) and ado-trastuzumab emtansine (Kadcyla^®). However, the therapeutic window for many ADCs that are currently in pre-clinical or clinical development remains narrow and further improvements may be required to enhance the therapeutic potential of these ADCs. Production of ADCs is an area where improvement is needed because current methods yield heterogeneous mixtures that may include 0–8 drug species per antibody molecule. Site-specific conjugation has been recently shown to eliminate heterogeneity, improve conjugate stability, and increase the therapeutic window. Here, we review and describe various site-specific conjugation strategies that are currently used for the production of ADCs, including use of engineered cysteine residues, unnatural amino acids, and enzymatic conjugation through glycotransferases and transglutaminases. In addition, we also summarize differences among these methods and highlight critical considerations when building next-generation ADC therapeutics.     

Anti‐EGFR antibody‐drug conjugate for triple‐negative breast cancer therapy

Triple‐negative breast cancers (TNBCs) are highly aggressive, metastatic and recurrent. Cytotoxic chemotherapies with limited clinical benefits and severe side effects are the standard therapeutic strategies, but, to date, there is no efficacious targeted therapy. Literature and our data showed that epidermal growth factor receptor (EGFR) is overexpressed on TNBC cell surface and is a promising oncological target. The objective of this study was to develop an antibody‐drug conjugate (ADC) to target EGFR⁺ TNBC and deliver high‐potency drug. First, we constructed an ADC by conjugating anti‐EGFR monoclonal antibody with mertansine which inhibits microtubule assembly via linker Sulfo‐SMCC. Second, we confirmed the TNBC‐targeting specificity of anti‐EGFR ADC by evaluating its surface binding and internalization in MDA‐MB‐468 cells and targeting to TNBC xenograft in subcutaneous mouse mode. The live‐cell and live‐animal imaging with confocal laser scanning microscopy and In Vivo Imaging System (IVIS) confirmed the TNBC‐targeting. Finally, both in vitro toxicity assay and in vivo anti‐cancer efficacy study in TNBC xenograft models showed that the constructed ADC significantly inhibited TNBC growth, and the pharmacokinetics study indicated its high circulation stability. This study indicated that the anti‐EGFR ADC has a great potential to against TNBC.     

Development of rabbit monoclonal and polyclonal antibodies for detection of site-specific histone modifications and their application in analyzing overall modification levels

In addition to DNA sequence information,site-specific histone modifications are another important determinant ofgene expression in a eukaryotic organism.We selected four modification sites in common histones that are known tosignificantly impact chromatin function and generated monoclonal or polyclonal antibodies that recognize each of thosesite-specific modifications.We used these antibodies to demonstrate that the site-specific histone modification levelsremain relatively constant in different organs of the same organism.We also compared the levels of selected histonemodifications among several representative organisms and found that site-specific modifications are highly variable amongdifferent organisms,providing new insight into the evolutionary divergence of specific histone modifications.     

Targeting of herbal bioactives through folate receptors: a novel concept to enhance intracellular drug delivery in cancer therapy

Targeted drug delivery through folate receptor (FR) has emerged as a most biocompatible, target oriented, and non-immunogenic cargoes for the delivery of anticancer drugs. FRs are highly overexpressed in many tumor cells (like ovarian, lung, breast, kidney, brain, endometrial, and colon cancer), and targeting them through conjugates bearing specific ligand with encapsulated nanodrug moiety is undoubtedly, a promising approach toward tumor targeting. Folate, being an endogenous ligand, can be exploited well to affect various cellular events occurring during the progress of tumor, in a more natural and definite way. Thus, the aim of the review lies in summarizing the advancements taken place in the drug delivery system of different therapeutics through FRs and to refine its role as an endogenous ligand, in targeting of synthetic as well as natural bioactives. The review also provides an update on the patents received on the folate-based drug delivery system.     

Development of an E. coli strain for cell-free ADC manufacturing

Recent advances in cell-free protein synthesis have enabled the folding and assembly of full-length antibodies at high titers with extracts from prokaryotic cells. Coupled with the facile engineering of the Escherichia coli translation machinery, E. coli based in vitro protein synthesis reactions have emerged as a leading source of IgG molecules with nonnatural amino acids incorporated at specific locations for producing homogeneous antibody–drug conjugates (ADCs). While this has been demonstrated with extract produced in batch fermentation mode, continuous extract fermentation would facilitate supplying material for large-scale manufacturing of protein therapeutics. To accomplish this, the IgG-folding chaperones DsbC and FkpA, and orthogonal tRNA for nonnatural amino acid production were integrated onto the chromosome with high strength constitutive promoters. This enabled co-expression of all three factors at a consistently high level in the extract strain for the duration of a 5-day continuous fermentation. Cell-free protein synthesis reactions with extract produced from cells grown continuously yielded titers of IgG containing nonnatural amino acids above those from extract produced in batch fermentations. In addition, the quality of the synthesized IgGs and the potency of ADC produced with continuously fermented extract were indistinguishable from those produced with the batch extract. These experiments demonstrate that continuous fermentation of E. coli to produce extract for cell-free protein synthesis is feasible and helps unlock the potential for cell-free protein synthesis as a platform for biopharmaceutical production.     

Effects of antibody,drug and linker on the preclinical and clinical toxicities of antibody-drug conjugates

Antibody-drug conjugates (ADCs) represent a new class of cancer therapeutics. Their design involves a tumor-specific antibody, a linker and a cytotoxic payload. They were designed to allow specific targeting of highly potent cytotoxic agents to tumor cells whilst sparing normal cells. Frequent toxicities that may be driven by any of the components of an ADC have been reported. There are currently more than 50 ADCs in active clinical development, and a further ～20 that have been discontinued. For this review, the reported toxicities of ADCs were analysed, and the mechanisms for their effects are explored in detail. Methods to reduce toxicities, including dosing strategies and drug design, are discussed. The toxicities reported for active and discontinued drugs are important to drive the rational design and improve the therapeutic index of ADCs of the future.     

Modular amino acids‐based chiral ligands for copper‐catalyzed enantioselective conjugation addition of diethylzinc to cyclic enones

New amino acid‐based modular chiral ligands were readily synthesized and used to catalyze the asymmetric conjugate addition of Et₂Zn to various cyclic enones in the presence of a variety of copper sources. Moderately high ee of up to 72% were obtained using ligand (S)‐ 1e under mild conditions. Chirality 2011. © 2010 Wiley‐Liss, Inc.     

抗体与小分子药物一体化的完美“联姻”——解析抗体-药物偶联物之定点偶联

基于单抗的靶向疗法已成为各种癌症的重要治疗手段,抗体与小分子药物一体化的联姻——抗体—药物偶联物（antibody-drug conjugates,ADC）新药获得了突破性进展。传统ADC是将药物与抗体的赖氨酸残基或链间二硫键还原而产生的半胱氨酸残基相偶联 而形成,其稳定性差,易发生聚集,且其中药物易脱落而产生非治疗性毒副作用。而应用近年发展起来的定点偶联技术所获ADC,除均 一性好外,还保留了母体单抗的药动学性质,毒副作用也远低于具有相同偶联比的传统ADC,极有可能发展成为新一代重磅药物。综述 4种ADC定点偶联方法。     

Influence of disulfide bond isoforms on drug conjugation sites in cysteine-linked IgG2 antibody-drug conjugates

Cysteine-linked antibody-drug conjugates (ADCs) produced from IgG2 monoclonal antibodies (mAbs) are more heterogeneous than ADCs generated from IgG1 mAbs, as IgG2 ADCs are composed of a wider distribution of molecules, typically containing 0 – 12 drug-linkers per antibody. The three disulfide isoforms (A, A/B, and B) of IgG2 antibodies confer differences in solvent accessibilities of the interchain disulfides and contribute to the structural heterogeneity of cysteine-linked ADCs. ADCs derived from either IgG2-A or IgG2-B mAbs were compared to better understand the role of disulfide isoforms on attachment sites and distribution of conjugated species. Our characterization of these ADCs demonstrated that the disulfide configuration affects the kinetics of disulfide bond reduction, but has minimal effect on the primary sites of reduction. The IgG2-A mAbs yielded ADCs with higher drug-to-antibody ratios (DARs) due to the easier reduction of its interchain disulfides. However, hinge-region cysteines were the primary conjugation sites for both IgG2-A and IgG2-B mAbs.     

Antibodies directed against receptor tyrosine kinases

Approximately 30 therapeutic monoclonal antibodies have already been approved for cancers and inflammatory diseases, and monoclonal antibodies continue to be one of the fastest growing classes of therapeutic molecules. Because aberrant signaling by receptor tyrosine kinases (RTKs) is a commonly observed factor in cancer, most of the subclasses of RTKs are being extensively studied as potential targets for treating malignancies. The first two RTKs that have been targeted by antibody therapy, with five currently marketed antibodies, are the growth factor receptors EGFR and HER2. However, due to systemic side effects, refractory patients and the development of drug resistance, these treatments are being challenged by emerging therapeutics. This review examines current monoclonal antibody therapies against RTKs. After an analysis of agents that have already been approved, we present an analysis of antibodies in clinical development that target RTKs. Finally, we highlight promising RTKs that are emerging as new oncological targets for antibody-based therapy.     

Insights from native mass spectrometry approaches for top- and middle- level characterization of site-specific antibody-drug conjugates

Antibody-drug conjugates (ADCs) have emerged as a family of compounds with promise as efficient immunotherapies. First-generation ADCs were generated mostly via reactions on either lysine side-chain amines or cysteine thiol groups after reduction of the interchain disulfide bonds, resulting in heterogeneous populations with a variable number of drug loads per antibody. To control the position and the number of drug loads, new conjugation strategies aiming at the generation of more homogeneous site-specific conjugates have been developed. We report here the first multi-level characterization of a site-specific ADC by state-of-the-art mass spectrometry (MS) methods, including native MS and its hyphenation to ion mobility (IM-MS). We demonstrate the versatility of native MS methodologies for site-specific ADC analysis, with the unique ability to provide several critical quality attributes within one single run, along with a direct snapshot of ADC homogeneity/heterogeneity without extensive data interpretation. The capabilities of native IM-MS to directly access site-specific ADC conformational information are also highlighted. Finally, the potential of these techniques for assessing an ADC's heterogeneity/homogeneity is illustrated by comparing the analytical characterization of a site-specific DAR4 ADC to that of first-generation ADCs. Altogether, our results highlight the compatibility, versatility, and benefits of native MS approaches for the analytical characterization of all types of ADCs, including site-specific conjugates. Thus, we envision integrating native MS and IM-MS approaches, even in their latest state-of-the-art forms, into workflows that benchmark bioconjugation strategies.     

Production of soluble and active microbial transglutaminase in Escherichia coli for site‐specific antibody drug conjugation

Applications of microbial transglutaminase (mTGase) produced from Streptomyces mobarensis (S. mobarensis) were recently extended from food to pharmaceutical industry. To use mTGase for clinical applications, like generation of site specific antibody drug conjugates, it would be beneficial to manufacture mTGase in Escherichia coli (E. coli). To date, attempts to express recombinant soluble and active S. mobarensis mTGase have been largely unsuccessful. mTGase from S. mobarensis is naturally expressed as proenzyme and stepwise proteolytically processed into its active mature form outside of the bacterial cell. The pro‐domain is essential for correct folding of mTGase as well as for inhibiting activity of mTGase inside the cell. Here, we report a genetically modified mTGase that has full activity and can be expressed at high yields in the cytoplasm of E. coli. To achieve this we performed an alanine‐scan of the mTGase pro‐domain and identified mutants that maintain its chaperone function but destabilize the cleaved pro‐domain/mTGase interaction in a temperature dependent fashion. This allows proper folding of mTGase and keeps the enzyme inactive during expression at 20°C, but results in full activity when shifted to 37°C due to loosen domain interactions. The insertion of the 3C protease cleavage site together with pro‐domain alanine mutants Tyr14, Ile24, or Asn25 facilitate high yields (30–75 mg/L), and produced an enzyme with activity identical to wild type mTGase from S. mobarensis. Site‐specific antibody drug conjugates made with the E .coli produced mTGase demonstrated identical potency in an in vitro cell assay to those made with mTGase from S. mobarensis.     

Development of a sensitive screening method for selecting monoclonal antibodies to be internalized by cells

Antibody–drug conjugates (ADCs), drugs developed by conjugation of an anticancer agent to a monoclonal antibody (mAb), have lately attracted attention in cancer therapy because ADCs can directly bind cancer cells and kill them. Although mAbs for ADCs must be internalized by the target cells, few methods are available for screening mAbs for their ability to be internalized by cells. We have developed a recombinant protein, termed DT3C, which consists of diphtheria toxin (DT) lacking the receptor-binding domain but containing the C1, C2, and C3 domains of Streptococcus protein G (3C). When a mAb–DT3C conjugate, which functions in vitro like an ADC, reduces the viability of cancer cells, the mAb being tested must have been internalized by the target cells. DT3C can thus be a tool to identify efficiently and easily mAbs that can be internalized by cells, thereby enhancing the development of promising ADCs.     

单克隆抗体药物与血液系统恶性肿瘤的治疗

单克隆抗体凭借其特异性强、副作用较小的优点,越来越广泛地应用于疾病的诊断与治疗。单克隆抗体药物在血液系统恶性肿瘤的治疗中也发挥了重要作用。目前,经美国食品与药品管理局(FDA)批准用于治疗血液系统恶性肿瘤的单克隆抗体药物已有六种,在临床取得良好的治疗效果。单克隆抗体药物主要通过对肿瘤细胞的直接杀伤作用、抗体依赖性细胞介导的细胞毒性反应(ADCC)、补体依赖性细胞毒性反应(CDC)和改变信号通路等机制达到治疗肿瘤的效果。另外,将单克隆抗体与放射性核素、化疗药物和毒素等偶联,用于肿瘤等疾病的靶向治疗研究,成为生物治疗领域的热点。该文对近年来国际上用于血液系统恶性肿瘤治疗的单克隆抗体药物进行了概括和总结,讨论了治疗性单克隆抗体药物存在的问题和应用前景。     

Targeting cell signaling pathways for drug discovery: an old lock needs a new key

In this age of targeted therapy, the failure of most current drug-discovery efforts to yield safe, effective, and inexpensive drugs has generated widespread concern. Successful drug development has been stymied by a general focus on target selection rather than clinical safety and efficacy. The very process of validating the targets themselves is inefficient and in many cases leads to drugs having poor efficacy and undesirable side effects. Indeed, some rationally designed drugs (e.g., inhibitors of receptor tyrosine kinases, tumor necrosis factor (TNF), cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF), bcr-abl, and proteasomes) are ineffective against cancers and other inflammatory conditions and produce serious side effects. Since any given cancer carries mutations in an estimated 300 genes, this raises an important question about how effective these targeted therapies can ever be against cancer. Thus, it has become necessary to rethink drug development strategies. This review analyzes the shortcomings of rationally designed target-specific drugs against cancer cell signaling pathways and evaluates the available options for future drug development.     

双特异性抗体在肿瘤治疗中的研究

双特异性抗体(bispecific antibody,BsAb)有两个抗原结合位点,其中一个位点可与靶细胞表面抗原结合,另一个位点则可与载荷物(如效应细胞,分子等)结合。将BsAb应用于肿瘤治疗,发挥抗肿瘤效应的思想已有二十多年历史,随着对效应细胞生物学了解的加深和抗体工程的飞速发展,各种形式的BsAb相继出现,多种BsAb药物已进入临床初期试验或治疗使用阶段。本文就BsAb的各种新形式及其在肿瘤治疗中的应用新进展作简要概述。     

Tailor-making of protein drugs by polymer conjugation for tumor targeting: A brief review on smancs

Chemical conjugation of poly(styrene-co-maleic acid) to an antitumor protein (neocarzinostatin) yielded an entirely new derivative designated as smancs (polystyrene-maleic acid conjugated neocarzinostatin). The purpose of the modification was to improve its pharmacological properties; the resulting conjugate exhibited much higher tumoritropism and lymphotropism, enhancedin vivo stability (about ten times), higher chemotherapeutic index (lower toxicity), and decreased antigenicity. Another advantage associated with this molecular engineering was an increased hydrophobicity. By this character it was solubilized in lipid contrast medium Lipiodol^R, which facilitated highly sensitive detection under x-ray-accompanying selective anticancer effectin situ. Three factors were responsible for such improvements: molecular size, hydrophobicity, and polyanionic nature. Most of the known drugs so far used as therapeutic agents are less than 1000 daltons, and those with larger molecular weights have been explored much less extensively. By polymer conjugation, the size of the drug can be extended to about 15,000 daltons (macromolecular therapeutics). The most outstanding characteristic of smancs (16,000 daltons) was the tumoritropicity, which may be a result of the highly developed neovasculature of solid tumors. Smancs as a prototype drug thus appears to lead the way in cancer drug targeting.