Antibody drug conjugates

Antibody drug conjugates (ADCs) have emerged as a viable option in targeted delivery of highly potent cytotoxic drugs in treatment of solid tumors. At the time of writing, only two ADCs have received regulatory approval with >40 others in clinical development. The first generation ADCs suffered from a lack of specificity in amino acid site-conjugations, yielding statistically heterogeneous stoichiometric ratios of drug molecules per antibody molecule. For the second generation ADCs, however, site-specific amino acid conjugation using enzymatic ligation, introduction of unnatural amino acids, and site-specific protein engineering hold promise to alleviate some of the current technical limitations. The rapid progress in technology platforms and antibody engineering has introduced novel linkers, site-specific conjugation chemistry, and new payload candidates that could possibly be exploited in the context of ADCs. A search using the Clinical Trial Database registry (www.clinicaltrials.gov), using the keyword ‘antibody drug conjugate’, yielded ~270 hits. The main focus of this article is to present a brief overview of the recent developments and current challenges related to ADC development.     

In-depth structural characterization of Kadcyla® (ado-trastuzumab emtansine) and its biosimilar candidate

The biopharmaceutical industry has become increasingly focused on developing biosimilars as less expensive therapeutic products. As a consequence, the regulatory approval of 2 antibody-drug conjugates (ADCs), Kadcyla® and Adcetris® has led to the development of biosimilar versions by companies located worldwide. Because of the increased complexity of ADC samples that results from the heterogeneity of conjugation, it is imperative that close attention be paid to the critical quality attributes (CQAs) that stem from the conjugation process during ADC biosimilar development process. A combination of physicochemical, immunological, and biological methods are warranted in order to demonstrate the identity, purity, concentration, and activity (potency or strength) of ADC samples. As described here, we performed extensive characterization of a lysine conjugated ADC, ado-trastuzumab emtansine, and compared its CQAs between the reference product (Kadcyla®) and a candidate biosimilar. Primary amino acid sequences, drug-to-antibody ratios (DARs), conjugation sites and site occupancy data were acquired and compared by LC/MS methods. Furthermore, thermal stability, free drug content, and impurities were analyzed to further determine the comparability of the 2 ADCs. Finally, biological activities were compared between Kadcyla® and biosimilar ADCs using a cytotoxic activity assay and a HER2 binding assay. The in-depth characterization helps to establish product CQAs, and is vital for ADC biosimilars development to ensure their comparability with the reference product, as well as product safety.     

Challenges and new frontiers in analytical characterization of antibody-drug conjugates

Antibody-drug conjugates (ADCs) are a growing class of biotherapeutics in which a potent small molecule is linked to an antibody. ADCs are highly complex and structurally heterogeneous, typically containing numerous product-related species. One of the most impactful steps in ADC development is the identification of critical quality attributes to determine product characteristics that may affect safety and efficacy. However, due to the additional complexity of ADCs relative to the parent antibodies, establishing a solid understanding of the major quality attributes and determining their criticality are a major undertaking in ADC development. Here, we review the development challenges, especially for reliable detection of quality attributes, citing literature and new data from our laboratories, highlight recent improvements in major analytical techniques for ADC characterization and control, and discuss newer techniques, such as two-dimensional liquid chromatography, that have potential to be included in analytical control strategies.     

抗体偶联药物的研究进展与质量控制

抗体偶联药物（antibody-drug conjugates,ADC）因其良好的靶向性及抗癌活性目前已成为抗肿瘤抗体药物研发的新热点和重要趋势,受到越来越多的关注。ADC药物由单克隆抗体、高效应的细胞毒性物质以及连接臂三部分组成,它将抗体的靶向性与细胞毒性药物的抗肿瘤作用相结合,可以降低细胞毒性抗肿瘤药物的不良反应,提高肿瘤治疗的选择性,还能更好地应对靶向单抗的耐药性问题。与传统单抗药物相比,因其结构复杂,ADC药物质量属性分析方法的建立具有更大的难度和特殊性。对抗体偶联药物的研发现状、质量属性分析方法和挑战以及质量控制要点进行了简要介绍,为ADC药物的研究和质量控制提供参考。     

抗体偶联药物的技术现状和展望

抗体偶联药物（antibody drug conjugate,ADC）通常由抗体通过链接体与毒素小分子偶联而成,同时具备抗体的高靶向性和小分子药物的高活性,使之作为一种新兴的靶向治疗手段,在肿瘤治疗领域展现出了优秀的疗效和潜力,成为药物研发领域的新热点。目前全球已有14款ADC药物获批上市,处于临床研究阶段的ADC候选药物分子超过140个。为了进一步提高ADC药物的安全性和有效性,近年来涌现出了各种新颖的技术。本文对ADC药物分子的关键元素,包括抗体、链接体、毒素小分子以及偶联技术等方面的最新研究进展进行总结,并讨论其优缺点。期望这些讨论能够帮助增加对ADC药物研究和开发更加系统的理解,为研发出更加高效和安全的ADC药物带来一些思考。     

Antibody-drug conjugates: recent advances in conjugation and linker chemistries

The antibody-drug conjugate (ADC), a humanized or human monoclonal antibody conjugated with highly cytotoxic small molecules (payloads) through chemical linkers, is a novel therapeutic format and has great potential to make a paradigm shift in cancer chemotherapy. Thisnewantibody-based molecular platform enables selective delivery of a potent cytotoxic payload to target cancer cells, resulting in improved efficacy, reduced systemic toxicity, and preferable pharmacokinetics (PK)/ pharmacodynamics (PD) and biodistribution compared to traditional chemotherapy. Boosted by the successes of FDA-approved Adcetris® and Kadcyla®, this drug class has been rapidly growing along with about 60 ADCs currently in clinical trials. In this article, we briefly review molecular aspects of each component (the antibody, payload, and linker) of ADCs, and then mainly discuss traditional and new technologies of the conjugation and linker chemistries for successful construction of clinically effective ADCs. Current efforts in the conjugation and linker chemistries will provide greater insights into molecular design and strategies for clinically effective ADCs from medicinal chemistry and pharmacology standpoints. The development of site-specific conjugation methodologies for constructing homogeneousADCs is an especially promising path to improving ADC design, which will open the way for novel cancer therapeutics.     

Impact of linker-drug on ion exchange chromatography separation of antibody-drug conjugates

ABSTRACT

Charge variants are important attributes of monoclonal antibodies, including antibody-drug conjugates (ADCs), because charge variants can potentially influence the stability and biological activity of these molecules. Ion exchange chromatography (IEX) is widely used for charge variants analysis of mAbs and offers the feasibility of fractionation for in-depth characterization. However, the conjugated linker-drug on ADCs could potentially affect the separation performance of IEX, considering IEX separation relies on surface charge distribution of analyte and involves the interaction between analyte surface and IEX stationary phase. Here, we investigated weak cation exchange chromatography (WCX) for its application in analyzing three ADCs (two broad distribution ADCs and an ADC with controlled conjugation sites) and the 2-drug/4-drug loaded species isolated from the two broad distribution ADCs using hydrophobic interaction chromatography. The major peaks in WCX profile were characterized via fraction collection followed by capillary electrophoresis-sodium dodecyl sulfate or peptide mapping. Results suggested that both the number of drug loads and conjugation sites could impact WCX separation of an ADC. The hypothesis was that the linker drugs could interfere with the ionic interaction between its surrounding amino acids on the mAb surface and column resin, which reduced the retention of ADCs on WCX column in this study. Our results further revealed that WCX brings good selectivity towards positional isomers, but limited resolution for different drug load, which causes the peak compositions of the two broad-distribution ADCs to be highly complex. We also compared results from WCX and imaged capillary isoelectric focusing (icIEF). Results showed that separation in icIEF was less influenced by conjugated linker drugs for the ADCs studied in this work, and better alignment was found between the two techniques for the ADC with controlled conjugate sites. Overall, this work provides insights into the complexity of WCX analysis of ADCs, which should be considered during method development and sample characterization.     

珍珠龙胆石斑鱼对7种蛋白源的表观消化率

研究测定了珍珠龙胆石斑鱼(Epinephelus fuscoguttatus♀×Epinephelus lanceolatu♂)对黄粉虫粉(TMM)、黑水虻虫粉(HIM)、乙醇梭菌蛋白(CAP)、荚膜甲基球菌蛋白(MCM)、小球藻粉(CVM)、棉籽浓缩蛋白(CPC)和秘鲁鱼粉(PFM)共7种蛋白源的表观消化率(ADCs)。试验配制1组含50%鱼粉的基础饲料,而7组试验饲料按70%的基础饲料和30%的蛋白源配制而成,8组饲料都加入0.1%氧化钇(Y₂O₃)作为外源标志物。将初始平均体重为(9.95±0.50) g的杂交石斑鱼幼鱼随机分配到0.3 m³的玻璃钢桶中,每个处理组设置3个重复(桶),每桶30尾鱼。经过5d的试验饲料饲喂驯化后,每天两次用虹吸法收集粪便样本。结果表明,7种蛋白源的干物质ADCs从高至低依次为:CVM>TMM=CAP=CPC>HIM=MCM=PFM。CVM的干物质、粗蛋白和大多数氨基酸(包括蛋氨酸和苏氨酸)的ADCs最高。而HIM的干物质、粗蛋白和大多数氨基酸的ADCs低于其他组。CAP的...     

Antibody–drug conjugates as novel anti-cancer chemotherapeutics

Over the past couple of decades, antibody–drug conjugates (ADCs) have revolutionized the field of cancer chemotherapy. Unlike conventional treatments that damage healthy tissues upon dose escalation, ADCs utilize monoclonal antibodies (mAbs) to specifically bind tumour-associated target antigens and deliver a highly potent cytotoxic agent. The synergistic combination of mAbs conjugated to small-molecule chemotherapeutics, via a stable linker, has given rise to an extremely efficacious class of anti-cancer drugs with an already large and rapidly growing clinical pipeline. The primary objective of this paper is to review current knowledge and latest developments in the field of ADCs. Upon intravenous administration, ADCs bind to their target antigens and are internalized through receptor-mediated endocytosis. This facilitates the subsequent release of the cytotoxin, which eventually leads to apoptotic cell death of the cancer cell. The three components of ADCs (mAb, linker and cytotoxin) affect the efficacy and toxicity of the conjugate. Optimizing each one, while enhancing the functionality of the ADC as a whole, has been one of the major considerations of ADC design and development. In addition to these, the choice of clinically relevant targets and the position and number of linkages have also been the key determinants of ADC efficacy. The only marketed ADCs, brentuximab vedotin and trastuzumab emtansine (T-DM1), have demonstrated their use against both haematological and solid malignancies respectively. The success of future ADCs relies on improving target selection, increasing cytotoxin potency, developing innovative linkers and overcoming drug resistance. As more research is conducted to tackle these issues, ADCs are likely to become part of the future of targeted cancer therapeutics.     

In Vitro and In Vivo Evaluation of Cysteine and Site Specific Conjugated Herceptin Antibody-Drug Conjugates

Antibody drug conjugates (ADCs) are monoclonal antibodies designed to deliver a cytotoxic drug selectively to antigen expressing cells. Several components of an ADC including the selection of the antibody, the linker, the cytotoxic drug payload and the site of attachment used to attach the drug to the antibody are critical to the activity and development of the ADC.The cytotoxic drugs or payloads used to make ADCs are typically conjugated to the antibody through cysteine or lysine residues. This results in ADCs that have a heterogeneous number of drugs per antibody. The number of drugs per antibody commonly referred to as the drug to antibody ratio (DAR), can vary between 0 and 8 drugs for a IgG₁ antibody. Antibodies with 0 drugs are ineffective and compete with the ADC for binding to the antigen expressing cells. Antibodies with 8 drugs per antibody have reduced in vivo stability, which may contribute to non target related toxicities.In these studies we incorporated a non-natural amino acid, para acetyl phenylalanine, at two unique sites within an antibody against Her2/neu. We covalently attached a cytotoxic drug to these sites to form an ADC which contains two drugs per antibody.We report the results from the first direct preclinical comparison of a site specific non-natural amino acid anti-Her2 ADC and a cysteine conjugated anti-Her2 ADC. We report that the site specific non-natural amino acid anti-Her2 ADCs have superior in vitro serum stability and preclinical toxicology profile in rats as compared to the cysteine conjugated anti-Her2 ADCs. We also demonstrate that the site specific non-natural amino acid anti-Her2 ADCs maintain their in vitro potency and in vivo efficacy against Her2 expressing human tumor cell lines. Our data suggests that site specific non-natural amino acid ADCs may have a superior therapeutic window than cysteine conjugated ADCs.     

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.     

A sensitive multidimensional method for the detection,characterization, and quantification of trace free drug species in antibody-drug conjugate samples using mass spectral detection

Conjugation processes and stability studies associated with the production and shelf life of antibody-drug conjugates (ADCs) can result in free (non-conjugated) drug species. These free drug species can increase the risk to patients and reduce the efficacy of the ADC. Despite stringent purification steps, trace levels of free drug species may be present in formulated ADCs, reducing the therapeutic window. The reduction of sample preparation steps through the incorporation of multidimensional techniques has afforded analysts more efficient methods to assess trace drug species. Multidimensional methods coupling size-exclusion and reversed phase liquid chromatography with ultra-violet detection (SEC-RPLC/UV) have been reported, but offer limited sensitivity and can limit method optimization. The current study addresses these challenges with a multidimensional method that is specific, sensitive, and enables method control in both dimensions via coupling of an on-line solid phase extraction column to RPLC with mass spectral detection (SPE-RPLC/MS). The proposed method was evaluated using an antibody-fluorophore conjugate (AFC) as an ADC surrogate to brentuximab vedotin and its associated parent maleimide-val-cit-DSEA payload and the derived N-acetylcysteine adduct formed during the conjugation process. Assay sensitivity was found to be 2 orders more sensitive using MS detection in comparison to UV-based detection with a nominal limit of quantitation of 0.30 ng/mL (1.5 pg on-column). Free-drug species were present in an unadulterated ADC surrogate sample at concentrations below 7 ng/mL, levels not detectable by UV alone. The proposed SPE-RPLC/MS method provides a high degree of specificity and sensitivity in the assessment of trace free drug species and offers improved control over each dimension, enabling straightforward integration into existing or novel workflows.     

An enzymatic deconjugation method for the analysis of small molecule active drugs on antibody-drug conjugates

Antibody-drug conjugates (ADCs) are complex therapeutic agents that use the specific targeting properties of antibodies and the highly potent cytotoxicity of small molecule drugs to selectively eliminate tumor cells while limiting the toxicity to normal healthy tissues. Two critical quality attributes of ADCs are the purity and stability of the active small molecule drug linked to the ADC, but these are difficult to assess once the drug is conjugated to the antibody. In this study, we report a enzyme deconjugation approach to cleave small molecule drugs from ADCs, which allows the drugs to be subsequently characterized by reversed-phase high performance liquid chromatography. The model ADC we used in this study utilizes a valine-citrulline linker that is designed to be sensitive to endoproteases after internalization by tumor cells. We screened several proteases to determine the most effective enzyme. Among the 3 cysteine proteases evaluated, papain had the best efficiency in cleaving the small molecule drug from the model ADC. The deconjugation conditions were further optimized to achieve complete cleavage of the small molecule drug. This papain deconjugation approach demonstrated excellent specificity and precision. The purity and stability of the active drug on an ADC drug product was evaluated and the major degradation products of the active drug were identified. The papain deconjugation method was also applied to several other ADCs, with the results suggesting it could be applied generally to ADCs containing a valine-citrulline linker. Our results indicate that the papain deconjugation method is a powerful tool for characterizing the active small molecule drug conjugated to an ADC, and may be useful in ensuring the product quality, efficacy and the safety of ADCs.     

Random Walk Simulation of the MRI Apparent Diffusion Coefficient in a Geometrical Model of the Acinar Tree

Apparent diffusion coefficient (ADC) measurement in the lung using gas magnetic resonance imaging is a promising technique with potential for reflecting changes in lung microstructure. Despite some recent impressive human applications, full interpretation of ADC measures remains an elusive goal, due to a lack of detailed knowledge about the structure dependency of ADC. In an attempt to fill this gap we have performed random walk simulations in a three-dimensional geometrical model of the lung acinus, the distal alveolated sections of the lung tree accounting for ∼90% of the total lung volume. Simulations were carried out adjusting model parameters after published morphological data for the rat peripheral airway system, which predict an ADC behavior as microstructure changes with lung inflation in partial agreement with measured ADCs at different airway pressures. The approach used to relate experimental ADCs to lung microstructural changes does not make any assumption about the cause of the changes, so it could be applied to other scenarios such as chronic obstructive pulmonary disease, lung development, etc. The work presented here predicts numerically for the first time ADC values measured in the lung from independent morphological measures of lung microstructure taken at different inflation stages during the breath cycle.     

Design and characterization of homogenous antibody-drug conjugates with a drug-to-antibody ratio of one prepared using an engineered antibody and a dual-maleimide pyrrolobenzodiazepine dimer

Most strategies used to prepare homogeneous site-specific antibody-drug conjugates (ADCs) result in ADCs with a drug-to-antibody ratio (DAR) of two. Here, we report a disulfide re-bridging strategy to prepare homogeneous ADCs with DAR of one using a dual-maleimide pyrrolobenzodiazepine (PBD) dimer (SG3710) and an engineered antibody (Flexmab), which has only one intrachain disulfide bridge at the hinge. We demonstrate that SG3710 efficiently re-bridge a Flexmab targeting human epidermal growth factor receptor 2 (HER2), and the resulting ADC was highly resistant to payload loss in serum and exhibited potent anti-tumor activity in a HER2-positive gastric carcinoma xenograft model. Moreover, this ADC was tolerated in rats at twice the dose compared to a site-specific ADC with DAR of two prepared using a single-maleimide PBD dimer (SG3249). Flexmab technologies, in combination with SG3710, provide a platform for generating site-specific homogenous PBD-based ADCs with DAR of one, which have improved biophysical properties and tolerability compared to conventional site-specific PBD-based ADCs with DAR of two.     

Antibody drug conjugates of cleavable amino-alkyl and aryl maytansinoids

Natural products have been used for many medicinal purposes for centuries. Antibody drug conjugates (ADCs) have utilized this rich source of small molecule therapeutics to produce several clinically useful treatments. ADCs based on the natural product maytansine have been successful clinically. The authors further the utility of the anti-cancer natural product maytansine by developing efficacious payloads and linker-payloads for conjugating to antibodies. The success of our approach was realized in the EGFRvIII targeting ADC EGFRvIII-16. The ADC was able to regress tumors in 2 tumor models (U251/EGFRvIII and MMT/EGFRvIII). When compared to a positive control ADC, the efficacy observed was similar or improved while the isotype control ADCs had no effect.     

Novel PIKK inhibitor antibody-drug conjugates: Synthesis and anti-tumor activity

Novel neolymphostin-based antibody-drug conjugate (ADC) precursors were synthesized either through amide couplings between both cleavable and non-cleavable linkers and neolymphostin derivatives, or through Cu(I)-catalyzed acetylene-azide click cycloadditon between non-cleavable linkers and neolymphostin acetal derivatives. These precursors were site-specifically conjugated to cysteine mutant trastuzumab-A114C to provide neolymphostin-based ADCs. Preliminary in vitro data indicated that the corresponding ADCs were active against HER2-expressing tumor cell lines, thus providing a proof-of-concept for using neolymphostin as ADC-based anticancer agents.     

Site-specific antibody-drug conjugation through an engineered glycotransferase and a chemically reactive sugar

Conjugation of small molecule drugs to specific sites on the antibody molecule has been increasingly used for the generation of relatively homogenous preparations of antibody-drug conjugates (ADCs) with physicochemical properties similar or identical to those of the naked antibody. Previously a method for conjugation of small molecules to glycoproteins through existing glycans by using an engineered glycotransferase and a chemically reactive sugar as a handle was developed. Here, for the first time, we report the use of this method with some modifications to generate an ADC from a monoclonal antibody, m860, which we identified from a human naïve phage display Fab library by panning against the extracellular domain of human HER2. M860 bound to cell surface-associated HER2 with affinity comparable to that of Trastuzumab (Herceptin®), but to a different epitope. The m860ADC was generated by enzymatically adding a reactive keto-galactose to m860 using an engineered glycotransferase and conjugating the reactive m860 to aminooxy auristatin F. It exhibited potent and specific cell-killing activity against HER2 positive cancer cells, including trastuzumab-resistant breast cancer cells. This unique ADC may have utility as a potential therapeutic for HER2 positive cancers alone or in combination with other drugs. Our results also validate the keto-galactose/engineered glycotransferase method for generation of functional ADCs, which could potentially also be used for preparation of ADCs targeting other disease markers.     

Site-specific antibody-drug conjugation through an engineered glycotransferase and a chemically reactive sugar

Conjugation of small molecule drugs to specific sites on the antibody molecule has been increasingly used for the generation of relatively homogenous preparations of antibody-drug conjugates (ADCs) with physicochemical properties similar or identical to those of the naked antibody. Previously a method for conjugation of small molecules to glycoproteins through existing glycans by using an engineered glycotransferase and a chemically reactive sugar as a handle was developed. Here, for the first time, we report the use of this method with some modifications to generate an ADC from a monoclonal antibody, m860, which we identified from a human naïve phage display Fab library by panning against the extracellular domain of human HER2. M860 bound to cell surface-associated HER2 with affinity comparable to that of Trastuzumab (Herceptin®), but to a different epitope. The m860ADC was generated by enzymatically adding a reactive keto-galactose to m860 using an engineered glycotransferase and conjugating the reactive m860 to aminooxy auristatin F. It exhibited potent and specific cell-killing activity against HER2 positive cancer cells, including trastuzumab-resistant breast cancer cells. This unique ADC may have utility as a potential therapeutic for HER2 positive cancers alone or in combination with other drugs. Our results also validate the keto-galactose/engineered glycotransferase method for generation of functional ADCs, which could potentially also be used for preparation of ADCs targeting other disease markers.     

Characterization of intact antibody-drug conjugates from plasma/serum in vivo by affinity capture capillary liquid chromatography-mass spectrometry

Antibody–drug conjugates (ADCs) are designed to facilitate the targeted delivery of cytotoxic drugs to improve their tumor fighting effects and minimize systemic toxicity. However, efficacy and safety can potentially be compromised due to the release of conjugated drugs from the ADC with time while in circulation, resulting in changes in the drug-to-antibody ratio (DAR). Current understanding of this process is limited because existing methods such as immunoassays fail to distinguish ADCs with different DARs. Here we demonstrate a novel method with bead-based affinity capture and capillary liquid chromatography–mass spectrometry to allow direct measurement of drug release by quantifying DAR distributions of the ADC in plasma/serum. This method successfully identified individual intact conjugated antibody species produced due to drug loss from ADCs (e.g., an engineered site-specific anti-MUC16 THIOMAB–drug conjugate) and measured the corresponding DAR distributions in vitro and in vivo. Information obtained can provide insights into the mechanisms involved in drug loss and help to optimize ADC therapeutics. Other potential applications of the method may include characterization of posttranslational modifications, protein adducts, and immunogenicity.