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.     

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。     

Proposed mechanism of off-target toxicity for antibody–drug conjugates driven by mannose receptor uptake

Antibody–drug conjugates (ADCs) are developed with the goal of increasing compound therapeutic index by specific and targeted delivery of a toxic payload to the site of action while considerably reducing damage to normal tissues. Yet, off-target hepatic toxicities have been reported for several ADC. Locations of these off-target toxicities coincide with the reported locations of cell surface mannose receptor (MR). The relative proportion of agalactosylated glycans on the Fc domain (G0F vs. G1F and G2F components) in monoclonal antibody (mAb)–based biotherapeutics is closer to some disease state IgG rather than to a normal serum-derived immunoglobulin. The lack of the terminal galactose on a G0F glycan creates an opportunity for the mAb to interact with soluble and cell surface MRs. MR is a known multi-domain lectin that specifically binds and internalizes glycoproteins and immune complexes with relatively high G0F content and has been found on the surface of various cell types, including immune cells of myeloid lineage, endothelial cells, and hepatic and splenic sinusoids. In this review paper it is proposed that the mechanism of the off-target toxicities for ADC biotherapeutics is at least in part driven by the carbohydrates, specifically agalactosylated glycans, such as G0F, their interactions with MR and resulting glycan-derived cellular uptake of ADCs. Several case studies are reviewed presenting corroborating information.     

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.     

Design,synthesis and evaluation of novel,potent DNA alkylating agents and their antibody-drug conjugates (ADCs)

Antibody-drug conjugates (ADCs) incorporating potent indolinobenzodiazepine (IGN) DNA alkylators as the cytotoxic payload are currently undergoing clinical evaluation. The optimized design of these payloads consists of an unsymmetrical dimer possessing both an imine and an amine effectively eliminating DNA crosslinking and demonstrating improved tolerability in mice. Here we present an alternate approach to generating DNA alkylating ADCs by linking the IGN monomer with a biaryl system which has a high DNA binding affinity to potentially enhance tolerability. These BIA ADCs were found to be highly cytotoxic in vitro and demonstrated potent antitumor activity in vivo.     

Potential drugs used in the antibody–drug conjugate (ADC) architecture for cancer therapy

Cytotoxic small-molecule drugs have a major influence on the fate of antibody–drug conjugates (ADCs). An ideal cytotoxic agent should be highly potent, remain stable while linked to ADCs, kill the targeted tumor cell upon internalization and release from the ADCs, and maintain its activity in multidrug-resistant tumor cells. Lessons learned from successful and failed experiences in ADC development resulted in remarkable progress in the discovery and development of novel highly potent small molecules. A better understanding of such small-molecule drugs is important for development of effective ADCs. The present review discusses requirements making a payload appropriate for antitumor ADCs and focuses on the main characteristics of commonly-used cytotoxic payloads that showed acceptable results in clinical trials. In addition, the present study represents emerging trends and recent advances of payloads used in ADCs currently under clinical trials.     

Antibody-drug conjugates in breast cancer: Marching from HER2-overexpression into HER2-low

The recent decade has witnessed a vigorous prosper of antibody-drug conjugates (ADCs) in solid tumors including breast cancer. Integrating the specificity of monoclonal antibodies and potency of cytotoxic drugs, ADCs are capable of delivering cytotoxic agents directly to tumor cells and surrounding accomplices with heterogeneous antigen expression by exerting the distinctive bystander effect. Up till now, three ADCs (T-DM1, T-DXd and SG) have attained the official approval and stepped into clinical practices in breast cancer, with numerous promising products in the pipeline. As an unprecedented breast cancer subgroup identified following solidified drug benefit, the cognitive and therapeutic paradigm of HER2-low population which was previously thought lacking definite targets and efficacious regimens has been thoroughly rewritten by ADCs, and several encouraging achievements are expected in ongoing trials. Herein, we discuss the contrived knowledge, latest advancements and future perspectives of ADCs in HER2-overexpressing and HER2-low breast cancer.     

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。     

Novel immunoconjugates comprised of streptonigrin and 17-amino-geldanamycin attached via a dipeptide-p-aminobenzyl-amine linker system

Cytotoxic agents streptonigrin and 17-amino-geldanamycin were linked to monoclonal antibodies (mAbs), forming antibody–drug conjugates (ADCs) for antigen-mediated targeting to cancer cells. The drugs were conjugated with a linker construct that is labile to lysosomal proteases and incorporates a valine-alanine-p-aminobenzyl (PAB)-amino linkage for direct attachment to the electron-deficient amine functional groups present in both drugs. The resulting ADCs release drug following internalization into antigen-positive cancer cells. The drug linkers were conjugated to mAbs cAC10 (anti-CD30) and h1F6 (anti-CD70) via alkylation of reduced interchain disulfides to give ADCs loaded with 4 drugs/mAb. The streptonigrin ADCs were potent and immunologically specific on a panel of cancer cell lines in vitro and in a Hodgkin lymphoma xenograft model. We conclude that streptonigrin ADCs are candidates for further research, and that the novel linker system used to make them is well-suited for the conjugation of cytotoxic agents containing electron-deficient amine functional groups.     

Minor groove binder antibody conjugates employing a water soluble beta-glucuronide linker

The minor groove binder beta-glucuronide drug-linker 3 was constructed from amino CBI 1 and determined to be a substrate for Escherichia coli beta-glucuronidase (EC 3.2.1.31), resulting in facile drug release. Compound 3 was conjugated to mAbs cAC10 (anti-CD30) and h1F6 (anti-CD70) to give antibody-drug conjugates (ADCs) with potencies comparable to that of free drug 1. The ADCs were largely monomeric at intermediate loading levels (4-5drug/mAb), in contrast to highly aggregated p-aminobenzylcarbamate dipeptide-based ADCs of 1 previously reported. Significant levels of immunologic specificity were observed with cAC10-3 by comparing antigen positive versus negative cell lines and binding versus non-binding control ADCs. The water soluble beta-glucuronide linker is stable in plasma and effectively delivers drugs to target cells leading to potent cytotoxic activities.     

Antibody-drug conjugates with HER2-targeting antibodies from synthetic antibody libraries are highly potent against HER2-positive human gastric tumor in xenograft models

HER2-ECD (human epidermal growth factor receptor 2 – extracellular domain) is a prominent therapeutic target validated for treating HER2-positive breast and gastric cancer, but HER2-specific therapeutic options for treating advanced gastric cancer remain limited. We have developed antibody-drug conjugates (ADCs), comprising IgG1 linked via valine-citrulline to monomethyl auristatin E, with potential to treat HER2-positive gastric cancer in humans. The antibodies optimally selected from the ADC discovery platform, which was developed to discover antibody candidates suitable for immunoconjugates from synthetic antibody libraries designed using antibody-antigen interaction principles, were demonstrated to be superior immunoconjugate targeting modules in terms of efficacy and off-target toxicity. In comparison with the two control humanized antibodies (trastuzumab and H32) derived from murine antibody repertoires, the antibodies derived from the synthetic antibody libraries had enhanced receptor-mediated internalization rate, which could result in ADCs with optimal efficacies. Along with the ADCs, two other forms of immunoconjugates (scFv-PE38KDEL and IgG1-AL1-PE38KDEL) were used to test the antibodies for delivering cytotoxic payloads to xenograft tumor models in vivo and to cultured cells in vitro. The in vivo experiments with the three forms of immunoconjugates revealed minimal off-target toxicities of the selected antibodies from the synthetic antibody libraries; the off-target toxicities of the control antibodies could have resulted from the antibodies’ propensity to target the liver in the animal models. Our ADC discovery platform and the knowledge gained from our in vivo tests on xenograft models with the three forms of immunoconjugates could be useful to anyone developing optimal ADC cancer therapeutics.     

Antibody–drug conjugates (ADCs) for cancer therapy: Strategies,challenges, and successes

Targeted delivery of therapeutic molecules into cancer cells is considered as a promising strategy to tackle cancer. Antibody–drug conjugates (ADCs), in which a monoclonal antibody (mAb) is conjugated to biologically active drugs through chemical linkers, have emerged as a promising class of anticancer treatment agents, being one of the fastest growing fields in cancer therapy. The failure of early ADCs led researchers to explore strategies to develop more effective and improved ADCs with lower levels of unconjugated mAbs and more-stable linkers between the drug and the antibody, which show improved pharmacokinetic properties, therapeutic indexes, and safety profiles. Such improvements resulted in the US Food and Drug Administration approvals of brentuximab vedotin, trastuzumab emtansine, and, more recently, inotuzumab ozogamicin. In addition, recent clinical outcomes have sparked additional interest, which leads to the dramatically increased number of ADCs in clinical development. The present review explores ADCs, their main characteristics, and new research developments, as well as discusses strategies for the selection of the most appropriate target antigens, mAbs, cytotoxic drugs, linkers, and conjugation chemistries.     

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.     

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.     

抗体偶联药物国内研发现状及企业布局分析

抗体偶联药物(antibody-drug conjugates,ADC)由单克隆抗体和小分子细胞毒药物通过连接子偶联而成,因在血液瘤、实体瘤等临床治疗中被证明是一种极有前景的技术手段,已经成为国内外抗体药物研发的新热门方向.系统阐述了ADC药物开发及工业化生产中的核心五要素,对国内ADC的研发管线进行了梳理,并对参与A...     

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.     

Trastuzumab-monomethyl auristatin E conjugate exhibits potent cytotoxic activity in vitro against HER2-positive human breast cancer

Targeted therapy using specific monoclonal antibodies (mAbs) conjugated to chemotherapeutic agents or toxins has become one of the top priorities in cancer therapy. Antibody–drug conjugates (ADCs) are emerging as a promising strategy for cancer-targeted therapy. In this study, trastuzumab, a humanized monoclonal anti-HER2 antibody, was reduced by dithiothreitol and conjugated to the microtubule-disrupting agent monomethyl auristatin E (MMAE) through a valine-citrulline peptide linker (trastuzumab-MC-Val-Cit-PABC-MMAE [trastuzumab-vcMMAE]). After conjugation, ADCs were characterized by using UV–vis, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and flow cytometry. The antitumor activity of the ADC was evaluated in breast cancer cells in vitro. In addition, ADCs were further characterized using purification by the protein A chromatography, followed by assessment using apoptosis and MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assays. Hydrophobic interaction chromatography was used to determine drug-to-antibody ratio species of ADCs produced. Our finding showed that approximately 5.12 drug molecules were conjugated to each mAb. H2L2, H2L, HL, H2, H, and L forms of ADCs were detected in nonreducing SDS-PAGE. The binding of trastuzumab-vcMMAE to HER2-positive cells was comparable with that of the parental mAb. The MTT assay showed that our ADCs induced significant cell death in HER2-positive cells, but not in HER2-negative cells. The ADCs produced was a mixture of species, unconjugated trastuzumab (14.147%), as well as trastuzumab conjugated with two (44.868%), four (16.886%), six (13.238%), and eight (10.861%) molecules of MMAE. These results indicated that MMAE-conjugated trastuzumab significantly increases the cytotoxic activity of trastuzumab, demonstrating high affinity, specificity, and antitumor activity in vitro. Trastuzumab-vcMMAE is an effective and selective agent for the treatment of HER2-positive breast tumors.     

Galacto‐conjugation of Navitoclax as an efficient strategy to increase senolytic specificity and reduce platelet toxicity

Pharmacologically active compounds with preferential cytotoxic activity for senescent cells, known as senolytics, can ameliorate or even revert pathological manifestations of senescence in numerous preclinical mouse disease models, including cancer models. However, translation of senolytic therapies to human disease is hampered by their suboptimal specificity for senescent cells and important toxicities that narrow their therapeutic windows. We have previously shown that the high levels of senescence‐associated lysosomal β‐galactosidase (SA‐β‐gal) found within senescent cells can be exploited to specifically release tracers and cytotoxic cargoes from galactose‐encapsulated nanoparticles within these cells. Here, we show that galacto‐conjugation of the BCL‐2 family inhibitor Navitoclax results in a potent senolytic prodrug (Nav‐Gal), that can be preferentially activated by SA‐β‐gal activity in a wide range of cell types. Nav‐Gal selectively induces senescent cell apoptosis and has a higher senolytic index than Navitoclax (through reduced activation in nonsenescent cells). Nav‐Gal enhances the cytotoxicity of standard senescence‐inducing chemotherapy (cisplatin) in human A549 lung cancer cells. Concomitant treatment with cisplatin and Nav‐Gal in vivo results in the eradication of senescent lung cancer cells and significantly reduces tumour growth. Importantly, galacto‐conjugation reduces Navitoclax‐induced platelet apoptosis in human and murine blood samples treated ex vivo, and thrombocytopenia at therapeutically effective concentrations in murine lung cancer models. Taken together, we provide a potentially versatile strategy for generating effective senolytic prodrugs with reduced toxicities.     

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.     

ADC药物的抗体组成及其作用靶点研究进展*

抗体药物偶联物(antibody-drug conjugates,ADC)是一类由单克隆抗体和小分子细胞毒性药物通过连接子偶联而成的新型生物治疗药物。与传统的细胞毒药物相比,ADC具有靶向性强、毒副作用小等优势,在临床上展现较好的治疗潜力。其中,抗体部分通过与肿瘤细胞表面的靶向抗原结合,精准地将小分子细胞毒性药物递送至肿瘤部位,从而实现肿瘤特异性杀伤效果,是影响ADC疗效的核心要素之一。对近年来ADC药物中抗体的组成及其作用靶点的研究进展进行了综述。