Polatuzumab vedotin or pinatuzumab vedotin plus rituximab in patients with relapsed or refractory non-Hodgkin lymphoma: final results from a phase 2 randomised study (ROMULUS).
Morschhauser Franck,Flinn Ian W,Advani Ranjana,Sehn Laurie H,Diefenbach Catherine,Kolibaba Kathryn,Press Oliver W,Salles Gilles,Tilly Hervé,Chen Andy I,Assouline Sarit,Cheson Bruce D,Dreyling Martin,Hagenbeek Anton,Zinzani Pier Luigi,Jones Surai,Cheng Ji,Lu Dan,Penuel Elicia,Hirata Jamie,Wenger Michael,Chu Yu-Waye,Sharman Jeff
The Lancet. Haematology
BACKGROUND:Antibody-drug conjugates (ADCs) polatuzumab vedotin (pola) and pinatuzumab vedotin (pina) showed clinical activity and tolerability in phase 1 trials. The aim of this multicentre, open-label, phase 2 study was to compare rituximab plus pola (R-pola) or pina (R-pina) in patients with relapsed or refractory diffuse large B-cell lymphoma and follicular lymphoma. METHODS:In this phase 2 randomised study at 39 investigational sites in six countries, patients were randomly assigned (1:1), by use of a dynamic hierarchical randomisation scheme, to receive R-pola or R-pina (375 mg/m rituximab plus 2·4 mg/kg ADCs) every 21 days until disease progression or unacceptable toxicity up to 1 year. Treatment allocations were not masked to the investigator, patients or sponsor after the patients were enrolled and randomly assigned. The primary objectives were safety and tolerability, and antitumour response. The study is registered with ClinicalTrials.gov, number NCT01691898, and is closed to accrual. FINDINGS:81 patients with diffuse large B-cell lymphoma and 42 with follicular lymphoma were recruited between Sept 27, 2012, and Oct 10, 2013, and were assigned to treatment. 81 patients with diffuse large B-cell lymphoma and 41 patients with follicular lymphoma were eligible for analysis. Of the 42 patients with diffuse large B-cell lymphoma who received R-pina, 25 (60%, 95% CI 43-74) achieved an objective response and 11 (26%, 95% CI 14-42) achieved a complete response. Of the 39 patients in this cohort who received R-pola, 21 (54%, 95% CI 37-70) achieved an objective response, and eight (21%, 95% CI 9-36) achieved a complete response. Of the 21 patients in the follicular lymphoma cohort who received R-pina, 13 (62%, 95% CI 38-82) achieved an objective response, and one (5%, 95% CI 0·1-24) achieved a complete response. Of the 20 patients in this cohort who received R-pola, 14 (70%, 95% CI 46-88) achieved an objective response, and nine (45%, 95% CI 23-68) achieved a complete response. In the diffuse large B-cell lymphoma cohort, grade 3-5 adverse events occurred in 33 (79%) of 42 patients receiving R-pina (most common were neutropenia [29%] and hyperglycaemia [10%]; nine [21%] grade 5 adverse events, five of which were infection-related), and in 30 (77%) of 39 patients receiving R-pola (most common were neutropenia [23%], anaemia [8%] and diarrhoea [8%]; no grade 5 adverse events). In the follicular lymphoma cohort, grade 3-5 adverse events occurred in 13 (62%) of 21 patients receiving R-pina (most common were neutropenia [29%] and hyperglycaemia [14%]; no grade 5 adverse events) and in ten (50%) of 20 patients receiving R-pola (most common were neutropenia [15%] and diarrhoea [10%]; one grade 5 adverse event). INTERPRETATION:R-pina and R-pola are potential treatment options in patients with relapsed or refractory diffuse large B-cell lymphoma and follicular lymphoma. Pola was selected by the study funder for further development in non-Hodgkin lymphoma, partly because of longer durations of response than pina, and an overall benefit-risk favouring R-pola. FUNDING:F Hoffmann-La Roche.
Chemoenzymatic synthesis of glycoengineered IgG antibodies and glycosite-specific antibody-drug conjugates.
Tang Feng,Wang Lai-Xi,Huang Wei
Glycoengineered therapeutic antibodies and glycosite-specific antibody-drug conjugates (gsADCs) have generated great interest among researchers because of their therapeutic potential. Endoglycosidase-catalyzed in vitro glycoengineering technology is a powerful tool for IgG Fc (fragment cystallizable) N-glycosylation remodeling. In this protocol, native heterogeneously glycosylated IgG N-glycans are first deglycosylated with a wild-type endoglycosidase. Next, a homogeneous N-glycan substrate, presynthesized as described here, is attached to the remaining N-acetylglucosamine (GlcNAc) of IgG, using a mutant endoglycosidase (also called endoglycosynthase) that lacks hydrolytic activity but possesses transglycosylation activity for glycoengineering. Compared with in vivo glycoengineering technologies and the glycosyltransferase-enabled in vitro engineering method, the current approach is robust and features quantitative yield, homogeneous glycoforms of produced antibodies and ADCs, compatibility with diverse natural and non-natural glycan structures, convenient exploitation of native IgG as the starting material, and a well-defined conjugation site for antibody modifications. Potential applications of this method cover a broad scope of antibody-related research, including the development of novel glycoengineered therapeutic antibodies with enhanced efficacy, site-specific antibody-drug conjugation, and site-specific modification of antibodies for fluorescent labeling, PEGylation, protein cross-linking, immunoliposome formation, and so on, without loss of antigen-binding affinity. It takes 5-8 d to prepare the natural or modified N-glycan substrates, 3-4 d to engineer the IgG N-glycosylation, and 2-5 d to synthesize the small-molecule toxins and prepare the gsADCs.
Improvement and extension of anti-EGFR targeting in breast cancer therapy by integration with the Avidin-Nucleic-Acid-Nano-Assemblies.
Roncato Francesco,Rruga Fatlum,Porcù Elena,Casarin Elisabetta,Ronca Roberto,Maccarinelli Federica,Realdon Nicola,Basso Giuseppe,Alon Ronen,Viola Giampietro,Morpurgo Margherita
Nowadays, personalized cancer therapy relies on small molecules, monoclonal antibodies, or antibody-drug conjugates (ADC). Many nanoparticle (NP)-based drug delivery systems are also actively investigated, but their advantage over ADCs has not been demonstrated yet. Here, using the Avidin-Nucleic-Acid-Nano-Assemblies (ANANAS), a class of polyavidins multifuctionalizable with stoichiometric control, we compare quantitatively anti-EGFR antibody(cetuximab)-targeted NPs to the corresponding ADC. We show that ANANAS tethering of cetuximab promotes a more efficient EGFR-dependent vesicle-mediated internalization. Cetuximab-guided ANANAS carrying doxorubicin are more cytotoxic in vitro and much more potent in vivo than the corresponding ADC, leading to 43% tumor reduction at low drug dosage (0.56 mg/kg). Advantage of cetuximab-guided ANANAS with respect to the ADC goes beyond the increase in drug-to-antibody ratio. Even if further studies are needed, we propose that NP tethering could expand application of the anti-EGFR antibody to a wider number of cancer patients including the KRAS-mutated ones, currently suffering from poor prognosis.
Gene-edited stem cells enable CD33-directed immune therapy for myeloid malignancies.
Borot Florence,Wang Hui,Ma Yan,Jafarov Toghrul,Raza Azra,Ali Abdullah Mahmood,Mukherjee Siddhartha
Proceedings of the National Academy of Sciences of the United States of America
Antigen-directed immunotherapies for acute myeloid leukemia (AML), such as chimeric antigen receptor T cells (CAR-Ts) or antibody-drug conjugates (ADCs), are associated with severe toxicities due to the lack of unique targetable antigens that can distinguish leukemic cells from normal myeloid cells or myeloid progenitors. Here, we present an approach to treat AML by targeting the lineage-specific myeloid antigen CD33. Our approach combines CD33-targeted CAR-T cells, or the ADC Gemtuzumab Ozogamicin with the transplantation of hematopoietic stem cells that have been engineered to ablate CD33 expression using genomic engineering methods. We show highly efficient genetic ablation of CD33 antigen using CRISPR/Cas9 technology in human stem/progenitor cells (HSPC) and provide evidence that the deletion of CD33 in HSPC doesn't impair their ability to engraft and to repopulate a functional multilineage hematopoietic system in vivo. Whole-genome sequencing and RNA sequencing analysis revealed no detectable off-target mutagenesis and no loss of functional p53 pathways. Using a human AML cell line (HL-60), we modeled a postremission marrow with minimal residual disease and showed that the transplantation of CD33-ablated HSPCs with CD33-targeted immunotherapy leads to leukemia clearance, without myelosuppression, as demonstrated by the engraftment and recovery of multilineage descendants of CD33-ablated HSPCs. Our study thus contributes to the advancement of targeted immunotherapy and could be replicated in other malignancies.
Nanoparticle conjugates of a highly potent toxin enhance safety and circumvent platinum resistance in ovarian cancer.
Qi Ruogu,Wang Yongheng,Bruno Peter M,Xiao Haihua,Yu Yingjie,Li Ting,Lauffer Sam,Wei Wei,Chen Qixian,Kang Xiang,Song Haiqin,Yang Xi,Huang Xing,Detappe Alexandre,Matulonis Ursula,Pepin David,Hemann Michael T,Birrer Michael J,Ghoroghchian P Peter
Advanced-stage epithelial ovarian cancers are amongst the most difficult to treat tumors and have proven to be refractory to most cytotoxic, molecularly targeted, or immunotherapeutic approaches. Here, we report that nanoparticle-drug conjugates (NDCs) of monomethyl auristatin E (MMAE) significantly increase loading on a per-vehicle basis as compared to antibody-drug conjugates (ADCs). Their intraperitoneal administration enabled triggered release of the active MMAE toxin to inhibit tumor growth and to extend animal survival to >90 days in a cell-line xenograft model of disseminated ovarian cancer. In a patient-derived xenograft model of advanced-stage and platinum-resistant ovarian cancer, an MMAE-based NDC doubled the duration of tumor growth inhibition as compared to cisplatin. NDCs of highly potent toxins thus introduce a translatable platform that may be exploited to maximize the safety and efficacy of cytotoxic chemotherapies, combining the best features of ADCs with those of nanoparticle-based therapeutics.
Antibody drug conjugates for treatment of breast cancer: Novel targets and diverse approaches in ADC design.
Trail Pamela A,Dubowchik Gene M,Lowinger Timothy B
Pharmacology & therapeutics
Breast cancer is a heterogeneous group of malignancies with a spectrum of molecular subtypes, pathologies and outcomes that together comprise the most common non-cutaneous cancer in women. Currently, over 80% of breast cancer patients are diagnosed at relatively early stages of disease where there are encouraging data on outcomes and long term survival. However, there is currently no curative option for those patients with metastatic disease and there is a substantial medical need to identify effective and safe treatment options for these patients. One approach to improve cancer therapy is by designing therapeutics directed against targets with differential levels of expression on malignant versus normal cells with the goal of improving tumor selectivity and reducing damage to normal tissues. Antibody drug conjugates (ADCs) are a rapidly evolving therapeutic class that exploits the target-selectivity of monoclonal antibodies (MAbs) to deliver cytotoxic drugs to antigen-expressing cells (Lambert & Morris, 2017; Senter, 2009; Thomas, Teicher, & Hassan, 2016; Trail, 2013). The regulatory approval of ADCs for both hematologic malignancies (brentuximab vedotin) (Younes et al., 2010) and solid tumors (ado-trastuzumab emtansine) (Amiri-Kordestani et al., 2014; Verma et al., 2012) clearly demonstrates the clinical potential of ADCs. This review will focus on targets under consideration for breast cancer directed ADCs and on the technology modifications being considered to improve ADC efficacy and safety.
Antibody-Drug Conjugates for Cancer Treatment.
Lambert John M,Berkenblit Anna
Annual review of medicine
The concept of exploiting the specific binding properties of monoclonal antibodies as a mechanism for selective delivery of cytotoxic agents to tumor cells is an attractive solution to the challenge of increasing the therapeutic index of cell-killing agents for treating cancer. All three parts of an antibody-drug conjugate (ADC)-the antibody, the cytotoxic payload, and the linker chemistry that joins them together-as well as the biologic properties of the cell-surface target antigen are important in designing an effective anticancer agent. The approval of brentuximab vedotin in 2011 for treating relapsed Hodgkin's lymphoma and systemic anaplastic large cell lymphoma, and the approval of ado-trastuzumab emtansine in 2013 for treating HER2-positive metastatic breast cancer, have sparked vigorous research in the field, with >65 ADCs currently in clinical evaluation. This review highlights the ADCs that are approved for marketing, in pivotal clinical trials, or in at least phase II clinical development for treating both hematologic malignancies and solid tumors.
Chemically triggered drug release from an antibody-drug conjugate leads to potent antitumour activity in mice.
Rossin Raffaella,Versteegen Ron M,Wu Jeremy,Khasanov Alisher,Wessels Hans J,Steenbergen Erik J,Ten Hoeve Wolter,Janssen Henk M,van Onzen Arthur H A M,Hudson Peter J,Robillard Marc S
Current antibody-drug conjugates (ADCs) target internalising receptors on cancer cells leading to intracellular drug release. Typically, only a subset of patients with solid tumours has sufficient expression of such a receptor, while there are suitable non-internalising receptors and stroma targets. Here, we demonstrate potent therapy in murine tumour models using a non-internalising ADC that releases its drugs upon a click reaction with a chemical activator, which is administered in a second step. This was enabled by the development of a diabody-based ADC with a high tumour uptake and very low retention in healthy tissues, allowing systemic administration of the activator 2 days later, leading to efficient and selective activation throughout the tumour. In contrast, the analogous ADC comprising the protease-cleavable linker used in the FDA approved ADC Adcetris is not effective in these tumour models. This first-in-class ADC holds promise for a broader applicability of ADCs across patient populations.
IKKα inactivation promotes Kras-initiated lung adenocarcinoma development through disrupting major redox regulatory pathways.
Song Na-Young,Zhu Feng,Wang Zining,Willette-Brown Jami,Xi Sichuan,Sun Zhonghe,Su Ling,Wu Xiaolin,Ma Buyong,Nussinov Ruth,Xia Xiaojun,Schrump David S,Johnson Peter F,Karin Michael,Hu Yinling
Proceedings of the National Academy of Sciences of the United States of America
Lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC) are two distinct and predominant types of human lung cancer. IκB kinase α (IKKα) has been shown to suppress lung SCC development, but its role in ADC is unknown. We found inactivating mutations and homologous or hemizygous deletions in the locus, which encodes IKKα, in human lung ADCs. The deletions significantly reduced the survival time of patients with lung ADCs harboring mutations. In mice, lung-specific ablation ( ) induces spontaneous ADCs and promotes Kras-initiated ADC development, accompanied by increased cell proliferation, decreased cell senescence, and reactive oxygen species (ROS) accumulation. IKKα deletion up-regulates NOX2 and down-regulates NRF2, leading to ROS accumulation and blockade of cell senescence induction, which together accelerate ADC development. Pharmacologic inhibition of NADPH oxidase or ROS impairs Kras-mediated ADC development in mice. Therefore, IKKα modulates lung ADC development by controlling redox regulatory pathways. This study demonstrates that IKKα functions as a suppressor of lung ADC in human and mice through a unique mechanism that regulates tumor cell-associated ROS metabolism.
Harnessing a catalytic lysine residue for the one-step preparation of homogeneous antibody-drug conjugates.
Nanna Alex R,Li Xiuling,Walseng Even,Pedzisa Lee,Goydel Rebecca S,Hymel David,Burke Terrence R,Roush William R,Rader Christoph
Current strategies to produce homogeneous antibody-drug conjugates (ADCs) rely on mutations or inefficient conjugation chemistries. Here we present a strategy to produce site-specific ADCs using a highly reactive natural buried lysine embedded in a dual variable domain (DVD) format. This approach is mutation free and drug conjugation proceeds rapidly at neutral pH in a single step without removing any charges. The conjugation chemistry is highly robust, enabling the use of crude DVD for ADC preparation. In addition, this strategy affords the ability to precisely monitor the efficiency of drug conjugation with a catalytic assay. ADCs targeting HER2 were prepared and demonstrated to be highly potent and specific in vitro and in vivo. Furthermore, the modular DVD platform was used to prepare potent and specific ADCs targeting CD138 and CD79B, two clinically established targets overexpressed in multiple myeloma and non-Hodgkin lymphoma, respectively.
A general approach for the site-selective modification of native proteins, enabling the generation of stable and functional antibody-drug conjugates.
Walsh Stephen J,Omarjee Soleilmane,Galloway Warren R J D,Kwan Terence T-L,Sore Hannah F,Parker Jeremy S,Hyvönen Marko,Carroll Jason S,Spring David R
Antibody-drug conjugates (ADCs) are a class of targeted therapeutics that utilize the specificity of antibodies to selectively deliver highly potent cytotoxins to target cells. Although recent years have witnessed significant interest in ADCs, problems remain with the standard linkage chemistries used for cytotoxin-antibody bioconjugation. These typically (1) generate unstable constructs, which may lead to premature cytotoxin release, (2) often give a wide variance in drug-antibody ratios (DAR) and (3) have poor control of attachment location on the antibody, resulting in a variable pharmacokinetic profile. Herein, we report a novel divinylpyrimidine (DVP) linker platform for selective bioconjugation covalent re-bridging of reduced disulfide bonds on native antibodies. Model studies using the non-engineered trastuzumab antibody validate the utility of this linker platform for the generic generation of highly plasma-stable and functional antibody constructs that incorporate variable biologically relevant payloads (including cytotoxins) in an efficient and site-selective manner with precise control over DAR. DVP linkers were also used to efficiently re-bridge both monomeric and dimeric protein systems, demonstrating their potential utility for general protein modification, protein stabilisation or the development of other protein-conjugate therapeutics.
Potential mechanisms of target-independent uptake and toxicity of antibody-drug conjugates.
Mahalingaiah Prathap Kumar,Ciurlionis Rita,Durbin Kenneth R,Yeager Ronnie L,Philip Binu K,Bawa Bhupinder,Mantena Srinivasa R,Enright Brian P,Liguori Michael J,Van Vleet Terry R
Pharmacology & therapeutics
Antibody-drug conjugates (ADCs) are a promising therapeutic modality for oncology indications. The concept of an ADC platform is to increase the therapeutic index (TI) of chemotherapeutics through more selective delivery of cytotoxic agents to tumor cells while limiting exposure to healthy normal cells. Despite the use of antibodies targeting antigens abundantly and/or exclusively expressed on cancer cells (i.e., target cells), dose limiting toxicities (DLTs) in normal cells/tissues are frequently reported even at suboptimal therapeutic doses. Although advancement of ADC technology has helped to optimize all three key components (i.e., mAb, linker, and payload), DLTs remain a key challenge for ADC development. Mechanisms of ADC toxicity in normal cells/tissues are not clearly understood, but the majority of DLTs are considered to be target-independent. In addition to linker-drug instability contributing to the premature release of cytotoxic drug (payload) in circulation, uptake/trafficking of intact ADCs by both receptor-dependent (FcγRs, FcRn and C-type lectin receptors), and-independent (non-specific endocytosis) mechanisms may contribute to off-target toxicity in normal cells. In this article, we review potential mechanisms of target-independent ADC uptake and toxicity in normal cells, as well as discuss components of ADCs which may influence these mechanisms. This information will provide a deeper understanding of the underlying mechanisms of ADC off-target toxicity and prove helpful toward improving the overall TI of the next generation of ADCs.
Antibody-Drug Conjugate-Based Therapeutics: State of the Science.
Birrer Michael J,Moore Kathleen N,Betella Ilaria,Bates Richard C
Journal of the National Cancer Institute
Antibody-drug conjugates (ADCs) are complex engineered therapeutics consisting of monoclonal antibodies, directed toward tumor-associated antigens, to which highly potent cytotoxic agents are attached using chemical linkers. This targeted drug delivery strategy couples the precision of the antibody targeting moiety with the cytocidal activity of the payload, which is generally too toxic on its own to be systemically administered. In this manner, ADCs confer a means to reduce off-target toxicities in patients by limiting the exposure of normal tissues to the payload, thus broadening the potential therapeutic window compared with traditional chemotherapy. The pace of ADC development is accelerating, with the number of investigational agents in human trials having more than tripled over the past 5 years, underscoring the enthusiasm for this transformative approach to cancer treatment. Here, we review the key structural elements of ADC design (antibody, linker, and payload), highlighting critical aspects and technological advances that have affected the clinical effectiveness of this class of biopharmaceuticals. The ADC field continues to evolve, including ongoing efforts aimed at improving target selection, developing payloads with varied mechanisms of action and increased potency, designing innovative bioconjugation strategies, as well as maximizing efficacy and tolerability in patients. An overview of the current clinical trial landscape is provided, with emphasis on the clinical experience of the four ADCs to have received regulatory approval to date, as well as additional promising candidates currently in late-stage clinical development in both solid tumor and hematological malignancies.
Monodisperse polysarcosine-based highly-loaded antibody-drug conjugates.
Viricel Warren,Fournet Guy,Beaumel Sabine,Perrial Emeline,Papot Sébastien,Dumontet Charles,Joseph Benoît
Antibody-drug conjugates (ADCs) convey highly potent anticancer drugs to antigen-expressing tumor cells, thereby sparing healthy tissues throughout the body. Pharmacokinetics and tolerability of ADCs are predominantly influenced by the drug-antibody ratio (DAR) of the conjugates, which is to-date limited to a value of 3-4 drugs per antibody in ADCs under clinical investigations. Here, we report the synthesis of monodisperse ( discrete) polysarcosine compounds and their use as a hydrophobicity masking entity for the construction of highly-loaded homogeneous β-glucuronidase-responsive antibody-drug conjugates (ADCs). The highly hydrophilic drug-linker platform described herein improves drug-loading, physicochemical properties, pharmacokinetics and antitumor efficacy of the resulting conjugates.
Antibody-drug conjugates for lung cancer in the era of personalized oncology.
Ricciuti Biagio,Lamberti Giuseppe,Andrini Elisa,Genova Carlo,De Giglio Andrea,Bianconi Vanessa,Sahebkar Amirhossein,Chiari Rita,Pirro Matteo
Seminars in cancer biology
With 9.6 million deaths in 2018, cancer represents one of the most common causes of death, both in men and women. Despite recent advances in the understanding of molecular mechanisms involved in cancer development and progression, treatment options are still limited. Limitations of traditional chemotherapy include the lack of selectivity and the unfavorable safety profile. The efficacy of targeted therapies (e.g., tyrosine kinase inhibitors) is also limited by their cytostatic action, which inhibits tumor cell proliferation without inducing tumor cell death, and by the risk of acquired resistance. Antibody-drug conjugates (ADCs), a newly developed class of engineered anticancer drugs, consist of recombinant monoclonal antibodies against tumor-specific antigens that are covalently bound to cytotoxic agents. They have been designed to overcome the limitations of traditional chemotherapy and targeted therapies by combining the target selectivity of monoclonal antibodies with the high potency of cytotoxic drugs. Currently, ADCs that have received regulatory approval include brentuximab vedotin for CD30-positive Hodgkin lymphoma and trastuzumab emtansine for human epidermal growth factor receptor 2-positive breast cancer. However, over 80 novel ADCs are actively being investigated in preclinical studies and early-phase clinical trials. In this review, we will provide a comprehensive overview of the biological rational, efficacy and safety of ADCs as therapeutic agents against non-small cell lung cancer and small cell lung cancer.
Therapeutic efficacy of a novel humanized antibody-drug conjugate recognizing plexin-semaphorin-integrin domain in the RON receptor for targeted cancer therapy.
Tong Xiang-Min,Feng Liang,Suthe Sreedhar Reddy,Weng Tian-Hao,Hu Chen-Yu,Liu Yi-Zhi,Wu Zhi-Gang,Wang Ming-Hai,Yao Hang-Ping
Journal for immunotherapy of cancer
BACKGROUND:Antibody-drug conjugates (ADCs) targeting the RON receptor, a tumorigenic factor contributing to cancer malignancy, has been considered as a novel strategy for cancer therapy. Here we describe a humanized antibody recognizing the RON plexin-semaphorin-integrin (PSI) domain with increased drug delivery capability for potential clinical application. METHOD:Monoclonal antibody PCM5B14 specific to the human and monkey RON PSI domain was generated and characterized by various immunological methods. Humanized antibody H5B14 was created by grafting PCM5B14 complementarity-determining regions into human IgG1/κ acceptor frameworks and conjugated with monomethyl auristatin E and duocarmycin to form two H5B14-based ADCs. Stability of H5B14-based ADCs in human plasma was measured using hydrophobic interaction chromatography. Various biochemical and biological assays were used to determine ADC- regulated RON internalization, cell viability, spheroid formation, and death of cancer stem-like cells. Efficacies of H5B14-based ADCs in vivo were validated using tumor xenograft models. Maximal tolerated doses of H5B14-based ADCs were established in mice. RESULTS:H5B14 was highly specific to the human RON PSI domain and superior over other anti-RON ADCs in induction of RON internalization in various cancer cell lines tested. H5B14-based ADCS had a drug to antibody ratio of ~ 3.70:1 and were stable in human plasma with a minimal dissociation within a 10-day period. Functionally, H5B14-mediated drug delivery decreased cell viability at early stages with an average IC at ~ 20 nM in multiple cancer cell lines examined. H5B14-based ADCs also inhibited spheroid formation and caused death of cancer stem-like cells with RON/CD44/ESA phenotypes. In vivo, H5B14-based ADCs in a single injection inhibited tumor xenograft growth mediated by multiple cancer cell lines. Tumoristatic concentrations calculated from xenograft tumor models were in the range of 0.63 to 2.0 mg/kg bodyweight. Significantly, H5B14-based ADCs were capable of eradicating tumors at variable levels across multiple xenograft models regardless their malignant statuses. Toxicologically, H5B14-based ADCs were well tolerated in mice up to 60 mg/kg. CONCLUSION:H5B14-based ADCs targeting the RON PSI domain are superior in inducing RON internalization, leading to robust drug delivery and overall inhibition and eradication of tumors in multiple xenograft models. These findings warrant H5B14-based ADCs for clinical trials in the future.
Antibody-drug conjugates in clinical trials for lymphoid malignancies and multiple myeloma.
Yu Bo,Liu Delong
Journal of hematology & oncology
Antibody-drug conjugates (ADC) represent a distinct family of chemoimmunotherapy agents. ADCs are composed of monoclonal antibodies conjugated to cytotoxic payloads via specialized chemical linkers. ADCs therefore combine the immune therapy with targeted chemotherapy. Due to the distinct biomarkers associated with lymphocytes and plasma cells, ADCs have emerged as a promising treatment option for lymphoid malignancies and multiple myeloma. Several ADCs have been approved for clinical applications: brentuximab vedotin, inotuzumab ozogamicin, moxetumomab pasudotox, and polatuzumab vedotin. More novel ADCs are under clinical development. In this article, we summarized the general principles for ADC design, and updated novel ADCs under various stages of clinical trials for lymphoid malignancies and multiple myeloma.
Elimination of melanoma by sortase A-generated TCR-like antibody-drug conjugates (TL-ADCs) targeting intracellular melanoma antigen MART-1.
Lai Jun,Wang Yun,Wu Shan-Shan,Ding Ding,Sun Ze-Yu,Zhang Ying,Zhou Jie,Zhou Zhan,Xu Ying-Chun,Pan Li-Qiang,Chen Shu-Qing
Most tumor-associated proteins are located inside tumor cells and thus are not accessible to current marketed therapeutic monoclonal antibodies or their cytotoxic conjugates. Human leukocyte antigen (HLA) class I can present peptides derived from intracellular tumor-associated proteins and somatically mutated proteins on the cell's surface, forming an HLA/peptide complex as tumor-specific antigens for T cell receptor (TCR) recognition. Therefore, HLA-mediated presentation of intracellular tumor antigen peptides provides a viable way to distinguish tumor cells from normal cells, which is important for broadening antigen selection, especially for antibody-drug conjugates (ADCs) regarding their highly cytotoxic payload. We applied sortase A-mediated conjugation to develop TCR-like ADCs (i.e., EA1 HL-vcMMAE) targeting intracellular MART-1 protein, a melanocyte-differentiating antigen specific for metastatic melanomas, via the cell surface HLA-A2/MART-1 peptide complex. Homogenous EA1 HL-vcMMAE (drug to antibody ratio of 4) efficiently eliminated melanoma cells in xenograft mouse models with no obvious toxicity at the therapeutic dosage. Trametinib, an MEK inhibitor serving as an HLA expression enhancing agent, augmented the TL-ADCs' efficacy both in vitro and in vivo by upregulating MART-1 peptide presentation, thus providing a strategy for overcoming the limitation of antigen presentation level for TL-ADCs. Hence, our findings validate the strategy of using sortase A-generated TL-ADCs to target tumor-specific intracellular proteins, with or without agents present, to increase presenting TCR epitope peptides.
Antibody drug conjugates (ADCs) charged with HDAC inhibitor for targeted epigenetic modulation.
Cini Elena,Faltoni Valentina,Petricci Elena,Taddei Maurizio,Salvini Laura,Giannini Giuseppe,Vesci Loredana,Milazzo Ferdinando Maria,Anastasi Anna Maria,Battistuzzi Gianfranco,De Santis Rita
We describe here two novel antibody-drug conjugates loaded with the HDAC inhibitor ST7612AA1 (IC equal to 0.07 μM on NCI-H460 cells), a thiol-based molecule with a moderate toxicity . Two payloads were prepared using cleavable and non-cleavable linkers. After anchoring to cetuximab through amide bond with lysines, the resulting HDAC inhibitor-antibody conjugates showed ability to recognize EGFR and efficient internalization in tumor cells. Both ADCs induced sensible increment of histones 3 and 4 and alpha-tubulin acetylation. Animal models of human solid tumors showed high anti-tumor efficacy of the conjugates without the toxicity generally observed with traditional ADCs delivering highly potent cytotoxic drugs. These compounds, the first ADCs charged with not highly cytotoxic warheads, are potentially suitable for epigenetic modulation, extending the ADC strategy to the targeted delivery of HDAC inhibitors with many possible therapeutic applications beyond cancer.
Antibody structure and engineering considerations for the design and function of Antibody Drug Conjugates (ADCs).
Hoffmann Ricarda M,Coumbe Ben G T,Josephs Debra H,Mele Silvia,Ilieva Kristina M,Cheung Anthony,Tutt Andrew N,Spicer James F,Thurston David E,Crescioli Silvia,Karagiannis Sophia N
Antibody-drug conjugates (ADCs) are emerging as effective tools in cancer therapy, combining the antibody's exquisite specificity for the target antigen-expressing cancer cell together with the cytotoxic potency of the payload. Much success stems from the rational design of "toxic warheads", chemically linked to antibodies, and from fine-tuning the intricate properties of chemical linkers. Here, we focus on the antibody moiety of ADCs, dissecting the impact of Fab, linkers, isotype and Fc structure on the anti-tumoral and immune-activating functions of ADCs. Novel design approaches informed by antibody structural attributes present opportunities that may contribute to the success of next generation ADCs.
Rapid conjugation of antibodies to toxins to select candidates for the development of anticancer Antibody-Drug Conjugates (ADCs).
Hoffmann Ricarda M,Mele Silvia,Cheung Anthony,Larcombe-Young Daniel,Bucaite Gintare,Sachouli Eirini,Zlatareva Iva,Morad Hassan O J,Marlow Rebecca,McDonnell James M,Figini Mariangela,Lacy Katie E,Tutt Andrew J N,Spicer James F,Thurston David E,Karagiannis Sophia N,Crescioli Silvia
Antibody-Drug Conjugates (ADCs) developed as a targeted treatment approach to deliver toxins directly to cancer cells are one of the fastest growing classes of oncology therapeutics, with eight ADCs and two immunotoxins approved for clinical use. However, selection of an optimum target and payload combination, to achieve maximal therapeutic efficacy without excessive toxicity, presents a significant challenge. We have developed a platform to facilitate rapid and cost-effective screening of antibody and toxin combinations for activity and safety, based on streptavidin-biotin conjugation. For antibody selection, we evaluated internalization by target cells using streptavidin-linked antibodies conjugated to biotinylated saporin, a toxin unable to cross cell membranes. For payload selection, we biotinylated toxins and conjugated them to antibodies linked to streptavidin to evaluate antitumour activity and pre-clinical safety. As proof of principle, we compared trastuzumab conjugated to emtansine via streptavidin-biotin (Trastuzumab-SB-DM1) to the clinically approved trastuzumab emtansine (T-DM1). We showed comparable potency in reduction of breast cancer cell survival in vitro and in growth restriction of orthotopic breast cancer xenografts in vivo. Our findings indicate efficient generation of functionally active ADCs. This approach can facilitate the study of antibody and payload combinations for selection of promising candidates for future ADC development.
A DNA-Interacting Payload Designed to Eliminate Cross-Linking Improves the Therapeutic Index of Antibody-Drug Conjugates (ADCs).
Miller Michael L,Shizuka Manami,Wilhelm Alan,Salomon Paulin,Reid Emily E,Lanieri Leanne,Sikka Surina,Maloney Erin K,Harvey Lauren,Qiu Qifeng,Archer Katie E,Bai Chen,Vitharana Dilrukshi,Harris Luke,Singh Rajeeva,Ponte Jose F,Yoder Nicholas C,Kovtun Yelena,Lai Katharine C,Ab Olga,Pinkas Jan,Keating Thomas A,Chari Ravi V J
Molecular cancer therapeutics
Tumor-selective delivery of cytotoxic agents in the form of antibody-drug conjugates (ADCs) is now a clinically validated approach for cancer treatment. In an attempt to improve the clinical success rate of ADCs, emphasis has been recently placed on the use of DNA-cross-linking pyrrolobenzodiazepine compounds as the payload. Despite promising early clinical results with this class of ADCs, doses achievable have been low due to systemic toxicity. Here, we describe the development of a new class of potent DNA-interacting agents wherein changing the mechanism of action from a cross-linker to a DNA alkylator improves the tolerability of the ADC. ADCs containing the DNA alkylator displayed similar potency, but improved bystander killing and efficacy, compared with those of the cross-linker. Thus, the improved tolerability and antitumor activity achieved in rodent models with ADCs of the novel DNA alkylator could provide an efficacious, yet safer option for cancer treatment. .
A Novel Anti-CD22 Anthracycline-Based Antibody-Drug Conjugate (ADC) That Overcomes Resistance to Auristatin-Based ADCs.
Yu Shang-Fan,Zheng Bing,Go MaryAnn,Lau Jeff,Spencer Susan,Raab Helga,Soriano Robert,Jhunjhunwala Suchit,Cohen Robert,Caruso Michele,Polakis Paul,Flygare John,Polson Andrew G
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE:We are interested in identifying mechanisms of resistance to the current generation of antibody-drug conjugates (ADC) and developing ADCs that can overcome this resistance. EXPERIMENTAL DESIGN:Pinatuzumab vedotin (anti-CD22-vc-MMAE) and polatuzumab vedotin (anti-CD79b-vc-MMAE) are ADCs that contain the microtubule inhibitor monomethyl auristatin E (MMAE) attached to the antibody by the protease-cleavable linker maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-vc-PAB). Early clinical trial data suggest that these ADCs have promising efficacy for the treatment of non-Hodgkin lymphoma (NHL); however, some patients do not respond or become resistant to the ADCs. Anthracyclines are very effective in NHL, but ADCs containing the anthracycline doxorubicin were not clinically efficacious probably due to the low drug potency and inadequate linker technology. The anthracycline analogue PNU-159682 is thousands of times more cytotoxic than doxorubicin, so we used it to develop a new class of ADCs. We used the same MC-vc-PAB linker and antibody in pinatuzumab vedotin but replaced the MMAE with a derivative of PNU-159682 to make anti-CD22-NMS249 and tested it for in vivo efficacy in xenograft tumors resistant to MMAE-based ADCs. RESULTS:We derived cell lines from in vivo xenograft tumors that were made resistant to anti-CD22-vc-MMAE and anti-CD79b-vc-MMAE. We identified P-gp (ABCB1/MDR1) as the major driver of resistance to the vc-MMAE-based conjugates. Anti-CD22-NMS249 was at least as effective as anti-CD22-vc-MMAE in xenograft models of the parental cell lines and maintained its efficacy in the resistant cell lines. CONCLUSIONS:These studies provide proof of concept for an anthracycline-based ADC that could be used to treat B-cell malignancies that are resistant to vc-MMAE conjugates.
Design, synthesis and evaluation of novel, potent DNA alkylating agents and their antibody-drug conjugates (ADCs).
Reid Emily E,Archer Katie E,Shizuka Manami,McShea Molly A,Maloney Erin K,Ab Olga,Lanieri Leanne,Wilhelm Alan,Ponte Jose F,Yoder Nicholas C,Chari Ravi V J,Miller Michael L
Bioorganic & medicinal chemistry letters
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.
Antibody-Drug Conjugates (ADCs) Derived from Interchain Cysteine Cross-Linking Demonstrate Improved Homogeneity and Other Pharmacological Properties over Conventional Heterogeneous ADCs.
Behrens Christopher R,Ha Edward H,Chinn Lawrence L,Bowers Simeon,Probst Gary,Fitch-Bruhns Maureen,Monteon Jorge,Valdiosera Amanda,Bermudez Abel,Liao-Chan Sindy,Wong Tiffany,Melnick Jonathan,Theunissen Jan-Willem,Flory Mark R,Houser Derrick,Venstrom Kristy,Levashova Zoia,Sauer Paul,Migone Thi-Sau,van der Horst Edward H,Halcomb Randall L,Jackson David Y
Conventional antibody-drug conjugates (ADCs) are heterogeneous mixtures of chemically distinct molecules that vary in both drugs/antibody (DAR) and conjugation sites. Suboptimal properties of heterogeneous ADCs have led to new site-specific conjugation methods for improving ADC homogeneity. Most site-specific methods require extensive antibody engineering to identify optimal conjugation sites and introduce unique functional groups for conjugation with appropriately modified linkers. Alternative nonrecombinant methods have emerged in which bifunctional linkers are utilized to cross-link antibody interchain cysteines and afford ADCs containing four drugs/antibody. Although these methods have been shown to improve ADC homogeneity and stability in vitro, their effect on the pharmacological properties of ADCs in vivo is unknown. In order to determine the relative impact of interchain cysteine cross-linking on the therapeutic window and other properties of ADCs in vivo, we synthesized a derivative of the known ADC payload, MC-MMAF, that contains a bifunctional dibromomaleimide (DBM) linker instead of a conventional maleimide (MC) linker. The DBM-MMAF derivative was conjugated to trastuzumab and a novel anti-CD98 antibody to afford ADCs containing predominantly four drugs/antibody. The pharmacological properties of the resulting cross-linked ADCs were compared with analogous heterogeneous ADCs derived from conventional linkers. The results demonstrate that DBM linkers can be applied directly to native antibodies, without antibody engineering, to yield highly homogeneous ADCs via cysteine cross-linking. The resulting ADCs demonstrate improved pharmacokinetics, superior efficacy, and reduced toxicity in vivo compared to analogous conventional heterogeneous ADCs.
Hydrophobic interaction chromatography (HIC) method development and characterization of resolved drug-load variants in site-specifically conjugated pyrrolobenzodiazepine dimer-based antibody drug conjugates (PBD-ADCs).
Janaratne Thamara,Wang Xiu C,Becker Calvin,Zhao Yanqun,Leanna Rob,Pritts Wayne
Journal of pharmaceutical and biomedical analysis
Antibody drug conjugates (ADCs) are heterogeneous biopharmaceutical products that demand extensive characterization to ensure batch consistency, safety, and efficacy. Hydrophobic interaction chromatography (HIC) is the state-of-the-art analytical tool to monitor conjugation-related critical quality attributes (CQAs) e.g. drug-load distribution and Drug-to-Antibody Ratio (DAR). For the next generation site-specific PBD-ADCs (PBD: pyrrolobenzodiazepine dimer), denaturing RP-HPLC (reverse-phase high-performance chromatography) is the current method to determine average DAR. In this manuscript, we have utilized native HIC for the first time to understand conjugation related CQAs in PBD-ADCs. In terms of the method development, the type of stationary phase and salt, coupled with reduction of the reactive imine in the PBD drug-linker to an amine form in the sample preparation, have played a key role in achieving the best HIC resolution for the drug-load variants. The established HIC conditions resolved DAR 0, DAR 1, and two DAR 2 peaks for PBD-ADCs. Extended characterization of the DAR 2 peaks confirmed that they have retained characteristically distinct antibody Fc N-glycan distributions (Fc = Fragment crystallization region). Therefore, the results support that the HIC conditions established for PBD-ADCs is valuable in not only determining DAR values but also other important attributes including native drug-load distribution and unique DAR 2 conformations existed as a result of the N-glycan heterogeneity.
Characterization of Ring-Opening Reaction of Succinimide Linkers in ADCs.
Zheng Kai,Chen Yan,Wang John,Zheng Laura,Hutchinson Matt,Persson Josefine,Ji Junyan
Journal of pharmaceutical sciences
A new class of highly potent biopharmaceutical drugs, antibody-drug conjugates (ADCs), has been proven to be clinically effective to treat oncologic diseases. ADCs contain 3 major components: the monoclonal antibody, cytotoxic drug, and chemical linker. THIOMAB™ drug conjugates and interchain-cysteine ADCs are common ADC platforms that apply thiol-maleimide chemistry via Michael addition to conjugate linker-drugs to cysteine residues. However, the resulting succinimide ring in the linker is susceptible to ring-opening reactions via hydrolysis, especially at high pH and elevated temperatures. Once the succinimide ring is opened, in vivo stability of the ADCs can be changed and the therapeutic activity will be altered. In this study, we investigated the impact of conjugation sites on succinimide ring opening for ADCs. A new methodology based on imaged capillary isoelectric focusing was developed to monitor the formation of succinimide ring-opened products. In addition, a reverse-phase high-performance liquid chromatography method was used to monitor site-specific ring-opening reactions. Our data confirmed that succinimide ring-opening rates in ADCs are conjugation-site dependent. With a good understanding of the conjugation site impact on final product's stability, it is potentially feasible to modify ring-opening rates in vitro to achieve desirable in vivo stability and biological activity.
Conjugation Site Analysis of Lysine-Conjugated ADCs.
Sang Hua,Wan Ning,Lu Gaoyuan,Tian Yang,Wang Guangji,Ye Hui
Methods in molecular biology (Clifton, N.J.)
Lysine-conjugated antibody-drug conjugates (ADCs) are formed by attaching cytotoxic drugs to reactive lysine residues of monoclonal antibodies (mAbs) through chemical linkers. During production, the payloads are conjugated nonspecifically to lysine residues in mAbs, resulting in a heterogeneous mixture of ADCs with both different number and conjugation sites of drug payloads per mAb. On account of the drug conjugation sites and levels that both have significant influences on physical and pharmaceutical properties of ADCs, a reliable and straightforward approach for conjugation site analysis for ADCs is highly demanded. Herein, we used a lysine-conjugated ADC, Trastuzumab-MCC-DM1 (T-DM1), as a model ADC, and described an integrative strategy that combines the signature ion fingerprinting method for rapid and reliable filtering of DM1-conjugated peptides, and the normalized area quantitation approach for accurately gauging the conjugation levels for each identified site. This approach is believed to be readily applicable to other maytansinoid derivatives-modified ADCs, and more importantly, universally applicable to lysine-conjugated ADCs for both the recognition of conjugation sites and the measurement of conjugation levels.
The Analysis of Key Factors Related to ADCs Structural Design.
Tang Haichao,Liu Yan,Yu Zhaojin,Sun Mingli,Lin Lu,Liu Wensi,Han Qiang,Wei Minjie,Jin Ying
Frontiers in pharmacology
Antibody-drug conjugates (ADCs) have developed rapidly in recent decades. However, it is complicated to map out a perfect ADC that requires optimization of multiple parameters including antigens, antibodies, linkers, payloads, and the payload-linker linkage. The therapeutic targets of the ADCs are expected to express only on the surface of the corresponding target tumor cells. On the contrary, many antigens usually express on normal tissues to some extent, which could disturb the specificity of ADCs and limit their clinical application, not to mention the antibody is also difficult to choose. It requires to not only target and have affinity with the corresponding antigen, but it also needs to have a linkage site with the linker to load the payloads. In addition, the linker and payload are indispensable in the efficacy of ADCs. The linker is required to stabilize the ADC in the circulatory system and is brittle to release free payload while the antibody combines with antigen. Also, it is a premise that the dose of ADCs will not kill normal tissues and the released payloads are able to fulfill the killing potency in tumor cells at the same time. In this review, we mainly focus on the latest development of key factors affecting ADCs progress, including the selection of antibodies and antigens, the optimization of payload, the modification of linker, payload-linker linkage, and some other relevant parameters of ADCs.
Optimizing Lysosomal Activation of Antibody-Drug Conjugates (ADCs) by Incorporation of Novel Cleavable Dipeptide Linkers.
Salomon Paulin L,Reid Emily E,Archer Katie E,Harris Luke,Maloney Erin K,Wilhelm Alan J,Miller Michael L,Chari Ravi V J,Keating Thomas A,Singh Rajeeva
Although peptide linkers are used in multiple clinical-stage ADCs, there are only few reports on optimizing peptide linkers for efficient lysosomal proteolysis and for stability in circulation. We screened multiple dipeptide linkers for efficiency of proteolysis and compared them to the dipeptide linkers currently being evaluated in the clinic: Val-Cit, Val-Ala, and Ala-Ala. Lead dipeptide linkers selected from the initial screen were incorporated into ADCs with indolinobenzodiazepine dimer (IGN) payloads to evaluate cellular processing, cytotoxic activity, plasma stability, and efficacy. ADCs with several dipeptide linkers bearing l-amino acids showed faster lysosomal processing in target cancer cells compared to the l-Ala-l-Ala linked ADC. These variances in linker processing rates did not result in different and activities among peptide linker ADCs, presumably due to accumulation of threshold cytotoxic catabolite levels for ADCs of several peptide linkers in the cell lines and xenografts tested. ADCs with l-amino acid dipeptide linkers exhibited superior cytotoxic potencies in multiple cell lines compared to an ADC with a d-Ala-d-Ala dipeptide linker and an ADC with a noncleavable linker. This work adds to the toolbox of stable, lysosomally cleavable peptide linkers for ADCs.
ADCs, as Novel Revolutionary Weapons for Providing a Step Forward in Targeted Therapy of Malignancies.
Amani Nooshafarin,Dorkoosh Farid Abedin,Mobedi Hamid
Current drug delivery
Antibody drug conjugates (ADCs), as potent pharmaceutical trojan horses for cancer treatment, provide superior efficacy and specific targeting along with low risk of adverse reactions compared to traditional chemotherapeutics. In fact, the development of these agents combines the selective targeting capability of monoclonal antibody (mAb) with high cytotoxicity of chemotherapeutics for controlling the neoplastic mass growth. Different ADCs (more than 60 ADCs) in preclinical and clinical trials were introduced in this novel pharmaceutical field. Various design-based factors must be taken into account for improving the functionality of ADC technology, including selection of appropriate target antigen and high binding affinity of fragment (miniaturized ADCs) or full mAbs (preferentially use of humanized or fully human antibodies compared to murine and chimeric ones), use of bispecific antibodies for dual targeting effect, linker engineering and conjugation method efficacy to obtain more controlled drug to antibody ratio (DAR). Challenging issues affecting therapeutic efficacy and safety of ADCs, including bystander effect, on- and off-target toxicities, multi drug resistance (MDR) are also addressed. 4 FDA-approved ADCs in the market, including ADCETRIS ®, MYLOTARG®, BESPONSA ®, KADCYLA®. The goal of the current review is to evaluate the key parameters affecting ADCs development.
TR1801-ADC: a highly potent cMet antibody-drug conjugate with high activity in patient-derived xenograft models of solid tumors.
Gymnopoulos Marco,Betancourt Oscar,Blot Vincent,Fujita Ryo,Galvan Diana,Lieuw Vincent,Nguyen Sophie,Snedden Jeanette,Stewart Christine,Villicana Jose,Wojciak Jon,Wong Eley,Pardo Raul,Patel Neki,D'Hooge Francois,Vijayakrishnan Balakumar,Barry Conor,Hartley John A,Howard Philip W,Newman Roland,Coronella Julia
cMet is a well-characterized oncogene that is the target of many drugs including small molecule and biologic pathway inhibitors, and, more recently, antibody-drug conjugates (ADCs). However, the clinical benefit from cMet-targeted therapy has been limited. We developed a novel cMet-targeted 'third-generation' ADC, TR1801-ADC, that was optimized at different levels including specificity, stability, toxin-linker, conjugation site, and in vivo efficacy. Our nonagonistic cMet antibody was site-specifically conjugated to the pyrrolobenzodiazepine (PBD) toxin-linker tesirine and has picomolar activity in cancer cell lines derived from different solid tumors including lung, colorectal, and gastric cancers. The potency of our cMet ADC is independent of MET gene copy number, and its antitumor activity was high not only in high cMet-expressing cell lines but also in medium-to-low cMet cell lines (40 000-90 000 cMet/cell) in which a cMet ADC with tubulin inhibitor payload was considerably less potent. In vivo xenografts with low-medium cMet expression were also very responsive to TR1801-ADC at a single dose, while a cMet ADC using a tubulin inhibitor showed a substantially reduced efficacy. Furthermore, TR1801-ADC had excellent efficacy with significant antitumor activity in 90% of tested patient-derived xenograft models of gastric, colorectal, and head and neck cancers: 7 of 10 gastric models, 4 of 10 colorectal cancer models, and 3 of 10 head and neck cancer models showed complete tumor regression after a single-dose administration. Altogether, TR1801-ADC is a new generation cMet ADC with best-in-class preclinical efficacy and good tolerability in rats.