Payload of T-DM1 binds to cell surface cytoskeleton-associated protein 5 to mediate cytotoxicity of hepatocytes

Endo, Yuichi; Takeda, Kazuyo; Mohan, Nishant; Shen, Yi; Jiang, Jiangsong; Rotstein, David S.; Wu, Wen Jin

doi:10.18632/oncotarget.26461

Cited by 24 publications

(19 citation statements)

References 37 publications

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“…This is required if the intent is to analyze live cells that have yet to change gene expression levels due to cytotoxic stress. Nonetheless, this study demonstrates that the amount of ADC utilized is comparable to that found in the study by Endo et al., 73 and could potentially be refined for future NLS-tagged therapeutics.…”

Section: Discussionsupporting

confidence: 78%

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A Novel Proteomic Method Reveals NLS Tagging of T-DM1 Contravenes Classical Nuclear Transport in a Model of HER2-Positive Breast Cancer

Lacasse

Beaudoin

Jean

et al. 2020

Molecular Therapy - Methods & Clinical Development

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The next breakthrough for protein therapeutics is effective intracellular delivery and accumulation within target cells. Nuclear localization signal (NLS)-tagged therapeutics have been hindered by the lack of efficient nuclear localization due to endosome entrapment. Although development of strategies for tagging therapeutics with technologies capable of increased membrane penetration has resulted in proportional increased potency, nonspecific membrane penetration limits target specificity and, hence, widespread clinical success. There is a long-standing idea that nuclear localization of NLS-tagged agents occurs exclusively via classical nuclear transport. In the present study, we modified the antibody-drug conjugate trastuzumab-emtansine (T-DM1) with a classical NLS linked to cholic acid (cell accumulator [Accum]) that enables modified antibodies to escape endosome entrapment and increase nuclear localization efficiency without abrogating receptor targeting. In parallel, we developed a proteomics-based method to evaluate nuclear transport. Accum-modified T-DM1 significantly enhanced cytotoxic efficacy in the human epidermal growth factor receptor 2 (HER2)-positive SKBR3 breast cancer system. We discovered that efficacy was dependent on the nonclassical importin-7. Our evaluation reveals that when multiple classical NLS tagging occurs, cationic charge build-up as opposed to sequence dominates and becomes a substrate for importin-7. This study results in an effective target cell-specific NLS therapeutic and a general approach to guide future NLS-based development initiatives.

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Section: Discussionsupporting

confidence: 78%

“…Endo et al. 73 used T-DM1 as bait to determine that DM1 binds a cell surface protein cytoskeleton-associated protein 5 (CKAP5) that is expressed on hepatocytes in the absence of HER2. A concentration of 250 μg/mL (∼1,667 nmol/L) T-DM1 was incubated with 1.5 × 10 6 cells.…”

Section: Discussionmentioning

confidence: 99%

A Novel Proteomic Method Reveals NLS Tagging of T-DM1 Contravenes Classical Nuclear Transport in a Model of HER2-Positive Breast Cancer

Lacasse

Beaudoin

Jean

et al. 2020

Molecular Therapy - Methods & Clinical Development

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“…As described, cells were checked to ensure cells were not undergoing apoptosis. Endo et al, used T-DM1 as bait to determine that DM1 binds a cell surface protein cytoskeleton-associated protein 5 (CKAP5) that is expressed on hepatocytes in the absence of HER2 (48). A concentration of 250 µg/mL (~1667 nmol/L) T-DM1 was incubated with 1.5 x 10 6 cells.…”

Section: Discussionmentioning

confidence: 99%

Proteomic-based evaluation of nuclear transport of NLS-tagged trastuzumab-emtansine with enhanced cytotoxic potency

Lacasse

Beaudoin

Jean³

et al. 2019

Preprint

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Nuclear transport receptors (NTRs) are the only proteins able to transport large molecular weight payloads into the nucleus. A dominant area of molecular therapeutic research is the extension of the use of NTRs to target the nucleus for the development of pharmaceuticals or as tools for investigating fundamental biological questions. Although several examples of synthesized peptides harbouring nuclear localization signal (NLS) sequences conjugated to various payloads exist in the literature, the assumption has been that transport occurs by classical nuclear localization via the NTRs importin-α and importin-β. This assumption is relevant to nuclear-targeted therapeutics that aim for full potential clinical impact. In addition, fundamental research can benefit from unbiased approaches to investigate the role of NTRs. Herein, we report the construction of a novel NLS-modified agent composed of trastuzumab-emtansine (T-DM1) coupled to cell accumulator (Accum), a technology that enables monoclonal antibodies to escape endosome entrapment and accumulate conjugated payloads in the nucleus without abrogating affinity or specificity to target antigens. Accum harbours a classical NLS sequence from SV-40 large T-antigen. We demonstrate that routing T-DM1 to the nucleus successfully increased cytotoxic potency in the HER2-positive cell line SKBR3. More importantly, through the development of a novel bait-prey proteomic approach, we show that the non-classical NTR importin 7 and not importin-α/importin-β was required for the cytotoxicity effect. This result was validated by siRNA knock down. Our findings also indicate that by discovering an unanticipated NTR regulator of an NLS-modified agent, this study demonstrates the utility of combining an unbiased proteomic approach to probe NTR function in mammalian cell system and, is a foresight for future NLS-based development initiatives.FRQS-funded Centre de recherche du CHUS. S.J. and J.V.L also hold faculty salary awards from FRQS. Dr. Myriam Badr for discussions about stability data. DECLARATION OF INTEREST STATEMENTNo potential conflicts of interest were disclosed Author ContributionsConception and design: V. L. prepared and characterized ADCs, designed the proteomic approach, performed all experiments, and co-wrote manuscript. S.B and S.J helped conceptualize experiments and to interpret data. J.V.L conceptualized and directed the overall approach and co-wrote the manuscript.

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“…For this we compared tubulin polymerization disruption in Her2-high HCC1954 cells treated with Trastuzumab-SB-DM1 or with T-DM1. The main mechanism of action of T-DM1 relies on the effects of the toxic payload DM1 which is known to cause microtubule disassembly 18 . We therefore labelled HCC1954 tumour cells treated with PBS, trastuzumab alone, T-DM1 or Trastuzumab-SB-DM1 with anti-IgG to confirm antibody internalization and with anti-β-tubulin and we compared tubulin formation between the two ADCs by confocal microscopy (Fig.…”

Section: Streptavidin-biotin Linking For Saporin-based Adc Generationmentioning

confidence: 99%

Rapid conjugation of antibodies to toxins to select candidates for the development of anticancer Antibody-Drug Conjugates (ADCs)

Hoffmann

Mele

Cheung

et al. 2020

Sci Rep

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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. Antibody-Drug Conjugates (ADCs) combine the high specificity of antibodies for their target antigens with the cytotoxic effects of payloads to more specifically guide toxic agents towards cancer cells 1. The field of ADCs has been undergoing significant expansion since the first agent gemtuzumab ozogamicin (Mylotarg), targeting CD20-expressing B cell lymphomas, was approved by the Food and Drug Administration (FDA) in 2000 2. However, despite the potential for this therapeutic modality and the launch of many clinical studies, attrition rates are high due to several challenges such as low efficacy and "on-target" toxicity on normal cells. Until now, only eight ADCs along with two immunotoxins have been approved by the regulatory agencies 1. In order to improve

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Payload of T-DM1 binds to cell surface cytoskeleton-associated protein 5 to mediate cytotoxicity of hepatocytes

Cited by 24 publications

References 37 publications

A Novel Proteomic Method Reveals NLS Tagging of T-DM1 Contravenes Classical Nuclear Transport in a Model of HER2-Positive Breast Cancer

A Novel Proteomic Method Reveals NLS Tagging of T-DM1 Contravenes Classical Nuclear Transport in a Model of HER2-Positive Breast Cancer

Proteomic-based evaluation of nuclear transport of NLS-tagged trastuzumab-emtansine with enhanced cytotoxic potency

Rapid conjugation of antibodies to toxins to select candidates for the development of anticancer Antibody-Drug Conjugates (ADCs)

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