ChAcNLS, a Novel Modification to Antibody-Conjugates Permitting Target Cell-Specific Endosomal Escape, Localization to the Nucleus, and Enhanced Total Intracellular Accumulation

Beaudoin, Simon; Rondeau, Andreanne; Martel, Olivier; Bonin, Marc-André; Lier, Johan E. van; Leyton, Jeffrey V.

doi:10.1021/acs.molpharmaceut.6b00075

Cited by 21 publications

(86 citation statements)

References 55 publications

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“…Cells grown on glass coverslips were stimulated with IL-5 (BD Biosciences, Ontario, Canada) at 50 ng/mL for the indicated time at 4 C and 37 C. Cells were then washed, fixed, and permeabilized as described previously. 32 Cells were then probed with 1/500 rabbit anti-IL-5Ra followed by washing in PBS and incubation with 1/500 secondary anti-murine AlexaFluor 647-labeled Fc polyclonal antibody (Life Technologies, Ontario, Canada). Nuclei were stained with Hoescht (1 mg/mL) for 5 min.…”

Section: Il-5ra Internalization and Expression Dynamics Following Expmentioning

confidence: 99%

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Targeting IL-5Rα with antibody-conjugates reveals a strategy for imaging and therapy for invasive bladder cancer

Paquette

Vilera-Perez²,

Beaudoin

et al. 2017

OncoImmunology

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Despite the high interest and concern due to an increasing incidence and death rate, patients who develop muscle invasive bladder cancer (MIBC) have few options available. However, the past decade has produced many candidate bladder tumor-specific markers but further development of these markers is still needed for creating effective targeted medications to solve this urgent need. Interleukin-5 receptor α-subunit (IL-5Rα) has recently been reported to be involved in MIBC progression. Thus, we aimed to validate IL-5Rα as a target for antibody-conjugates to better manage patients with MIBC. Patients were recruited and their tumors were processed for IL-5Rα immunohistochemical analysis. NOD/SCID mice were also heterotopically implanted with the human MIBC HT-1376 and HT-B9 cell lines and established xenografts immunohistochemically evaluated for IL-5Rα and compared against patient tumors. Using the mAb A14, an antibody-drug conjugate (ADC) and a radiolabeled immunoconjugate (RIC) were developed by conjugating to vinblastine and to the positron emitter copper-64 (Cu), respectively. As a proof-of-concept for ADC and RIC efficacy, cytotoxicity and positron emission tomography (PET) imaging in tumor-bearing mice were performed, respectively. In addition, as rapid internalization and accumulation are important components for effective antibody-conjugates, we evaluated these aspects in response to IL-5 and Cu-A14 treatments. Our findings suggest that although IL-5Rα protein expression is preferentially increased in MIBC, it is rapid IL-5Rα-mediated internalization allowing vinblastine-A14 to have cytotoxic activity andCu-A14 to detect MIBC tumors . This is the first report to elucidate the potential of IL-5Rα as an attractive MIBC target for antibody-conjugate applications.

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Section: Il-5ra Internalization and Expression Dynamics Following Expmentioning

confidence: 99%

“…Trypsin also served for the removal of surface IL-5Ra and bound 64 Cu-A14 as previously performed. 32 Radioactive supernatant (termed extracellular in this study) was removed and saved. Cells were then washed and centrifuged.…”

Section: Preparation Of 64 Cu-a14mentioning

confidence: 99%

Targeting IL-5Rα with antibody-conjugates reveals a strategy for imaging and therapy for invasive bladder cancer

Paquette

Vilera-Perez²,

Beaudoin

et al. 2017

OncoImmunology

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“…Nuclear staining was performed with Hoechst (1:10,000) concurrently for 1 h at RT. Cells were washed, processed, and images acquired as previously described (22).…”

Section: Immunofluorescence Confocal Microscopymentioning

confidence: 99%

“…We set out to develop an approach to identify NTRs and test their uniqueness or redundancy to import an NLS-modified agent in live cells. We present the technology cell accumulator (Accum), which harnesses a two-step movement of endosome escape coupled to nuclear translocation (22).…”

Section: Introductionmentioning

confidence: 99%

“…Accum-modified mAbs conjugated to radionuclides retain high target affinity and specificity yet can accumulate the delivered payload at increased intracellular levels relative to unmodified mAbs and mAbs modified with the NLS but no cholic acid (22,23). Following receptor-mediated internalization, Accum enables mAbs to escape endosomes by utilizing cholic acid to activate acid-sphingomyelinase and produce ceramide, which destabilizes endosomal membranes (23).…”

Section: Introductionmentioning

confidence: 99%

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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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Unlocking Natural Potential: Antibody‐Drug Conjugates With Naturally Derived Payloads for Cancer Therapy

Jiang,

Nik Nabil,

et al. 2024

Phytotherapy Research

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Natural compound‐derived chemotherapies remain central to cancer treatment, however, they often cause off‐target side effects that negatively impact patients' quality of life. In contrast, antibody‐drug conjugates (ADCs) combine cytotoxic payloads with antibodies to specifically target cancer cells. Most approved and clinically investigated ADCs utilize naturally derived payloads, while those with conventional synthetic molecular payloads remain limited. This review focuses on approved ADCs that enhance the efficacy of naturally derived payloads by linking them with antibodies. We provide an overview of the core components of ADCs, their working mechanisms, and FDA‐approved ADCs featuring naturally derived payloads, such as calicheamicin, camptothecin, dolastatin 10, maytansine, pyrrolbenzodiazepine (PBD), and the immunotoxin Pseudomonas exotoxin A. This review also explores recent clinical advancements aimed at broadening the therapeutic potential of ADCs, their applicability in treating heterogeneously composed tumors and their potential use beyond oncology. Additionally, this review highlights naturally derived payloads that are currently being clinically investigated but have not yet received approval. By summarizing the current landscape, this review provides insights into promising avenues for exploration and contributes to the refinement of treatment protocols for improved patient outcomes.

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ChAcNLS, a Novel Modification to Antibody-Conjugates Permitting Target Cell-Specific Endosomal Escape, Localization to the Nucleus, and Enhanced Total Intracellular Accumulation

Cited by 21 publications

References 55 publications

Targeting IL-5Rα with antibody-conjugates reveals a strategy for imaging and therapy for invasive bladder cancer

Targeting IL-5Rα with antibody-conjugates reveals a strategy for imaging and therapy for invasive bladder cancer

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

Unlocking Natural Potential: Antibody‐Drug Conjugates With Naturally Derived Payloads for Cancer Therapy

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