Engineered Thio-Trastuzumab-DM1 Conjugate with an Improved Therapeutic Index to Target Human Epidermal Growth Factor Receptor 2–Positive Breast Cancer

Junutula, Jagath R.; Flagella, Kelly M.; Graham, Richard; Parsons, Kathryn L.; Ha, Edward; Raab, Helga; Bhakta, Sunil; Nguyen, Trung Van; Dugger, Debra L.; Li, Guangmin; Mai, Elaine; Phillips, Gail D. Lewis; Hiraragi, Hajime; Fuji, Reina N.; Tibbitts, Jay; Vandlen, Richard; Spencer, Susan D.; Scheller, Richard H.; Polakis, Paul; Sliwkowski, Mark X.

doi:10.1158/1078-0432.ccr-10-0987

Cited by 261 publications

(224 citation statements)

References 24 publications

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“…These values returned to levels similar to control rats by day 14. Similar elevations in liver enzymes have been observed with thioTMAb-mpeo-DM1 (89 mg/kg or greater) and Trastuzumab-MCC-DM1 (20 mg/kg or greater) (15,30). Minimal hematologic changes included elevated neutrophils in rats treated with 60 or 90 mg/kg Her HC-A114pAF-NC-D1 on study day 5 (Table S4), similar to what has previously been observed with thioTMAbmpeo-DM1 (30).…”

Section: Nonnative Amino Acid Conjugates Demonstrate Superior In Vivosupporting

confidence: 83%

“…Test article-related findings in the lungs included dosedependent congestion, alveolar edema, and increased alveolar macrophages. Similar findings were observed in rats treated with thioTMAb-mpeo-DM1 (30). Additional findings in rats treated with 60 or 90 mg/kg of Her HC-A114pAF-NC-D1 included subacute inflammation in the thymus and decreased lymphocytes in the thymus and spleen.…”

Section: Nonnative Amino Acid Conjugates Demonstrate Superior In Vivosupporting

confidence: 80%

“…Test article-related findings in the kidney were primarily in the 60 and 90 mg/kg-treated animals and included interstitial inflammation, congestion, and tubular basophilia, signifying tubular regeneration. The interstitial inflammation was also observed in rats treated with 127 mg/kg of thioTMAb-mpeo-DM1 (30). In the liver, test article-related findings included dosedependent congestion (all dose levels) and degeneration and/or necrosis (90 mg/kg only).…”

Section: Nonnative Amino Acid Conjugates Demonstrate Superior In Vivomentioning

confidence: 85%

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A general approach to site-specific antibody drug conjugates

Tian

Manibusan

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

283

274

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Using an expanded genetic code, antibodies with site-specifically incorporated nonnative amino acids were produced in stable cell lines derived from a CHO cell line with titers over 1 g/L. Using anti-5T4 and anti-Her2 antibodies as model systems, site-specific antibody drug conjugates (NDCs) were produced, via oxime bond formation between ketones on the side chain of the incorporated nonnative amino acid and hydroxylamine functionalized monomethyl auristatin D with either protease-cleavable or noncleavable linkers. When noncleavable linkers were used, these conjugates were highly stable and displayed improved in vitro efficacy as well as in vivo efficacy and pharmacokinetic stability in rodent models relative to conventional antibody drug conjugates conjugated through either engineered surface-exposed or reduced interchain disulfide bond cysteine residues. The advantages of the oximebonded, site-specific NDCs were even more apparent when lowantigen-expressing (2+) target cell lines were used in the comparative studies. NDCs generated with protease-cleavable linkers demonstrated that the site of conjugation had a significant impact on the stability of these rationally designed prodrug linkers. In a single-dose rat toxicology study, a site-specific anti-Her2 NDC was well tolerated at dose levels up to 90 mg/kg. These experiments support the notion that chemically defined antibody conjugates can be synthesized in commercially relevant yields and can lead to antibody drug conjugates with improved properties relative to the heterogeneous conjugates formed by nonspecific chemical modification.A ntibody drug conjugates (ADCs) are emerging as a new class of anticancer therapeutics that combine the efficacy of small-molecule therapeutics with the targeting ability of an antibody (Ab) (1, 2). By combining these two components into a single molecular entity, highly cytotoxic small-molecule drugs (SMDs) can be delivered to cancerous target tissues, thereby enhancing efficacy while reducing the potential systemic toxic side effects of the SMD. Conventional ADCs are typically produced by conjugating the SMD to the Ab through the side chains of either surface-exposed lysines or free cysteines generated through reduction of interchain disulfide bonds (3, 4). Because antibodies contain many lysine and cysteine residues, conventional conjugation typically produces heterogeneous mixtures that present challenges with respect to analytical characterization and manufacturing. Furthermore, the individual constituents of these mixtures exhibit different pharmacology with respect to their pharmacokinetic, efficacy, and safety profiles, hindering a rational approach to optimizing this modality (5).Recently, it was reported that the pharmacological profile of ADCs may be improved by applying site-specific conjugation technologies that make use of surface-exposed cysteine residues engineered into antibodies (THIOMABS) that are then conjugated to the SMD, resulting in site-specifically conjugated ADCs (TDCs) with defined Ab-drug ratios. Rel...

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Section: Nonnative Amino Acid Conjugates Demonstrate Superior In Vivosupporting

confidence: 83%

Section: Nonnative Amino Acid Conjugates Demonstrate Superior In Vivosupporting

confidence: 80%

Section: Nonnative Amino Acid Conjugates Demonstrate Superior In Vivomentioning

confidence: 85%

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A general approach to site-specific antibody drug conjugates

Tian

Manibusan

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

283

274

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“…The present effort aims to test a pretargeted approach in a clinically relevant model that features an internalizing target. 49 Not only has HER2 been exploited as a signaling target via the therapeutic antibody trastuzumab, 50 it has been further utilized as a drug delivery vector in the antibody-drug conjugate trastuzumab emtansine 51 and in clinical imaging of HER2-positive breast cancer. 40–43 It should be noted, however, that HER2 is a somewhat unusual internalizing oncology target in that it exhibits extremely high expression levels and that trastuzumab binding does not down-regulate surface HER2, but instead passively recycles after HER2 endocytosis.…”

Section: Discussionmentioning

confidence: 99%

Site-specific conjugation allows modulation of click reaction stoichiometry for pretargeted SPECT imaging

Mandikian¹,

Rafidi²,

Adhikari³

et al. 2018

mAbs

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Antibody pretargeting is a promising strategy for improving molecular imaging, wherein the separation in time of antibody targeting and radiolabeling can lead to rapid attainment of high contrast, potentially increased sensitivity, and reduced patient radiation exposure. The inverse electron demand Diels-Alder ‘click’ reaction between trans-cyclooctene (TCO) conjugated antibodies and radiolabeled tetrazines presents an ideal platform for pretargeted imaging due to rapid reaction kinetics, bioorthogonality, and potential for optimization of both slow and fast clearing components. Herein, we evaluated a series of anti-human epidermal growth factor receptor 2 (HER2) pretargeting antibodies containing distinct molar ratios of site-specifically incorporated TCO. The effect of stoichiometry on tissue distribution was assessed for pretargeting TCO-modified antibodies (monitored by 125I) and subsequent accumulation of an 111In-labeled tetrazine in a therapeutically relevant HER2+tumor-bearing mouse model. Single photon emission computed tomography (SPECT) imaging was also employed to assess tumor imaging at various TCO-to-monoclonal antibody (mAb) ratios. Increasing TCO-to-mAb molar ratios correlated with increased in vivo click reaction efficiency evident by increased tumor distribution and systemic exposure of 111In-labeled tetrazines. The pharmacokinetics of TCO-modified antibodies did not vary with stoichiometry. Pretargeted SPECT imaging of HER2-expressing tumors using 111In-labeled tetrazine demonstrated robust click reaction with circulating antibody at ~2 hours and good tumor delineation for both the 2 and 6 TCO-to-mAb ratio variants at 24 hours, consistent with a limited cell-surface pool of pretargeted antibody and benefit from further distribution and internalization. To our knowledge, this represents the first reported systematic analysis of how pretargeted imaging is affected solely by variation in click reaction stoichiometry through site-specific conjugation chemistry.

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“…Then all samples were washed using FACS buffer, followed by FACS analysis using a BD FACSCalibur system (BD Biosciences). 46 …”

Section: Egfr Binding Measurements By Elisa and Flow Cytometrymentioning

confidence: 99%

Preclinical studies of a Pro-antibody-drug conjugate designed to selectively target EGFR-overexpressing tumors with improved therapeutic efficacy

Yang¹,

Guo²,

Chen³

et al. 2016

mAbs

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Antibody-drug conjugates (ADCs) have exhibited potent clinical benefits in cancer therapy. However, development of ADCs against epidermal growth factor receptor (EGFR) has limitations because of wide expression of EGFR in both normal and tumor tissues. Previously, we developed an anti-EGFR proteaseactivated antibody (pro-antibody), termed as PanP, which remains inert against EGFR until activated by tumor-specific protease. Herein, we for the first time report a new class of pro-antibody-drug conjugate (PDC) against EGFR, denoted as PanP-DM1. It has been designed to selectively target the EGFRoverexpressing tumor cells and exert greater anti-tumor activity compared with PanP. Our data showed that PanP-DM1 also could be selectively activated by tumor-specific protease 'uPA'. Furthermore, activated PanP-DM1 was potently cytotoxic against EGFR-overexpressing tumor cell lines in vitro. Crucially, our data indicated that PanP-DM1 was significantly more effective in eradicating EGFR-overexpressing tumors in vivo. Additionally, toxicity was preliminarily evaluated in mice as measured by body weight loss. In summary, our study suggests that PanP-DM1, a novel pro-antibody-drug conjugate, has cancer-selectivity, efficacy and safety profile that supports its potential use for EGFR-overexpressing tumors.

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Engineered Thio-Trastuzumab-DM1 Conjugate with an Improved Therapeutic Index to Target Human Epidermal Growth Factor Receptor 2–Positive Breast Cancer

Cited by 261 publications

References 24 publications

A general approach to site-specific antibody drug conjugates

A general approach to site-specific antibody drug conjugates

Site-specific conjugation allows modulation of click reaction stoichiometry for pretargeted SPECT imaging

Preclinical studies of a Pro-antibody-drug conjugate designed to selectively target EGFR-overexpressing tumors with improved therapeutic efficacy

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