Antibody Conjugates with Unnatural Amino Acids

Hallam, Trevor J.; Wold, Erik D.; Wahl, Alan F.; Smider, Vaughn V.

doi:10.1021/acs.molpharmaceut.5b00082

Cited by 105 publications

(86 citation statements)

References 109 publications

(245 reference statements)

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“…Heterogeneous ADCs have been shown in some cases to possess suboptimal activity and pharmacokinetic properties compared with homogeneous ADC molecules (41)(42)(43). Site-specific drug conjugation to create homogeneous ADCs has been carried out through introduction of non-natural amino acids and functional groups that facilitate orthogonal chemistries (44)(45)(46). Such chemical handles have been incorporated into recombinantly produced antibodies using cell-based (44,47) or cell-free (14) methods, introduced cysteine residues (41), lysine analog incorporation (48,49), or enzymatic conjugation using formylglycine converting enzyme or trans-glutaminase (39).…”

Section: Discussionmentioning

confidence: 99%

Targeted Drug Delivery with an Integrin-Binding Knottin–Fc–MMAF Conjugate Produced by Cell-Free Protein Synthesis

Currier

Ackerman

Kintzing

et al. 2016

Molecular Cancer Therapeutics

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Antibody-drug conjugates (ADC) have generated significant interest as targeted therapeutics for cancer treatment, demonstrating improved clinical efficacy and safety compared with systemic chemotherapy. To extend this concept to other tumor-targeting proteins, we conjugated the tubulin inhibitor monomethyl-auristatin-F (MMAF) to 2.5F-Fc, a fusion protein composed of a human Fc domain and a cystine knot (knottin) miniprotein engineered to bind with high affinity to tumorassociated integrin receptors. The broad expression of integrins (including avb3, avb5, and a5b1) on tumor cells and their vasculature makes 2.5F-Fc an attractive tumor-targeting protein for drug delivery. We show that 2.5F-Fc can be expressed by cellfree protein synthesis, during which a non-natural amino acid was introduced into the Fc domain and subsequently used for site-specific conjugation of MMAF through a noncleavable linker. The resulting knottin-Fc-drug conjugate (KFDC), termed 2.5F-Fc-MMAF, had approximately 2 drugs attached per KFDC. 2.5F-Fc-MMAF inhibited proliferation in human glioblastoma (U87MG), ovarian (A2780), and breast (MB-468) cancer cells to a greater extent than 2.5F-Fc or MMAF alone or added in combination. As a single agent, 2.5F-Fc-MMAF was effective at inducing regression and prolonged survival in U87MG tumor xenograft models when administered at 10 mg/kg two times per week. In comparison, tumors treated with 2.5F-Fc or MMAF were nonresponsive, and treatment with a nontargeted control, CTRL-Fc-MMAF, showed a modest but not significant therapeutic effect. These studies provide proofof-concept for further development of KFDCs as alternatives to ADCs for tumor targeting and drug delivery applications.

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Section: Discussionmentioning

confidence: 99%

Targeted Drug Delivery with an Integrin-Binding Knottin–Fc–MMAF Conjugate Produced by Cell-Free Protein Synthesis

Currier

Ackerman

Kintzing

et al. 2016

Molecular Cancer Therapeutics

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“…Thus, there is currently intense research ongoing to develop strategies for the manufacturing of site-specifically conjugated, homogeneous ADCs. These strategies not only include the introduction of unique handles for chemical conjugation, such as by incorporation of unnatural amino acids (20) or by site-directed mutagenesis (21), but also various enzymatic approaches, including the use of bacterial transglutaminase (22), formylglycine-generating enzyme (FGE; ref. 23), and S. aureus sortase A (24).…”

Section: Introductionmentioning

confidence: 99%

Highly Potent, Anthracycline-based Antibody–Drug Conjugates Generated by Enzymatic, Site-specific Conjugation

Stefan

Gébleux

Waldmeier

et al. 2017

Molecular Cancer Therapeutics

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Antibody-drug conjugates (ADC) are highly potent and specific antitumor drugs, combining the specific targeting of mAbs with the potency of small-molecule toxic payloads. ADCs generated by conventional chemical conjugation yield heterogeneous mixtures with variable pharmacokinetics, stability, safety, and efficacy profiles. To address these issues, numerous site-specific conjugation technologies are currently being developed allowing the manufacturing of homogeneous ADCs with predetermined drug-to-antibody ratios. Here, we used sortase-mediated antibody conjugation (SMAC) technology to generate homogeneous ADCs based on a derivative of the highly potent anthracycline toxin PNU-159682 and a noncleavable peptide linker, using the anti-HER2 antibody trastuzumab (part of Kadcyla) and the anti-CD30 antibody cAC10 (part of Adcetris). Characterization of the resulting ADCs in vitro and in vivo showed that they were highly stable and exhibited potencies exceeding those of ADCs based on conventional tubulin-targeting payloads, such as Kadcyla and Adcetris. The data presented here suggest that such novel and highly potent ADC formats may help to increase the number of targets available to ADC approaches, by reducing the threshold levels of target expression required.

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“…Orthogonal aminoacyl‐tRNA synthetase/tRNA pairs that enable the incorporation of diverse ncAAs have been discovered,4 and the site‐specific incorporation of ncAAs bearing bioorthogonal reactive groups provides an attractive, and potentially general, strategy for generating ADCs 5. However, the ncAAs that have been incorporated into antibodies so far, which contain ketones or azides, are not optimal for bioconjugation.…”

mentioning

confidence: 99%

Rapid and Efficient Generation of Stable Antibody–Drug Conjugates via an Encoded Cyclopropene and an Inverse‐Electron‐Demand Diels–Alder Reaction

2018

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Homogeneous antibody–drug conjugates (ADCs), generated by site‐specific toxin linkage, show improved therapeutic indices with respect to traditional ADCs. However, current methods to produce site‐specific conjugates suffer from low protein expression, slow reaction kinetics, and low yields, or are limited to particular conjugation sites. Here we describe high yielding expression systems that efficiently incorporate a cyclopropene derivative of lysine (CypK) into antibodies through genetic‐code expansion. We express trastuzumab bearing CypK and conjugate tetrazine derivatives to the antibody. We show that the dihydropyridazine linkage resulting from the conjugation reaction is stable in serum, and generate an ADC bearing monomethyl auristatin E that selectively kills cells expressing a high level of HER2. Our results demonstrate that CypK is a minimal bioorthogonal handle for the rapid production of stable therapeutic protein conjugates.

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Antibody Conjugates with Unnatural Amino Acids

Cited by 105 publications

References 109 publications

Targeted Drug Delivery with an Integrin-Binding Knottin–Fc–MMAF Conjugate Produced by Cell-Free Protein Synthesis

Targeted Drug Delivery with an Integrin-Binding Knottin–Fc–MMAF Conjugate Produced by Cell-Free Protein Synthesis

Highly Potent, Anthracycline-based Antibody–Drug Conjugates Generated by Enzymatic, Site-specific Conjugation

Rapid and Efficient Generation of Stable Antibody–Drug Conjugates via an Encoded Cyclopropene and an Inverse‐Electron‐Demand Diels–Alder Reaction

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