Location Matters: Site of Conjugation Modulates Stability and Pharmacokinetics of Antibody Drug Conjugates

Strop, Pavel; Liu, Shuhui; Dorywalska, Magdalena; Delaria, Kathy; Dushin, Russell G.; Tran, Thomas-Toan; Ho, Wei-Hsien; Farias, Santiago; Casas, Meritxell Galindo; Abdiche, Yasmina; Zhou, Dahui; Chandrasekaran, Ramalakshmi Y.; Samain, Caroline; Loo, Carole; Rossi, Andrea; Rickert, Mathias; Krimm, Stellanie; Wong, Teresa; Chin, Sherman M.; Yu, Jessica; Dilley, Jeanette; Chaparro‐Riggers, Javier; Filzen, Gary F.; O’Donnell, Christopher J; Wang, Fang; Myers, Jeremy S.; Pons, Jaume; Shelton, David L.; Rajpal, Arvind

doi:10.1016/j.chembiol.2013.01.010

Cited by 434 publications

(452 citation statements)

References 18 publications

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“…Recent advances in conjugation chemistry have led to the production of ADCs with homogeneous drug loading and improved stability (53). These technologies use nonnatural amino acids, engineered cysteines, or transglutaminases to attach the warhead linker to specific sites on the antibody (54,55). The first published data using site-specific conjugation-i.e., THIOMAB technology-to generate homogenous DAR2 conjugates demonstrated significant improvements in TI compared to stochastically conjugated DAR4 ADCs (56).…”

Section: Stabilitymentioning

confidence: 99%

Antibody Drug Conjugates: Nonclinical Safety Considerations

Hinrichs

Dixit

2015

AAPS J

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Abstract. Antibody drug conjugates (ADCs) are biopharmaceutical molecules consisting of a cytotoxic small molecule covalently linked to a targeted protein carrier via a stable cleavable or noncleavable linker. The process of conjugation yields a highly complex molecule with biochemical properties that are distinct from those of the unconjugated components. The impact of these biochemical differences on the safety and pharmacokinetic (PK) profile of the conjugate must be considered when determining the types of nonclinical safety studies required to support clinical development of ADCs. The hybrid nature of ADCs highlights the need for a science-based approach to safety assessment that incorporates relevant aspects of small and large molecule testing paradigms. This thinking is reflected in current regulatory guidelines, where sections pertaining to conjugates allow for a flexible approach to nonclinical safety testing. The aim of this article is to review regulatory expectations regarding early assessment of nonclinical safety considerations and discuss how recent advances in our understanding of ADCmediated toxicity can be used to guide the types of nonclinical safety studies needed to support ADC clinical development. The review will also explore nonclinical testing strategies that can be used to streamline ADC development by assessing the safety and efficacy of next generation ADC constructs using a rodent screen approach.

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Section: Stabilitymentioning

confidence: 99%

Antibody Drug Conjugates: Nonclinical Safety Considerations

Hinrichs

Dixit

2015

AAPS J

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“…Although this approach may be useful for generating site-specifically conjugated ADCs, THIOMABS produced using this process are not directly amenable to conjugation, but instead, require a multistep process that includes decapping of the engineered cysteine residues, which inevitably results in the partial breaking and reformation of structurally important internal disulfide bonds. Site-specific ADCs generated by enzymatic modification also have demonstrated improved stability and pharmacokinetics; however, a surface-exposed transglutamase tag (LLQG) needs to be engineered into antibodies at a permissive site (8).…”

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confidence: 99%

A general approach to site-specific antibody drug conjugates

Tian

Manibusan

et al. 2014

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

284

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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“…Although we achieved this goal with two heavy chain tags, at that time our best conjugating light chain-tagged antibody returned low titers compared to untagged antibodies and was deemed unmanufacturable as a result. However, because other groups reported improved pharmacokinetics or in vivo efficacy by using ADCs conjugated to the light chain, 15,16 we refocused our efforts on identifying useful tag insertions in that region. As a next step, we tried computer-modeling approaches to predict optimal light chain tag site locations based on solvent accessibility as assessed by IgG crystal structures.…”

Section: Discussionmentioning

confidence: 99%

“…16 They tested the glutamine tag at ~90 sites identified as surface accessible within the constant domains of an IgG1. Of these sites, 12 yielded highly conjugated ADCs with good biophysical properties, translating to a success rate of ~13%, similar to that of our own unbiased scanning method (Table 1).…”

Section: Discussionmentioning

confidence: 99%

Antibody-drug conjugate library prepared by scanning insertion of the aldehyde tag into IgG1 constant regions

Huang

Kim

Bañas

et al. 2018

mAbs

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The advantages of site-specific over stochastic bioconjugation technologies include homogeneity of product, minimal perturbation of protein structure/function, and – increasingly – the ability to perform structure activity relationship studies at the conjugate level. When selecting the optimal location for site-specific payload placement, many researchers turn to in silico modeling of protein structure to identify regions predicted to offer solvent-exposed conjugatable sites while conserving protein function. Here, using the aldehyde tag as our site-specific technology platform and human IgG1 antibody as our target protein, we demonstrate the power of taking an unbiased scanning approach instead. Scanning insertion of the human formylglycine generating enzyme (FGE) recognition sequence, LCTPSR, at each of the 436 positions in the light and heavy chain antibody constant regions followed by co-expression with FGE yielded a library of antibodies bearing an aldehyde functional group ready for conjugation. Each of the variants was expressed, purified, and conjugated to a cytotoxic payload using the Hydrazinyl Iso-Pictet-Spengler ligation to generate an antibody-drug conjugate (ADC), which was analyzed in terms of conjugatability (assessed by drug-to-antibody ratio, DAR) and percent aggregate. We searched for insertion sites that could generate manufacturable ADCs, defined as those variants yielding reasonable antibody titers, DARs of ≥ 1.3, and ≥ 95% monomeric species. Through this process, we discovered 58 tag insertion sites that met these metrics, including 14 sites in the light chain, a location that had proved refractory to the placement of manufacturable tag sites using in silico modeling/rational approaches.

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Location Matters: Site of Conjugation Modulates Stability and Pharmacokinetics of Antibody Drug Conjugates

Cited by 434 publications

References 18 publications

Antibody Drug Conjugates: Nonclinical Safety Considerations

Antibody Drug Conjugates: Nonclinical Safety Considerations

A general approach to site-specific antibody drug conjugates

Antibody-drug conjugate library prepared by scanning insertion of the aldehyde tag into IgG1 constant regions

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