Reducing hydrophobicity of homogeneous antibody-drug conjugates improves pharmacokinetics and therapeutic index

Lyon, Robert P.; Bovee, Tim D.; Doronina, Svetlana O.; Burke, Patrick; Hunter, Joshua H.; Neff-LaFord, Haley; Jonas, Mechthild; Anderson, Martha E.; Setter, Jocelyn R.; Senter, Peter D.

doi:10.1038/nbt.3212

Cited by 412 publications

(452 citation statements)

References 16 publications

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“…Using this approach, an ADC is produced that is homogeneous and bears an average of 16 total drugs, split evenly between the two drug linkers. The carrier utilizes two recent advancements for the construction of ADCs with improved pharmacological activity: a self‐stabilizing maleimide (mDPR) to minimize drug‐linker deconjugation in vivo,2a and a PEG 24 stretcher to enable high drug loading without concomitant hydrophobicity‐induced ADC aggregation 6…”

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confidence: 99%

“…B) A multiplexing drug carrier 4 bearing Cys(SiPr) and Cys(Acm) groups that can be unmasked using orthogonal conditions. The carrier also contains a PEG 24 group to mask drug‐linker hydrophicity6 and a self‐stabilizing maleimide (mDPR)2a for antibody attachment. C) Homogeneous dual‐drug ADCs prepared using 4 bear 16 total drugs, split evenly (8+8) between the two component drugs.…”

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confidence: 99%

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Orthogonal Cysteine Protection Enables Homogeneous Multi‐Drug Antibody–Drug Conjugates

et al. 2016

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A strategy for the preparation of homogeneous antibody–drug conjugates (ADCs) containing multiple payloads has been developed. This approach utilizes sequential unmasking of cysteine residues with orthogonal protection to enable site‐specific conjugation of each drug. In addition, because the approach utilizes conjugation to native antibody cysteine residues, it is widely applicable and enables high drug loading for improved ADC potency. To highlight the benefits of ADC dual drug delivery, this strategy was applied to the preparation of ADCs containing two classes of auristatin drug‐linkers that have differing physiochemical properties and exert complementary anti‐cancer activities. Dual‐auristatin ADCs imparted activity in cell line and xenograft models that are refractory to ADCs comprised of the individual auristatin components. This work presents a facile method for construction of potent dual‐drug ADCs and demonstrates how delivery of multiple cytotoxic warheads can lead to improved ADC activities. Lastly, we anticipate that the conditions utilized herein for orthogonal cysteine unmasking are not restricted to ADCs and can be broadly utilized for site‐specific protein modification.

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Orthogonal Cysteine Protection Enables Homogeneous Multi‐Drug Antibody–Drug Conjugates

et al. 2016

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“…Together, ADC stability and hydrophobicity are linked to in vivo pharmacokinetic and pharmacodynamic outcomes, including ADC half-life, efficacy, and tolerability. 19–21 Therefore, we determined the relative retention time (RRT) of the ADCs in our panel as measured by hydrophobic interaction chromatography (HIC), a typical measure of hydrophobicity. The RRT is calculated by dividing the retention time of the DAR 2 ADC by that of the unconjugated antibody.…”

Section: Resultsmentioning

confidence: 99%

“…In general, current trends in the ADC field suggest that constructs with low RRTs offer superior performance compared to more hydrophobic constructs. 19–21 The aldehyde tag scan approach allowed us to identify 43 heavy chain-tagged RED-106 ADC variants (23% of conjugatable tag insertions) displaying RRT values of ≤ 1.3. Of these, 21 had RRT values of ≤ 1.2, and 12 had values of 1, where no change in retention time was detected upon conjugation.…”

Section: Resultsmentioning

confidence: 99%

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

Huang

Kim

Bañas

et al. 2018

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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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“…16-19 Moreover, understanding the effect of the site of conjugation, drug loading, and drug-linker design on ADC PK has enabled mitigation of accelerated clearance observed with some ADCs. 20-22 Mechanistic PK/pharmacodynamic (PD) 23-26 and multi-scale models 13-15 have been proposed to improve translatability from preclinical species to the clinic. However, implementation and calibration of these multi-scale models require a substantial amount of in vitro and in vivo data that may not be available when human PK predictions are first required, which is typically during early stages of drug development.…”

Section: Introductionmentioning

confidence: 99%

Prediction of non-linear pharmacokinetics in humans of an antibody-drug conjugate (ADC) when evaluation of higher doses in animals is limited by tolerability: Case study with an anti-CD33 ADC

Figueroa

Leipold

Leong

et al. 2018

mAbs

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For antibody-drug conjugates (ADCs) that carry a cytotoxic drug, doses that can be administered in preclinical studies are typically limited by tolerability, leading to a narrow dose range that can be tested. For molecules with non-linear pharmacokinetics (PK), this limited dose range may be insufficient to fully characterize the PK of the ADC and limits translation to humans. Mathematical PK models are frequently used for molecule selection during preclinical drug development and for translational predictions to guide clinical study design. Here, we present a practical approach that uses limited PK and receptor occupancy (RO) data of the corresponding unconjugated antibody to predict ADC PK when conjugation does not alter the non-specific clearance or the antibody-target interaction. We used a 2-compartment model incorporating non-specific and specific (target mediated) clearances, where the latter is a function of RO, to describe the PK of anti-CD33 ADC with dose-limiting neutropenia in cynomolgus monkeys. We tested our model by comparing PK predictions based on the unconjugated antibody to observed ADC PK data that was not utilized for model development. Prospective prediction of human PK was performed by incorporating in vitro binding affinity differences between species for varying levels of CD33 target expression. Additionally, this approach was used to predict human PK of other previously tested anti-CD33 molecules with published clinical data. The findings showed that, for a cytotoxic ADC with non-linear PK and limited preclinical PK data, incorporating RO in the PK model and using data from the corresponding unconjugated antibody at higher doses allowed the identification of parameters to characterize monkey PK and enabled human PK predictions.

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Reducing hydrophobicity of homogeneous antibody-drug conjugates improves pharmacokinetics and therapeutic index

Cited by 412 publications

References 16 publications

Orthogonal Cysteine Protection Enables Homogeneous Multi‐Drug Antibody–Drug Conjugates

Orthogonal Cysteine Protection Enables Homogeneous Multi‐Drug Antibody–Drug Conjugates

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

Prediction of non-linear pharmacokinetics in humans of an antibody-drug conjugate (ADC) when evaluation of higher doses in animals is limited by tolerability: Case study with an anti-CD33 ADC

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