Limits...
Aldehyde tag coupled with HIPS chemistry enables the production of ADCs conjugated site-specifically to different antibody regions with distinct in vivo efficacy and PK outcomes.

Drake PM, Albers AE, Baker J, Banas S, Barfield RM, Bhat AS, de Hart GW, Garofalo AW, Holder P, Jones LC, Kudirka R, McFarland J, Zmolek W, Rabuka D - Bioconjug. Chem. (2014)

Bottom Line: It is becoming increasingly clear that site-specific conjugation offers significant advantages over conventional conjugation chemistries used to make antibody-drug conjugates (ADCs).This chemistry results in a stable C-C bond between the antibody and the cytotoxin payload, providing a uniquely stable connection with respect to the other linker chemistries used to generate ADCs.We demonstrate that in a panel of ADCs with aldehyde tags at different locations, the site of conjugation has a dramatic impact on in vivo efficacy and pharmacokinetic behavior in rodents; this advantage translates to an improved safety profile in rats as compared to a conventional lysine conjugate.

View Article: PubMed Central - PubMed

Affiliation: Redwood Bioscience , 5703 Hollis Street, Emeryville, California 94608, United States.

ABSTRACT
It is becoming increasingly clear that site-specific conjugation offers significant advantages over conventional conjugation chemistries used to make antibody-drug conjugates (ADCs). Site-specific payload placement allows for control over both the drug-to-antibody ratio (DAR) and the conjugation site, both of which play an important role in governing the pharmacokinetics (PK), disposition, and efficacy of the ADC. In addition to the DAR and site of conjugation, linker composition also plays an important role in the properties of an ADC. We have previously reported a novel site-specific conjugation platform comprising linker payloads designed to selectively react with site-specifically engineered aldehyde tags on an antibody backbone. This chemistry results in a stable C-C bond between the antibody and the cytotoxin payload, providing a uniquely stable connection with respect to the other linker chemistries used to generate ADCs. The flexibility and versatility of the aldehyde tag conjugation platform has enabled us to undertake a systematic evaluation of the impact of conjugation site and linker composition on ADC properties. Here, we describe the production and characterization of a panel of ADCs bearing the aldehyde tag at different locations on an IgG1 backbone conjugated using Hydrazino-iso-Pictet-Spengler (HIPS) chemistry. We demonstrate that in a panel of ADCs with aldehyde tags at different locations, the site of conjugation has a dramatic impact on in vivo efficacy and pharmacokinetic behavior in rodents; this advantage translates to an improved safety profile in rats as compared to a conventional lysine conjugate.

Show MeSH

Related in: MedlinePlus

Hydrophobic interaction chromatography analysis demonstratestheclean conversion of LC-, CH1-, and CT-tagged antibodies into homogeneousADCs. Unconjugated antibody (black) elutes as one peak. After conjugationto HIPS-Glu-PEG2-maytansine, the ADC (green) elutes as a diconjugatedmaterial (right). This clean separation of conjugated from unconjugatedmaterial allows for conjugate enrichment and simple determinationof DAR. α-HER2-DM1 was included as a comparator.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4215875&req=5

fig3: Hydrophobic interaction chromatography analysis demonstratestheclean conversion of LC-, CH1-, and CT-tagged antibodies into homogeneousADCs. Unconjugated antibody (black) elutes as one peak. After conjugationto HIPS-Glu-PEG2-maytansine, the ADC (green) elutes as a diconjugatedmaterial (right). This clean separation of conjugated from unconjugatedmaterial allows for conjugate enrichment and simple determinationof DAR. α-HER2-DM1 was included as a comparator.

Mentions: Trastuzumab antibodies modified to contain the aldehydetag in either the light chain (LC), the CH1 domain (Tag C), or atthe heavy chain C-terminus (CT, Tag G) were producedin bulk pools of cells overexpressing human FGE. In terms of Cys tofGly conversion efficiency, we achieved 86%, 92%, and 98% conversionat the LC, CH1, and CT aldehyde tag sites, respectively, as measuredby a mass spectrometric method.9 The conjugationreaction was carried out by treating the fGly-tagged antibody with8–10 equiv of HIPS-Glu-PEG2-maytansine in 50 mM sodium citrate,50 mM NaCl pH 5.5 containing 0.85% DMA and 0.085% Triton X-100 at37 °C, and the progress of the reaction was tracked by analyticalhydrophobic interaction chromatography (HIC). Upon completion, theexcess payload was removed by tangential flow filtration and the unconjugatedantibody was removed by preparative HIC. These reactions were highyielding, with >90% conjugation efficiency at the CH1 and CT tagsites,and 75% conjugation efficiency at the LC tag site. HIC analysis ofthe final product highlights the facile analytics (Figure 3), which are a major benefit of site-specific conjugationapproaches as compared to global conjugation strategies. SEC analysisof the conjugates demonstrated minimal aggregation (SI Figure S1).


Aldehyde tag coupled with HIPS chemistry enables the production of ADCs conjugated site-specifically to different antibody regions with distinct in vivo efficacy and PK outcomes.

Drake PM, Albers AE, Baker J, Banas S, Barfield RM, Bhat AS, de Hart GW, Garofalo AW, Holder P, Jones LC, Kudirka R, McFarland J, Zmolek W, Rabuka D - Bioconjug. Chem. (2014)

Hydrophobic interaction chromatography analysis demonstratestheclean conversion of LC-, CH1-, and CT-tagged antibodies into homogeneousADCs. Unconjugated antibody (black) elutes as one peak. After conjugationto HIPS-Glu-PEG2-maytansine, the ADC (green) elutes as a diconjugatedmaterial (right). This clean separation of conjugated from unconjugatedmaterial allows for conjugate enrichment and simple determinationof DAR. α-HER2-DM1 was included as a comparator.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215875&req=5

fig3: Hydrophobic interaction chromatography analysis demonstratestheclean conversion of LC-, CH1-, and CT-tagged antibodies into homogeneousADCs. Unconjugated antibody (black) elutes as one peak. After conjugationto HIPS-Glu-PEG2-maytansine, the ADC (green) elutes as a diconjugatedmaterial (right). This clean separation of conjugated from unconjugatedmaterial allows for conjugate enrichment and simple determinationof DAR. α-HER2-DM1 was included as a comparator.
Mentions: Trastuzumab antibodies modified to contain the aldehydetag in either the light chain (LC), the CH1 domain (Tag C), or atthe heavy chain C-terminus (CT, Tag G) were producedin bulk pools of cells overexpressing human FGE. In terms of Cys tofGly conversion efficiency, we achieved 86%, 92%, and 98% conversionat the LC, CH1, and CT aldehyde tag sites, respectively, as measuredby a mass spectrometric method.9 The conjugationreaction was carried out by treating the fGly-tagged antibody with8–10 equiv of HIPS-Glu-PEG2-maytansine in 50 mM sodium citrate,50 mM NaCl pH 5.5 containing 0.85% DMA and 0.085% Triton X-100 at37 °C, and the progress of the reaction was tracked by analyticalhydrophobic interaction chromatography (HIC). Upon completion, theexcess payload was removed by tangential flow filtration and the unconjugatedantibody was removed by preparative HIC. These reactions were highyielding, with >90% conjugation efficiency at the CH1 and CT tagsites,and 75% conjugation efficiency at the LC tag site. HIC analysis ofthe final product highlights the facile analytics (Figure 3), which are a major benefit of site-specific conjugationapproaches as compared to global conjugation strategies. SEC analysisof the conjugates demonstrated minimal aggregation (SI Figure S1).

Bottom Line: It is becoming increasingly clear that site-specific conjugation offers significant advantages over conventional conjugation chemistries used to make antibody-drug conjugates (ADCs).This chemistry results in a stable C-C bond between the antibody and the cytotoxin payload, providing a uniquely stable connection with respect to the other linker chemistries used to generate ADCs.We demonstrate that in a panel of ADCs with aldehyde tags at different locations, the site of conjugation has a dramatic impact on in vivo efficacy and pharmacokinetic behavior in rodents; this advantage translates to an improved safety profile in rats as compared to a conventional lysine conjugate.

View Article: PubMed Central - PubMed

Affiliation: Redwood Bioscience , 5703 Hollis Street, Emeryville, California 94608, United States.

ABSTRACT
It is becoming increasingly clear that site-specific conjugation offers significant advantages over conventional conjugation chemistries used to make antibody-drug conjugates (ADCs). Site-specific payload placement allows for control over both the drug-to-antibody ratio (DAR) and the conjugation site, both of which play an important role in governing the pharmacokinetics (PK), disposition, and efficacy of the ADC. In addition to the DAR and site of conjugation, linker composition also plays an important role in the properties of an ADC. We have previously reported a novel site-specific conjugation platform comprising linker payloads designed to selectively react with site-specifically engineered aldehyde tags on an antibody backbone. This chemistry results in a stable C-C bond between the antibody and the cytotoxin payload, providing a uniquely stable connection with respect to the other linker chemistries used to generate ADCs. The flexibility and versatility of the aldehyde tag conjugation platform has enabled us to undertake a systematic evaluation of the impact of conjugation site and linker composition on ADC properties. Here, we describe the production and characterization of a panel of ADCs bearing the aldehyde tag at different locations on an IgG1 backbone conjugated using Hydrazino-iso-Pictet-Spengler (HIPS) chemistry. We demonstrate that in a panel of ADCs with aldehyde tags at different locations, the site of conjugation has a dramatic impact on in vivo efficacy and pharmacokinetic behavior in rodents; this advantage translates to an improved safety profile in rats as compared to a conventional lysine conjugate.

Show MeSH
Related in: MedlinePlus