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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.

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Payload location does not influence in vitro potency ofaldehyde-taggedα-HER2 ADCs against NCI-N87 target cells. NCI-N87 cells, whichoverexpress HER2, were used as targets for in vitro cytotoxicity ina 6 day assay. Free maytansine (gray line) was included as a positivecontrol, and an isotype control ADC (orange line) was used as a negativecontrol to indicate specificity. α-HER2 HIPS-Glu-PEG2-maytansineADCs bearing the aldehyde tag on the light chain (LC, green), or onthe CH1 (red) or C-terminal (CT, blue) regions ofthe heavy chain showed comparable activity. α-HER2-DM1 was includedas a comparator. IC50 values (reflecting the antibody concentrationsexcept in the case of the free drug) were measured as follows: freemaytansine, 214 pM; isotype control ADC, could not be determined;LC ADC 87 pM; CH1 ADC, 132 pM; CT ADC 114 pM, α-HER2-DM1, 54.7pM.
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fig5: Payload location does not influence in vitro potency ofaldehyde-taggedα-HER2 ADCs against NCI-N87 target cells. NCI-N87 cells, whichoverexpress HER2, were used as targets for in vitro cytotoxicity ina 6 day assay. Free maytansine (gray line) was included as a positivecontrol, and an isotype control ADC (orange line) was used as a negativecontrol to indicate specificity. α-HER2 HIPS-Glu-PEG2-maytansineADCs bearing the aldehyde tag on the light chain (LC, green), or onthe CH1 (red) or C-terminal (CT, blue) regions ofthe heavy chain showed comparable activity. α-HER2-DM1 was includedas a comparator. IC50 values (reflecting the antibody concentrationsexcept in the case of the free drug) were measured as follows: freemaytansine, 214 pM; isotype control ADC, could not be determined;LC ADC 87 pM; CH1 ADC, 132 pM; CT ADC 114 pM, α-HER2-DM1, 54.7pM.

Mentions: As a first measure ofefficacy, the LC, CH1, and CT tagged α-Her2 ADCs were testedin vitro against the HER2-overexpressing cell line, NCI-N87. Freemaytansine and α-HER2-DM1 (DAR 3.4) were used as comparators.All three of the α-HER2 HIPS-Glu-PEG2-maytansine ADC conjugatesshowed excellent in vitro cytotoxicity that was on par with free maytansineand α-HER2-DM1 (Figure 5). By contrast,the isotype control CT-tagged conjugate showed essentially no activity,as expected.


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)

Payload location does not influence in vitro potency ofaldehyde-taggedα-HER2 ADCs against NCI-N87 target cells. NCI-N87 cells, whichoverexpress HER2, were used as targets for in vitro cytotoxicity ina 6 day assay. Free maytansine (gray line) was included as a positivecontrol, and an isotype control ADC (orange line) was used as a negativecontrol to indicate specificity. α-HER2 HIPS-Glu-PEG2-maytansineADCs bearing the aldehyde tag on the light chain (LC, green), or onthe CH1 (red) or C-terminal (CT, blue) regions ofthe heavy chain showed comparable activity. α-HER2-DM1 was includedas a comparator. IC50 values (reflecting the antibody concentrationsexcept in the case of the free drug) were measured as follows: freemaytansine, 214 pM; isotype control ADC, could not be determined;LC ADC 87 pM; CH1 ADC, 132 pM; CT ADC 114 pM, α-HER2-DM1, 54.7pM.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Payload location does not influence in vitro potency ofaldehyde-taggedα-HER2 ADCs against NCI-N87 target cells. NCI-N87 cells, whichoverexpress HER2, were used as targets for in vitro cytotoxicity ina 6 day assay. Free maytansine (gray line) was included as a positivecontrol, and an isotype control ADC (orange line) was used as a negativecontrol to indicate specificity. α-HER2 HIPS-Glu-PEG2-maytansineADCs bearing the aldehyde tag on the light chain (LC, green), or onthe CH1 (red) or C-terminal (CT, blue) regions ofthe heavy chain showed comparable activity. α-HER2-DM1 was includedas a comparator. IC50 values (reflecting the antibody concentrationsexcept in the case of the free drug) were measured as follows: freemaytansine, 214 pM; isotype control ADC, could not be determined;LC ADC 87 pM; CH1 ADC, 132 pM; CT ADC 114 pM, α-HER2-DM1, 54.7pM.
Mentions: As a first measure ofefficacy, the LC, CH1, and CT tagged α-Her2 ADCs were testedin vitro against the HER2-overexpressing cell line, NCI-N87. Freemaytansine and α-HER2-DM1 (DAR 3.4) were used as comparators.All three of the α-HER2 HIPS-Glu-PEG2-maytansine ADC conjugatesshowed excellent in vitro cytotoxicity that was on par with free maytansineand α-HER2-DM1 (Figure 5). By contrast,the isotype control CT-tagged conjugate showed essentially no activity,as expected.

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