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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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Aldehyde-taggedHIPS conjugates are stable in plasma at 37 °C,but payload attachment plays a role. We tested the plasma stabilityof LC-, CH1-, and CT-tagged antibodies conjugated using HIPS-Glu-PEG2to either (A) Alexa Fluor 488 (AF488) or (B) maytansine. Conjugateswere incubated in rat plasma at 37 °C for up to 13 d. When analyzedby ELISA for total payload and total antibody, we observed no lossof total payload signal relative to total antibody signal for theAF488 conjugates, regardless of tag placement. For the maytansineconjugates, we observed evidence that some deconjugation occurredover time at 37 °C. The stability differed according to tag placement,with the CT-tag showing the highest conservation of payload-to-antibodysignal (84%), followed by CH1 (72%), and LC (65%).
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fig4: Aldehyde-taggedHIPS conjugates are stable in plasma at 37 °C,but payload attachment plays a role. We tested the plasma stabilityof LC-, CH1-, and CT-tagged antibodies conjugated using HIPS-Glu-PEG2to either (A) Alexa Fluor 488 (AF488) or (B) maytansine. Conjugateswere incubated in rat plasma at 37 °C for up to 13 d. When analyzedby ELISA for total payload and total antibody, we observed no lossof total payload signal relative to total antibody signal for theAF488 conjugates, regardless of tag placement. For the maytansineconjugates, we observed evidence that some deconjugation occurredover time at 37 °C. The stability differed according to tag placement,with the CT-tag showing the highest conservation of payload-to-antibodysignal (84%), followed by CH1 (72%), and LC (65%).

Mentions: In a parallel set of experiments using a differentantibody backbonebut the same three tag placements, we tested the stability of aldehyde-taggedHIPS conjugates in plasma at 37 °C. We tested antibodies carryingthe HIPS-Glu-PEG2 linker attached to either a fluorophore (Alexa Fluor488, AF488) or cytotoxin payload (maytansine). The purpose of testingtwo payloads was to explore how differences in payload attachmentto the linker (e.g., ester vs aryl amide bond, see SI Figure S2) can affect stability. The results indicatedthat the HIPS conjugation chemistry is highly stable; specifically,for the AF488 conjugates, we saw no loss of payload signal over 12d at 37 °C in rat plasma, regardless of tag placement (Figure 4A). However, this stability did not completely translateto the maytansine conjugates, which did show some loss of payloadsignal over time (Figure 4B). The amount ofpayload loss differed according to tag placement, with the CT siteshowing the greatest stability, followed by the CH1 and LC sites.We hypothesize that the differences in stability between the AF488and maytansine conjugates are related to the differences in the chemicallinkages connecting the payload to the PEG2 portion of the linker(SI Figure S2). While the AF488 is attachedto the PEG2 by a stable aryl amide bond, the ester bond that connectsthe maytansine payload is a known chemical liability at high pH. Therefore,the differences that we observed in the stability of the three maytansineADCs might reflect distinct microenvironments at the three attachmentsites that influence the differential hydrolysis of the ester bond.


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)

Aldehyde-taggedHIPS conjugates are stable in plasma at 37 °C,but payload attachment plays a role. We tested the plasma stabilityof LC-, CH1-, and CT-tagged antibodies conjugated using HIPS-Glu-PEG2to either (A) Alexa Fluor 488 (AF488) or (B) maytansine. Conjugateswere incubated in rat plasma at 37 °C for up to 13 d. When analyzedby ELISA for total payload and total antibody, we observed no lossof total payload signal relative to total antibody signal for theAF488 conjugates, regardless of tag placement. For the maytansineconjugates, we observed evidence that some deconjugation occurredover time at 37 °C. The stability differed according to tag placement,with the CT-tag showing the highest conservation of payload-to-antibodysignal (84%), followed by CH1 (72%), and LC (65%).
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Related In: Results  -  Collection

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

fig4: Aldehyde-taggedHIPS conjugates are stable in plasma at 37 °C,but payload attachment plays a role. We tested the plasma stabilityof LC-, CH1-, and CT-tagged antibodies conjugated using HIPS-Glu-PEG2to either (A) Alexa Fluor 488 (AF488) or (B) maytansine. Conjugateswere incubated in rat plasma at 37 °C for up to 13 d. When analyzedby ELISA for total payload and total antibody, we observed no lossof total payload signal relative to total antibody signal for theAF488 conjugates, regardless of tag placement. For the maytansineconjugates, we observed evidence that some deconjugation occurredover time at 37 °C. The stability differed according to tag placement,with the CT-tag showing the highest conservation of payload-to-antibodysignal (84%), followed by CH1 (72%), and LC (65%).
Mentions: In a parallel set of experiments using a differentantibody backbonebut the same three tag placements, we tested the stability of aldehyde-taggedHIPS conjugates in plasma at 37 °C. We tested antibodies carryingthe HIPS-Glu-PEG2 linker attached to either a fluorophore (Alexa Fluor488, AF488) or cytotoxin payload (maytansine). The purpose of testingtwo payloads was to explore how differences in payload attachmentto the linker (e.g., ester vs aryl amide bond, see SI Figure S2) can affect stability. The results indicatedthat the HIPS conjugation chemistry is highly stable; specifically,for the AF488 conjugates, we saw no loss of payload signal over 12d at 37 °C in rat plasma, regardless of tag placement (Figure 4A). However, this stability did not completely translateto the maytansine conjugates, which did show some loss of payloadsignal over time (Figure 4B). The amount ofpayload loss differed according to tag placement, with the CT siteshowing the greatest stability, followed by the CH1 and LC sites.We hypothesize that the differences in stability between the AF488and maytansine conjugates are related to the differences in the chemicallinkages connecting the payload to the PEG2 portion of the linker(SI Figure S2). While the AF488 is attachedto the PEG2 by a stable aryl amide bond, the ester bond that connectsthe maytansine payload is a known chemical liability at high pH. Therefore,the differences that we observed in the stability of the three maytansineADCs might reflect distinct microenvironments at the three attachmentsites that influence the differential hydrolysis of the ester bond.

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