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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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α-HER2 HIPS-Glu-PEG2-maytansine ADCs are highly stable invivo regardless of tag placement. BALB/c mice were dosed with 5 mg/kgof aldehyde-tagged α-HER2 HIPS-Glu-PEG2-maytansine ADCs conjugatedto either the light chain (LC), or to the CH1 or C-terminal (CT) regions of the heavy chain. α-HER2-DM1 was includedas a comparator. Plasma was sampled at the time points indicated andassayed by ELISA. Area under the curve (AUC) was determined usingGraphPad Prism and is reported in Table 4.
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fig7: α-HER2 HIPS-Glu-PEG2-maytansine ADCs are highly stable invivo regardless of tag placement. BALB/c mice were dosed with 5 mg/kgof aldehyde-tagged α-HER2 HIPS-Glu-PEG2-maytansine ADCs conjugatedto either the light chain (LC), or to the CH1 or C-terminal (CT) regions of the heavy chain. α-HER2-DM1 was includedas a comparator. Plasma was sampled at the time points indicated andassayed by ELISA. Area under the curve (AUC) was determined usingGraphPad Prism and is reported in Table 4.

Mentions: We wanted to determineif the differential efficacy of our ADC panel was a function of distinctpharmacokinetic profiles. Mice were dosed with 5 mg/kg of LC-, CH1-,or CT-tagged ADC, or with trastuzumab or α-HER2-DM1 as comparators.Plasma was collected from the mice and analyzed by ELISA to quantitatethe total ADC and total antibody concentrations. To measure totalADC, analytes were captured with an anti-human Fab-specific antibodyand detected with an anti-maytansine antibody. To measure total antibody,analytes were captured with an anti-human IgG-specific antibody anddetected with an anti-human Fc-specific antibody. The measured concentrationsover time were fit to a two-compartment model by nonlinear regressionto determine half-lives (Table 3). The totalantibody half-life for each aldehyde-tagged ADC was the same as, orlonger than, trastuzumab, suggesting that aldehyde tag insertion andHIPS conjugation did not change the basic PK properties of the antibody.By contrast, the total antibody half-life of the α-HER2-DM1conjugate was significantly shorter, suggesting that the nonspecificconjugation chemistry (which leads to overconjugated species) hada negative effect on PK. We also measured the conjugated antibody(total ADC) half-lives, which showed that the CT-tagged ADC, whichconferred the biggest survival benefit to tumor-bearing mice, alsodemonstrated longest total half-life. The conjugate half-lives ofthe α-HER2-DM1, and the CH1- and LC-tagged ADCs, were shorterthan the CT-tagged conjugate. These numbers clearly indicate thatthe conjugation site played a key role in governing ADC half-lives.In all cases, the aldehyde-tagged conjugates were stable in the circulation,with percent area under the curve ratios of total ADC to total antibodyconcentrations comparable to or better than the α-HER2-DM1 conjugate(Figure 7).


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)

α-HER2 HIPS-Glu-PEG2-maytansine ADCs are highly stable invivo regardless of tag placement. BALB/c mice were dosed with 5 mg/kgof aldehyde-tagged α-HER2 HIPS-Glu-PEG2-maytansine ADCs conjugatedto either the light chain (LC), or to the CH1 or C-terminal (CT) regions of the heavy chain. α-HER2-DM1 was includedas a comparator. Plasma was sampled at the time points indicated andassayed by ELISA. Area under the curve (AUC) was determined usingGraphPad Prism and is reported in Table 4.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: α-HER2 HIPS-Glu-PEG2-maytansine ADCs are highly stable invivo regardless of tag placement. BALB/c mice were dosed with 5 mg/kgof aldehyde-tagged α-HER2 HIPS-Glu-PEG2-maytansine ADCs conjugatedto either the light chain (LC), or to the CH1 or C-terminal (CT) regions of the heavy chain. α-HER2-DM1 was includedas a comparator. Plasma was sampled at the time points indicated andassayed by ELISA. Area under the curve (AUC) was determined usingGraphPad Prism and is reported in Table 4.
Mentions: We wanted to determineif the differential efficacy of our ADC panel was a function of distinctpharmacokinetic profiles. Mice were dosed with 5 mg/kg of LC-, CH1-,or CT-tagged ADC, or with trastuzumab or α-HER2-DM1 as comparators.Plasma was collected from the mice and analyzed by ELISA to quantitatethe total ADC and total antibody concentrations. To measure totalADC, analytes were captured with an anti-human Fab-specific antibodyand detected with an anti-maytansine antibody. To measure total antibody,analytes were captured with an anti-human IgG-specific antibody anddetected with an anti-human Fc-specific antibody. The measured concentrationsover time were fit to a two-compartment model by nonlinear regressionto determine half-lives (Table 3). The totalantibody half-life for each aldehyde-tagged ADC was the same as, orlonger than, trastuzumab, suggesting that aldehyde tag insertion andHIPS conjugation did not change the basic PK properties of the antibody.By contrast, the total antibody half-life of the α-HER2-DM1conjugate was significantly shorter, suggesting that the nonspecificconjugation chemistry (which leads to overconjugated species) hada negative effect on PK. We also measured the conjugated antibody(total ADC) half-lives, which showed that the CT-tagged ADC, whichconferred the biggest survival benefit to tumor-bearing mice, alsodemonstrated longest total half-life. The conjugate half-lives ofthe α-HER2-DM1, and the CH1- and LC-tagged ADCs, were shorterthan the CT-tagged conjugate. These numbers clearly indicate thatthe conjugation site played a key role in governing ADC half-lives.In all cases, the aldehyde-tagged conjugates were stable in the circulation,with percent area under the curve ratios of total ADC to total antibodyconcentrations comparable to or better than the α-HER2-DM1 conjugate(Figure 7).

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