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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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Toxicokinetic analysis demonstrated that theα-HER2 CT ADCwas more stable in rats than the α-HER2-DM1. The same animalsthat were analyzed for indicators of toxicity (Figure 8) were used to assess the toxicokinetic profiles of the α-HER2CT ADC and α-HER2-DM1 analytes. Plasma was sampled at the timepoints indicated and assayed by ELISA.
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fig9: Toxicokinetic analysis demonstrated that theα-HER2 CT ADCwas more stable in rats than the α-HER2-DM1. The same animalsthat were analyzed for indicators of toxicity (Figure 8) were used to assess the toxicokinetic profiles of the α-HER2CT ADC and α-HER2-DM1 analytes. Plasma was sampled at the timepoints indicated and assayed by ELISA.

Mentions: Finally,we wanted to get an initial assessment of the safety profile of oursite-specific conjugates. Accordingly, we conducted a single doseexploratory toxicology study in Sprague–Dawley rats. Animals(5/group) received a 6, 20, or 60 mg/kg dose of CT-tagged α-HER2followed by a 12 day observation period. As a comparator, we usedthe conventional α-HER2-DM1 at the same doses. Body weight andfood intake were assessed at days 0, 1, 4, 8, and 11. Blood was drawnon days 5 and 12 for clinical chemistry, hematology, and toxicokinetic(TK) analysis. Additional TK blood samples were drawn at 8 h and onday 9. All of the animals in the α-HER2-DM1 60 mg/kg group diedon day 5 (Table 5). This mortality was consistentwith the known preclinical safety profile of the analogue, T-DM1.29 By contrast, no mortality was observed in theα-HER2 CT ADC groups, even at 60 mg/kg. With respect to bodyweight (Figure 8A), treatment with 6 or 20mg/kg of α-HER2 CT ADC had no effect, while treatment with 60mg/kg of α-HER2 CT ADC reduced rat body weight to a similarextent as treatment with 20 mg/kg of α-HER2-DM1. With respectto clinical chemistry, levels of both alanine aminotransferase (ALT)and aspartate aminotransferase (AST) were essentially unchanged inrats treated with 6 or 20 mg/kg of α-HER2 CT ADC, but were somewhatelevated in rats treated with 20 mg/kg of α-HER2-DM1 (Figure 8B and C). Levels of both enzymes were elevated atthe 60 mg/kg dose for both compounds, although to a lesser extentwith the α-HER2 CT ADC. Increased ALT and AST levels are indicatorsof liver toxicity, the former a much more specific marker than thelatter. Increases in these enzymes are consistent with known toxicityprofiles of maytansine and maytansine conjugates. With respect tohematology, platelet counts were essentially unchanged in rats treatedwith 6 or 20 mg/kg of α-HER2 CT ADC, but were decreased in ratstreated with 20 mg/kg of α-HER2-DM1 (Figure 8D). Platelet counts were decreased at the 60 mg/kg dose forboth compounds, although to a lesser extent with the α-HER2CT ADC. Decreases in platelet counts at day 5 postdose are generallyindicative of localized tissue damage, rather than bone marrow toxicity,which takes longer to manifest due to the long half-life of platelets.For all surviving animals, the clinical chemistry and hematology indicatorsof toxicity observed at day 5 were essentially resolved by day 12(SI Figure S3). In total, the data indicatedthat the α-HER2 CT ADC was less toxic to rats than the α-HER2-DM1at the same doses; however, as expected, the target organs were thesame. Furthermore, toxicokinetic analyses of the total ADC and totalantibody concentrations showed that the α-HER2 CT ADC was stablein vivo, showing similar profiles in the rat as were observed in themouse. Notably, the α-HER2-DM1 total ADC was cleared fasterthan the α-HER2 CT ADC total ADC (Figure 9).


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)

Toxicokinetic analysis demonstrated that theα-HER2 CT ADCwas more stable in rats than the α-HER2-DM1. The same animalsthat were analyzed for indicators of toxicity (Figure 8) were used to assess the toxicokinetic profiles of the α-HER2CT ADC and α-HER2-DM1 analytes. Plasma was sampled at the timepoints indicated and assayed by ELISA.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4215875&req=5

fig9: Toxicokinetic analysis demonstrated that theα-HER2 CT ADCwas more stable in rats than the α-HER2-DM1. The same animalsthat were analyzed for indicators of toxicity (Figure 8) were used to assess the toxicokinetic profiles of the α-HER2CT ADC and α-HER2-DM1 analytes. Plasma was sampled at the timepoints indicated and assayed by ELISA.
Mentions: Finally,we wanted to get an initial assessment of the safety profile of oursite-specific conjugates. Accordingly, we conducted a single doseexploratory toxicology study in Sprague–Dawley rats. Animals(5/group) received a 6, 20, or 60 mg/kg dose of CT-tagged α-HER2followed by a 12 day observation period. As a comparator, we usedthe conventional α-HER2-DM1 at the same doses. Body weight andfood intake were assessed at days 0, 1, 4, 8, and 11. Blood was drawnon days 5 and 12 for clinical chemistry, hematology, and toxicokinetic(TK) analysis. Additional TK blood samples were drawn at 8 h and onday 9. All of the animals in the α-HER2-DM1 60 mg/kg group diedon day 5 (Table 5). This mortality was consistentwith the known preclinical safety profile of the analogue, T-DM1.29 By contrast, no mortality was observed in theα-HER2 CT ADC groups, even at 60 mg/kg. With respect to bodyweight (Figure 8A), treatment with 6 or 20mg/kg of α-HER2 CT ADC had no effect, while treatment with 60mg/kg of α-HER2 CT ADC reduced rat body weight to a similarextent as treatment with 20 mg/kg of α-HER2-DM1. With respectto clinical chemistry, levels of both alanine aminotransferase (ALT)and aspartate aminotransferase (AST) were essentially unchanged inrats treated with 6 or 20 mg/kg of α-HER2 CT ADC, but were somewhatelevated in rats treated with 20 mg/kg of α-HER2-DM1 (Figure 8B and C). Levels of both enzymes were elevated atthe 60 mg/kg dose for both compounds, although to a lesser extentwith the α-HER2 CT ADC. Increased ALT and AST levels are indicatorsof liver toxicity, the former a much more specific marker than thelatter. Increases in these enzymes are consistent with known toxicityprofiles of maytansine and maytansine conjugates. With respect tohematology, platelet counts were essentially unchanged in rats treatedwith 6 or 20 mg/kg of α-HER2 CT ADC, but were decreased in ratstreated with 20 mg/kg of α-HER2-DM1 (Figure 8D). Platelet counts were decreased at the 60 mg/kg dose forboth compounds, although to a lesser extent with the α-HER2CT ADC. Decreases in platelet counts at day 5 postdose are generallyindicative of localized tissue damage, rather than bone marrow toxicity,which takes longer to manifest due to the long half-life of platelets.For all surviving animals, the clinical chemistry and hematology indicatorsof toxicity observed at day 5 were essentially resolved by day 12(SI Figure S3). In total, the data indicatedthat the α-HER2 CT ADC was less toxic to rats than the α-HER2-DM1at the same doses; however, as expected, the target organs were thesame. Furthermore, toxicokinetic analyses of the total ADC and totalantibody concentrations showed that the α-HER2 CT ADC was stablein vivo, showing similar profiles in the rat as were observed in themouse. Notably, the α-HER2-DM1 total ADC was cleared fasterthan the α-HER2 CT ADC total ADC (Figure 9).

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