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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 placementmodifies the in vivo efficacy of aldehyde-taggedα-HER2 ADCs against NCI-N87 xenografts in mice. CB.17 SCID mice(8/group) were implanted subcutaneously with NCI-N87 cells. When thetumors reached ∼113 mm3, the animals were givena single 5 mg/kg dose of trastuzumab alone, an isotype ADC, or anα-HER2 HIPS-Glu-PEG2-maytansine ADC conjugated to either thelight chain (LC), or the CH1 or C-terminal (CT) regionsof the heavy chain. α-HER2-DM1 was included as a comparator.(A) Tumor growth was monitored twice weekly. (B) The differences inefficacy among the tag placements tested were reflected in survivalcurves. Animals were euthanized when tumors reached 800 mm3.
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fig6: Payload placementmodifies the in vivo efficacy of aldehyde-taggedα-HER2 ADCs against NCI-N87 xenografts in mice. CB.17 SCID mice(8/group) were implanted subcutaneously with NCI-N87 cells. When thetumors reached ∼113 mm3, the animals were givena single 5 mg/kg dose of trastuzumab alone, an isotype ADC, or anα-HER2 HIPS-Glu-PEG2-maytansine ADC conjugated to either thelight chain (LC), or the CH1 or C-terminal (CT) regionsof the heavy chain. α-HER2-DM1 was included as a comparator.(A) Tumor growth was monitored twice weekly. (B) The differences inefficacy among the tag placements tested were reflected in survivalcurves. Animals were euthanized when tumors reached 800 mm3.

Mentions: The in vivo efficacy of LC-, CH1-,and CT-tagged α-HER2 ADCswas explored using a staged NCI-N87 xenograft model in SCID mice.Trastuzumab alone and an isotype control CT-tagged HIPS-Glu-PEG2-maytansineADC were used as negative controls, and α-HER2-DM1 (DAR 3.4)was included as a comparator. All compounds were administered as asingle 5 mg/kg dose at the onset of the study. While the tumors continuedto grow in mice treated with either trastuzumab or the isotype controlADC, a single dose of α-HER2-targeted ADC was sufficient tostop tumor growth for ∼30 days in treated animals (Figure 6A). When tumors did eventually begin to grow back,it became clear that the tumor sizes were larger in animals treatedwith the CH1-tagged ADC as compared to those treated with LC- or CT-taggedADCs (p < 0.026 and 0.016, respectively, two-tailed t-test at day 67). In order to investigate this effect,we looked at the log10 cell kill for tumors dosed withthe various treatments (Table 3). Indeed, theresults suggested that treatment with the CH1-tagged ADC killed fewertumor cells as compared to treatment with the other ADCs. Furthermore,the CT-tagged ADC appeared to be the most efficacious conjugate resultingin the highest log10 cell kill. This increased potencytranslated into a significant survival advantage for animals treatedwith the CT-tagged ADC (Figure 6B).


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 placementmodifies the in vivo efficacy of aldehyde-taggedα-HER2 ADCs against NCI-N87 xenografts in mice. CB.17 SCID mice(8/group) were implanted subcutaneously with NCI-N87 cells. When thetumors reached ∼113 mm3, the animals were givena single 5 mg/kg dose of trastuzumab alone, an isotype ADC, or anα-HER2 HIPS-Glu-PEG2-maytansine ADC conjugated to either thelight chain (LC), or the CH1 or C-terminal (CT) regionsof the heavy chain. α-HER2-DM1 was included as a comparator.(A) Tumor growth was monitored twice weekly. (B) The differences inefficacy among the tag placements tested were reflected in survivalcurves. Animals were euthanized when tumors reached 800 mm3.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Payload placementmodifies the in vivo efficacy of aldehyde-taggedα-HER2 ADCs against NCI-N87 xenografts in mice. CB.17 SCID mice(8/group) were implanted subcutaneously with NCI-N87 cells. When thetumors reached ∼113 mm3, the animals were givena single 5 mg/kg dose of trastuzumab alone, an isotype ADC, or anα-HER2 HIPS-Glu-PEG2-maytansine ADC conjugated to either thelight chain (LC), or the CH1 or C-terminal (CT) regionsof the heavy chain. α-HER2-DM1 was included as a comparator.(A) Tumor growth was monitored twice weekly. (B) The differences inefficacy among the tag placements tested were reflected in survivalcurves. Animals were euthanized when tumors reached 800 mm3.
Mentions: The in vivo efficacy of LC-, CH1-,and CT-tagged α-HER2 ADCswas explored using a staged NCI-N87 xenograft model in SCID mice.Trastuzumab alone and an isotype control CT-tagged HIPS-Glu-PEG2-maytansineADC were used as negative controls, and α-HER2-DM1 (DAR 3.4)was included as a comparator. All compounds were administered as asingle 5 mg/kg dose at the onset of the study. While the tumors continuedto grow in mice treated with either trastuzumab or the isotype controlADC, a single dose of α-HER2-targeted ADC was sufficient tostop tumor growth for ∼30 days in treated animals (Figure 6A). When tumors did eventually begin to grow back,it became clear that the tumor sizes were larger in animals treatedwith the CH1-tagged ADC as compared to those treated with LC- or CT-taggedADCs (p < 0.026 and 0.016, respectively, two-tailed t-test at day 67). In order to investigate this effect,we looked at the log10 cell kill for tumors dosed withthe various treatments (Table 3). Indeed, theresults suggested that treatment with the CH1-tagged ADC killed fewertumor cells as compared to treatment with the other ADCs. Furthermore,the CT-tagged ADC appeared to be the most efficacious conjugate resultingin the highest log10 cell kill. This increased potencytranslated into a significant survival advantage for animals treatedwith the CT-tagged ADC (Figure 6B).

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