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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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Related in: MedlinePlus

Together, the aldehydetag and HIPS chemistry allow for stablecytotoxic payload conjugation at precise locations across the antibodysurface. (Top) We inserted the aldehyde tag (red) at one locationin the light chain (LC) and seven locations (labeled A–G) inthe heavy chain. Antibodies bearing these tags were produced and analyzedas the first step in making ADCs conjugated at different sites. (Bottom)HIPS-Glu-PEG2-maytansine 20 served as the linker (in black) and thecytotoxic payload (in blue) for ADCs used in these studies.
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fig2: Together, the aldehydetag and HIPS chemistry allow for stablecytotoxic payload conjugation at precise locations across the antibodysurface. (Top) We inserted the aldehyde tag (red) at one locationin the light chain (LC) and seven locations (labeled A–G) inthe heavy chain. Antibodies bearing these tags were produced and analyzedas the first step in making ADCs conjugated at different sites. (Bottom)HIPS-Glu-PEG2-maytansine 20 served as the linker (in black) and thecytotoxic payload (in blue) for ADCs used in these studies.

Mentions: As a first step toward generating a library of antibodiescarrying the aldehyde tag at various locations, we surveyed the humanIgG1 crystal structure15 to identify exposed,relatively unstructured areas within the heavy and light chain constantregions. Our design principle was to install the tag at locationsthat minimally perturbed the native IgG structure but remained accessiblefor conjugation. We incorporated each tag once into either the heavyor light chain, such that each antibody would bear two aldehyde groups.Specifically, we chose one internal site in the light chain, one atthe C-terminus and seven internal sites (three inthe CH1, two in the CH2, one in the CH3 domains) of the heavy chain(Figure 2, top). For the purpose of this discussionwe have represented these heavy chain sites in alphabetical orderaccording to their occurrence from N- to C-terminus (Tags A-G), while the light chain tag is designatedas “LC”. The selected tag sites were cloned into theconstant regions of a prototype human IgG1 heavy chain and kappa lightchain. Proteins were produced transiently in bulk pools of cells overexpressinghuman FGE to ensure efficient conversion of Cys to fGly within theconsensus sequence and resulting tagged antibodies were purified usingProtein A affinity columns and stored in PBS.


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)

Together, the aldehydetag and HIPS chemistry allow for stablecytotoxic payload conjugation at precise locations across the antibodysurface. (Top) We inserted the aldehyde tag (red) at one locationin the light chain (LC) and seven locations (labeled A–G) inthe heavy chain. Antibodies bearing these tags were produced and analyzedas the first step in making ADCs conjugated at different sites. (Bottom)HIPS-Glu-PEG2-maytansine 20 served as the linker (in black) and thecytotoxic payload (in blue) for ADCs used in these studies.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Together, the aldehydetag and HIPS chemistry allow for stablecytotoxic payload conjugation at precise locations across the antibodysurface. (Top) We inserted the aldehyde tag (red) at one locationin the light chain (LC) and seven locations (labeled A–G) inthe heavy chain. Antibodies bearing these tags were produced and analyzedas the first step in making ADCs conjugated at different sites. (Bottom)HIPS-Glu-PEG2-maytansine 20 served as the linker (in black) and thecytotoxic payload (in blue) for ADCs used in these studies.
Mentions: As a first step toward generating a library of antibodiescarrying the aldehyde tag at various locations, we surveyed the humanIgG1 crystal structure15 to identify exposed,relatively unstructured areas within the heavy and light chain constantregions. Our design principle was to install the tag at locationsthat minimally perturbed the native IgG structure but remained accessiblefor conjugation. We incorporated each tag once into either the heavyor light chain, such that each antibody would bear two aldehyde groups.Specifically, we chose one internal site in the light chain, one atthe C-terminus and seven internal sites (three inthe CH1, two in the CH2, one in the CH3 domains) of the heavy chain(Figure 2, top). For the purpose of this discussionwe have represented these heavy chain sites in alphabetical orderaccording to their occurrence from N- to C-terminus (Tags A-G), while the light chain tag is designatedas “LC”. The selected tag sites were cloned into theconstant regions of a prototype human IgG1 heavy chain and kappa lightchain. Proteins were produced transiently in bulk pools of cells overexpressinghuman FGE to ensure efficient conversion of Cys to fGly within theconsensus sequence and resulting tagged antibodies were purified usingProtein A affinity columns and stored in PBS.

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