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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 tagcoupled with HIPS chemistry yields site-specificallymodified antibodies carrying a payload attached through a stable C–Cbond. (A) A formylglycine-generating enzyme (FGE) recognition sequenceis inserted at the desired location along the antibody backbone usingstandard molecular biology techniques. Upon expression, FGE, whichis endogenous to eukaryotic cells, catalyzes the conversion of theCys within the consensus sequence to a formylglycine residue (fGly).(B) Antibodies carrying aldehyde moieties (in red, 2 per antibody)are reacted with a Hydrazino-iso-Pictet-Spengler(HIPS) linker and payload to generate a site-specifically conjugatedADC. (C) The HIPS chemistry proceeds through an intermediate hydrazoniumion followed by intramolecular alkylation with a nucleophilic indoleto generate a stable C–C bond.
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fig1: Aldehyde tagcoupled with HIPS chemistry yields site-specificallymodified antibodies carrying a payload attached through a stable C–Cbond. (A) A formylglycine-generating enzyme (FGE) recognition sequenceis inserted at the desired location along the antibody backbone usingstandard molecular biology techniques. Upon expression, FGE, whichis endogenous to eukaryotic cells, catalyzes the conversion of theCys within the consensus sequence to a formylglycine residue (fGly).(B) Antibodies carrying aldehyde moieties (in red, 2 per antibody)are reacted with a Hydrazino-iso-Pictet-Spengler(HIPS) linker and payload to generate a site-specifically conjugatedADC. (C) The HIPS chemistry proceeds through an intermediate hydrazoniumion followed by intramolecular alkylation with a nucleophilic indoleto generate a stable C–C bond.

Mentions: Our platform for site-specific ADC production is built upontheincorporation of formylglycine (fGly), a non-natural amino acid, intothe protein sequence. To install fGly (Figure 1), a short consensus sequence, CXPXR—where X is usually serine,threonine, alanine, or glycine—is inserted at the desired locationin the conserved regions of antibody heavy or light chains using standardmolecular biology cloning techniques.9 This“tagged” construct is produced recombinantly in cellsthat coexpress the formylglycine-generating enzyme (FGE), which cotranslationallyconverts the cysteine within the tag into an fGly residue, generatingan antibody expressed with two aldehyde tags per molecule.10 The aldehyde functional group serves as a chemicalhandle for bioorthogonal conjugation.11 We developed the Hydrazino-iso-Pictet-Spengler(HIPS) ligation to connect the payload to fGly, resulting in the formationof a stable, covalent C–C bond between the cytotoxin payloadand the antibody.12 This C–C bondis expected to be stable to physiologically relevant challenges encounteredby the ADC during circulation and FcRn recycling, e.g., proteases,low pH, and reducing reagents. Due to the modular nature of our platform,we can produce antibodies bearing the aldehyde tag at a variety oflocations, enabling us to empirically discover ideal conjugation sites.In this work, we tested the effects of inserting the aldehyde tagat one site in the light chain and seven sites in the heavy chain.We present in-depth biophysical and functional characterization ofthree of the resulting ADCs made by conjugation to maytansine payloadsvia a HIPS linker.6,13,14 We observed that HIPS conjugation produces physiologically stableconjugates; however, modulating the conjugation site had a pronouncedeffect on antibody efficacy and PK, and resulted in ADCs with an improvedsafety profile as compared to a conventional lysine-conjugated ADC.


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 tagcoupled with HIPS chemistry yields site-specificallymodified antibodies carrying a payload attached through a stable C–Cbond. (A) A formylglycine-generating enzyme (FGE) recognition sequenceis inserted at the desired location along the antibody backbone usingstandard molecular biology techniques. Upon expression, FGE, whichis endogenous to eukaryotic cells, catalyzes the conversion of theCys within the consensus sequence to a formylglycine residue (fGly).(B) Antibodies carrying aldehyde moieties (in red, 2 per antibody)are reacted with a Hydrazino-iso-Pictet-Spengler(HIPS) linker and payload to generate a site-specifically conjugatedADC. (C) The HIPS chemistry proceeds through an intermediate hydrazoniumion followed by intramolecular alkylation with a nucleophilic indoleto generate a stable C–C bond.
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Related In: Results  -  Collection

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

fig1: Aldehyde tagcoupled with HIPS chemistry yields site-specificallymodified antibodies carrying a payload attached through a stable C–Cbond. (A) A formylglycine-generating enzyme (FGE) recognition sequenceis inserted at the desired location along the antibody backbone usingstandard molecular biology techniques. Upon expression, FGE, whichis endogenous to eukaryotic cells, catalyzes the conversion of theCys within the consensus sequence to a formylglycine residue (fGly).(B) Antibodies carrying aldehyde moieties (in red, 2 per antibody)are reacted with a Hydrazino-iso-Pictet-Spengler(HIPS) linker and payload to generate a site-specifically conjugatedADC. (C) The HIPS chemistry proceeds through an intermediate hydrazoniumion followed by intramolecular alkylation with a nucleophilic indoleto generate a stable C–C bond.
Mentions: Our platform for site-specific ADC production is built upontheincorporation of formylglycine (fGly), a non-natural amino acid, intothe protein sequence. To install fGly (Figure 1), a short consensus sequence, CXPXR—where X is usually serine,threonine, alanine, or glycine—is inserted at the desired locationin the conserved regions of antibody heavy or light chains using standardmolecular biology cloning techniques.9 This“tagged” construct is produced recombinantly in cellsthat coexpress the formylglycine-generating enzyme (FGE), which cotranslationallyconverts the cysteine within the tag into an fGly residue, generatingan antibody expressed with two aldehyde tags per molecule.10 The aldehyde functional group serves as a chemicalhandle for bioorthogonal conjugation.11 We developed the Hydrazino-iso-Pictet-Spengler(HIPS) ligation to connect the payload to fGly, resulting in the formationof a stable, covalent C–C bond between the cytotoxin payloadand the antibody.12 This C–C bondis expected to be stable to physiologically relevant challenges encounteredby the ADC during circulation and FcRn recycling, e.g., proteases,low pH, and reducing reagents. Due to the modular nature of our platform,we can produce antibodies bearing the aldehyde tag at a variety oflocations, enabling us to empirically discover ideal conjugation sites.In this work, we tested the effects of inserting the aldehyde tagat one site in the light chain and seven sites in the heavy chain.We present in-depth biophysical and functional characterization ofthree of the resulting ADCs made by conjugation to maytansine payloadsvia a HIPS linker.6,13,14 We observed that HIPS conjugation produces physiologically stableconjugates; however, modulating the conjugation site had a pronouncedeffect on antibody efficacy and PK, and resulted in ADCs with an improvedsafety profile as compared to a conventional lysine-conjugated ADC.

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