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. 2014 Jul 16;25(7):1331-41.
doi: 10.1021/bc500189z. Epub 2014 Jun 23.

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

Penelope M Drake  1 , Aaron E Albers, Jeanne Baker, Stefanie Banas, Robyn M Barfield, Abhijit S Bhat, Gregory W de Hart, Albert W Garofalo, Patrick Holder, Lesley C Jones, Romas Kudirka, Jesse McFarland, Wes Zmolek, David Rabuka
Affiliations

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

Penelope M Drake et al. Bioconjug Chem. .

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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Figures

Figure 1
Figure 1
Aldehyde tag coupled with HIPS chemistry yields site-specifically modified antibodies carrying a payload attached through a stable C–C bond. (A) A formylglycine-generating enzyme (FGE) recognition sequence is inserted at the desired location along the antibody backbone using standard molecular biology techniques. Upon expression, FGE, which is endogenous to eukaryotic cells, catalyzes the conversion of the Cys 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 conjugated ADC. (C) The HIPS chemistry proceeds through an intermediate hydrazonium ion followed by intramolecular alkylation with a nucleophilic indole to generate a stable C–C bond.
Figure 2
Figure 2
Together, the aldehyde tag and HIPS chemistry allow for stable cytotoxic payload conjugation at precise locations across the antibody surface. (Top) We inserted the aldehyde tag (red) at one location in the light chain (LC) and seven locations (labeled A–G) in the heavy chain. Antibodies bearing these tags were produced and analyzed as the first step in making ADCs conjugated at different sites. (Bottom) HIPS-Glu-PEG2-maytansine 20 served as the linker (in black) and the cytotoxic payload (in blue) for ADCs used in these studies.
Figure 3
Figure 3
Hydrophobic interaction chromatography analysis demonstrates the clean conversion of LC-, CH1-, and CT-tagged antibodies into homogeneous ADCs. Unconjugated antibody (black) elutes as one peak. After conjugation to HIPS-Glu-PEG2-maytansine, the ADC (green) elutes as a diconjugated material (right). This clean separation of conjugated from unconjugated material allows for conjugate enrichment and simple determination of DAR. α-HER2-DM1 was included as a comparator.
Figure 4
Figure 4
Aldehyde-tagged HIPS conjugates are stable in plasma at 37 °C, but payload attachment plays a role. We tested the plasma stability of LC-, CH1-, and CT-tagged antibodies conjugated using HIPS-Glu-PEG2 to either (A) Alexa Fluor 488 (AF488) or (B) maytansine. Conjugates were incubated in rat plasma at 37 °C for up to 13 d. When analyzed by ELISA for total payload and total antibody, we observed no loss of total payload signal relative to total antibody signal for the AF488 conjugates, regardless of tag placement. For the maytansine conjugates, we observed evidence that some deconjugation occurred over time at 37 °C. The stability differed according to tag placement, with the CT-tag showing the highest conservation of payload-to-antibody signal (84%), followed by CH1 (72%), and LC (65%).
Figure 5
Figure 5
Payload location does not influence in vitro potency of aldehyde-tagged α-HER2 ADCs against NCI-N87 target cells. NCI-N87 cells, which overexpress HER2, were used as targets for in vitro cytotoxicity in a 6 day assay. Free maytansine (gray line) was included as a positive control, and an isotype control ADC (orange line) was used as a negative control to indicate specificity. α-HER2 HIPS-Glu-PEG2-maytansine ADCs bearing the aldehyde tag on the light chain (LC, green), or on the CH1 (red) or C-terminal (CT, blue) regions of the heavy chain showed comparable activity. α-HER2-DM1 was included as a comparator. IC50 values (reflecting the antibody concentrations except in the case of the free drug) were measured as follows: free maytansine, 214 pM; isotype control ADC, could not be determined; LC ADC 87 pM; CH1 ADC, 132 pM; CT ADC 114 pM, α-HER2-DM1, 54.7 pM.
Figure 6
Figure 6
Payload placement modifies the in vivo efficacy of aldehyde-tagged α-HER2 ADCs against NCI-N87 xenografts in mice. CB.17 SCID mice (8/group) were implanted subcutaneously with NCI-N87 cells. When the tumors reached ∼113 mm3, the animals were given a single 5 mg/kg dose of trastuzumab alone, an isotype ADC, or an α-HER2 HIPS-Glu-PEG2-maytansine ADC conjugated to either the light chain (LC), or the CH1 or C-terminal (CT) regions of the heavy chain. α-HER2-DM1 was included as a comparator. (A) Tumor growth was monitored twice weekly. (B) The differences in efficacy among the tag placements tested were reflected in survival curves. Animals were euthanized when tumors reached 800 mm3.
Figure 7
Figure 7
α-HER2 HIPS-Glu-PEG2-maytansine ADCs are highly stable in vivo regardless of tag placement. BALB/c mice were dosed with 5 mg/kg of aldehyde-tagged α-HER2 HIPS-Glu-PEG2-maytansine ADCs conjugated to either the light chain (LC), or to the CH1 or C-terminal (CT) regions of the heavy chain. α-HER2-DM1 was included as a comparator. Plasma was sampled at the time points indicated and assayed by ELISA. Area under the curve (AUC) was determined using GraphPad Prism and is reported in Table 4.
Figure 8
Figure 8
α-HER2 CT ADC is less toxic than the α-HER2-DM1 at the same doses in Sprague–Dawley rats. Animals (5/group) received a 6, 20, or 60 mg/kg dose of α-HER2 CT ADC (shown in blue) or α-HER2-DM1 (shown in red), followed by a 12 day observation period. Body weight (A) was monitored at the times indicated. Alanine aminotransferase (B), aspartate aminotransferase (C), and platelet counts (D) were assessed at day 5 postdose. Due to premature death (for 2 animals in the α-HER2-DM1 60 mg/kg group) or sample collection errors (platelet-specific, for animals in the vehicle control and both 6 mg/kg groups), some groups comprise fewer than 5 data points.
Figure 9
Figure 9
Toxicokinetic analysis demonstrated that the α-HER2 CT ADC was more stable in rats than the α-HER2-DM1. The same animals that were analyzed for indicators of toxicity (Figure 8) were used to assess the toxicokinetic profiles of the α-HER2 CT ADC and α-HER2-DM1 analytes. Plasma was sampled at the time points indicated and assayed by ELISA.
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