Mass Spectrometric Characterization of Transglutaminase Based Site-Specific Antibody–Drug Conjugates

Antibody drug conjugates (ADCs) are becoming an important new class of therapeutic agents for the treatment of cancer. ADCs are produced through the linkage of a cytotoxic small molecule (drug) to monoclonal antibodies that target tumor cells. Traditionally, most ADCs rely on chemical conjugation methods that yield heterogeneous mixtures of varying number of drugs attached at different positions. The potential benefits of site-specific drug conjugation in terms of stability, manufacturing, and improved therapeutic index has recently led to the development of several new site-specific conjugation technologies. However, detailed characterization of the degree of site specificity is currently lacking. In this study we utilize mass spectrometry to characterize the extent of site-specificity of an enzyme-based site-specific antibody–drug conjugation technology that we recently developed. We found that, in addition to conjugation of the engineered site, a small amount of aglycosylated antibody present in starting material led to conjugation at position Q295, resulting in approximately 1.3% of off-target conjugation. Based on our detection limits, we show that Q295N mutant eliminates the off-target conjugation yielding highly homogeneous conjugates that are better than 99.8% site-specific. Our study demonstrates the importance of detailed characterization of ADCs and describes methods that can be utilized to characterize not only our enzyme based conjugates, but also ADCs generated by other conjugation technologies

Site-Dependent Degradation of a Non-Cleavable Auristatin-Based Linker-Payload in Rodent Plasma and Its Effect on ADC Efficacy

<div><p>The efficacy of an antibody-drug conjugate (ADC) is dependent on the properties of its linker-payload which must remain stable while in systemic circulation but undergo efficient processing upon internalization into target cells. Here, we examine the stability of a non-cleavable Amino-PEG6-based linker bearing the monomethyl auristatin D (MMAD) payload site-specifically conjugated at multiple positions on an antibody. Enzymatic conjugation with transglutaminase allows us to create a stable amide linkage that remains intact across all tested conjugation sites on the antibody, and provides us with an opportunity to examine the stability of the auristatin payload itself. We report a position-dependent degradation of the C terminus of MMAD in rodent plasma that has a detrimental effect on its potency. The MMAD cleavage can be eliminated by either modifying the C terminus of the toxin, or by selection of conjugation site. Both approaches result in improved stability and potency <i>in vitro</i> and <i>in vivo</i>. Furthermore, we show that the MMAD metabolism in mouse plasma is likely mediated by a serine-based hydrolase, appears much less pronounced in rat, and was not detected in cynomolgus monkey or human plasma. Clarifying these species differences and controlling toxin degradation to optimize ADC stability in rodents is essential to make the best ADC selection from preclinical models. The data presented here demonstrate that site selection and toxin susceptibility to mouse plasma degradation are important considerations in the design of non-cleavable ADCs, and further highlight the benefits of site-specific conjugation methods.</p></div

Protease inhibition studies of the PEG6-C2-MMAD degradation in mouse plasma.

<p>“Yes” indicates the same extent of cleavage as observed in plasma without inhibitors, “partial” indicates reduced cleavage compared to uninhibited plasma, while “no” indicates that no degradation was observed. All assays were carried out at pH 7.4.</p><p>Protease inhibition studies of the PEG6-C2-MMAD degradation in mouse plasma.</p

Effect of Attachment Site on Stability of Cleavable Antibody Drug Conjugates

The systemic stability of the antibody–drug linker is crucial for delivery of an intact antibody–drug conjugate (ADC) to target-expressing tumors. Linkers stable in circulation but readily processed in the target cell are necessary for both safety and potency of the delivered conjugate. Here, we report a range of stabilities for an auristatin-based payload site-specifically attached through a cleavable valine-citrulline-<i>p</i>-aminobenzylcarbamate (VC-PABC) linker across various sites on an antibody. We demonstrate that the conjugation site plays an important role in determining VC-PABC linker stability in mouse plasma, and that the stability of the linker positively correlates with ADC cytotoxic potency both in vitro and in vivo. Furthermore, we show that the VC-PABC cleavage in mouse plasma is not mediated by Cathepsin B, the protease thought to be primarily responsible for linker processing in the lysosomal degradation pathway. Although the VC-PABC cleavage is not detected in primate plasma in vitro, linker stabilization in the mouse is an essential prerequisite for designing successful efficacy and safety studies in rodents during preclinical stages of ADC programs. The divergence of linker metabolism in mouse plasma and its intracellular cleavage offers an opportunity for linker optimization in the circulation without compromising its efficient payload release in the target cell

Stability studies of site-specific non-cleavable ADCs.

<p>a) Positions of conjugation sites on an antibody. b) Structure of the PEG6-C2-MMAD non-cleavable payload conjugated to the glutamine tag on the antibody, and its cleavage product. The glutamine residue is shown in blue. c) Structure of the PEG6-C2-Aur3377 non-cleavable payload conjugated to the glutamine tag shown in blue.</p

Degradation of the C-terminal portion of the PEG6-C2-MMAD payload in the plasma of different species.

<p>Degradation is calculated as percentage of payload cleaved. Calculations are based on DAR values obtained from HIC analysis of the Site A-PEG6-2-MMAD conjugate before and after incubation in plasma for 4.5 days in three independent experiments.</p

Comparative efficacy studies of non-cleavable ADCs.

<p>Comparison of <i>in vitro</i> cytotoxic activities of untreated and <i>in vivo</i>-exposed non-cleavable conjugates against BxPC3 cells (M1S1+++). a) C16 Site A-PEG6-C2-MMAD conjugate. b) C16 Site I-PEG6-C2-MMAD conjugate. c) C16 Site A-PEG6-C2-Aur3377 conjugate. d) <i>In vivo</i> comparison of the three conjugates in the BxPC3 xenograft model, along with a negative control conjugate NCC Site F-PEG6-C2-MMAD. All compounds were given as a single dose at 10 mg/kg.</p

Mass spectrometric analysis of non-cleavable conjugates.

<p>The Fig labels represent experimentally observed masses for conjugates before (upper panel) and after (lower panel) <i>in vivo</i> exposure. a) Intact mass deconvolution of C16 Site A-PEG6-C2-MMAD conjugate. The metabolic products of the DAR 2 species show a mass loss from either 1 x 186 Da (one payload) or 2 x 186 Da (both payloads). b) Intact mass of C16 Site I-PEG6-C2-MMAD conjugate. The metabolic product shows a 186 Da loss from one of the conjugated payloads. c) Intact mass of C16 Site A-PEG6-C2-Aur3377 conjugate. The <i>in vivo</i> exposed conjugate shows no mass shift compared to the untreated compound.</p

Stability of the non-cleavable PEG6-C2-MMAD and PEG6-C2-Aur3377 conjugates under <i>in vitro</i> and <i>in vivo</i> conditions.

<p>The percentage of intact MMAD was calculated as the ratio of treated DAR to untreated DAR. Degradation of the C-terminal portion of the PEG6-C2-MMAD payload is considered equivalent to payload removal for DAR determination purposes. Calculations are based on DAR values obtained from HIC analysis for most conjugates, except for Site G, H, and I conjugates for which the percentage of intact MMAD was determined by mass spec analysis. For mouse plasma stability, values reported are averages of three independent experiments. For mouse <i>in vivo</i> stability, samples collected from individually dosed mice were pooled to obtain the measurement.</p><p>Stability of the non-cleavable PEG6-C2-MMAD and PEG6-C2-Aur3377 conjugates under <i>in vitro</i> and <i>in vivo</i> conditions.</p

Synthesis of Amino-PEG6-C2-Aur3377.

<p>Reagents and conditions: a) Aur-3377, Fmoc-AmPeg6C2-CO2H, HATU, <i>i</i>-Pr₂NEt, DMF; b) LiOH, H₂O-DMF.</p