APLP2 and LDLR interactions with PCSK9 and their regulation of PCSK9 function.

<p>(A and B) Western blot showing APLP2, PCSK9, or Transferrin receptor (TFNR) levels in input fraction (I), IC or J16 immunoprecipitated samples (IP Ab.) in the absence or presence of 5F6 Fab or 12E3 Fab, as indicated. (B) Quantification of (A); shown as average APLP2 normalized to PCSK9 of 3 independent experiments with SEM. (C and D) J16 coIPs of PCSK9 from Neg or LDLR siRNA treated HepG2 cells with IC control, as indicated. (D) Quantification of (C); shown as average APLP2 normalized to PCSK9 from 3 independent experiments with SEM. (E, F, and G) Western blot of LDLR, APOER2, or TFNR in siRNA treated cells following treatment with PCSK9 at 0, 20, 50, or 100 μg/ml. (F) LDLR levels from (E) quantified as percent LDLR degradation of untreated cells and normalized to Neg siRNA samples. Shown as average with SEM from 4 independent experiments. (G) Same as F, but measuring APOER2 levels.</p

Determining which of APP or APLP2 affects PCSK9 internalization.

<p>(A, B, and C) PCSK9-488 internalization in the presence of mIC and hIC antibodies (top row), mIC and J16 (middle top row), hIC and 5F6 (middle bottom row), or 5F6 and J16 (bottom row) in (A) Neg, (B) APLP2, or (C) APP siRNA treated HepG2 cells. Internalized human and mouse antibodies shown in blue and red, respectively. Scale bars, 10 μM. (D) Quantification of (A, B, and C) shown as average fluorescent signal of PCSK9-488 per cell normalized to IC with SEM of 3 independent experiments.</p

ApoB/LDL effects on PCSK9 internalization and function.

<p>(A and B) PCSK9-488 internalization in the presence of J16 (top row), LDL and J16 (middle row), or LDL+5F6+J16 (bottom row) in APLP2 siRNA treated cells. Dotted line indicates background signal, as measured by IC alone. Scale bars, 10 μM. (B) Quantification of (A) shown as average+SEM of J16 fluorescent signal per cell in APLP2 siRNA treated cells from 3 independent experiments. Dotted line indicates average IC background levels. Scale bars, 10 μM. (C) Representative western blot showing APLP2, LDLR, ApoB, Transferrin receptor (TFNR) levels in input fraction (I), IC or J16 immunoprecipitated samples (IP Ab.) under pH 7.4 or pH 6.0 conditions with increasing concentrations of ApoB, as indicated. (D) Western blot showing recombinant ApoB, LDLR-ECD, or PCSK9 in anti-LDLR immunoprecipitated samples at pH 6.0, with or without 5F6 Fab, as indicated. All experiments were performed independently at least 3 times and representative data are shown here.</p

PCSK9-488 internalization and LDLR levels <i>in vivo</i>.

<p>PCSK9-488 (green) internalization in mouse liver in the presence of mIC and hIC (top row), mIC and J16 (middle top row), hIC and 5F6 (middle bottom row), or 5F6 and J16 (bottom row). LDLR (red) and DAPI (blue) staining shown, as indicated. Scale bars, 10 μM.</p

PCSK9 follows both direct and indirect internalization routes.

<p>(A) Western blot of liver lysates taken from negative siRNA or APLP2 siRNA treated mice showing relative APLP2 levels as compared to Actin loading control. (B) Internalization of J16 bound PCSK9 in liver in negative or APLP2 siRNA treated mice. Scale bars, 10 μM. (C) Schematic depicting interactions of the proposed direct and indirect PCSK9 internalization routes. At the cell surface, PCSK9 can bind directly to LDLR or APLP2; PCSK9 binding to APLP2 requires LDLR/APLP2 interactions. For both direct routes, following endocytosis, PCSK9 bridges LDLR to APLP2, and APLP2 mediates lysosomal delivery of the complex. Indirect PCSK9 internalization is mediated via LDL. PCSK9 binds LDL, and LDL binds LDLR at the cell surface. Following endocytosis, PCSK9 can potentially bridge dissociated LDL to LDLR.</p

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

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