FRET Reagent Reveals the Intracellular Processing of Peptide-Linked Antibody–Drug Conjugates

Despite the recent success of antibody–drug conjugates (ADCs) in cancer therapy, a detailed understanding of their entry, trafficking, and metabolism in cancer cells is limited. To gain further insight into the activation mechanism of ADCs, we incorporated fluorescence resonance energy transfer (FRET) reporter groups into the linker connecting the antibody to the drug and studied various aspects of intracellular ADC processing mechanisms. When comparing the trafficking of the antibody–FRET drug conjugates in various different model cells, we found that the cellular background plays an important role in how the antigen-mediated antibody is processed. Certain tumor cells showed limited cytosolic transport of the payload despite efficient linker cleavage. Our FRET assay provides a facile and robust assessment of intracellular ADC activation that may have significant implications for the future development of ADCs

FRET Reagent Reveals the Intracellular Processing of Peptide-Linked Antibody–Drug Conjugates

Despite the recent success of antibody–drug conjugates (ADCs) in cancer therapy, a detailed understanding of their entry, trafficking, and metabolism in cancer cells is limited. To gain further insight into the activation mechanism of ADCs, we incorporated fluorescence resonance energy transfer (FRET) reporter groups into the linker connecting the antibody to the drug and studied various aspects of intracellular ADC processing mechanisms. When comparing the trafficking of the antibody–FRET drug conjugates in various different model cells, we found that the cellular background plays an important role in how the antigen-mediated antibody is processed. Certain tumor cells showed limited cytosolic transport of the payload despite efficient linker cleavage. Our FRET assay provides a facile and robust assessment of intracellular ADC activation that may have significant implications for the future development of ADCs

An Anti-B7-H4 Antibody–Drug Conjugate for the Treatment of Breast Cancer

B7-H4 has been implicated in cancers of the female reproductive system and investigated for its possible use as a biomarker for cancer, but there are no preclinical studies to demonstrate that B7-H4 is a molecular target for therapeutic intervention of cancer. We provide evidence that the prevalence and expression levels of B7-H4 are high in different subtypes of breast cancer and that only a few normal tissues express B7-H4 on the cell membrane. These profiles of low normal expression and upregulation in cancer provide an opportunity for the use of antibody–drug conjugates (ADCs), cytotoxic drugs chemically linked to antibodies, for the treatment of B7-H4 positive cancers. We have developed an ADC specific to B7-H4 that uses a linker drug consisting of a potent antimitotic, monomethyl auristatin E (MMAE), linked to engineered cysteines (THIOMAB) via a protease labile linker. We will refer to ADCs that use the THIOMAB format as TDCs to help distinguish the format from standard MC-vc-MMAE ADCs that are conjugated to the interchain disulfide bonds. Anti-B7-H4 (h1D11)-MC-vc-PAB-MMAE (h1D11 TDC) produced durable tumor regression in cell line and patient-derived xenograft models of triple-negative breast cancer. It also binds rat B7-H4 with similar affinity to human and allowed us to test for target dependent toxicity in rats. We found that our anti-B7-H4 TDC has toxicity findings similar to untargeted TDC. Our results validate B7-H4 as an ADC target for breast cancer and support the possible use of this TDC in the treatment of B7-H4⁺ breast cancer

SdgB glycosylation protects SDR proteins from cleavage by human neutrophil-derived cathepsin G.

<p>(<b>A</b>) Live, in tact WT or Δ<i>sdgB</i> USA300 bacteria were incubated in the presence or absence of human neutrophil lysosomal extracts (NLE). Culture supernatants were immunoblotted with a mAb against the A-domain of ClfA (9E10) to detect cleaved ClfA fragments released from the bacteria. (<b>B</b>) Live, in tact WT or Δ<i>sdgB</i> cells were incubated in the presence or absence of lysosomal extracts from human THP1 cells or mouse RAW cells and culture supernatants were immunoblotted with anti-ClfA. (<b>C</b>) Live, intact WT or Δ<i>sdgB</i> cells were incubated with a panel of purified human neutrophil serine proteases, ie. neutrophil elastase (NE), cathepsin G (CatG), proteinase-3 (P3), and neutrophil serine protease-4 (NSP4). (<b>D</b>) <b>Δ</b><i>sdgB</i> cells were treated with human neutrophil lysosomal extract in the presence or absence of a biochemical inhibitor of cathepsin G. (<b>E</b>) WT or various Sdg-mutant strains were treated with purified human cathepsin G. (<b>B-E</b>) Culture supernatants were analyzed by immunoblotting as in (A) to detect released ClfA fragments. (<b>F</b>) Live bacteria of WT, <b>Δ</b><i>sdgB</i>, or Δ<i>sdgB</i> complemented with exogenous SdgB (p<i>sdgB</i>) were treated with purified human cathepsin G. Culture supernatants (Sup) or cell wall preparations (CWP) were immunoblotted with mAb against the A-domain of ClfA (S4675), SdrD (17H4), or IsdA (2D3). In addition to S4675, another mAb against the A-domain of ClfA (9E10) showed similar results (not shown). (<b>G</b>) Human cathepsin G inhibits adherence of glycosylation-deficient <i>S. aureus</i> to human fibrinogen. Live WT or Δ<i>sdgB</i> USA300 bacteria were pre-incubated with cathepsin G, and allowed to adhere to fibrinogen-precoated plates. Bacterial adhesion was quantified by measuring the amount of bacterial ATP associated with the plates.</p

Recognition of SdgB-dependent epitope by human antibodies.

<p>(<b>A</b>) Four different human IgG preparations were reacted with plate-bound CWP from WT or Δ<i>sdgB</i> USA300 by ELISA. To calculate the specific anti-staphylococcal IgG content, data were normalized using a calibration curve with known IgG concentrations of a mAb against peptidoglycan, which has the same reactivity with both USA300 strains by ELISA. Data are expressed as µg/mL of anti-staphylococcal IgG in the serum. The reduction in reactivity observed for CWP from Δ<i>sdgB</i> (red bars) as compared to wild-type CWP (black bars) reflects IgG specific for SdgB-dependent epitopes. Asterisks indicate significant differences (p < 0.05) from WT CWP. (<b>B</b>) CWP from WT, Δ<i>sdgA</i>, or Δ<i>sdgB</i>, Δ<i>sdgAΔsdgB</i> USA300 were immunoblotted with rF1 and three additional human mAbs (SD2, SD3, and SD4) from different patients. All four mAbs showed similar epitope specificity.</p

mAb rF1 exhibits robust binding to and killing of <i>S. aureus</i> bacteria.

<p>(<b>A-C</b>) Bacteria were preopsonized with huIgG1 mAbs rF1 (squares), 4675 anti-ClfA (triangles), or anti-herpes virus gD (circles). (<b>A</b>) Binding of mAbs to WT (USA300-Δ<i>spa</i>) bacteria was assessed by flow cytometry, and expressed as mean fluorescent intensity (MFI). (<b>B</b>) CFSE-labeled, preopsonized WT (USA300-Δ<i>spa</i>) bacteria were incubated with human PMN. Bacterial uptake was expressed as % of CFSE-positive PMN, after gating for CD11b-positive cells by flow cytometry. (<b>C</b>) Preopsonized WT (USA300-Δ<i>spa</i>) bacteria were incubated with PMN to assess bacterial killing. Numbers of viable CFU per mL are representative of at least three experiments. (<b>D</b>) Flow cytometry analysis of binding of rF1 to <i>S. aureus</i> from various infected tissues. Homogenized tissues were double stained with mAb rF1 (X-axis), and with anti-peptidoglycan mAb 702 to distinguish bacteria from tissue debris (Y-axis) (left panel; gate indicated by arrow), followed by gating of bacteria to generate histogram figures. (<b>E</b>) Binding of rF1 to various staphylococcal and non-staphylococcal Gram-positive bacterial species by flow cytometry. <i>Red lines</i>, rF1; <i>blue lines</i>, isotype control mAb anti-gD; <i>green lines</i>, control without mAb. (See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003653#ppat.1003653.s001" target="_blank">Figure S1</a>).</p

Strains, plasmids, and antibodies.

<p>Abbreviations: MRSA, methicillin-resistant <i>Staphylococcus aureus</i>; MSSA, methicillin-sensitive <i>Staphylococcus aureus</i>; VISA, vancomycin intermediate-resistant <i>Staphylococcus aureus</i>; mAb, monoclonal antibody. *NARSA, network on Antibiotic Resistance in <i>Staphylococcus aureus</i> (NARSA) Program supported under NIAID/NIH Contract No. HHSN272200700055C.</p

SdgB is the key rF1 epitope-modifying enzyme.

<p>(<b>A</b>) SdgB is necessary for rF1 reactivity. Cell wall lysates from WT and various putative glycosyltransferase mutants were immunoblotted with mAbs rF1, anti-ClfA (9E10), anti-SdrD (17H4) or anti-panSDR (9G4 α-SDR; recognizes the unmodified SDR-domain. (<b>B</b>) Complementation of Δ<i>sdgB</i> with exogenous SdgB confers rF1 reactivity. Cell wall lysates from WT, glycosyltransferase mutants, and the SdgB-complemented strain were immunoblotted with rF1, anti-ClfA, and anti-SDR mAbs as in (A). (<b>C</b>) Binding of rF1 to whole USA300 bacteria requires SdgB. Binding of mAbs to Δ<i>sdgB</i> USA300 was assessed by flow cytometry as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003653#ppat-1003653-g001" target="_blank">Figure 1A</a>. (<b>D</b>) rF1-mediated killing of USA300 activity requires SdgB. Wild-type USA300 bacteria preopsonized with rF1 (closed square) or anti-gD (closed circle), and Δ<i>sdgB</i> preopsonized with rF1 (closed triangle) or anti-gD (open circle), were incubated with PMN, and bacterial killing was determined as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003653#ppat-1003653-g001" target="_blank">Figure 1C</a>. (<b>E</b>) MBP-SDR-His construct was expressed in WT, Δ<i>sdgA</i>, Δ<i>sdgB</i>, or Δ<i>sgdAΔsdgB S. aureus</i>, and whole cell lysates were immunoblotted with rF1, anti-His and anti-SDR. (<b>F</b>) Preliminary model for step-wise glycosylation of SDR-proteins by SdgB and SdgA. SDR-domains are first glycosylated by SdgB, which appends sugar modifications creating the epitope of mAb rF1. SdgA further modifies these epitopes with additional sugar moieties (left panel). The Δ<i>sdgA S. aureus</i> mutant shows that SdgA-mediated modifications do not influence rF1-binding activity (middle panel). In Δ<i>sdgB or</i> Δ<i>sgdAΔsdgB S. aureus</i>, the unmodified SDR-region is now recognized by the anti-pan-SDR mAb (9G4).</p

mAb rF1 binds to a family of serine-aspartate-repeat (SDR)-proteins.

<p>(<b>A</b>) rF1-reactivity with USA300 CWP is sensitive to proteinase-K (PK) treatment. Lysostapahin-derived CWP from WT (USA300-Δ<i>spa</i>) bacteria was left untreated (lane 1) or treated with 10 µg/mL PK for 1 hour (lane 2), and immunoblotted with rF1. (<b>B</b>) rF1-reacitivty is dependent on the presence of SDR-proteins. CWPs from WT, indicated deletion strains of various combinations of SDR-family proteins <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003653#ppat.1003653-Fitzgerald1" target="_blank">[12]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003653#ppat.1003653-McAleese1" target="_blank">[40]</a>, and a Δ<i>spa</i> strain as control for non-specific binding, were immunoblotted with rF1. The lower molecular weight bands (∼50 kDa) were due to non-specific IgG binding to protein A. (<b>C</b>) rF1 also binds to additional SDR-proteins from <i>S. epidermidis</i>. Cell lysates from <i>S. epidemidis</i> were immunoprecipitated with rF1 (lane 1) or an isotype-control mAb (lane 2) and immunoblotted with rF1 mAb. Identities of rF1-reactive bands were revealed by mass-spectrometry of the same lysates (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003653#ppat.1003653.s002" target="_blank">Figure S2</a>). (<b>D</b>) Alignment of SDR-proteins revealed by mass-spectrometry from <i>S. aureus</i> and <i>S. epidermidis</i>. SDR-regions are indicated by red hatches. Three truncation mutants of clumping factor A (ClfA) that were fused with maltose-binding protein (MBP) are also shown. (<b>E</b>) SDR-region is sufficient for rF1 reactivity. CWPs from <i>S. aureus</i> expressing truncated recombinant constructs were immunoblotted with anti-MBP mAb or rF1 mAb.</p