Article Cite This: Biochemistry 2019, 58, 2642−2652
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Tumor-Targeting Immune System Engagers (ISErs) Activate Human Neutrophils after Binding to Cancer Cells Andre ́ J. G. Pötgens,*,† Anne C. Conibear,‡,§ Claudia Altdorf,† Clarissa Hilzendeger,† and Christian F. W. Becker*,‡ †
Syntab Therapeutics GmbH, 52074 Aachen, Germany Faculty of Chemistry, Institute of Biological Chemistry, University of Vienna, 1090 Vienna, Austria
‡
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S Supporting Information *
ABSTRACT: Immune system engagers (ISErs) make up a new class of immunotherapeutics against cancer. They comprise two or more tumortargeting peptides and an immune-stimulating effector peptide connected by inert polymer linkers. They are produced by solid phase peptide synthesis and share the specific targeting activities of antibodies (IgGs) but are much smaller in size and exploit a different immune-stimulating mechanism. Two ISErs (Y-9 and Y-59) that bind to the cancer cell markers integrin α3 and EphA2, respectively, are analyzed here with respect to their immune cell stimulation. We have previously shown that they activate formyl peptide receptors on myeloid immune cells and induce respiratory burst in neutrophils and myeloid chemotaxis in solution. It remained, however, unclear whether these molecules can stimulate immune cells while bound to tumor cells, an essential step in the hypothesized mode of action. Here, we demonstrate that ISEr Y-9 induced respiratory burst and caused a change in the shape of neutrophils when bound to the surface of protein A beads as a model of tumor cells. More importantly, tumor cell lines carrying receptor-bound Y-9 or Y-59 also activated neutrophils, evidenced by a significant change in shape. Interestingly, similar activation was induced by the supernatants of the cells incubated with ISEr, indicating that ISErs released from tumor cells, intact or degraded into fragments, significantly contributed to immune stimulation. These findings provide new evidence for the mode of action of ISErs, namely by targeting cancer cells and subsequently provoking an innate immune response against them.
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macrophages. Activation of FPR-1 by N-formyl peptides is known to lead to rapid effects such as chemo-attraction of the target cells associated with a change in shape from round to irregular (thought to be necessary for extravasation in vivo), activation of the phagocyte NADPH oxidase (respiratory burst), and degranulation leading to the release of toxic enzymes.4−10 We already have demonstrated these effects on myeloid cells with ISErs in solution.2,3 In addition, there are reports of N-formyl peptides inducing cytokine expression (TNFα, IL-1β, IL-6, and IL-8) and nitric oxide production and facilitating phagocytosis,11−19 suggesting that these additional or secondary effects might also contribute to the anticancer activity of ISErs. ISErs containing N-formyl peptides are designed to accumulate inside tumors and modulate their immune microenvironment, largely governed by tumor-associated macrophages and other myeloid cells (reviewed in refs 20−23), to become more hostile to tumors. A precedent for this approach was published by Sandberg et al. in 1983;24 they found higher numbers of macrophages in guinea pig tumors and (nonsignificant) decreases in tumor weight after injecting antitumor antibodies conjugated to N-formyl peptide fMLF,
mmune therapies against cancer are still rapidly developing. They comprise the use of therapeutic molecules, such as immune checkpoint inhibitors, antibodies against tumorspecific antigens, and vaccines, and cellular therapies, including those utilizing genetically modified immune cells.1 Despite tremendous successes, there is still a need for novel types of (immune) therapy approaches against cancer. Most immune therapies currently focus on modifying T-lymphocyte functions. In contrast, we have previously described a new modality termed immune system engagers (ISErs) that follow a different mode of action, in which they primarily target innate immune cells, in particular myeloid cells. Elucidating the mode of action of ISErs, ∼5−7 kDa synthetic molecules comprising at least two specific binder peptides that target them to cancer cell markers and an effector peptide that activates immune cells (Figure 1A), is critical for their potential use as therapeutics. ISErs bearing binder peptides targeting the integrin α 3 chain (CD49c) and erythropoietin-producing hepatoma receptor A2 (EphA2) have been described, and their cell binding properties have been analyzed in previous work, showing binding affinities (KD) of 1−100 nM.2,3 These ISErs also contain an N-formyl peptide-based effector moiety that targets and activates formyl peptide receptors (FPRs), most probably the high-affinity receptor FPR-1, present on several myeloid cell types: neutrophilic granulocytes, monocytes, and © 2019 American Chemical Society
Received: February 28, 2019 Revised: May 15, 2019 Published: May 22, 2019 2642
DOI: 10.1021/acs.biochem.9b00169 Biochemistry 2019, 58, 2642−2652
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Biochemistry
Figure 1. (A) ISEr concept. ISErs are synthetic peptide compounds that mimic the functional moieties of IgG antibodies in binding specifically to cancer cells (binder peptide) and stimulating an immune response (effector peptide). (B) Modes of action tested. (i) Can ISErs bound to protein A beads activate immune cells? (ii) Can ISErs bound to tumor cells activate immune cells? (iii) Can ISErs (complete or degraded into fragments) released by tumor cells activate immune cells? (C) Compounds used in this study. B9 (black rod) is a peptide binding integrin α3. B59 (white rod) is a peptide binding EphA2. F2 (gray rod) is an agonist peptide for formyl peptide receptors that activates myeloid immune cells. Compounds bearing a Biotin tag (hexagon) are designated with b. F2 with two PEG chains (helices) is called Y-scaffold or Yb-scaffold. The following ISErs were used: Y9 (two B9 peptides), Yb-9 (biotin at the C-terminus of F2), bY-9 (biotin replacing the N-terminal formyl group), Y-sc9 (scrambled B9 peptides and unable to bind integrin α3), Y-59, Yb-59 (C-terminal biotin), Tet-Yb-59 (four B59 peptides), and Yb-sc59 (two scrambled B59 peptides).
which were shown in vitro to be chemotactic for monocytes and macrophages. Our initial tests in animals demonstrated the ability of a single dose of 100 nmol (0.5 mg) of locally applied ISEr Y-9 to inhibit the establishment of tumors from inoculated tumor cells in guinea pigs. Furthermore, no systemic immune toxicity was observed after repeated subcutaneous dosing of 7 × 800 nmol of ISEr Y-9 in healthy mice.2 On the basis of independent data for the effector and binder moieties of ISErs in solution, a mode of action was proposed to explain these results. Here, we will use in vitro methods to demonstrate an important aspect of this mechanism, namely that ISErs are still able to stimulate immune cells after binding to tumor cells. We test two scenarios of how ISErs might work after successfully targeting tumor cells in this study (Figure 1B). In one scenario, ISErs bind to their target cell surface marker and remain bound. Immune cells already present in the tumor microenvironment are then activated by the effector peptide presented on the tumor cells (Figure 1B-ii). Although FPRs typically internalize along with their bound ligands, this is not a prerequisite for activation as internalization occurs more slowly (within minutes) than signaling (within seconds).25 This study will test whether immobilizing the effector on a particle (e.g., a cell or a bead, Figure 1B-i) still allows for signaling via the FPR−ligand complex. A second scenario (Figure 1B-iii) considers that compounds bound to cell surface markers can be released again. Depending on their binding affinity and especially the off rate, release of ISErs can lead to a concentration gradient around tumor cells and inside a tumor. Tumor cell-associated proteases might also cleave the ISEr into smaller fragments, potentially resulting in small N-formylated peptides being released. Small N-
formylated peptides have a much higher potency in activating immune cells than the original ISErs.2,3 Independently, or at the same time, ISErs could also be internalized and subsequently released again intact, as ISEr fragments or as part of extracellular vesicles.26 Via these routes, immune cell activation in the tumor environment could also be induced by freely diffusing ISErs or their fragments (Figure 1B-iii). Here, we utilize a series of biotinylated and nonbiotinylated compounds [ISErs equipped with tumor binding peptides and effector peptides as well as control compounds that contain scrambled binding peptides, effector peptides alone, and binder peptides alone (Figure 1C)] to test the different scenarios. These compounds were immobilized on beads or bound to tumor cells and subsequently tested for their capacity to activate immune cells.
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MATERIALS AND METHODS ISErs and Related Compounds. ISErs and ISEr components (effector and binder peptides) used in this study were synthesized by solid phase peptide synthesis and chemoselective ligations as described previously.2,3 The ISErs comprised binder peptide B9, B59 or scrambled versions, effector peptide F2, and PEG linkers. ISErs as well as control compounds (binder peptides, effector peptides, and effector− PEG conjugates), some of them carrying a biotin, used in this study are depicted schematically in Figure 1C. Full sequences and chemical structures are as described previously.2,3 Cell-Based and Bead-Based Assays. Cell lines were obtained from LGC/ATCC or DSMZ and cultured according to the provider’s guidelines. Cell identity was not tested. Mycoplasma contamination was checked by polymerase chain reaction every month, and all cell lines were always found to be 2643
DOI: 10.1021/acs.biochem.9b00169 Biochemistry 2019, 58, 2642−2652
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Biochemistry
Yb-scaffold, 5 μL samples of beads were taken shortly before addition to the leukocyte activation assay and added to 95 μL of streptavidin-PerCP-Cy5.5 (BD Biosciences catalog no. 551419) diluted 1:400 in peptide blocking buffer [0.9% NaCl, 10 mM Hepes (pH 7.3), 5 mM KCl, 3% BSA, and 5% FCS]. After incubation on ice for 30 min, 1 mL of phosphatebuffered saline (PBS) was added, and the beads were pelleted, resuspended in 100 μL of PBS, and analyzed by flow cytometry. In the evaluation, single (non-aggregated) beads were gated in the FSC/SSC plot and PerCP-Cy5.5 fluorescence levels were compared between differently coated beads. Binding of ISErs to Cell Lines. For use in experiments, the adherent cell lines (A431, PC-3, and L-929) were detached from the flasks using trypsin/EDTA (PAN Biotech) diluted 1:1 with PBS and resuspended in complete medium containing 10% FCS. K562 cells growing in suspension were taken from the flasks directly. Cells were washed, blocked and incubated with 1 μM solutions of the ISErs or control compounds (100 nM Tet-Yb-59) for 30 min on ice, and washed again as described previously.2,3 Cells incubated with ISErs were washed three times (Y-9 family) or four times (Y-59 family), including tube changes in every washing step, to prevent carryover of compounds via tube walls. Cells incubated with ISErs or their supernatants after extended incubations in assay buffer on ice or at 37 °C were added to the immune stimulation assays. More details about tumor cell incubations, washing steps, and flow cytometric analysis of the leukocyte/ tumor cell mixtures can be found on page 3 of the Supporting Information. Demonstration of ISErs Binding to Cells. Cell samples (5 μL, 100000 cells) were taken from the 125 μL cell suspensions just before being added to the leukocyte activation assay, or after a 10 or 20 min incubation in assay buffer on ice or at 37 °C, and were added to 95 μL of streptavidin-PerCPCy5.5 (see above) diluted 1:400 in blocking buffer. After a 30 min incubation on ice, 1 mL of PBS was added, and cells were pelleted, resuspended in 100 μL of PBS and Hoechst 33252 (0.5 μg/mL), and analyzed by flow cytometry (10000 events were acquired per sample). Gated live and single cells were compared in overlay histograms showing PerCP-Cy5.5 fluorescence. Streptavidin Beads. Streptavidin beads (Dynabeads MyOne Streptavidin T1, Invitrogen, 1 μm diameter, theoretical binding capacity of 400 pmol of biotinylated peptide/mg) were washed three times in 1 mL of assay buffer, and portions of the washed beads were resuspended in samples containing ISErs or control compounds in assay buffer. After 30 min incubations on ice, the beads were pelleted (1 min at 12000 rpm and 4 °C in an Eppendorf centrifuge). Supernatants were added to leukocyte activation assays. In some cases, beads incubated with ISErs were washed three times with assay buffer and then also added to leukocyte activation assays.
negative. All cell-based and bead-based assays were performed three times unless otherwise stated. Flow cytometry data were obtained with a FACS Canto II instrument (Beckton Dickinson), and flow cytometry data were analyzed using Flowing Software (http://flowingsoftware.btk.fi/). Quantitative flow cytometry data were exported to Excel and then graphically and statistically evaluated using GraphPad Prism 5.0. Isolation of Human Leukocytes and the Leukocyte Activation Assay. Crude human leukocyte isolates were isolated at room temperature (RT) from heparinized blood taken from healthy volunteers (laboratory staff) using dextran sedimentation and hypotonic lysis to remove erythrocytes as described previously.3 NADPH oxidase induction assays [dihydrorhodamine (DHR) oxidation in neutrophils] were performed in Hepes-buffered Hank’s balanced salt solution with bovine serum albumin (BSA) and EDTA (assay buffer) with DHR and catalase, as described in refs 3, 27, and 28. This assay measures the intracellular oxidation of DHR to fluorescent rhodamine on a single-cell basis using flow cytometry. In the presence of myeloperoxidase (MPO), DHR is oxidized by hydrogen peroxide that forms from oxygen radicals generated by phagocyte NADPH oxidase.28 This DHR oxidation assay was selected on the basis of its single-cell sensitivity, its high reproducibility, and its ability to indicate sufficient activation of neutrophils, including generation of toxic compounds.29,30 Cytochalasin B (SigmaAldrich catalog no. C6762, 21 mM stock in dimethyl sulfoxide) was added to a final concentration of 21 μM only where indicated. In addition to DHR oxidation (percentages of rhodamine-positive cells as readout), neutrophil forward scatter (mean neutrophil FSC) was also used as an indicator of leukocyte activation. In experiments involving ISEr-loaded beads, leukocyte samples were preincubated in 200 μL volumes of assay buffer with the additives at 1.25× final concentrations and 50 μL volumes at 5× final concentrations were added as stimuli. In experiments involving cell lines incubated with ISErs, leukocytes were preincubated in 125 μL volumes of assay buffer with the additives at 2× final concentrations and 125 μL volumes at 2× final concentrations were added as stimuli. Binding of ISErs to Protein A Beads. Protein A beads (Dynabeads 10001D, Invitrogen, 30 mg/mL slurry) with a diameter of 2.8 μm and a theoretical binding capacity of 8 μg of IgG/mg (53 pmol/mg) were first coated with a custommade rabbit polyclonal and affinity-purified antibody against B92 and then incubated with Y-9, Yb-9, or control substances in assay buffer as indicated. Beads that had been incubated with the compound and washed were added to leukocyte activation assays. To monitor the efficiency of binding of the ISEr to the beads, the supernatants of the ISEr/bead incubations were tested in comparison with the ISEr solutions before bead incubation. To test the release of ISEr from the beads incubated with the compound, the washed beads were further incubated in assay buffer for 20 min on ice or at 37 °C and pelleted. Next, the supernatants were added to the immune assays. Preparations, washing and incubation steps, and special precautions in measuring bead-containing samples by flow cytometry are described in more detail on page 2 of the Supporting Information. Demonstration of ISErs Binding to Beads. To demonstrate the binding of biotinylated compounds such as Yb-9, bY-9, or B9b to the beads or the absence of binding of
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RESULTS Sensitive Detection of Neutrophil Stimulation by ISErs To Investigate the Mechanism of Action. Respiratory burst of neutrophils was measured in a dihydrorhodamine (DHR) oxidation assay and showed a steep dose−effect curve for ISErs in the presence of cytochalasin B. For the ISErs Y-9 and Yb-9 (Figure 1C), activation started at concentrations of 100 nM and a maximum level of positive cells of 80% was reached at 1 μM (Figure 2A), 2644
DOI: 10.1021/acs.biochem.9b00169 Biochemistry 2019, 58, 2642−2652
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Figure 2. Neutrophil activation assays in the presence (●) or absence (○) of cytochalasin B (CytoB). (A) Induction of NADPH oxidase activity in human neutrophils. (B) Increase in forward scatter (FSC, shape change) of neutrophils in response to Yb-9. Shown are means and standard deviations of three measurements. FSC values were normalized (unstimulated samples = 1). See Figure S1 for more information.
Figure 3. Neutrophil activation by protein A beads loaded with the antibody (AB) and ISErs. Differently coated protein A beads (0.5 mg) were tested for their stimulatory effect on human neutrophils in (A) the DHR oxidation assay and (B) the shape change assay, both performed in the absence of cytochalasin B. Data from a series of experiments were compiled in these two graphs. Beads carrying Yb-9 were tested five times, those with Y-9 four times, and those with B9b three times. Statistical analysis (paired t test) was performed on only these groups of samples. Beads carrying bY-9 or B9 were tested twice. FSC values were normalized against unstimulated samples from the same donor tested the same day. (C and D) Activation of neutrophils by supernatants (20 min incubations in assay buffer on ice or at 37 °C) from 0.5 mg of protein A beads coated with the antibody with Yb-9, Y-9, or B9. (E and F) Activation by 0.5 mg of beads coated with the antibody with either Yb-9 or B9, which was added directly after washing, or pelleted and resuspended after an extended incubation period in assay buffer of 20 min on ice or at 37 °C. All conditions shown in panels C−F were tested three times with leukocytes from different donors each time. Paired t tests showed significant differences at a p < 0.05 (one asterisk) or p < 0.01 (two asterisks) level. For calibration of the effects of Y-9 and Yb-9 in both assay types (performed without cytochalasin B), see Figure 2 and Figure S1H.
in agreement with previously reported results.1,2 Cytochalasin B prevents the formation of a desensitized FPR receptor complex with the cytoskeleton and augments some effects induced by N-formylated peptides.31 DHR oxidation assays are therefore usually performed in the presence of cytochalasin B.2,3,27,28 Here, we show that in the absence of cytochalasin B, the dose−effect curve had a shallower slope, but the first signs of neutrophil activation were already observed at 30 nM (Figure 2A). An MPO-independent cytochrome c reduction assay, which directly measures oxygen radical formation, was tested in comparison with the DHR oxidation assay for studying the response to Y-9.32 As the cytochrome c assay showed thresholds similar to those of the DHR oxidation assay
(Figure S1M,N), we relied on the latter for the reasons explained above. We also tested the addition of exogenous MPO, which can rescue the DHR oxidation assay in the absence of endogenous MPO, for example, in MPO-deficient patients and cell models.30 However, MPO addition did not increase the sensitivity of this assay (Figure S1L). A parameter for activation of neutrophils even more sensitive to low ISEr concentrations proved to be the increase of their forward scatter (FSC) in flow cytometry measurements. The change in the shape of neutrophils with increased light scattering properties is a reaction of neutrophils to stimuli such as IL8, leukotriene B4 (LTB4), platelet-activating factor (PAF), complement factor 5a (C5a), Gro-α/CXCL-1, stromal cell2645
DOI: 10.1021/acs.biochem.9b00169 Biochemistry 2019, 58, 2642−2652
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Biochemistry derived factor 1α (SDF-1α/CXCL12), and fMLF.4,7,33,34 This change in shape from round to irregular cells is thought to be physiologically relevant for neutrophils to extravasate from circulation toward the site of bacterial infection. In Figure 2B and Figure S1H, a first increase in FSC was visible at 1 nM Y-9 or Yb-9, while maximum effects were reached at 30 nM. This high sensitivity was achieved only in the absence of cytochalasin B. In a series of experiments, maximum FSC increases of 1.7−2.2-fold relative to the control (buffer only) were achieved with Y-9 or Yb-9 concentrations of 30−300 nM. For examples of FSC and morphology changes, see Figure S1. To confirm that FPR-1 is the main signaling pathway for ISErs as previously proposed with formyl peptide effectors, we used an anti-FPR-1 antibody. Y-9 and Y-scaffold (PEGylated formyl peptide) both inhibited binding of this antibody to monocytes and granulocytes, while activation of neutrophils by Y-9 was inhibited by prior incubation of these cells with the anti-FPR-1 antibody but not by the isotype control (Figure S2A,B), demonstrating that ISErs exert their effects mainly through this receptor. Protein A Beads Loaded with Y-9 and Yb-9 Activate Neutrophils. Protein A beads, coated with a rabbit-derived anti-B9 antibody, were used to determine whether surfacebound ISErs are able to stimulate neutrophils as well as when in solution. Y-9 or Yb-9 (both ISErs contain two B9 peptides binding the anti-B9 antibody) were successfully loaded onto protein A beads as demonstrated by a depletion assay. Y-9 and Y-9b (but not control substance Y-scaffold lacking a B9 peptide) were loaded on beads from solution, as demonstrated by a reduced effector activity in the DHR oxidation assay in the supernatants of the beads (Figure S3A,B). Biotinylated Yb-9 could also be detected on beads via streptavidin staining (Figure S3C,D). The presence of Y-9 on beads was determined indirectly due to the lack of either a biotin moiety or an available secondary antibody. Protein A beads loaded with ISErs Y-9 and Yb-9 were then used in neutrophil activation assays. As shown in panels A and B of Figure 3, the beads loaded with Y-9 or Yb-9 significantly (p < 0.05 when compared to control B9b-loaded beads) activated neutrophils in neutrophil shape change assays, and those loaded with Yb-9 also did so in DHR oxidation assays (Y-9-loaded beads vs B9b-loaded beads: 0.05 < p < 0.10). The addition of 0.5 mg of ISEr-coated beads caused on average 10−12% positive neutrophils (Figure 3A) and 40−50% increases in neutrophil FSC (Figure 3B). Details about the assays using protein A beads can be found in Figure S4. The level of activation in both assay types was dependent on the amounts of ISEr-loaded beads added (Figure S5A) and on the density of loading of Yb-9 on protein A beads (Figure S5B). When tested in an alternative respiratory burst assay based on cytochrome c reduction, Y-9- and Yb-9-loaded protein A beads induced a positive response, which was not observed for B9bloaded beads (Figure S5C, panel C). The latter result was anticipated as B9b does not contain the effector moiety F2. These are the first indications that activation of neutrophils by surface-bound ISErs, as predicted in the mechanism of action, is possible. Apart from the activation of neutrophils by ISEr-loaded beads shown in Figure 3, we noted that naked beads (washed in only assay buffer) also caused a dose-dependent oxidase activation (Figure S5A, panel A). This effect was diminished but not completely prevented after the beads had been coated with the antibody. Beads covered with the antibody only or
with the antibody and inactive compounds such as binder peptides B9 and B9b or with bY-9 (N-formyl group replaced by a biotin, thus inactive) caused some oxidase induction when applied in large amounts (0.5−1 mg of beads), but much stronger oxidase induction was always caused by the same amounts of beads coated with Y-9 or Yb-9 (for examples and numbers, see panels A and E of Figure 3; panels A and B of Figure S5 and their legends). At the same time, naked beads led to very little if any increase in FSC when 0.5 or 1 mg was applied (Figure 3B,F; Figure S5A), suggesting that the change in the shape of neutrophils is a more sensitive readout for immune activation by ISErs bound to protein A beads than induction of NADPH oxidase. In an attempt to circumvent the problem of protein A beads (both naked and antibody-coated) causing background activation of phagocyte NADPH oxidase, and to prevent elution of the bead-bound ISEr (see below), we also performed similar assays with streptavidin beads. Even though these beads were able to deplete Yb-9 and other biotinylated compounds from solution, the Yb-9-coated beads did not activate neutrophils as strongly as ISEr-loaded protein A beads (Figure S6A−C). This might be based on the fact that complexation of biotinylated ISErs with soluble streptavidin protein also inhibited their immune-stimulating activities (Figure S6D−F). Release of the ISEr from Protein A Beads? To ensure that the activation by ISEr-loaded protein A beads is caused by immobilized effectors, the contribution of ISEr released from the beads within 20 min at 37 °C in assay buffer was tested. Bead samples coated with the antibody and incubated with Yb9 or Y-9 (active ISErs) or B9b (biotinylated binder peptide without an immune effector) were either added directly to the activation assay or incubated at 37 °C or on ice for 20 min before pelleting the beads. Supernatants of the beads as well as the resuspended beads were assayed (Figure 3C−F). The supernatants after incubation on ice did not exhibit any measurable activity. However, supernatants of Yb-9-loaded beads incubated for 20 min at 37 °C did lead to some increase in FSC (Figure 3D and Figure S5C, panel A), but the increase was smaller than that obtained with Yb-9-coated beads directly (Figure 3B and Figure S5C, panel A), indicating some release of the ISEr from the beads. Supernatants of Yb-9-coated beads incubated at 37 °C increased FSC values significantly (p < 0.05) when compared with the values for 37 °C supernatants from B9b-coated beads and highly significantly (p < 0.01) when compared with the values of 0 °C supernatants of the same Yb-9-coated beads. No DHR oxidation was found using the same supernatants, confirming the results presented above that the increase in FCS is the more sensitive assay (Figure 3C and Figure S5C, panel E). To confirm that the absence of DHR oxidation by the bead supernatants indeed reflects a lack of NADPH oxidase induction, supernatants of experiments at 37 °C were also tested in the cytochrome c reduction assay, in which all supernatants gave no positive signal (Figure S5C, panel F). Yb-9 and Y-9 beads that were incubated for 20 min at 0 or 37 °C led to activation levels equal to those of freshly washed beads in both assay types (Figure 3E,F), indicating little loss of surface-bound ISErs during these 20 min incubations. Efficient release of active Y-9 from protein A beads was observed after a 150 min incubation in a solution of 100 μM B9 peptide. A clear immune activation by displaced Y-9 in the bead supernatant was observed that allowed for an estimation of the binding capacity of these beads for Y-9 (Figure S7). 2646
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Biochemistry Activation of Neutrophils by Tumor Cells Incubated with Integrin-Specific ISErs. Having shown that immobilized ISErs can induce neutrophil activation, we moved on to investigate whether tumor cells bearing ISErs on their surface could elicit similar responses. We used four cell lines, two of which were known to bind Y-9 and Yb-9 to a high level (A431 and PC-3; see also refs 2 and 3) and two of which do not to bind them or only to a very low level (K562 and L-929). In addition to Y-9 and Yb-9, we also employed a number of control compounds (see Figure 1C), which are not supposed to bind to cells (e.g., carrying a scrambled binder peptide or no binder peptide: Y-sc9 and Yb-scaffold) or do bind but lack a functional F2 effector peptide (B9 and B9b). Cells incubated with Y-9 and Yb-9 were added to leukocyte activation assays after extensive washing. Binding of Yb-9 (and also B9b) to A431 and PC-3 cells (but not or to a low level on K562 and L-929 cells) was confirmed by streptavidin staining (Figure S8). Y-9 and Yb-9 incubated A431 and PC-3 cells did not induce NADPH oxidase activity (Figure S9A,D) but did cause a change in the shape of the neutrophils (Figure 4 and Figure S9F,H). FSC values of neutrophils were significantly (p < 0.05) increased after incubation with A431 and PC-3 cells carrying Y-9 or Yb-9 but not by the same cells incubated with
control compounds or by control K562 or L-929 cells incubated with any of the compounds (Figure 4A). Activation of Neutrophils by Integrin-Specific ISErs Released from Cells. The biotin of Yb-9 (and B9b) could not be detected on the surface of A431 and PC-3 cells after 20 min upon incubation at 37 °C (Figure S10) but could still be detected when the cells were incubated on ice. Thus, the activation of neutrophils by A431 or PC-3 cells incubated with Yb-9 occurred either quite fast by cell-bound ISEr or was induced by material released from the cells or by a combination of both. To study the contribution of activity released by cells during the assay, the A431 and PC-3 cells incubated with the ISEr or a control were incubated for 20 min at 37 °C (controls on ice) in assay buffer and then pelleted at 4 °C. The supernatants were added to the neutrophil activation assay. The 37 °C supernatants of A431 and PC-3 cells incubated with Yb-9 led to a significant (p < 0.05) increase in neutrophil FSC (Figure 4B). The increases in FSC induced by the 37 °C supernatants of A431 or PC-3 cells incubated with Y-9 were not quite significant due to large standard deviations (p < 0.10). The 37 °C supernatants of untreated or controlincubated A431 and PC-3 cells did not lead to any noticeable increase, indicating that no neutrophil-activating, endogenous substances were released by these cells. None of the 0 °C supernatants induced any change in neutrophil FSC (Figure 4C), and no NADPH oxidase activation was noticed with supernatants of 0 or 37 °C incubations (not shown). The FSC increases induced by A431 and PC-3 cells incubated with the ISEr added directly to the assay and the increases induced by their 37 °C supernatants (compare Figure 4A with Figure 4B) appear to be very similar, ranging between 1.2- and 1.6-fold. Therefore, the major part, if not all, of the neutrophil activation induced by tumor cells incubated with Y9 or Yb-9 is probably caused by ISErs or ISEr fragments released from the cells during the 20 min assay. The release of immune-activating material from the cells incubated with Y-9 and Yb-9 at 37 °C correlated with the disappearance of the cell surface signals of Yb-9 at 37 °C (Figure S10), suggesting that the dissociation of these ISErs from the receptors at 37 °C is the major cause of activity release. An estimation of the amounts of Y-9 and Yb-9 that were released from the tumor cells is presented after Figure S10 (page 17 of the Supporting Information). This calculation suggests that between 1.8 × 105 and 7.5 × 105 ISEr molecules were bound to and released again from every A431 or PC-3 cell. Activation of Neutrophils by Tumor Cells Incubated with EphA2-Specific ISErs. In a fashion similar to that described above for the Y-9 family of ISErs, the EphA2-specific Y-59 ISErs were also tested for their ability to activate neutrophils after binding to tumor cells. PC-3 cells were used because of their high expression levels of EphA2,3,35 while K562 cells were used as a negative control. Biotinylated ISErs, such as Yb-59 (KD = 69 nM), its four-armed sibling Tet-Yb-59 [with a higher binding affinity (KD = 1.2 nM); see ref 3], and a control compound Yb-sc59 [with a scrambled binder peptide (Figure 1C)], were used because their presence and fate on cell membranes could be monitored. Binding of Yb-59 and Tet-Yb-59 to PC-3 cells, but not to K562 cells, was confirmed by streptavidin staining (Figure S11A). Yb-sc59 also bound PC-3 cells (not K562 cells) after incubation at high concentrations [>500 nM (data not shown)], indicating that the scrambled binder peptide retained some unspecific affinity and is not an ideal negative control. It
Figure 4. Change in the shape of neutrophils induced by cell lines incubated with integrin binding ISErs and supernatants thereof. Panel A shows the effects of direct incubation of leukocytes with 2−2.5 million tumor cells incubated with the ISErs or control substances. Panels B and C show the effects of the supernatants of 2−2.5 million ISEr-incubated cells after incubation for 20 min at 37 °C (B) or 0 °C (C). Neutrophil FSC values from three experiments (four experiments for A431 cells in panel A) were normalized (the mean FSC of untreated samples was taken as 1), and average values with standard deviations are shown. The legend in panel C is also to be used in panel B. Asterisks indicate significant differences at the p < 0.05 level (paired t test). More information and controls can be found in Figure S9. 2647
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after the extensive incubation and centrifugation steps, evidenced by Hoechst-positive events in flow cytometry (not shown)] and later released in the presence of neutrophils. PC3 cells incubated with the ISEr did not release sufficient amounts of ISErs after a 20 min incubation on ice to induce any significant effects on neutrophils (Figure S12A). However, after 20 min at 37 °C, activity was released from PC-3 cells loaded with Yb-59 and Tet-Yb-59, causing significant (p < 0.05) increases in FCS (Figure 5C). Supernatants collected at 37 °C from PC-3 cells incubated with Yb-sc59 and from K562 cells incubated with Yb-59 or Tet-Yb-59 caused minor, nonsignificant increases in neutrophil FSC. No increase was observed for untreated PC-3 or K562 cells or their 37 °C supernatants. Neither PC-3 cells incubated with Yb-59 or TetYb-59 nor their 37 °C supernatants caused any induction of NADPH oxidase (Figure S12B,C). A calibration series with known ISEr concentrations was used to estimate the amount of Yb-59 ISErs released from cells at 37 °C (Figure S13 and page 22 of the Supporting Information). These calculations suggest that between 7.5 × 104 and 7.5 × 105 ISErs per PC-3 cell were bound and released again. These numbers are much higher than published data for the numbers of EphA2 receptors on PC-3 cells (1.6 × 104 per cell35). The detection of neutrophil activation with supernatants of PC-3 cells incubated with Yb59 or Tet-Yb-59 at 37 °C (but not at 0 °C) was not accompanied by a faster disappearance of cell surface signals at this temperature [as monitored by streptavidin staining (Figure 5A)]. A situation more complex than just faster dissociation of complete ISErs at 37 °C than at 0 °C could be surmised. The nature of the material released from cells will be studied further below. Biotinylation Status of ISErs Released from Cells. In an attempt to detect enzymatic or metabolism-driven cleavage that led to release of an immune-active substance from the ISErs (e.g., N-formylated peptide), we used streptavidin beads that can capture biotinylated ISErs and cleavage products but not nonbiotinylated compounds. Test reactions with low to subnanomolar concentrations of biotinylated ISErs and control compounds (Yb-scaffold and F2b peptide) showed removal of all biotinylated species from solutions using 0.1 mg of these beads (Figure 6A,B). In contrast, nonbiotinylated ISErs, Yscaffold and F2 peptide, were not removed by streptavidin beads, excluding unspecific adsorption. Supernatants obtained after 20 min incubations at 37 °C of A431 or PC-3 cells carrying Yb-9 were treated with streptavidin beads, leading to almost complete removal of the FSC-increasing activity from the supernatants (Figure 6C,D). In contrast, no activity was removed with streptavidin beads from the supernatants of Y-9-treated A431 cells. These data indicate that the immune stimulant (formyl peptide F2) within Yb-9 was still connected to biotin after release from the tumor cells. In contrast, supernatants obtained after 20 min incubations at 37 °C of PC-3 cells with Yb-59 contained immune-stimulating activity that could not be removed with streptavidin beads (Figure 6D), which is similar to the activity in supernatants of cells carrying nonbiotinylated Y-59. This is a first indication that cleavage of Yb-59 occurs on the cell surface or inside of cells, by which the F2 effector peptide becomes separated from biotin.
should be noted that other negative control compounds such as Yb-scaffold and Y-sc9 were also incubated with PC-3 and K562 cells, not leading to any binding or activation of immune cells (Figure 4). Incubation of PC-3 cells loaded with Yb-59 or Tet-Yb-59 at 37 °C did not lead to a faster loss of their biotin signals from the cell surface than at 0 °C (Figure 5A and Figure
Figure 5. ISErs binding EphA2. Analysis of binding to PC-3 cells and immune activation by cells incubated with the ISEr and their supernatants. (A) Quantitation of the binding levels of biotinylated ISErs on PC-3 cells either directly after four washing steps (t = 0) or after an additional 20 min incubation on ice or at 37 °C. The mean geometrical fluorescence values were normalized to the values of the respective negative control sample (PC-3 cells not incubated with any ISEr), which therefore all have a value of 1. Means and standard deviations of three experiments are shown. More details are shown in Figure S11. (B) Induction of a change in the shape of neutrophils by cell lines incubated with the ISEr. (C) Induction of the change in the shape of neutrophils by 37 °C supernatants of cells incubated with the ISEr. Cells loaded with the ISEr were incubated for 20 min at 37 °C, and the supernatants of the pelleted cells were used in the assay. Means and standard deviations of three experiments with normalized values (in which the mean FSC of an untreated sample was taken as 1) are shown in panels B and C. The legend in panel B is also to be used in panel C. One asterisk indicates significant effects at the p < 0.05 level (paired t test), and two asterisks indicate p < 0.01. More data about this experiment can be found in Figure S12, and for calibration of the effects, see Figure S13.
S11B), unlike the situation observed with Yb-9 (Figure S10). In contrast, control ISEr Yb-sc59 disappeared from the cell surface faster at 37 °C than on ice [p < 0.01 (Figure 5A and Figure S11B)]. Highly significant (p < 0.01) increases in granulocyte FSC were achieved after incubation with PC-3 cells carrying Yb-59 or Tet-Yb-59 (Figure 5B), while smaller increases were achieved with PC-3 cells incubated with Yb-sc59 (p < 0.05). Even K562 cells incubated with Yb-59 (p < 0.05) or Tet-Yb-59 (p < 0.10) activated neutrophils, even though K562 cells did not show any binding of Yb-59 or Tet-Yb-59 (Figure S11A). A possible explanation is that small amounts of these ISErs could have been taken up by dead K562 cells [that always occurs
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DISCUSSION In this study, we demonstrated using a bead model that immobilized ISErs can stimulate human neutrophils. This was 2648
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Figure 6. Depletion by streptavidin beads of low concentrations of biotinylated ISErs and control compounds and of activity released from tumor cells incubated with the ISEr. (A and B) Change in the shape of neutrophils induced by six different biotinylated or nonbiotinylated compounds diluted directly from the stocks, as such (white bars) and after incubation with streptavidin beads and pelleting of the beads (black bars). In panel A, concentrations (depicted in nanomolar, final concentrations in the 250 μL assay volume; 125 μL volumes of twice these concentrations were incubated with beads) were chosen such that they induced an almost maximum change in shape (1.6−1.8-fold). In panel B, concentrations were chosen that caused a minor change in shape (1.1−1.5-fold). Shown are means and standard deviations of normalized values (unstimulated samples = 1) of three experiments. (C and D) Depletion by streptavidin beads of activity released by A431 (C) and PC-3 (D) cells incubated with the ISEr. Naive tumor cells (no agent) or tumor cells incubated with an ISEr (Yb-9, Y-9, Yb-59, or Y-59) were thoroughly washed and then incubated for 20 min at 37 °C in 125 μL of assay buffer. The supernatants were either directly added to the immune stimulation assay (white bars) or incubated with streptavidin beads and added to the assay after pelleting the beads (black bars). Shown are means and standard deviations of normalized values (unstimulated samples = 1) of three (PC-3 with Y-59 and Yb-9), four (all A431 incubations), or five (PC-3 with Yb-59 and no agent) experiments. *p < 0.05; **p < 0.01 (paired t test).
this is the first demonstration of activation of formyl peptide receptors by surface-bound N-formyl peptides. Additional mechanisms appear to be involved in FPR activation when ISErs are bound to tumor cells. These involve release of ISErs or parts thereof, which are also able to activate neutrophils and therefore complicated the assessment of the contribution of cell surface-bound ISErs to immune stimulation. Here, we demonstrate immune cell activation, as evidenced by a change in the shape of neutrophils/an increase in FSC, by supernatants of A431 and PC-3 cells incubated with the ISEr. This indicates that a second scenarionamely the release of immune-activating molecules from ISEr-coated tumor cells, not necessarily including complete ISERsis part of the ISEr mode of action. No or very little direct activation or release of neutrophil-activating material was observed from cells incubated with the ISEr that do not possess the respective receptors for Y-9 and Y-59, such as K562 cells. No activating species were released from A431 or PC-3 cells incubated with several control compounds (nonbinding or lacking the N-formyl peptide effector). Thus, activation was specifically mediated by the F2 peptide within ISErs binding to A431 and PC-3 tumor cells via their specific binding peptides B9 and B59, and this activity was released at 37 °C but not at 0 °C. Already in the 1980s,24,36 studies had shown that chemotactic activity was released from tumor cells preincubated with fMLF-conjugated antibodies, preferentially at 37 °C but not at 4 °C. Interestingly, the possibility that free fMLP peptide generated by cell-derived enzymes might have been shed by the cells instead of the complete fMLF−IgG conjugate was already mentioned in these studies. A clear difference between the two ISEr families that bind different cell surface receptors (Y-9 and Y-59) tested here became obvious when comparing the temperature dependence
confirmed by induction of NADPH oxidase (respiratory burst) and, more sensitively, by increased neutrophil forward scatter. The contribution of the ISEr released from protein A beads to the activation was minimal. A downside of the protein A bead model was the activation of neutrophil NADPH oxidase by untreated beads. This might be attributed to the bacterial origin of protein A, which in itself could stimulate myeloid immune cells. In contrast, untreated protein A beads did not cause a change in the shape of neutrophils, which therefore seems to be a more specific marker for neutrophil activation through FPRs than activation of NADPH oxidase. Background activation was kept to a minimum by coating the beads with an antibody and ISErs or control compounds and by adding ≤0.5 mg of the coated beads to the assays. Studies by our group and others have indicated that with an increase in molecular size, the potency of N-formyl peptide F2 decreased.2,3,24,36 We have furthermore found that complexing the biotinylated F2 peptide (F2b), the F2b−PEG conjugate (Yb-scaffold), or biotinylated ISErs (Yb-9 and Yb-59) to soluble streptavidin also led to a loss of potency (Figure S6). This effect is reminiscent of the findings of Sklar et al.37 who found that a fluorescein-labeled N-formyl peptide no longer bound its receptors after being attached to an anti-fluorescein antibody. Therefore, binding of ISErs to a large particle, such as a bead or a cell, could lead to a further reduction or even complete loss of activity. On the other hand, adhesion to opsonized particles usually enhances activation of neutrophils and respiratory burst,38 so the surface-bound N-formyl peptide might also induce activation through adhesion. We indeed could demonstrate that N-formyl peptide F2, incorporated in an ISEr molecule, can still activate neutrophils when bound to the surface of protein A beads. To the best of our knowledge, 2649
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of release and/or processing of ISErs on tumor cells. Whereas Yb-9 demonstrated very slow dissociation and/or processing at 0 °C, its biotin signal disappeared from the cell membranes within 20 min at 37 °C. The rapid loss of the biotin signal from the outer leaflet of the cell membrane at physiological temperature correlated with the release of immune-activating substances from cells incubated with the ISEr at 37 °C but not at 0 °C. Internalization and release of intact ISEr or degradation products thereof cannot be excluded, but we have discovered no evidence of cleavage, at least not between the F2 peptide and the biotin within Yb-9. In contrast to the Y9 family, ISErs Yb-59 and Tet-Yb-59 (or at least their biotin tags) disappeared from the cell surface at about the same rate on ice as at 37 °C. Nevertheless, more neutrophil-stimulating activity was found in the 37 °C supernatants of PC-3 cells carrying these ISErs than those generated on ice. The species responsible for this activity could not be captured by streptavidin beads, indicating a release of nonbiotinylated ISEr fragments with neutrophil-stimulating activity from PC-3 cells. A potential tumor cell-specific cleavage of intact ISErs that leads to possibly 100-fold more potent smaller fragments containing (or consisting solely of) the immune effector peptide F2 would add an additional level of tumor specificity to this class of therapeutics as it would create a chemotactic gradient required for neutrophil attraction. This scenario is speculative and will have to be thoroughly investigated; however, it encourages the design of novel ISErs that allow the controlled release of more potent immune effector species. Here, we have estimated the concentrations of neutrophil activators released in our assays to be equivalent to 3−10 nM Y-9 or 1−10 nM Yb-59 released from 2−2.5 × 106 cells in a 0.25 mL volume. Concentrations of at least 30 nM would be necessary to induce a positive response in the DHR oxidation assay, explaining why we could observe neutrophil activation only by increased forward scatter. Whether these levels of activation (induced by ISErs or fragments released from cells) would suffice for an antitumor immune reaction is questionable. We expect, however, that much higher local concentrations of ISErs or fragments released from cells would be achieved in solid tumors that might allow for full activation of neutrophils that includes respiratory burst and degranulation. ISErs specifically target tumors in vivo, and the numbers and densities of tumor cells (and tumor stromal cells that might also express the receptors binding ISErs) are much higher in situ than what can be achieved in the in vitro setting.
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Article
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.9b00169. More detailed information about some methods, 13 additional figures, and estimations of amounts of ISErs that were bound and released again from beads and from tumor cells (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*Syntab Therapeutics GmbH, Pauwelsstrasse 17, D-52074 Aachen, Germany. E-mail:
[email protected]. Telephone: (+49) 241 99032932. *Faculty of Chemistry, Institute of Biological Chemistry, University of Vienna, Währinger Str. 42, 1090 Wien, Austria. E-mail:
[email protected]. Telephone: (+43) 1 4277 70510. ORCID
Anne C. Conibear: 0000-0002-5482-6225 Christian F. W. Becker: 0000-0002-8890-7082 Present Address §
A.C.C.: School of Biomedical Sciences, University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia.
Author Contributions
A.J.G.P., A.C.C., and C.F.W.B. designed research. A.C.C., C.A., C.H., and A.J.G.P. performed research and analyzed data. A.J.G.P., A.C.C., and C.F.W.B. wrote the paper. Funding
This work was financially supported by the Bundesministerium fü r Bildung and Forschung (BMBF, KMU-innovativ-6, Förderkennzeichen 0315918) and by the European Union and NRW government [NRW-EU Ziel 2, (EFRE), 005-09080112]. The research leading to these results has received funding from the Mahlke-Obermann Stiftung and the European Union’s 7th FP under Grant Agreement 609431. Notes
The authors declare the following competing financial interest(s): C.F.W.B. is a co-founder and partner of Syntab Therapeutics GmbH.
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ACKNOWLEDGMENTS The authors thank Manuel Felkl for assistance with the synthesis and analysis of the compounds. Frank Tacke and the members of his research group are kindly acknowledged for their feedback and support.
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ABBREVIATIONS ISEr, immune system engager; EphA2, erythropoietin-producing hepatoma receptor A2; FPR-1, (N-)formyl peptide receptor 1; DHR, dihydrorhodamine 123; FSC, forward scatter; MPO, myeloperoxidase
CONCLUSION
We have demonstrated that ISErs, after binding to beads and to their specific receptors on tumor cells, are still able to stimulate innate immune cells. This finding is fundamental to the mode of action of ISErs as tumor cells decorated with these molecules can provoke an innate immune response. We also found evidence that an immune-stimulating species is released from target cells. Further studies to elucidate the molecular nature of these species are required, a crucial prerequisite for determining if such effects can be employed for modulating the therapeutic potential of ISErs.
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