Targeting Gastrin-Releasing Peptide Receptors on Small Cell Lung Cancer Cells with a Bispecific Molecule that Activates Polyclonal T Lymphocytes

Small cell lung cancer (SCLC) is characterized by rapid progression, early metastasis, and high mortality. This neuroendocrine carcinoma expresses unique molecular genetics and cellular regulation pathways (1). SCLC cells produce a variety of neuroendocrine peptides. These neuroendocrine peptides as well as their receptors are attractive targets for anticancer treatment. Gastrin-releasing peptide (GRP) is a neuroendocrine peptide and a member of the mammalian bombesin-like peptide family. SCLC cells are known to produce bombesin-like peptides that function as autocrine growth factors to promote growth of this tumor (2). GRP receptor (GRP-R) is expressed in a majority of SCLC cell lines and has limited distribution in normal human tissues (3–5). We have developed an immunotherapeutic approach to target GRP-R for the treatment of SCLC and other GRP-R-expressing malignancies (6).

CTLs are potent immune effector cells. They can be activated through their T-cell receptor (TCR) to effectively kill a target cell. Antigen recognition is mediated by either the α/β chain or γ/δ chain of the TCR. The α/β chain forms a heterodimer that is closely associated with a cluster of transmembrane peptides to form a CD3 complex on the T-cell surface (7). In patients with advanced cancer, host T cells are functionally suppressed and unable to mount an effective attack on cancer cells. There are several explanations for the mechanisms by which cancer cells escape the host immune system, including lack of the recognition antigens on the cancer cells and inadequate activation of immune trigger molecules on host immune effector cells (8).

One strategy to redirect cytotoxic T cells toward cancer cells is to develop a bispecific molecule that can bind to TCR and a tumor-associated antigen. OKT3 is an anti-CD3 monoclonal antibody specific for TCR. Several groups have constructed bispecific antibodies consisting of an anti-CD3 antibody and a tumor-specific antibody for targeted immunotherapy. A bispecific antibody, OKT3xTrastuzumab (anti-CD3xanti-HER2), has been shown in preclinical studies to effectively mediate the killing of HER2-positive breast cancer cells. A phase I trial in patients with advanced and metastatic breast cancer and hormone-refractory prostate cancer has been reported recently. Partial and minor responses have been observed in both prostate and breast cancer patients (9). A recombinant single-chain bispecific antibody (anti-CD3xanti-CD19) has been shown to effectively activate autologous T cells from patients with advanced B-cell lymphoma or leukemia and effectively kill the autologous B cells at a low effector to target cell ratio (10). An anti-CD3xanti-PSA bispecific antibody has been shown to inhibit prostate cancer growth in a mouse model (11). A phase I clinical trial with a HEA125xOKT3 bispecific antibody has been reported. The treatment was given as weekly i.p. injections and was well tolerated. All 10 patients with advanced ovarian carcinoma had a response to the treatment with temporary inhibition of the accumulation of malignant ascites (12).

We have constructed a bispecific molecule with an anti-CD3 monoclonal antibody and a synthetic bombesin/GRP antagonist Antag2. The bispecific molecule OKT3xAntag 2 specifically binds to SCLC cells and activates TCR, effectively mediating the inhibition of SCLC growth by human T cells in vitro as well as in vivo.

Cell culture. Two human SCLC cell lines (H345 and DMS273) were maintained in serum-free RPMI 1640 containing 1 × 10⁻⁸ mol/L hydrocortisone, 5 μg/mL insulin, 10 μg/mL transferrin, 1 × 10⁻⁸ mol/L β-estradiol, and 3 × 10⁻⁸ mol/L selenium (HITES medium). All chemical reagents were purchased from Sigma Chemical Co. (St. Louis, MO). H345 was purchased from the American Type Culture Collection (Rockville, MD). DMS273 cell line was established from the pleural fluid of a patient with SCLC in 1980 at Dartmouth Medical School and has been used in our laboratory for a variety of in vitro and in vivo studies (6, 13).

Construction of the bispecific molecule. The chemical conjugation procedure was described previously (6). A bombesin antagonist (Cys⁵-D-Phe⁶, Leu-NHEt¹³, des-Met¹⁴)BN(5-14), was custom synthesized by BACHEM, Inc. (Torrance, CA). It contains a free sulfhydryl group at the NH₂ terminus of the peptide. Anti-CD3 monoclonal antibody (OKT3) was purchased from Ortho Biotech (Raritan, NJ). A cross-linker, SPDP (N-succinimidyl 3-[2-pyridyldithio] propionate; Pierce, Rockford, IL), was mixed with intact IgG2a murine OKT3 for 1 hour and then mixed with Antag 2 at a molar ratio of 1:10. The free peptide was removed from the conjugated bispecific molecule by centrifugation through a size exclusion apparatus (Millipore, Billerica, MA).

Preparation of T lymphocytes. Buffy-coat cells from unselected healthy donors were obtained from the San Diego Blood Bank (San Diego, CA). Peripheral blood mononuclear cells were separated by Ficoll-Hypaque density centrifugation. Nonadherent cells were collected after incubating the peripheral blood mononuclear cells in DMEM containing 0.2% bovine serum albumin at 37°C, 5% CO₂ for 2 hours. The lymphocytes were cultured in RPMI 1640 containing 10% FCS and 100 units/mL interleukin-2 (IL-2; Chiron Therapeutics, Emeryville, CA) for 3 to 5 days. The phenotype of the peripheral blood lymphocytes was analyzed by flow cytometry (FACScan, BD Biosciences, San Jose, CA).

Binding of bispecific molecule to SCLC cells. Two SCLC cell lines (H345 and DMS273) and peripheral blood mononuclear cells were stained with the bispecific molecule using an indirect immunofluorescence staining method as described (6). In addition, free bombesin, Antag2, or angiotensin (a negative control peptide) at 10 μmol/L final concentration was added before incubation of the cells with the bispecific molecule to determine specificity of binding. All samples were analyzed by flow cytometry.

T-cell activation and proliferation. T-cell proliferation was measured by a standard thymidine incorporation assay. Fresh peripheral blood lymphocytes (2.5 × 10⁴ per well) were seeded into a 96-well plate and cultured for 72 hours, with either an unconjugated OKT3, a bispecific molecule, or a control antibody mouse IgG2a. IL-2 at 100 units/mL was added in the culture for 72 hours. Cells were harvested by a Tomtec cell harvester (Perkin-Elmer, Downers Grove, IL) and counted in a liquid scintillation counter. All assays were done in triplicate.

Cytotoxicity assay. Antibody-dependent cellular cytotoxicity (ADCC) was measured by a standard thymidine incorporation assay. DMS273 cells (2.5 × 10³ per well) were seeded into a 96-well plate. Effector T cells were added at E/T ratios of 5:1, 2.5:1, and 1.25:1. The bispecific molecule, unconjugated OKT3 or mouse IgG2a (negative control) was added in different concentrations. The controls included tumor cells alone and T cells alone with or without OKT3. The cell mixtures were incubated in RPMI 1640 containing 2.5% FCS for 72 hours. [³H]thymidine was added during the last 8 hours of incubation. Cells were harvested, and [³H]thymidine was counted in a liquid scintillation beta counter. All assays were done in triplicate. The inhibition of thymidine incorporation was calculated as: [1 − (experimental counts per minute − T cells alone counts per minute) / tumor cells alone counts per minute] × 100%.

IFNγ assay. In 96-well plates, 5 × 10³ SCLC cells were mixed with 1 × 10⁵ T cells in the presence of the bispecific molecule, OKT3, or mouse IgG2a at 0.1 μg/mL. After 48 hours of culture, the plate was centrifuged, and 100 μL of supernatant was collected from each well. The amount of human IFNγ in the supernatant was determined by an ELISA OptEIA assay according to the manufacturer's instructions (BD PharMingen, San Diego, CA). Absorbance at 450 nm was measured by a microplate spectrophotometer (Molecular Devices, Sunnyvale, CA), and the results were calculated using the SOFTmax program (Molecular Devices). All assays were done in duplicate.

Analysis of SCLC apoptosis and necrosis. SCLC cells were mixed in a 96-well plate with T cells at an E/T ratio of 2:1 or 1:1 in the presence of the bispecific molecule or mouse IgG2a and cultured for 4, 24, and 48 hours. At the end of the culture, cells were washed and immediately stained with Annexin V/FITC, propidium iodide, and anti-CD45-FITC. All samples were acquired and analyzed by flow cytometry using Cellquest software.

Western blot analysis of caspase-3, caspase-9, and poly(ADP-ribose) polymerase cleavage. DMS273 or H345 cells were mixed with T cells at 2:1 or 1:1 ratio with or without the bispecific molecule in a 96-well microplate. After culture for 48 hours, cells were washed thrice, centrifuged, collected, and transferred to a microtube. Whole-cell lysates were prepared from cell pellets as described (6). The protein content in the whole-cell lysate was determined; 40 μg of protein were loaded to each lane of 10% and 12% SDS-polyacrylamide gel. After electrophoresis, the gel was transferred to a nitrocellulose membrane. The full-length as well as the cleaved caspase-3, caspase-9, and poly(ADP-ribose) polymerase (PARP) were measured by an Apoptosis Sampler kit (Cell Signaling, Inc., Beverly, MA), according to the manufacturer's instructions. The signals were detected by exposing the membrane to a Kodak film after incubating the membrane with chemiluminescent reagent (Pierce Chemical Co., Rockford, IL).

Targeted immunotherapy in vivo. Ten- to 12-week-old NOD.CB17-Prkdc^scid (nonobese diabetic/Prkdc severe combined immunodeficient) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The human SCLC xenograft mouse model was established in our laboratory as previously described (6). Mice received a single fraction of whole-body irradiation (350 cGy) 6 hours before the experiment. Each mouse was injected with 1 × 10⁶ DMS273 cells i.p. Human T lymphocytes were isolated from an unselected healthy donor and cultured with 100 units/mL IL-2 for 3 days. Before injection, T cells were incubated (armed) with either the bispecific molecule (1 μg per 1 × 10⁷ cells) or OKT3 (1 μg per 1 × 10⁷ cells) for 20 minutes. The cells were washed to remove the excess bispecific molecule or OKT3 and resuspended to 5 × 10⁶ per 0.3 mL PBS for injection. Control mice received only DMS273 cells. One group of mice received bispecific molecule–armed T cells, and the other group of mice received OKT3-armed T cells.

In the first experiment, mice in the two treatment groups received two injections with 5 × 10⁶ T cells (days 4 and 10). In the second experiment, the mice in the treatment groups received i.p. injections of 5 × 10⁶ T cells on days 3, 7, and 14. Mice were examined daily for general health, activity, signs of illness, and visible tumor growth. In the first experiment, the mice were sacrificed at day 40, whereas the second experiment examined earlier time points of days 20 and 30. The peritoneal cavities were washed with 5 mL PBS to collect peritoneal exudate cells. Peritoneal exudate cells from each mouse were counted and stained with anti-human CD45-FITC (marker for T cells), anti-human CD56-PE (marker for DMS273 cell), and anti-mouse CD45-PE for flow cytometry analysis. Peritoneal exudate tumor cells (PETC) were calculated as: peritoneal exudate cells count × % human CD56⁺/human CD45⁻. After the peritoneal wash, the peritoneal cavity was opened and carefully examined. All visible tumors were dissected and collectively weighed for each mouse. The overall tumor burden was determined by both the gross tumor weight and PETC.

Statistics. The Student's t test was used to analyze the statistical significance of the in vitro experiments. Significance was considered when P < 0.05 (two-sided analysis). The results are presented as mean ± SD. For the in vivo study, one-way ANOVA analysis for multiple experimental groups was done. P < 0.05 was considered as significant. The Student's t test was then used to analyze differences between two treatment groups.

Binding of the bispecific molecule to SCLC cells and T cells. The bispecific molecule bound to both H345 and DMS273 SCLC cells (Fig. 1). The binding was specific and partially blocked by preincubating SCLC cells with an excess amount of free bombesin or Antag2 (data not shown). The bispecific molecule bound to peripheral blood lymphocytes showing that the chemical conjugation process did not interfere with the binding of OKT3. Peripheral blood monocytes did not bind to either OKT3 or the bispecific molecule.

T-cell proliferation. Peripheral blood lymphocytes separated from normal donors consisted of >80% T cells. The immunophenotype of these cells was 81 ± 7% CD3, 52 ± 13% CD4, 34 ± 10% CD8, 17 ± 6% CD56, 8 ± 4% CD25, and 19 ± 11% CD69. Cell viability was >90%. [³H]thymidine incorporation (counts per minute) into T cells after 72 hours culture is shown in Fig. 2. Without IL-2, there was little increase in thymidine uptake into T cells (Fig. 2A). There was no increase in thymidine uptake into T cells in the presence of a control mouse IgG2a antibody. Both OKT3 and the bispecific molecule increased [³H]thymidine uptake into T cells in a dose-dependent pattern (Fig. 2B). There was no significant difference in thymidine uptake into T cells stimulated by the OKT3 or the bispecific molecule, again suggesting that chemical conjugation had no effect on the biological function of OKT3.

Growth inhibition of SCLC cells in vitro. The bispecific molecule–mediated inhibition of DMS273 cell growth was dose dependent and occurred at a low E/T ratio as shown in Fig. 3. At an E/T ratio of 2.5:1, the inhibition increased from a low concentration of bispecific molecule of 0.1 to 10 ng/mL and did not increase further at a higher concentration of 100 ng/mL. The bispecific molecule–mediated growth inhibition was specific and could be partially blocked by preincubating DMS273 cells with an excess of Antag2 (data not shown). There was no growth inhibition when DMS273 cells were cultured with the bispecific molecule alone.

The result of bispecific molecule–mediated growth inhibition of DMS273 cells by T cells from six healthy donors, at an E/T ratio of 2.5:1, is summarized in Fig. 4. An average of 63 ± 18% inhibition of thymidine uptake was observed in the presence of the bispecific molecule compared with an average of 28 ± 11% inhibition in the presence of the OKT3 alone and 7 ± 14% inhibition in the control condition (P < 0.001). The activity of T cells varied among individual donor. However, at a higher E/T ratio above 5:1, >80% growth inhibition was observed from all donors.

Analysis of SCLC cell apoptosis and necrosis during the cytotoxicity assay. The SCLC cells in dot plots were defined as CD45/FITC–negative cells (dark black dots). Apoptotic SCLC cells were defined as Annexin V positive and propidium iodide negative, whereas necrotic SCLC cells were defined as propidium iodide positive and Annexin V negative. As shown in Fig. 5A, the apoptotic H345 cells increased from 12% at 4 hours to 32% at 48 hours, and the necrotic H345 cells increased from 14% at 4 hours to 20% at 48 hours. As shown in Fig. 5B, the apoptotic DMS273 cells increased from 12% at 4 hours to 26% at 48 hours, and the necrotic DMS273 cells increased from 15% at 4 hours to 21% at 48 hours.

The summary of flow cytometry analysis using T cells from four different donors is presented in Fig. 6. After a 24-hour ADCC assay, there was a significant increase in both apoptosis and necrosis of SCLC cells in the presence of the bispecific molecule when compared with a control antibody. The results suggest that apoptosis might be an important mechanism in target cell death at a lower E/T ratio of ADCC. This observation is important in the clinical setting, wherein the E/T ratio is usually low at the actual tumor site.

Production of IFNγ by bispecific molecule–activated T cells. There was little increase of IFNγ production from T cells incubated with either OKT3 or bispecific molecule in the absence of target SCLC cells. There was an increased production of IFNγ by T cells incubated with OKT3 and SCLC cells. However, there was a much greater increase of IFNγ production by T cells incubated with the bispecific molecule and SCLC cells. The results of IFNγ production from T cells are summarized in Fig. 7.

Cleavage of caspase-3, caspase-9, and PARP. At baseline, both H345 and DMS273 cells expressed the full-length caspase-3 (35 kDa) or PARP (116 kDa) without any detectable level of cleaved caspase-3 (17-19 kDa) and PARP (89 kDa). Caspase-9 was not detected in either H345 or DMS273 cells at baseline. As Fig. 8 shows, after 48-hour ADCC, a cleaved PARP band (89 kDa) was detected in H345 cells in the presence of the bispecific molecule but not in the presence of a control antibody. In DMS273 cells, there was a significant increase in signal intensity of a cleaved PARP in the presence of the bispecific molecule compared with the control antibody. A cleaved caspase-3 at 17 kDa was detected in both H345 and DMS273 cells in the presence of the bispecific molecule. In DMS273 cells, there was no detectable full-length caspase-3 in the presence of bispecific molecule and very faint band of cleaved caspase-3 in the presence of a control antibody. In H345 cells, there was a very strong signal of cleaved caspase-3 in the presence of the bispecific molecule and a much weaker band of cleaved caspase-3 in the presence of a control antibody. The detection of both cleaved caspase-3 and PARP is the biochemical marker of cell apoptosis. There was no detectable cleaved caspase-9.

Targeted immunotherapy in vivo. All control mice developed visible tumors at days 30 and 40. Some mice in the control group and the OKT-armed T cell–treated group developed clinical signs of jaundice and weight loss by day 30. Of mice treated with the bispecific molecule–armed T cells, in the second experiment, there were seven of eight mice with no visible tumor at day 20 and five of eight mice with no visible tumor at day 30, whereas only one mouse had no tumor after 40 days in the first experiment. As shown in Table 1, in experiment 1, the gross tumor weights in the bispecific molecule–treated group showed a 58% reduction compared with the control group, whereas no decrease was observed in the OKT3-armed T cell–treated group. In experiment 2, mice that received three treatments with the bispecific molecule–armed T cells had a >90% tumor weight reduction on day 20 and 87% on day 30. There was a significant difference in both experiments when analyzing all three groups by one-way ANOVA analysis. The PETC in control mice increased from days 20 to 30 in the second experiment, whereas the PETC in mice treated with the bispecific molecule–armed T cells were significantly decreased and remained stable from days 20 to 30.

We further compared the groups treated with OKT-3-armed T cells versus bispecific molecule–armed T cells for differences in tumor weights and PETC by the Student's t test. Both tumor weight and PETC had significant differences in favor of the bispecific molecule–armed T cell–treated group in the two outcomes with Ps = 0.02 and 0.05 in the first experiment. For experiment 2, at day 20, only one of eight animals treated with bispecific molecule–armed T cells had visible tumor, whereas three of eight animals treated with OKT-3-armed T cells had visible tumors. The difference in the average weights of these groups was not significant (P = 0.1). However, the PETC at day 20 was significantly reduced in the bispecific molecule–treated group (P = 0.04). Comparing the tumor weights between the two treated groups on day 30, there was a significant difference (P = 0.02).

Treatment options for SCLC are limited. Although the majority of SCLC tumors are sensitive to both radiation and chemotherapy initially, they eventually become refractory. Targeted immunotherapy is an attractive approach that is being pursued actively in many clinical settings. Bombesin/GRP-R as a target for cancer treatment has been explored not only for SCLC but also for other tumors, including breast and prostate cancers. Growth inhibition of GRP-R-expressing tumors by potent bombesin/GRP antagonists; antibody against bombesin, drug, or radiolabeled bombesin/GRP analogues; and antisense oligodeoxynucleotide to GRP-R have been reviewed (14).

Antibodies and antibody conjugates against tumor surface antigens play an increasing role in targeted immunotherapy. In general, two mechanisms are responsible for antibody-mediated target cell destruction, including ADCC and complement-dependent cytotoxicity. Bispecific molecules or bispecific antibodies can activate and redirect CTLs to mediate tumor cell lysis. ADCC is the most important mechanism for T-cell cytotoxicity. In this study, we have shown that a bispecific molecule (OKT3xAntag2) can mediate the binding and killing of SCLC cells, both in vitro and in vivo, by activating allogeneic T cells through the CD3/TCR complex. The specific cytotoxicity does not require a costimulatory pathway but is, however, dependent on the activation of the T cells with IL-2.

T-cell activation in vivo is a more complex process, involving the recognition of the MHC and the presence of a costimulatory signal, such as CD28, antigen-presenting cells, and the activating cytokines, such as IL-2 (15). Whether bispecific molecules or bispecific antibodies are capable of activating T cells in vivo without additional costimulatory or MHC restriction is a subject of debate and research. There are numerous reports supporting the concept that bispecific antibodies can activate autologous T cells to kill the target cells, without costimulatory or prior stimulation (9, 10, 12, 16, 17). Davol et al. reported on a bispecific antibody (anti-CD3xanti-HER2/neu) given i.v. without a costimulatory factor and cytokine activation. Several patients with metastatic breast cancer and hormone-refractory prostate cancer had partial and minor response after eight infusions. Antitumor activity of the host immune cells was observed even after a month of the last infusion (9). Loffler et al. reported on a bispecific antibody (anti-CD19xanti-CD3) that effectively mediated the lysis of autologous B cells in the absence of a costimulatory signal and independent of IL-2. In their report, the anti-CD19xanti-CD3 mediated the depletion of autologous B cells in all four healthy donors and the depletion of autologous primary lymphoma cells in 22 of 25 patients (10). Marme et al. reported on a bispecific antibody (HEA125xOKT3) that inhibited malignant ascites production in advanced ovarian cancer. The bispecific antibody was given as weekly i.p. injections without cytokine activation of T cells or other costimulatory signals. A clinical response was observed in all 10 patients treated with the bispecific antibody, including eight complete inhibitions of malignant ascites production. The duration of response was relatively short, with a median time to progression of 10.5 weeks. A decrease of serum CA125 (a tumor mark for ovarian cancer) was observed in three patients (12). These data support that T-cell activation can be achieved by a bispecific antibody in vivo without a costimulatory signal.

We also explored the possible mechanisms of T cell–mediated cytotoxicity. Activated T cells can kill target cells through two major mechanisms: activation of death receptor (Fas and tumor necrosis factor–ralated apoptosis-inducing ligand) pathways and the secretion of perforin/granzymes–mediated pathway (18). Both mechanisms lead to the activation of caspases and subsequent cell apoptosis. In our study, the detection of cleaved caspase-3 and PARP is suggestive of SCLC apoptosis induced by the bispecific molecule and activated T cells. This activation of the caspase pathway partly contributes to target cell death. Caspase-3, an executioner caspase, can be activated by either an extrinsic (caspase-8) or an intrinsic (caspase-9) pathway. The extrinsic pathway signals through the Fas receptor on the target cell surface and the Fas ligand on the T-cell surface, resulting in the activation of caspase-8. The caspase-8, an initiator caspase, can cleave the downstream executioner caspases, such as caspase-3 (19). SCLC cells differ from non-SCLC cells in the mechanisms of apoptosis and response to chemotherapy (20). The overexpression of Bcl-2 can be detected in the majority of SCLC cells and has been associated with the resistance to chemotherapy (21, 22). The expression of caspase-8 and caspase-10 is frequently lost in SCLC cells (23, 24). Both H345 and DMS273 cells lack the expression of Fas receptor on their cell surface, as determined by flow cytometry and Western blot. Fas/FasL–mediated apoptosis pathway probably does not function in these two SCLC cell lines. Granzyme B produced by cytotoxic T cells can directly activate caspase-dependent pathways, including caspase-2, caspase-3, caspase-7, caspase-8, caspase-9, and caspase-10, and directly cleave a full-length Bid to a truncated Bid (25). A truncated Bid translocates to the mitochondria, recruits Bax and Bak, and results in the release of cytochrome c, Smac/Diablo, and apoptosis-inducing factor, leading to subsequent caspase activation and DNA fragmentation (26). Additional studies of granzyme production and the regulation of Bcl-2 family proteins during the bispecific molecule–mediated ADCC are warranted. PARP is an important nuclear enzyme involved in DNA repair, replication, and transcription. Cleavage of PARP by caspase-3 or caspase-7 deregulates the synthesis of ADP-ribose polymers in response to a damaged DNA (27). The detection of cleaved PARP is a biochemical hallmark of cell apoptosis.

We have previously constructed two bispecific molecules targeting GRP-R and CD64 on human immune effector cells, including monocytes, macrophages, and cytokine-activated neutrophils (14). Both bispecific molecule effectively mediated the lysis of targeted SCLC cells at a high E/T ratio. In this report, we have shown that the anti-CD3–based bispecific molecule activates T cells and mediates target cell destruction at a much lower E/T ratio. In our mouse SCLC xenograft model, the bispecific molecule–armed T cells significantly reduced SCLC tumor burdens. Repetitive infusions of ex vivo bispecific molecule–armed T cells at days 3, 7, and 14 eliminated tumor growth in five of eight mice at day 30. T cells and macrophages are the most common host immune effector cells present in tumor stroma (28, 29). The recruitment of these important immune effector cells with one or more bispecific molecule in combination may increase therapeutic efficacy in GRP-R-positive tumors.

We thank Dr. Ingo Schmidt-Wolf (Bonn, Germany) for critically reading the article and providing helpful suggestions.