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. Author manuscript; available in PMC: 2020 Dec 7.
Published in final edited form as: Mol Pharm. 2015 May 19;12(6):2151–2157. doi: 10.1021/acs.molpharmaceut.5b00132

Glypican-3 Targeted Human Heavy Chain Antibody as a Drug Carrier for Hepatocellular Carcinoma Therapy

Hirofumi Hanaoka †, Tadanobu Nagaya †, Kazuhide Sato †, Yuko Nakamura †, Rira Watanabe †, Toshiko Harada †, Wei Gao ‡, Mingqian Feng ‡, Yen Phung ‡, Insook Kim §, Chang H Paik ∥, Peter L Choyke †, Mitchell Ho ‡, Hisataka Kobayashi †,*
PMCID: PMC7720675  NIHMSID: NIHMS1648561  PMID: 25955255

Abstract

Glypican-3 (GPC3) represents an attractive target for hepatocellular carcinoma (HCC) therapy because it is highly expressed in HCC but not in adult normal tissue. Recently, high affinity anti-GPC3 antibodies have been developed; however, full antibodies may not penetrate evenly into tumor parenchyma, reducing their effectiveness. In this study, we compared a whole IgG antibody, anti-GPC3 YP7, with an anti-GPC3 human heavy chain antibody, HN3, with regard to their relative therapeutic effects. Both YP7 and HN3 bound to GPC3-positive A431/G1 cells and were internalized by the cells by in vitro evaluation with 125I- and 111In-radiolabeling antibodies. In vivo biodistribution and tumor accumulation was performed with 111In-labeled antibodies, and intratumoral microdistribution was evaluated using fluorescently labeled antibodies (IR700). HN3 showed similar high tumor accumulation but superior homogeneity within the tumor compared with YP7. Using the same IR700 conjugated antibodies photoimmunotherapy (PIT) was performed in vitro and in a tumor-bearing mouse model in vivo. PIT with IR700-HN3 and IR700-YP7 demonstrated that comparable results could be achieved despite of low reaccumulation 24 h after the first NIR light exposure. These results indicated that a heavy-chain antibody, HN3, showed more favorable characteristics than YP7, a conventional IgG, as a therapeutic antibody platform for designing molecularly targeted agents against HCC.

Keywords: heavy-chain antibody, intratumoral distribution, hepatoma, Glypican-3, photoimmunotherapy

Graphical Abstract

graphic file with name nihms-1648561-f0001.jpg

INTRODUCTION

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, especially in Asia, and is the third leading cause of cancer death. While surgical resection or liver transplantation are the only curative treatments for HCC, only 15% of patients are eligible for these treatments because HCC frequently grows as multiple foci.1,2 Percutaneous ethanol injection, radiofrequency ablation, and transarterial chemoembolization are minimally invasive techniques that have shown efficacy in reducing tumor volume; however, they have had only a modest impact on patient survival.2,3 Although HCC is highly resistant to conventional systemic therapies, new targeted drugs offer some hope for the future.4,5

Glypican-3 (GPC3) represents an attractive target for HCC therapy because it is highly expressed in HCC but not in normal tissue.6–9 The GPC3 core protein is a 70 kDa protein. GPC3 has been suggested as a target for antibody and cell-based immunotherapies.10,11 High affinity anti-GPC3 antibodies have been developed; however, because of their relatively large size, conventional IgG antibodies may not penetrate evenly into the tumor parenchyma, reducing their effectiveness. Thus, genetically engineered, relatively small heavy-chain antibodies (78 kDa, approximately half of the 150 kDa IgG size) may improve the intratumoral distribution of the conjugate.12 An anti-GPC3 human heavy-chain antibody, HN3 was developed as a new type of antibody format by Ho et al.13 HN3 is composed of CH2 and CH3 of the human Fc domain (hFc) and the VH domain, which bind cell surface-associated GPC3. It has demonstrated high and specific binding to GPC3 and thus has potential as a therapeutic antibody for HCC.

In addition to being a potential form of monotherapy, HN3 also has the possibility of becoming an antibody-conjugate. For instance, antibody-photosensitizer conjugates (APC) have been shown to be effective after they have been injected intravenously and then exposed to near-infrared light, a process known as “near-infrared photoimmunotherapy” (NIR-PIT). Typically, a NIR-PIT conjugate consists of an antibody bound to the photosensitizing phthalocyanine dye, IRDye700DX (IR700), and has been shown to be effective with a variety of different antibodies.14

In this study, we investigated HN3 as a candidate of therapeutic antibody-conjugates and compared it to a whole IgG anti-GPC3 antibody, YP7. Using a GPC3 expressing cell line, in vitro binding and internalization, in vivo biodistribution, tumor accumulation, and intratumoral microdistribution were evaluated. In addition, NIR-PIT was performed with IR700-HN3 and IR700-YP7 in vitro and in a tumor-bearing mouse model in vivo.

EXPERIMENTAL SECTION

Chemical Agents.

Anti-GPC3 mouse mAb YP7 (IgG) and HN3 (VH-hFc) were developed in house.13,15 A431/G1, a cell line stably expressing human GPC3, was established.15 IR700 NHS ester was obtained from LI-COR Bioscience (Lincoln, NE, USA). Other reagents were of reagent grade and were used as received.

Synthesis of IR700-Conjugated Antibodies.

The antibodies YP7 and HN3 were conjugated with dyes as previously described.14 YP7 (1.0 mg, 6.8 nmol) or HN3 (0.70 mg, 8.9 nmol) was incubated with IR700 NHS ester (60.2 µg, 30.8 nmol) in 0.1 mol/L Na2HPO4 (pH 8.5) at room temperature for 1 h. The mixture was purified with a Sephadex G25 column (PD-10; GE Healthcare, Piscataway, NJ, USA). The protein concentration was determined with the Coomassie Plus protein assay kit (Thermo Fisher Scientific Inc., Rockford, IL, USA) by measuring the absorption at 595 nm with spectroscopy (8453 Value System; Agilent Technologies, Santa Clara, CA, USA). The concentration of IR700 was determined by absorption at 689 nm to confirm the number of IR700 molecules conjugated to each antibody. The synthesis was controlled so that an average of three IR700 molecules were conjugated to a single antibody molecule.

Radiolabeling.

125I-YP7 and 125I-HN3 were prepared using the Iodo-Gen procedure. Briefly, 100 µg of YP7 or HN3 was added to each Iodogen coated vial with 0.5 M phosphate buffer, pH 7.2, and labeled with 37 MBq of 125I-NaI at room temperature. After 5 min, the 125I-labeled products were purified with a PD-10 column. The specific activity of the radiolabeled YP7 and HN3 was 218 and 148 MBq/mg, respectively. Quality control was performed with size exclusion-HPLC with TSK SWxl G3000 (TosoHaas, Philadelphia, PA, USA) and mobile phase 0.067 M PBS with 100 mM KCl.

For preparing 111In-labeled antibodies, 2-(4-isothiocyanato-benzyl)-diethylenetriaminepentaacetic acid (SCN-Bn-DTPA; Macrocyclics, Dallas, TX, USA) was conjugated to YP7 and HN3. Typically, SCN-Bn-DTPA in dimethylformamide was added to the mAb at 5–10 mg/mL in 0.1 mol/L Na2HPO4 (pH 8.5) at a molar ratio of 5:1. After incubation at 37 °C for 20 h, DTPA-mAbs were purified with a PD-10 column. Following this, 40 µL of 111InCl3 was incubated in 60 µL of 0.25 M acetate buffer (pH 5.5) for 5 min at room temperature followed by incubation with 10 µg of the DTPA-conjugated antibody for 1 h at room temperature. The labeled antibodies were purified with a PD-10 column. The specific activity of the radiolabeled YP7 and HN3 was 259 and 344 MBq/mg, respectively.

Binding and Internalization Assay.

A431/G1 cells (1 × 105) were placed into 6-well plates and then incubated with medium containing 125I-HN3 (2 kBq/0.1 µg) and 111In-DTPA-HN3 (2 kBq/0.01 µg) or containing 125I-YP7 (2 kBq/0.1 µg) and 111In-DTPA-YP7 (2 kBq/0.01 µg) overnight at 4 °C. After washing with PBS, the cells were incubated with antibody (Ab) free medium for 0, 1, 6, and 24 h at 37 °C or 24 h at 4 °C. After washing with PBS, the cells were lysed with 0.2 N NaOH and radioactivity of the cell fraction was measured with a gamma counter (Wizard 2480, PerkinElmer, Shelton, CT, USA). The ratio of binding of 111In-labeled antibody to 125I-labeled antibody (In/I ratio) at each time point was calculated. After internalization, radiometabolites of 111In-labeled antibodies stably stay in the cell, while radiometabolites of 125I-labeled antibodies are eliminated from the cell by dehalogenation immediately after being internalized into early endosome due to oxidizing enzymes.16 Therefore, bound 125I count to cells represents surface-bound amount of antibody; in contrast, bound 111In count to cells represents total-bound (surface-bound and internalized) amount of antibody. Thus, increased In/I ratio indicates internalization of an antibody. To detect the subcellular localization of IR700-HN3 and IR700-YP7, fluorescence microscopy was also performed (BX61; Olympus America, Melville, NY, USA). A431/G1 cells (1 × 104) were plated on cover-glass-bottomed dishes. Cells were incubated with IR700-HN3 (6.85 µg/mL) or IR700-YP7 (10 µg/mL) for 1 and 6 h at 37 °C. The filter was set to detect IR700 fluorescence with a 590–650 nm excitation filter since IR700 has subabsorption peak at 600–650 nm, and a 665–740 nm band-pass emission filter. Transmitted light differential interference contrast (DIC) images were also acquired before the fluorescence imaging.

Biodistribution Study.

All in vivo procedures were conducted in compliance with the Guide for the Care and Use of Laboratory Animal Resources (1996), US National Research Council, and approved by the local Animal Care and Use Committee. Six- to eight-week-old female homozygote athymic nude mice were purchased from Charles River (NCI-Frederick).

Two million A431/G1 cells were injected subcutaneously in the right dorsum of the mice. In order to determine tumor volume, the greatest longitudinal diameter (length) and the greatest transverse diameter (width) were determined with an external caliper. Tumor volume based on caliper measurements was calculated by the following formula: tumor volume = length × width2 × 0.5. Tumors reaching approximately 40 mm3 in volume were selected for the study.

111In-DTPA-HN3 or 111In-DTPA-YP7 (10 kBq/5.0 µg/200 µL in PBS/mouse) was injected via the tail vein of tumor-bearing mice, and the biodistribution was determined at 1, 6, 24, and 72 h after injection. Organs of interest were excised and weighed, and the radioactivity counts were determined with a gamma counter using the injected dose as a standard. The uptake data were calculated as the percentage injected dose per gram of tissue (%ID/g).

Fluorescence Microscopy Studies.

Tumor xenografts were excised from nude mice 24 h after injection of IR700-HN3 or IR700-YP7 and subsequently embedded in OCT compound. Frozen sections (50 µm thick) were prepared, and fluorescence was assessed using fluorescence microscopy. DIC images were also acquired.

In Vitro Photoimmunotherapy.

A red light-emitting diode (LED) light source, which emits light at 690 ± 20 nm wavelength (L690–66–60, Marubeni America Co., Santa Clara, CA, USA) was used for NIR light irradiation during NIR-PIT experiments. Power density was measured with an optical power meter (PM 100, Thorlabs, Newton, NJ, USA). Exposure of the LED light for 1 min was calculated to represent 2.5 J/cm2.17 A431/G1 cells (1 × 105) were placed into 24-well plates and incubated for 24 h at 37 °C. Cells were incubated with IR700-HN3 (6.85 µg/mL) or IR700-YP7 (10 µg/mL) for 1 h at 37 °C. After washing with PBS, PBS was replaced and cells were irradiated with NIR light at 0.1, 0.25, 0.5, 1, 2, and 4 J/cm2. Cells incubated with an APC but no NIR light exposure and cells receiving NIR light exposure at 4 J/cm2 without an APC were also prepared as controls. Plates were washed with PBS and cytotoxicity was determined using propidium iodide (PI) as a stain for dead cells. Fluorescence from cells was measured using a flow cytometer (FACS Calibur, BD BioSciences, San Jose, CA, USA) and CellQuest software (BD BioSciences). Binding of an APC to the cell without NIR light exposure was also determined by FACS.

In Vivo Therapeutic Studies.

A431/G1-tumor bearing mice were randomly assigned to one of 4 groups (8–9 mice per group). (1) No treatment (control); (2) 68.5 µg of IR700-HN3 i.v., no NIR light exposure (HN3 only); (3) 68.5 µg of IR700-HN3 i.v., NIR light exposure at 50 J/cm2 on day 1 after injection and 100 J/cm2 on day 2 and day 3 after injection (HN3 PIT); (4) 100 µg of IR700-YP7 i.v., NIR light exposure at 50 J/cm2 on day 1 after injection and 100 J/cm2 on day 2 and day 3 after injection (YP7 PIT). Mice were monitored daily and tumor volumes were measured three times a week until the tumor volume reached 2000 mm2, whereupon the mice were euthanized with carbon dioxide. Fluorescence images of IR700-YP7 or IR700-HN3 injected mice were obtained before and after NIR light irradiation. Mice were anesthetized with 2% isoflurane, and fluorescence imaging was obtained with a Pearl Imager (LI-COR Biosciences) using the 700 nm fluorescence channels for IR700.

Statistical Analysis.

Data are expressed as means ± SEM from a minimum of four experiments, unless otherwise indicated. Statistical analysis was performed using the unpaired t test for comparing differences between two groups and the one-way ANOVA followed by Tukey’s honestly significant difference (HSD) test for comparing differences between multiple groups. Differences were considered statistically significant when p values were less than 0.05.

RESULTS

Binding and Internalization Assay.

Radiolabeled YP7 and HN3 with both 125I and 111In demonstrated excellent binding to A431/G1 cells (Figure 1B,C). The ratio of binding of 111In-labeled antibody to 125I-labeled antibody (In/I ratio) at 37 °C changed little with time for YP7, whereas that for HN3 increased with time (Figure 1D). The stable In/I ratio for HN3 uptake did not increase at 4 °C, but did at 37 °C suggests that the internalization of HN3 depends on biological activity of A431/G1 cells. Furthermore, the internalization rate of HN3 was much faster than that of YP7. Fluorescence microscopy study showed that IR700-HN3 showed stronger intracellular dot-like signal that represented internalized APC fraction than IR700-YP7 at both 1 and 6 h postincubation (Figure 1E). Therefore, morphological internalization observed under fluorescence microscope is consistent with calculated internalization based on In/I ratio.

Figure 1.

Figure 1.

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In vitro binding and internalization assay with GPC-3 positive A431/G1 cells. (A) Schematic structures of a heavy chain antibody (e.g., HN3) compared with a whole IgG (e.g., YP7). Percentage binding of (B) 111In-DTPA-HN3 and 125I-HN3 or (C) 111In-DTPA-YP7 and 125I-YP7 after incubation overnight at 4 °C, then incubated with Ab-free medium for 0, 1, 6, and 24 h at 37 °C or 24 h at 4 °C. (D) The ratio of binding of 111In-labeled antibody to 125I-labeled antibody (In/I ratio). (E) Serial DIC (left row) and fluorescence microscopy (right row) images after incubation with IR700-HN3 or IR700-YP7 for 1 and 6 h. IR700-HN3 yields stronger dot-like fluorescent signal in the cytoplasm than IR700-YP7 at both 1 and 6 h post-incubation. Bar = 20 µm. All data reported as mean ± SEM (n = 5).

Biodistribution Studies.

Results of the in vivo biodistribution studies with 111In-DTPA-YP7 and 111In-DTPA-HN3 were expressed as a percentage of injected dose per gram (Figure 2). The initial blood clearance of 111In-DTPA-HN3 was significantly faster than that of 111In-DTPA-YP7, although radioactivity was retained in the body at 72 h after injection of 111In-DTPA-HN3. Compared with YP7, HN3 distributed to the kidney immediately after injection, whereas uptake of YP7 in the kidney was delayed. Although tumor accumulation of 111In-DTPA-HN3 was low at 6 h after injection, it became almost as high level as that of 111In-DTPA-YP7 at 24 and 72 h after injection.

Figure 2.

Figure 2.

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Biodistribution of (A) 111In-DTPA-HN3 and (B) 111In-DTPA-YP7 in tumor-bearing mice. Data were calculated as the percentage injected dose per gram of tissue and represented as the mean ± SEM (n = 4 or 5). Significant differences were observed compared to YP7 (*p < 0.05, #p < 0.01).

Fluorescence Microscopy Studies.

Ex vivo fluorescence imaging demonstrated high accumulation of IR700-YP7 and IR700-HN3 in the tumors at 24 h after injection (Figure 3). Both of the APCs demonstrated uptake within A431/G1 tumors. In general, IR700-HN3 distributed in tumors with better homogeneity than IR700-YP7 throughout the tumors as reported in other small antibody fragments12 indicating higher penetration of HN3 due to its smaller size.

Figure 3.

Figure 3.

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Serial DIC (upper row) and fluorescence microscopy (lower row) images of tumor xenografts 24 h after injection of (A) IR700-HN3 or (B) IR700-YP7. Representative DIC and fluorescence microscope images are shown. IR700-HN3 fluorescence is homogeneously shown throughout tumors; however, IR700-YP7 fluorescence is shown to be stronger in hypervascular area (H) than in hypovascular area (L).

In Vitro Near-Infrared Photoimmunotherapy.

IR700-HN3 bound to cells with approximately equivalent or slightly higher binding compared with IR700-YP7 (Figure 4A). IR700-HN3 by itself without NIR exhibited minimal cell death (Figure 4B) that might cause due to antigrowth activity of HN3,13 whereas cell death was not observed with IR700-YP7 by itself. The ratio of dead cells to live cells increased with increasing doses of NIR light (Figure 4B). These results indicated that NIR-PIT can be effective with IR700-HN3 and IR700-YP7.

Figure 4.

Figure 4.

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Flow cytometry analysis of (A) IR700-HN3 or IR700-YP7 binding to A431/G1 and (B) PI staining after irradiation with NIR light at 0.1, 0.25, 0.5, 1, 2, and 4 J/cm2 of A431/G1 cells. Data are represented as the mean ± SEM (n = 3 or 4). Significant differences in cell killing were observed compared to no treatment controls (*p < 0.05, #p < 0.01).

In Vivo Therapeutic Studies.

A431/G1 tumors were visualized by fluorescence 1 day after intravenous injection of IR700-YP7 or IR700-HN3 (Figure 5A). The fluorescence intensity in the tumor decreased to background level immediately after NIR light irradiation. Reaccumulation of IR700-YP7 in the tumor bed was clearly observed within a day, whereas that of IR700-HN3 was barely visible.

Figure 5.

Figure 5.

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Therapeutic effect of NIR-PIT. (A) A typical IR700 fluorescence image of APC injected tumor-bearing mice. Images were obtained before and immediately after day 1 NIR irradiation and before day 2 NIR irradiation. Yellow arrows depict the tumor. (B) Tumor growth inhibition by NIR-PIT in A431/G1 tumors. Data are reported as mean ± SEM (8 or 9 mice in each group). Tumor growth was significantly inhibited in NIR-PIT treated mice compared to untreated control mice (*p < 0.05).

The results of NIR-PIT are shown in Figure 5B. Tumor growth in treated mice was significantly inhibited by PIT with either IR700-HN3 or IR700-YP7 compared with that of untreated or no-NIR-light-exposure control group (p < 0.05).

DISCUSSION

The glypican, GPC3, is an emerging target for HCC therapy based on its high expression levels and high tumor to background ratios. Several GPC3 antibodies have been developed for HCC therapy. Recently a human heavy-chain antibody, HN3, was developed by Ho et al.13 HN3 can mediate antibody-dependent cell-mediated cytotoxicity (ADCC) activity since it possesses CH2 and CH3 regions of the human Fc domain. Thus, HN3 could constitute a potential therapeutic antibody for the treatment of HCC. In binding assays, 111In-DTPA-HN3 and IR700-HN3 bound to the GPC3-positive tumor cell line, A431/G1, with at least equal affinity as 111In- YP7 and IR700-YP7 indicating that HN3 exhibits an equal affinity for GPC3 as the full intact antibody, YP7, whose results agree with Kd value of them (YP7, 0.3 nM;15 HN3, 0.7 nM13). The lower immunoreactive fraction of 125I-HN3 compared with 125I-YP7 is probably due to tyrosine residues at critical positions for antigen binding on HN3 that would be subject to iodination with Iodogen. Therefore, modification of HN3, such as radiolabeling or drug or toxin conjugation, would be preferably performed by conjugating via amino groups of lysine on HN3.

Heavy-chain antibodies, by virtue of their smaller size, are expected to improve the intratumoral distribution compared to the full antibody.12 Ex vivo fluorescence imaging demonstrated that IR700-HN3 distributed in tumors with better homogeneity compared with IR700-YP7. Since most of antibody-based anticancer drugs are effective only when the antibody-conjugates bind to target cells, homogeneous distribution is a desirable property for therapeutic antibodies. Homogenous distribution of antibody would also be favorable for radioimmunotherapy since it would permit better dosimetry within the tumor.

Efficient internalization of the antibody is important for using molecularly targeted therapy such as antibody-drug conjugates (ADC) or antibody-toxin conjugates since drugs and toxins are most effective after internalization and following catabolism. As shown in Figure 1B–E, HN3 was internalized faster than YP7, suggesting that HN3 might be a superior platform for designing an ADC. Internalization of antibodies would also benefit radioimmunotherapy as there would be longer retention of therapeutic radiometals within cells.18,19 As opposed to many other antibody fragments, HN3 can still induce ADCC activity when bound on the cell surface as Fc is retained in its structure. Thus, HN3 has several desirable features that favor it over conventional IgG-based antibodies.

NIR-PIT with IR700-HN3 and IR700-YP7 led to comparable cell death in vitro and in vivo. Thus, IR700-HN3 is a potential NIR-PIT agent for HCC treatment. Our previous data demonstrates that PIT is more effective with repeated NIR light exposures, even with a single injection of the APC17 since circulating APC reaccumulates in the tumor after PIT. In this study, reaccumulation of IR700-YP7 was observed within 24 h after NIR-PIT, whereas reaccumulation of IR700-HN3 was minimal. The faster clearance of HN3 compared to YP7 in part explains this.20 However, IR700-HN3 might bind and kill a little larger number of target cells after the first exposure of NIR light than IR700-YP7 due to its homogeneous distribution, and thus, the low reaccumulation of IR700-HN3 might be caused in part by a reduced number of surviving target cells that are accessible to a low concentration of HN3 in the circulation after the first exposure of NIR light. Since the overall effectiveness of NIR-PIT with IR700-HN3 and IR700-YP7 was comparable, it is likely that the enhanced effects of IR700-HN3 after the first NIR exposure were compensated by the effects of the second and third NIR dose with IR700-YP7. Although renal accumulation of IR700-HN3 was higher than that of IR700-YP7, nephrotoxicity should be minimal because the kidneys are not exposed with the NIR light.

An obvious limitation of NIR-PIT for HCC therapy is the inability to apply NIR light externally. Because of pigments in the liver, NIR light penetration is minimal. Therefore, for NIR-PIT to be effective for HCC therapy, light probes would have to be placed within tumors using either catheters through the vessels or needles through the skin after the administration of IR700-HN3. This would be a procedure comparable to conventional interventional angiography or focal laser ablation of HCC with the difference that much lower energy of light would be required and that the penetration of NIR light would enable a larger treatment area with NIR-PIT than thermal injury due to laser ablation. To evaluate the efficacy of NIR-PIT using a needle-based light diffuser, it will be better to use an orthotopic liver tumor model.21,22 We might try this in a future study. In the case of surgery, it would be possible to expose NIR light during surgery. Combining NIR-PIT using IR700-HN3 with fluorescence-guided surgery could yield a superior therapeutic outcome that may be similar to the results of combination therapy of ultraviolet light irradiation with fluorescence-guided surgery.23

CONCLUSIONS

A human heavy-chain antibody targeting GPC3, HN3, demonstrated rapid internalization, rapid blood clearance resulting in high tumor-to-blood accumulation ratio, and improved homogeneity within the tumor compared with the conventional full IgG antibody, YP7. NIR-PIT with IR700-HN3 showed a therapeutic effect in tumor-bearing mice despite low reaccumulation of APC 24 h after the first NIR light exposure. These findings indicate that HN3 could be a promising platform for designing molecularly targeted agents against HCC.

ACKNOWLEDGMENTS

This research was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research. This project has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. K.S. is supported with JSPS Research Fellowship for Japanese Biomedical and Behavioral Researchers at NIH.

Footnotes

The authors declare no competing financial interest.

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