Expression of c-erb-B2 oncoprotein as a neoantigen strategy to repurpose anti-neu antibody therapy in a model of melanoma

In this study, we tested a novel approach of “repurposing” a biomarker typically associated with breast cancer for use in melanoma. HER2/neu is a well characterized biomarker in breast cancer for which effective anti-HER2/neu therapies are readily available. We constructed a lentivirus encoding c-erb-B2 (the animal homolog to HER2/neu). This was used to transfect B16 melanoma in vitro for use in an orthotopic preclinical mouse model, which resulted in expression of c-erb-B2 as a neoantigen target for anti-c-erb-B2 monoclonal antibody (7.16.4). The c-erb-B2-expressing melanoma was designated B16/neu. 7.16.4 produced statistically significant in vivo anti-tumor responses against B16/neu. This effect was mediated by NK-cell antibody-dependent cell-mediated cytotoxicity. To further model human melanoma (which expresses <5% HER2/neu), our c-erb-B2 encoding lentivirus was used to inoculate naïve (wild-type) B16 tumors in vivo, resulting in successful c-erb-B2 expression. When combined with 7.16.4, anti-tumor responses were again demonstrated where approximately 40% of mice treated with c-erb-B2 lentivirus and 7.16.4 achieved complete clinical response and long-term survival. For the first time, we demonstrated a novel strategy to repurpose c-erb-B2 as a neoantigen target for melanoma. Our findings are particularly significant in the contemporary setting where newer anti-HER2/neu antibody-drug candidates have shown increased efficacy.


Introduction
2][3] However, up to 40 to 50% of these patients still die from melanoma or develop resistance to currently available immune checkpoint inhibitors. 4,5While there are other targeted agents available for melanomas with BRAF mutations, durability of their response is limited. 6Adverse events from immunotherapy or targeted therapies also limit their use, and there are populations of patients in whom immune checkpoint blockade is contraindicated, such as those with organ transplants. 7Therefore, novel therapies and approaches are needed to continue to improve outcomes for melanoma patients and overcome emerging mechanisms of resistance.
For melanoma and other cancers, there is much investigation into uncovering neoantigen biomarkers and an array of strategies to therapeutically target such neoantigens, including adoptive effector cell therapy and antigen-based vaccines. 8,9While the goal of these innovative approaches is to develop mainstream clinically effective treatments, this may not be the case for many early therapeutics or may take decades to become realized through the industrial pipeline.
While targeted agents speci c for melanoma are limited (namely BRAF and MEK inhibitors), there are other cancers that have different targeted drugs with long-term durability.For primary, locally advanced, and metastatic HER2/neu + breast cancer, as examples, patients may bene t from anti-HER2 monoclonal antibodies (i.e., trastuzumab and pertuzumab) and the newer antibody-drug conjugates (ADCs), including ado-trastuzumab (T-DM1 or emtansine) and trastuzumab deruxtecan (T-DXd). 10,11These more contemporary agents have been shown to be effective even when the HER2 expression is low because the chemotherapeutic component of the ADC is delivered to the tumor targets in order to generate their effect. 12 this preclinical study, the goal was to determine whether the animal homolog of HER2/neu (namely the c-erb-B2 oncoprotein) could be "repurposed" as a neoantigen target for melanoma.It has been shown that human melanoma overexpresses HER2/neu in less than 5% of cases, and thus anti-HER2 therapies are not currently approved or used for melanoma. 13However, the rationale of this study was that if c-erb-B2 could be expressed in melanoma tumors, then anti-c-erb-B2 monoclonal antibodies would generate effective anti-tumor responses through recognition of repurposed anti-c-erb-B2 as a neoantigen target.
Our hypothesis was that the use of a c-erb-B2-encoding lentivirus vector would be effective in expressing c-erb-B2 as a neoantigen for anti-c-erb-B2 monoclonal antibody and lead to effective anti-tumor responses in an orthotopic mouse model of melanoma.
Original Western blots with a positive neu control using the c-erb-B2 expressing mouse cell line MMC (derived from mammary cells of female BALB/c mice) are included in the supplementary materials (Supplementary Fig. 1). Figure 1C shows a cropped version of this Western blot, which highlights the cerb-B2 expression in our newly created c-erb-B2-expressing cell line compared to naïve B16.Similar to human melanoma, naïve B16 showed only approximately 1% endogenous c-erb-B2 (neu) expression on ow cytometry.This was signi cantly increased with pLenti6.3 c-erb-B2 transfection to over 95% surface expression of c-erb-B2.The c-erb-B2-expressing B16 was designated "B16/neu."We also con rmed via gene sequencing that the c-erb-B2 insert represented the oncogenic variant of neu, as opposed to the wildtype phenotype (Fig. 1E). 14,15Expression of the oncogenic c-erb-B2 variant was important for our model in order to better replicate human breast cancer, where oncogenic HER2 overexpression is the target of anti-HER2 therapies. 16ti-neu monoclonal antibody (7.16.4) has no effect on B16/neu in vitro.
Compared to naïve B16, the newly created B16/neu cell line showed increased growth and cell viability in vitro as measured by the Cyquant assay.This was observed throughout all time points up to 96 hours.When the mouse anti-rat c-erb-B2 monoclonal antibody 7.16.4 was added to the cell culture at increasing dose concentrations, there was no signi cant effect on B16/neu growth in vitro (Fig. 2).As expected, there was also no effect from 7.16.4 on naïve (non-c-erb-B2-expressing wild-type) B16.
Interestingly, the absence of effect of 7.16.4 on B16/neu was contrary to our group's previous investigation of 7.16.4 in c-erb-B2 + mouse breast cancer (using the neu + MMC cell line). 17However, naïve B16 does not normally express oncogenic c-erb-B2 (as shown in Fig. 1D), and its oncogenicity is driven by other melanoma-speci c mutations. 18,19Thus, neutralization of B16/neu growth by 7.16.4 in vitro was not expected.
To test our hypothesis that 7.16.4 could generate anti-tumor responses against B16/neu in vivo, B16/neu tumors were grown orthotopically within the dorsal skin of both male and female C57BL/6 mice.Although prior studies including our own have not demonstrated sex-based differences in B16 growth, 20,21 we accounted for possible sex-based differences in the setting of the newly transfected c-erb-B2, which is most often associated with breast cancer cells in female subjects.Equal numbers of male and female mice were used for each experiment.Experiments were completed in triplicate to assess for reproducibility, and data were pooled for analysis.
For tumor subcuticular inoculation, 1 x 10 5 B16/neu cells were injected in 0.1 ml of PBS with a 23-gauge needle.Treatments were initiated when tumors reached 5 mm 3 in their longest dimension (approximately 7-10 days post-inoculation).As controls, naïve B16 at the same tumor inoculation dosage was included.The antibody 7.16.4 was administered intraperitoneally (ip) at 400 µg in 0.2 ml of PBS every 3 days until mice reached the prede ned endpoints listed in the Methods.As an antibody control, isotype IgG2a was used at the same frequency and dosage ip (400 µg).Tumor measurements were taken every 5-6 days.
Figure 3 shows the tumor growth (A) and survival analysis (B) for the control and experimental groups.
As expected, there was no effect of 7.16.4 on naïve B16.There was slower tumor growth observed with the B16/neu controls (PBS or isotype) compared to naïve B16, but these differences were not statistically signi cant (p = 0.08 for PBS control, p = 0.09 for isotype control).When compared to isotype controls, B16/neu tumors treated with 7.16.4showed statistically signi cant decreased tumor growth (p = < 0.000001) and improved survival with 5/12 (41.2%) tumors showing complete tumor response and regression (p = < 0.0001).Mice that obtained complete tumor response, of which 2 were female and 3 were male, did not have tumor volumes that exceeded 10 mm 3 during the course of the experiment.This suggested that tumor growth kinetics beyond 10 mm 3 was su cient to overcome the anti-tumor effects of 7.16.4.Antibody therapy was stopped 60 days after tumor inoculation, and the mice with complete response showed long-term survival (over 90 days) and were then humanely euthanized.
Anti-neu 7.16.4responses are mediated by NK-cell antibody-dependent cell-mediated cytotoxicity.Whereas 7.16.4 did not demonstrate a neutralizing effect on B16/neu in vitro (as shown in Fig. 2), we hypothesized that the mechanism by which in vivo c-erb-B2 expression facilitated anti-tumor responses from 7.16.4(as shown in Fig. 3) was obtained by NK-cell antibody-dependent cell-mediated cytotoxicity (ADCC).To test our hypothesis, we included anti-NK cell monoclonal antibody (NK 1.1) at 200 µg injected every 3 days ip for C57BL/6 mice bearing B16/neu tumors.The NK 1.1 injections started 1 week before B16/neu inoculation to deplete NK cells prior to tumor growth and 7.16.4treatment.
The addition of NK 1.1 inhibited the effect of 7.16.4 on B16/neu tumors (Fig. 4).Mice treated with NK 1.1 and 7.16.4 or NK 1.1 alone (control) showed similar growth rates (Fig. 4A) and survival (Fig. 4B) to the negative (PBS) controls.Equal numbers of male and female mice were used, and experiments were completed in triplicate.Again, mice treated with 7.16.4only demonstrated superior statistically signi cant responses for both tumor growth (p = < 0.000001 compared to 7.16.4+ NK 1.1) and survival with 37.5% achieving complete clinical response and long-term survival (p = 0.0289).
Whole tumors were removed from mice treated with NK 1.1 +/-7.16.4 and from non-responders that were treated with 7.16.4.Resected tumors were sectioned and analyzed by IHC for NK cells.Mice treated with 7.16.4only (7.16.4 control) showed a qualitatively signi cant higher number of stained NK cells within the harvested B16/neu tumors (Fig. 4C) compared to mice treated with NK 1.1 +/-7.16.4 (Fig. 4D), which showed essentially no NK cells within the tumor parenchyma.This provided pathological con rmatory evidence that NK 1.1 effectively inhibited NK cell activity via NK cell depletion, and that the in vivo antitumor mechanism of action of 7.16.4 was NK-cell ADCC.IHC for NK cells was also performed on normal (non-tumor bearing) C57BL/6 splenic tissue as a control, showing expectedly high levels of NK cells within the spleen parenchyma (Fig. 4E).
In vivo transfection of naïve B16 melanoma tumors with c-erb-B2 lentivirus combined with 7.16.4generates anti-tumor responses.
The presented experiments have thus far utilized B16/neu that was developed in vitro using our c-erb-B2 lentivirus (pLenti6.3c-erb-B2).To better model human melanoma that does not normally express high levels of HER2/neu, we tested the in vivo injection of naïve B16 tumors with pLenti6.3 c-erb-B2.We hypothesized that in vivo transfection of naïve B16 tumors with pLenti6.3 c-erb-B2 would generate c-erb-B2 as a neoantigen target for 7.16.4 and allow for the repurposing of anti-c-erb-B2 (7.16.4) therapy.
To obtain the optimal dose of in vivo pLenti6.3c-erb-B2, we tested a linear set of viral doses from 6 x 10 5 pfu to 1 x 10 7 pfu.These doses were injected into naïve B16 tumors when they grew to a minimum of 5 mm 3 .Virus was injected every 3 days.Aliquots of the different viral dosages were delivered in 50 µl PBS.
At smaller tumor sizes (5-10 mm 3 ), 1-2 injections into the tumor were performed.As tumors grew beyond 10 mm 3 , 3-5 injections were performed to cover as much of the tumor volume as possible.Virus injections were performed for a total of 4 intratumoral inoculations (or 12 days).
In vivo surface expression of c-erb-B2 as a neoantigen target for 7.16.4 was quanti ed by ow cytometry after resecting and processing the tumors.c-erb-B2 expression using this approach was shown to be dose dependent, with the highest dose (1 x 10 7 pfu) yielding approximately 45% successful expression of c-erb-B2 in vivo.This was the selected dose of pLenti6.3 c-erb-B2 used in this set of experiments.As a virus control, normal or "blank" pLenti6.3 was used at the same intratumoral dose and schedule.
Virus and 7.16.4injections were given every 3 days until endpoints were met.Similar to our prior experiments, equal numbers of male and female mice were used, and experiments were completed in triplicate, again to meets standard for rigor and reproducibility.
Figures 5A and B show the tumor growth and survival data, respectively.Similar to our experiments with B16/neu (derived in vitro) and 7.16.4,the in vivo inoculation of naïve (non-c-erb-B2) B16 tumors generated c-erb-B2 as a neoantigen target for 7.16.4.Naïve B16 tumors treated with pLenti6.3 c-erb-B2 and 7.16.4showed statistically signi cant responses compared to isotype controls.These responses were demonstrated by decreased tumor growth (p = < 0.000001 ) and improved long-term survival (p = 0.0015).Similar to our experiments with B16/neu derived in vitro, our in vivo approach yielded a 41.2% complete clinical response (5/12 mice, 3 female and 2 male).These mice showed long-term survival to over 90 days.Again, tumors in these mice did not progress beyond 10 mm 3 during the treatment period.

Discussion
Herein, we demonstrated the feasibility and e cacy of repurposing c-erb-B2 as a neoantigen target via a c-erb-B2-encoding lentivirus vector.To our knowledge, this represents the rst preclinical approach to utilize c-erb-B2 as a neoantigen in melanoma.Importantly, there were no sex-based differences even with c-erb-B2 most commonly being associated with breast cancer in females.c-erb-B2 expression was successfully generated both in vitro and in vivo, and anti-c-erb-B2 antibody produced highly signi cant responses with > 40% complete clinical response against B16, a highly aggressive melanoma tumor cell line.Responses appeared to be mediated via NK-cell ADCC, which is a well-established mechanism of antibody-mediated tumor killing.
Our preclinical model holds promise for future clinical relevance, particularly given the success of targeted anti-HER2/neu treatments.Anti-HER2/neu monoclonal antibodies (trastuzumab and pertuzumab) are part of the standard of care for all stages of breast cancer, including rst line treatment for metastatic disease as well as for neoadjuvant treatment in resectable T2 primary tumors with or without lymph node metastases. 22,23Within the last decade, novel HER2-based antibody-drug conjugates (including ado-trastuzumab and trastuzumab deruxtecan) have been developed and shown to be even more effective than the monoclonal antibodies alone.][26] Importantly, the anti-HER2/neu ADCs have been shown to effective in low HER2-expressing cancers because the chemotherapeutic component of the ADCs exerts its anti-tumor effect after targeted delivery to the HER2 + tumors, even if the HER2 expression is low. 27Altogether, this results in the bystander effect that is characteristic of ADCs, but not of monoclonal antibodies. 28Our model aims to take advantage of these currently available therapies and repurposes them for cancers that do not express HER2/neu as a therapeutic biomarker, such as melanoma.If successful, our novel approach may establish anti-HER2/neu therapies for cancers that have limited effective systemic options and open up known effective treatments to a larger number of cancer patients.
While we have demonstrated the effectiveness of our approach in a preclinical animal model, additional adjustments would need to be considered when translated to human therapy.We utilized the oncogenic (as opposed to wild-type) variant of c-erb-B2, comprising the entire gene sequence.Applying a similar approach to human therapy may increase the oncogenicity or metastatic potential of human cancers, which would be a counter-productive consequence of our approach.Thus, our future investigations will determine whether attenuated c-erb-B2 expression could generate similar responses.0][31] However, utilization of just the extracellular domain may be su cient to repurpose anti-cerb-B2 targeted therapy.The extracellular domain has been shown in breast cancer models to generate immune responses without increasing tumorigenicity, which occurs through signal transduction mediated by the transmembrane and intracellular components. 32Therefore, one of our future investigations will determine whether a virus vector encoded with only the extracellular c-erb-B2 sequence can yield similar anti-tumor responses.Re ning our strategy in this manner may provide the most optimal combination of neoantigen recognition without increasing tumorigenicity, which will be critically important when translating our approach to human cancer studies.
Our chosen viral vector was the lentivirus pLenti6.3.While our recombinant plasmid pLenti6.3c-erb-B2 was able to generate the B16/neu cell line in vitro (Fig. 1) and produce a high level of transfection and anti-tumor effect in vivo in naïve B16 tumors (Fig. 5), it is important to note that this vector does not result in cell lysis. 33,34As a non-oncolytic plasmid, pLenti6.3 has the advantage of limiting c-erb-B2 expression within the injected tumor targets and thereby minimizes neoantigen spread to bystander tissues or to distant tissues should the plasmid enter the bloodstream during intratumoral inoculations.While there is an intrinsically subjective component to the technique of intratumoral injections or vaccinations (where precision may vary from provider to provider), use of pLenti6.3 likely restricts c-erb-B2 expression to the site of injection.
We recognize that our data are early and are derived from a preclinical animal model of primary melanoma.Much of the morbidity and mortality for human melanoma stems from locally advanced, intransit, and metastatic disease that are unresectable and unresponsive to current immunotherapies and targeted therapies. 35,36Whether our biomarker repurposing approach can be applied to regional or distant metastases was not addressed by these experiments.However, intratumoral injection of immunogenic viruses comprise a feasible, effective, and approved method for treating melanoma.Speci cally, Talimogene Laherparepvec (T-VEC) is a herpes simplex virus oncolytic therapy that selectively replicates within melanoma and produces granulocyte macrophage colony-stimulating factor (GM-CSF) to enhance systemic anti-tumor immune responses. 37,38T-VEC can be injected into cutaneous melanoma tumors, but it can also be used for regional and distant metastases that can be accessed via image-guided percutaneous injections. 39Thus, there is an established precedent for intratumoral virus injections for melanoma at both cutaneous (super cial) and metastatic (deep) anatomical locations.
We also recognize that our strategy did not produce complete clinical responses in all animal subjects.However, the complete response rate was still quite high (> 40%) and appeared to be associated with tumor growth kinetics.If tumor growth was able to plateau (at approximately 10 mm 3 ), then the success of our approach was signi cantly higher than if tumors grew more rapidly and outpaced any potential anti-tumor responses.This observation would represent another potential barrier of our approach to human clinical translation, where advanced disease tends to be larger or bulkier than small primary tumors.Nonetheless, our mouse model represents a promising starting point for our novel strategy and demonstrates proof of principle that c-erb-B2 can be repurposed as a neoantigen target for effective antic-erb-B2 therapies.
In conclusion, our approach to repurpose HER2/neu may be a timely, innovative strategy for melanoma and potentially other cancers that do not intrinsically overexpress oncogenic HER2/neu.Because of persistent limitations and emerging mechanisms of resistance to current therapies for melanoma, novel treatments are needed.In this study, the innovation of our approach is highlighted by the repurposing of contemporary effective therapies to malignancies for which they are not currently approved.Future preclinical studies are in development to determine whether our approach can be successful for regional or metastatic melanoma, larger primary tumors, and other cancers that do not normally express c-erb-B2.

Animals
Male and female C57BL/6 mice (6-8 weeks old) were purchased from the Jackson Laboratory (Bar Harbor, ME).Prior to use, mice were allowed to acclimate to the animal facility for 1 week.
Animal care and use were in accordance with institutional guidelines and approved under Mayo Clinic IACUC protocol A00005532-23: Expressing the oncoprotein neu on B16 melanoma as a therapeutic target for dynamic control of tumor vessels.
When animal experiments were completed, mice were humanely euthanized via a 30-70% per minute displacement of cage/chamber air with compressed CO2.This was con rmed with cervical dislocation per our IACUC approved protocol.

Reagents
FCS was obtained from Gemini Bioproducts (Woodland, CA).RPMI 1640, PBS, penicillin-streptomycin, Lglutamine, and β-mercaptoethanol were obtained from Life Technologies Inc. (Grand Island, NY).The monoclonal antibody 7.16.4,a mouse IgG2a antibody reactive with the rat neu oncogene-encoded p185 molecule, was obtained from Invitrogen (Thermo Fisher Scienti c, Carlsbad, CA) and has been previously described by our group. 17The IgG2a isotype control antibody was also obtained from Invitrogen.
Enhanced chemiluminescence reagents and enhanced chemiluminescence lm were obtained from Amersham International (Oakville, Ontario, Canada).
Construction of the B16 c-erb-B2 cell line (B16/neu) A cDNA PCR amplicon corresponding to the full length c-erb-B2 gene was inserted into pLenti6.3(Thermo Fisher Scienti c, Carlsbad, CA) by NEBuilder® HiFi seamless cloning (NEB, Ipswich, MA) in order to generate the plasmid pLenti6.3c-erb-B2.High quantities of our lentivirus were generated by transfection of 293FT cells with pLenti6.3 c-erb-B2 and ViraPower™ packaging mix at a ratio of 1:3 using Lipofectamine 3000 per the manufacturer's instructions (Thermo Fisher Scienti c, Carlsbad, CA).Filtered fresh virus was added to naïve (wild-type) B16 cells at a 1:5 ratio in the presence of 6 µg/ml polybrene for 24 hr, and then media was replaced.At 72 hr post infection, infected B16 cells were selected with 10 µg/ml blasticidin for 2 weeks to generate the stable B16 c-erb-B2 cell line, which was designated B16/neu.Quantitative PCR Two-step quantitative reverse transcriptase-mediated real-time PCR (qPCR) was used to measure the relative abundance of c-erb-B2 mRNA from equal amounts of cDNA (10 ng) of B16 and B16/neu using the TaqMan™ gene expression assay (Rn00566561_m1) and POLR2A (Mm00839502_m1) (Thermo Fisher Scienti c, Carlsbad, CA).Data were normalized to the endogenous control POLR2A, 40 and mRNA abundance was calculated using the ΔΔCT method. 40

Flow cytometry
For tumors, single cell suspensions were generated using a mouse tumor dissociation kit in combination with the gentleMACS™ Octo Desiccator (Miltenyi Biotec, Auburn, CA) per manufacturer protocol.Cell lines were harvested with a cell stripper dissociation reagent (Corning, Manassa, VA).Single cell solutions were washed twice in PBS and resuspended in 1X FACS buffer at 1 x 10 7 cells/ml.Prior to staining, the monoclonal antibody 7.16.4 and IgG2a isotype control were rst conjugated to DyLight™ 650 using the Lightning-Link antibody labeling kit (Novus Biologicals, Centennial, CO).Antibodies were added at 0.5 ug per 100 µl of cell sample and incubated for 30 min on ice, alongside a set of unstained samples.Samples were washed 4X with FACS buffer with centrifugations at 400G @ 5 min each.After the nal wash, samples were resuspended in 0.5 ml FACS buffer with the viability dye SYTOX Green (1:1000), except for selected controls.Fluorescence was detected with a Attune NxT Cytometer (Thermo Fisher Scienti c, Carlsbad, CA) and gated on viability.Plots and calculations were analyzed with the FCS Express software.

Cell proliferation in vitro
Cells were plated at 3000 cells per well in a total volume of 200 µl in quintuple per condition/time point, using black, clear bottom 96 well plates.At each time point, cell proliferation was measured using the CyQUANT direct cell proliferation assay (C3501) according to manufacturer instructions (Thermo Fisher Scienti c, Carlsbad, CA) and a SpectraMax M5 spectrophotometer (Ther Devices, San Jose, CA).For baseline (time = 0), cells were allowed to attach for 3 hr before measuring with the CyQuant assay.Background values of wells containing no cells ("no cell wells") was subtracted from all data prior to plotting values.

Assessment of in vivo
Three perpendicular axes of the tumors were measured approximately every 5-6 days using external digital calipers (Control Co.).Tumor volume was calculated using the formula ½ × length × width × height.Mice that died or were euthanized due to morbidity, tumor ulceration, or tumor reaching the size endpoint (2,000 mm 3 ) were classi ed as events.Measurements were performed by a lab member who was blinded to the treatment group.All in vivo experiments were performed in triplicate for replication.Group/treatment randomization was performed on the basis of cage position on the rack(s) within our vivarium.Cages were assigned a numerical designation.For each group, a cage was selected randomly from the pool of all cages.To establish blinding within each experiment involving tumor response in animals, tasks were completed by different members of the lab.These tasks included tumor cell preparation, tumor cell inoculation, treatment administration, and measurement of tumor response (tumor growth and survival).Following experiment completion, groups were unblinded in order to analyze the data.Naïve B16 and B16/neu were grown in vitro, and cell viability was assessed via the Cyquant assay.Overall, B16/neu showed higher cell viability at all time points compared to naïve B16.When the anti-cerb-B2 monoclonal antibody 7.16.4 was added in increasing doses to B16/neu, no statistically signi cant effects were observed on cell growth at any of the tested doses.
ImmunohistochemistryB16 tumor histologic sections were stained with standard hematoxylin and eosin and for NK cells [antimouse NK1.1 Monoclonal Antibody (PK136) -1:50 dilution, Invitrogen, ThermoFisher Scienti c].Stained sections were scanned with an Aperio Scanscope XT (Leica Microsystems Inc., Buffalo Grove, IL) and evaluated using the Aperio Spectrum software.Statistical Analysisdata.B.S. performed the statistical analysis.E.G. drafted the initial manuscript.Each author participated in the critical revision and nal approval of the manuscript.

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