Therapeutic potential of FLT4-targeting peptide in acute myeloid leukemia

Therapeutic potential of FLT4-targeting peptide in acute myeloid leukemia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Therapeutic potential of FLT4-targeting peptide in acute myeloid leukemia Ji Yoon Lee, Soojin Park, A-Reum Han, Hee-Sun Hwang, Hee-Je Kim This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2041166/v2 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Feb, 2023 Read the published version in Cancer Immunology, Immunotherapy → Version 2 posted 9 You are reading this latest preprint version Show more versions Abstract Fms-related tyrosine kinase-4 (FLT4) is involved in tumor progression. Previously, we found that dysfunctional natural killer (NK) cells with low interferon-gamma (IFN-γ) were restored in acute myeloid leukemia (AML) by the FLT4 antagonist MAZ51. In this work, we developed 12 peptides targeting FLT4 for clinical application and ultimately selected 4 of them to examine whether they restored the frequency of lymphocytes, especially T cells and NK cells, and high IFN-γ expression, as MAZ51 treatment did in our previous study. Although clinical data from using intracellular kinase domain–targeting peptides are currently available, peptides targeting FLT4 to modulate immune cells have not been fully elucidated. In this study, we focus on novel peptide 4 (P4) from the intracellular domain of FLT4 because it had dominant negative activity. Similar to MAZ51, high IFN-γ levels were expressed in AML-mononuclear cells (MNCs) exposed to P4. In addition, T and NK cell levels were restored, as were high IFN-γ levels, in a leukemic environment when P4 was co-cultured with cytosine β-D-arabinofuranoside. Interestingly, the frequency of regulatory T cells was significantly decreased by P4, implying that the peptide plays a role in modulating the tumor niche. Overall, we demonstrated the therapeutic value of functionally modulating lymphocytes using a peptide targeting FLT4 and propose the development of advanced therapeutic approaches against AML by using immune cells. Fms-related tyrosine kinase-4 (FLT4) natural killer (NK) interferon-gamma (IFN-γ) acute myeloid leukemia (AML) mononuclear cells (MNCs) Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Acute myeloid leukemia (AML) is defined as an increase in myeloid lineage cells with aberrant genetic changes that result in abnormal hematopoietic and immune procedures [ 1 ]. To cure it, numerous trials of immune checkpoint inhibitors, small molecules, therapeutic antibodies, blocking peptides, and chimeric antigen receptor-T cells have been conducted [ 2 , 3 , 4 ]. Because most small molecules have a narrow therapeutic window and cause serious toxicity to normal tissues, therapeutic peptides, which have high specificity and good solubility, are regarded as promising alternatives for clinical application in treating both leukemia and solid tumors. Peptide-based combinational therapy is emerging as an important strategy for treating cancer [ 4 , 5 ]. Whereas large proteins, including monoclonal antibodies, have some drawbacks such as difficulty being delivered into the tumor and non-specific uptake into the reticuloendothelial system, peptides have many advantages, including their small size, easy synthesis, and access to the tumor [ 5 , 6 ]. The biggest benefit of peptides is that they can be applied directly in clinical trials using diverse combination regimens [ 7 ]. Many peptide therapeutics, including various Fc-fusion peptides, mutated peptides, and extracellular domain–targeting peptides, have been reported to have pros and cons in their pathophysiologic function and offer advanced opportunities to fine tune their activity spectrum [ 8 , 9 , 10 ]. Because synthetic peptides are small, with a 12/18-mer structure, they can penetrate cells and function as bioactive peptides that target internalized signaling pathways, making them highly efficient in regulating molecular movements. Veli-Matti L et al. reported the crystal structure of VEGF-C/Fms-related tyrosine kinase-4 (FLT4) and provided mechanistic insights about its binding and activation [ 11 ]. Using that crystal structure information, we selected several domains as targeted binding sites: the VEGF-C binding sites into the FLT4 extracellular domain (D1-2) and FLT4 intracellular domain (D4-5). To inhibit FLT4 kinase activity, its ability to bind to its ligand should be inhibited through the extracellular domain or the homodimer structure of the intracellular kinase domain. Inhibiting FLT4 activity by blocking its phosphorylation increased the expression of IFN-γ in immune cells, suggesting that FLT4 inhibition offers the potential benefit of cancer regression in tumor microenvironments [ 12 ]. Despite the biological importance of the intracellular kinase domain in immune cell activation, studies about the signal domains for targeting peptides are still sparse compared with studies about the extracellular domains for targeting peptides [ 13 , 14 ]. FLT4 (also known as VEGFR-3) is a representative mediator of lymphangiogenesis via its ligands VEGF-C and –D [ 15 ]. The VEGF-C/FLT4 axis is a gatekeeper signaling pathway for starting lymphangiogenesis in both hematological malignancies and solid tumors [ 16 , 17 ]. Receptor tyrosine kinases (RTKs) were the first kinases to be considered in cancer research, and FLT4, which is an RTK with the VEGF family, is a popular standard target for treating leukemia. Because the VEGF-C/FLT4 axis is involved in the high rate of growth and survival of leukemia cells, as well as with dysfunctional natural killer (NK) cells,[ 18 , 19 ] our group previously focused on the role of the FLT4 inhibitor MAZ51 in restoring NK cell functioning, and we demonstrated the therapeutic effects of blocking FLT4 by showing that it increased IFN-γ in NK cells in AML [ 20 , 21 , 22 ]. In this work, we designed novel peptides targeting FLT4 by means of an intracellular kinase domain–mediated effect that could replicate the effect of MAZ51 in previous studies, and we confirmed that it inhibits each downstream event, suggesting its clinical applicability. Therefore, this study demonstrates that our FLT4-targeting peptide synergistically restored dysfunctional AML-NK cells when it was administered together with cytosine β-D-arabinofuranoside (ara-C), and it shows that the therapeutic potential of the peptide involves the activation of immune cells suppressed by AML. Materials And Methods Peptides The FLT4 peptide ligand used in this experiment inhibits FLT4 protein activity. Its sequence is listed in Table I, and the sequences of the other tested peptides are listed in Supplementary table I. The peptides were designed by randomly cutting peptides with sizes not exceeding 18-mers based on sequences in Thr446 and Lys516 of the D5 intracellular region that were bound by strong disulfide bonds in the FLT4 region (Peptron, Daejeon, Korea). In the experiments, 20 mg/kg of the peptides were used in vivo and 25 µM were used in vitro . The FLT4 peptide was dissolved in distilled water (D.W.). NK cells expanded ex vivo with P4 were intravenously injected, and additional P4 was intraperitoneally injected into tumor-bearing mice. Human Samples All experiments were conducted with the approval of the Catholic Human Ethics Review Committee (KC19TESI0462). Bone marrow (BM) and PB samples were collected from 23 patients with primary AML according to the AML subtype classification designated by the World Health Organization. BM- and PB-mononuclear cells (MNCs) were fractionated by density gradient centrifugation using Ficoll-Paque® Plus (GE Healthcare Life Sciences, Piscataway, NJ, USA, 17-1440-03). The clinical characteristics and experimental information for the AML patients enrolled in this study are listed in Table 2 . The collected BM and PB MNCs (1 x 10 6 cells) were grown in a humidified incubator containing 5% CO 2 at 37°C and cultured in RPMI 1640 medium (Gibco) supplemented with 10% FBS (Gibco) for use in the peptide (Peptron, 25 µM stock in D.W.), IL-2 (Peprotech, 212-12-20UG, 500 U/ml), and IL-15 (Peprotech, 210-15-50UG, 50 ng/ml) treatments. Table 1 Sequence of the peptides selected by binding to FLT4 Petide ID Peptide Sequence P4, 440 RQALTCTAYGVP P6, 440 mutation RQATAYGVPLPLS P11, 121 mutation KSIDNEWRKGCMPREVA P12, 121 mutation KSIDNEWRKLGCMPREVA Table 2 Clinical characteristics and experimental information for AML patients Patients Cell source Age at diagnosis Sex WBS count [10^9/L] cytogenetic anomalies 1 BM 50 F 116.6 46, XX[ 20 ] 2 BM 20 M 190.2 46, XY[ 20 ] 3 BM 33 M 2.78 46, XY, t(6;9) (p23;q34) [ 18 ] /46, xy[ 2 ] 4 BM 71 M 2.33 46, XY[ 20 ] 5 BM, PB 62 M 61.47 46, XY[ 20 ] 6 PB 41 M 208.55 46, XY, t(9;22) (q34;q11,2) [ 2 ] /53, idem, + 3, +8, + 10, +10, + 15, +19, +der(22)t9;22)[ 18 ] 7 PB 84 M 345.38 45,X, -Y[ 20 ] 8 PB 59 M 10.84 46,XY, inv(16) (p13,1q22) [ 20 ] 9 PB 52 F 406.24 46, XX, t(9;22) (q34;q11,2) [ 18 ] /47, idem, + 19[ 2 ] 10 PB 47 M 335.41 46, XY, inv(9) (p12q13) [ 20 ] Flow Cytometry And Mscs Sorting The collected BM MNCs and PB MNCs were incubated together in 100 µl of PBS. The following human antibodies were used: APC-labeled mouse anti-human CD56 (555518; BD Biosciences), FITC-labeled mouse anti-human CD3 (555339; BD Biosciences), and PE-labeled anti-human FLT4 (356204; BioLegend, San Diego, CA, USA), along with APC mouse IgG1, κ isotype control (555751; BD Biosciences), FITC mouse IgG2a, κ isotype control (555573; BD Biosciences), PE mouse IgG1, κ isotype control (FC; 400114; BioLegend), and PE mouse IgG1, κ isotype control (559320; BD Biosciences). The collection and analysis of intracellular IFN-γ expression were performed as described above. In the in vivo model, PB, spleen, and liver cells were obtained from the AML mice on day 18 post-injection with C1498 cells. BM cells were flushed from the mouse femurs, suspended in 100 µl of Hanks' Balanced Salt Solution (Welgene, Gyeongsangbuk-do, South Korea) and 1% FBS (Gibco), and incubated with the antibodies. After being washed, the cells were collected using a FACSCanto II flow cytometer (BD Biosciences), and the data were analyzed using FlowJo software (v10; Tree Star Inc., Ashland, OR, USA). PE-labeled mouse anti-mouse NK-1.1 (553165; BD Biosciences), perCP-conjugated mouse CD8 (ThermoFisher, 45-0081-82), APC anti-mouse CD4 antibody (100412; BioLegend) was used. For Treg cell analysis, FITC anti-mouse CD4 antibody (553729, BD Biosciences) APC rat anti-mouse CD25 (558643; BD Biosciences), FITC anti-mouse TCR γ/δ antibody (118106; BioLegend), biotin-labeled FLT4 monoclonal antibody (AFL4; 13-5988-82; Invitrogen, Carlsbad, CA, USA), APC-labeled IFN-γ monoclonal antibody (XMG1.2; 17-7311-82; Invitrogen), FOXP3 monoclonal antibody (FJK-16s; PE, eBioscience™; 12-5773-82; Invitrogen), PE mouse IgG2a, κ isotype control (553457; BD Biosciences), rat IgG2a κ isotype control (eBR2a), biotin (13-4321-82; Invitrogen), rat IgG1 κ isotype control (eBRG1), and APC (17-4301-82; Invitrogen) were used. To measure IFN-γ expression in the cells, we used phorbol myristate acetate (PMA; P1585; 50 ng/mL; Sigma), calcium ionophore (A23187, 500 ng/mL; Sigma), and Protein Transport Inhibitor (554724; GolgiStop™; 2 µM; BD Biosciences) to stimulate the cells for 5 hours. Then, the cells were washed with cold fixation and permeabilization solution (554722; BD Biosciences). The biotin signals were detected using the streptavidin-APC conjugate (17-4317-82; Invitrogen) as the secondary antibody. The flow cytometry data were analyzed using the appropriate isotype and unstained controls. For NK isolation, isolation of NK cells was performed by magnetic-activated cell sorting (MACS) using the NK MicroBead Kit (130-092-657, Miltenyi Biotec) Statistical analysis All data are presented as the mean ± standard error (SE). The means were used to compare the data. The results were analyzed by the Kruskal-Wallis test. GraphPad Prism ver. 7 software was used for the analyses. P-values of < 0.05 were considered to indicate statistical significance. Results Peptides targeting FLT4 increased IFN-γ expressions on AML-NK cells in BM/PB We designed functional peptides based on previous reports [ 11 ] on the crystal structure and mechanistic insights of VEGF-C/FLT4 for peptide binding and activation. Based on this, 4 peptides were selected to further examine their function in immune cells. P4 and P6 are targeting intracellular signal domain, while peptide 11 and 12 are linked to binding site of FLT4 as a competitor against VEGF-C (Fig. 1 a). Detailed loci for these peptides were depicted in Fig. 1 b. To screen proper peptides in increasing the expression of IFN-γ on mononuclear cells (MNCs) in AML patients, 12 peptides were designed and tested their effects in MNCs with dose-dependent manners. The transcription level of IFN-γ was increased by P4, P6, and P11 in a dose-dependent manner, compared to that of other type of peptides (Fig. 1 c). Consistently, the expression of IFN-γ in protein level showed a concentration–dependent increase in P4, P6, P11, and P12 ( Fig. S1 ). In protein level, IFN-γ level was increased in CD3 − CD56 + cytokine released NK cells, compared to CD3 − CD16 + cytolytic NK cells, regardless of peptide types. It also was found that the 50uM concentration of peptide was better to be as inducer for IFN-γ expression than 25uM concentration (Fig. 1 d). Next, to investigate the role of IFN-γ in NK cells, NK cells were isolated by MACS kit for NK cells. At least purity of 96.5% should be maintained to further study after sorting NK cells. As shown in Fig. 1 e, the expression of FLT4 in MNCs was highly increased in AML patients, compared to that of normal donors. To acquire accurate data, ELISpot assay for IFN-γ was performed using isolated NK cells from leukapheresis. PMA (2.5ng/ml) and calcium ionophore (25ng/ml) group displayed that P4, P11, and P12 showed significantly increase in IFN-γ spot forming cells, compared to that of DMSO group (Fig. 1 f). A representative image for results was provided in Fig. 1 f right panel . From present data, we found that designed 4 peptides (including 4, 6, 11, and 12) may similar or superior efficacy with FLT4 antagonist, MAZ51 in increasing the expression of IFN-γ in vitro treatment. Effects Of Peptides On Increasing The Frequency Of Aml-nk Cells And -t Cells Next, we examined that peptides have a role to support cell survival and to increase the frequency of immune cells such as NK and T cells. As adjuvant materials, IL-2 (500U/ml) and IL-15 (50ng/ml) were treated with peptides and were used in culture of AML-BM/PB cells. Data showed that BM-NK cells were dramatically increased in only P4 at 21 days after culture (13.8 ± 4.4%), and P12 from 18 days (in P12, 11.3 ± 3.8%). However, PB-NK cells were significantly increased by culturing all peptides at day 21 (in P4, 10.9 ± 3.7%; in P6, 9.1 ± 2.9%; in P11, 6.7 ± 2.3%; in P12, 9.0 ± 2.9%)(Fig. 2 a), suggesting the relevance of immune cells releasing IFN-γ by peptide 4 in PB and BM cells. In CD3 T cells, peptide 4 only showed significant increase in cell frequency from 12 days after culture both in IL-2/IL-15 treated group of BM, compared to non-treated group (in BM, at day 18, 49.9 ± 9.7%, at day 21, 49.4 ± 13.5%) Meanwhile, CD3 T of PB cells were increased in peptide 4 treated group with significant difference at day 21 only (50.0 ± 6.5%)(Fig. 2 b ) . Other peptides were detected with no significant difference in cells. Since AML-MNCs exhibit a rapid apoptosis when cultured in in vitro due to defects in the dynamic niche, cells spontaneously result in necrosis without proliferation by cytokine [ 23 , 24 ]. Despite of this property, PB-, BM-NK and PB-T cells were dramatically expanded by peptide 4 under exposure of cytokines such as IL-2 and IL-15 at day 21. It suggests the role of peptide in proliferating immune cells by interfering with FLT4 kinase activity in AML [ 21 , 25 ]. Both in NK and T cell viability, no significant differences in all peptides treated groups were measured for 21 days (Fig. 2 a and data not shown). The Increase Of Nk And T Cells Using Peptides In Aml Mice To examine whether peptide has an effect to increase the in vivo immune network, all P4, P6, P11, and P12 were intraperitonealy (i.p.) injected for 5 days into C57BL/6J mouse with induction of C1498 cells. At 1 week post-injection, tissues including PB, BM, and spleen from sacrificed tumor-bearing mice were collected and subjected to FACS analysis. The experimental design was presented in Fig. 3 a. FACS data from spleen showed that peptide 4 only significantly increased the frequency of IFN-γ from lymphocytes such as CD4 T, CD8 T, and NK cells at day 7, compared to that of leukemic mice. Similarly, PB-NK cells were also peaked their frequency with significance difference, compared to that of leukemic group only when peptide 4 was treated (Fig. 3 b). In case of peptide 6, the frequency of NK and CD4 T cells in spleen has increased patterns at day 7, compared to that of leukemic mice group. Due to consistency of the results by P4, we ultimately decided to use further experiments in vivo to investigate the restoration of lymphocytes using the combination of P4 as an effector and ara-C for high stimulation of immune cells. The Effects Of Peptide With Conventional Chemotherapy On The Expression Of Ifn-γ From Leukemic Mice Next, to address effects of peptide with conventional chemotherapeutic agents, a syngeneic leukemic mouse model was developed using C1498 leukemic cells and ex vivo cultured NK cells with or without peptide were used. Due to the importance of functional NK production with high IFN-γ in AML, ex vivo expanded NK cells with functionality and proliferation is regarded as an essential cell source in anti-tumor therapeutic strategies [ 21 , 26 , 27 ] Thus, we applied the novel P4 in ex vivo expanded NK cells for 3 days and then NK cells were injected into tumor bearing mice with exogenous P4 and ara-C together. It was used for in vivo experiments based on the protocol shown in Fig. 4 a. The tissues including PB, BM, and spleen from each group were collected and subjected to FACS analysis. Results in PB and BM showed that IFN-γ expression was increased in NK cells in the ara-C treatment and the NK cells injected group co-cultured with the ex vivo peptide (Fig. 4 b-c). Also, the frequency of CD3 including CD4/CD8 in PB was high peaked in peptide only treated group and NK cells combinational treated group ( Fig. S2 ). The γδ CD3 T cells in PB were significantly increased in peptide only group and NK cells without peptide stimulation injected group ( Fig. S2, PB ). However, the γδ CD8 T cells in BM were dramatically decreased in peptide and ara-C combinational treated group and the NK cells injected group co-cultured with the ex vivo peptide ( Fig. S2, BM ). In the case of the spleen, where lymphocyte activity is most expected, the frequency of lymphocytes was rather reduced by peptide treatment. These data provided that activation and proliferation of immune cells by peptide treatment can be altered depends on tissues types of AML. Although the diversity exists, we interestingly found that CD4 + CD25 + Foxp3 + Treg cells in PB, BM and spleen were dramatically decreased in all peptides and ara-C treated groups, compared to that of leukemic group (Fig. 4 b-d). It implied the role for P4 functioning in increasing IFN-γ, is known as helping factor for cytolysis, from the activation of CD3 and NK cells along with regression of Treg cells in tumor microenvironments. Discussion Our previous studies [ 20 , 21 ] provided evidence that blocking FLT4 enhances IFN-γ expression in dysfunctional NK cells by showing that MAZ51 treatment provided functional restoration. Based on that evidence, we developed novel peptides that can bind soluble FLT4 and replace MAZ51, and we found that P4 effectively increased IFN-γ by inhibiting FLT4 activation. Thus, these Fc targeting peptides found in tumor microenvironments are emerging as therapeutic candidates for treating tumors directly or indirectly via access to immune cells [ 28 ]. Chang et al. [ 29 ] confirmed the antagonistic action of several peptides as an antibody concept that can suppress FLT4 activity by binding soluble FLT4, and they showed the therapeutic potential of peptides by inhibiting FLT4 kinase activity in tumor progression. However, therapeutic suggestions for a peptide that inhibits FLT4 activity in the dysfunctional immune cells in tumor microenvironments are still lacking, and this field remains to be developed. In 2013, the Kari A group assessed the crystal structure of human FLT4 and revealed that homotypic interactions in D5 of the Fc domain of FLT4 (Thr444, Thr446, Glu426, and Lys516) are essential for FLT4 activation [ 11 ]. Because the signaling domain of FLT4 is susceptible to disruption by homodimers, FLT4 activation can be hampered without antibodies by continuing interactions with other molecules such as peptides that target the wild-type intracellular domain of FLT4 [ 30 ]. Therefore, we carefully assumed that a peptide with a dominant negative action that binds to the wild-typed subunit could make an inactive oligomer that inhibits signal activation [ 31 ]. Another possibility in activating both NK cells and T cells in vivo to restore their IFN-γ expression is that the peptide binding with FLT4 is present in all cells, such as macrophages, dendritic cells, and even tumor cells, and thus accelerates their defensive activity, leading to more efficient antigen processing and presentation to CD4 and CD8 cells, which in turn confers anti-tumor effects [ 32 , 33 ]. Aberrant expression of FLT4 on immune cells leads to dysfunctions that can decrease abnormal signaling by blocking the FLT4 signal cascade using the instability of the homodimer structure, which ultimately results in functional immune cell restoration. That process is similar to the action of antibodies. Although potent monoclonal antibodies are significantly compromised in their ability to confer pathogen protection in in vitro assays, they can produce robust immunity in vivo . Thus, pathologic protection depends on the acting FcγR engagement and disparity between immunity in vitro and in vivo .[ 34 ] In the case of tumor-associated antigens, i.e., FLT4 in NK cells, a strong signal cascade can suppress the immune response by blocking high IFN-γ expression [ 20 , 21 , 22 ]. We finally showed that blocking the signal domain of aberrant FLT4 with P4 effectively increased the IFN-γ expressed by immune cells, including NK cells. Unlike MAZ51, P4 was restricted to FLT4 signaling, with no effects on other VEGFRs [ 35 ]. IFN-γ also has strong, well-known anti-lymphatic and anti-angiogenic effects, consistently causing vessel regression both in murine and human tumors, and is considered a primary anti-cancer cytokine and lytic helping factor [ 33 , 36 ]. Despite the unraveling evidence for P4 in the in vivo cascade for immune cell stimulation, increased IFN-γ in CD4 and CD8 T cells was detected, along with a low frequency of CD4 + CD25 + Foxp3 + Treg cells, in this study (Fig. 4 b-d). Consistent with H Nishikawa’s data, the ex vivo cultured NK cells in this study also functioned as effectors to increase IFN-γ expression and induce immunity in tumor microenvironments [ 36 , 37 ]. In the future, we will investigate the anti-tumor effects of P6, P11, and P12 and compare them with that of P4 in leukemic mice. Although our in vivo study was incomplete because we did not test P6, P11, and P12, our present results (and our previous reports) show that the peptides we designed to target FLT4 can restore low-functioning CD56 + CD3 − AML-NK cells, which express high levels of the lymphatic marker FLT4. Additionally, they reveal high IFN-γ expression in the T cells and NK cells of leukemic mice treated with P4. Our experiments show that both AML-NK cells and AML leukemic cells have increased expression of the lymphatic marker FLT-4, suggesting the relevance of AML-NK cell activation via the anti-lymphatic cascade. Taken together, these results suggest that the intracellular domain of a peptide targeting FLT4 can be used in combination with conventional chemotherapy to increase IFN-γ expression in NK cells and T cells. Furthermore, these results provide clues about advanced therapeutic approaches that could be used to correct the tumor microenvironment. Declarations Author Contributions JY Lee, S Park, AR Han, and HS Hwang performed the experiments and analyzed the data. JY Lee and HJ Kim wrote the manuscripts. Funding This work was financially supported by a grant from the National Research Foundation (NRF, 2018R1D1A1A09083557), Ministry of Education, Republic of Korea. Ethics approval and consent to participate All experiments were performed with authorization from the Institutional Review Board for Human Research at the Catholic University of Korea (KC19TESI0462). The patients/participants provided their written informed consent to participate in this study. All protocols for testing the animals were approved by the Catholic University of Korea's Institutional Animal Care and Use Committee (CUMC-2019-0135-01) Conflict of Interest The authors declare no conflicts of interest. References Chen SJ, Shen Y, Chen Z. A panoramic view of acute myeloid leukemia. Nat Genet. 2013;45(6):586-587. Majeti R, Chao MP, Alizadeh AA et al. CD47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells. Cell. 2009;138(2):286-299. Kampen KR, Scherpen FJG, Mahmud H et al. VEGFC Antibody Therapy Drives Differentiation of AML. Cancer Res. 2018;78(20):5940-5948. Augsberger C, Hanel G, Xu W et al. 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Myeloid cell-based delivery of IFN-gamma reprograms the leukemia microenvironment and induces anti-tumoral immune responses. EMBO Mol Med. 2021;13(10):e13598. Horwitz JA, Bar-On Y, Lu CL et al. Non-neutralizing Antibodies Alter the Course of HIV-1 Infection In Vivo. Cell. 2017;170(4):637-648 e610. Kirkin V, Thiele W, Baumann P et al. MAZ51, an indolinone that inhibits endothelial cell and tumor cell growth in vitro, suppresses tumor growth in vivo. Int J Cancer. 2004;112(6):986-993. Nishikawa H, Kato T, Tawara I et al. IFN-gamma controls the generation/activation of CD4+ CD25+ regulatory T cells in antitumor immune response. J Immunol. 2005;175(7):4433-4440. Cluff E, Magdaleno CC, Fernandez E et al. Hypoxia-inducible factor-1 alpha expression is induced by IL-2 via the PI3K/mTOR pathway in hypoxic NK cells and supports effector functions in NKL cells and ex vivo expanded NK cells. Cancer. Immunol Immunother 2022. Additional Declarations No competing interests reported. Supplementary Files SupplementarymethodsfigureandTableCII20220916.docx Cite Share Download PDF Status: Published Journal Publication published 10 Feb, 2023 Read the published version in Cancer Immunology, Immunotherapy → Version 2 posted Editorial decision: Major revision 25 Dec, 2022 Reviews received at journal 20 Dec, 2022 Reviews received at journal 28 Sep, 2022 Reviewers agreed at journal 23 Sep, 2022 Reviewers agreed at journal 21 Sep, 2022 Reviewers invited by journal 20 Sep, 2022 Editor assigned by journal 17 Sep, 2022 Submission checks completed at journal 17 Sep, 2022 First submitted to journal 16 Sep, 2022 You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2041166","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[{"code":1,"date":"2022-09-12 14:47:26","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":141244862,"identity":"f5f6f52c-c6eb-404b-b1f3-4e7e5de8af9e","order_by":0,"name":"Ji Yoon Lee","email":"","orcid":"","institution":"CHA University","correspondingAuthor":false,"prefix":"","firstName":"Ji","middleName":"Yoon","lastName":"Lee","suffix":""},{"id":141244863,"identity":"29aed832-79cb-40ff-a4bd-7bbb1e5021b5","order_by":1,"name":"Soojin Park","email":"","orcid":"","institution":"Catholic University of Korea","correspondingAuthor":false,"prefix":"","firstName":"Soojin","middleName":"","lastName":"Park","suffix":""},{"id":141244864,"identity":"0967958b-2e2a-499d-b07a-2acd278074f1","order_by":2,"name":"A-Reum Han","email":"","orcid":"","institution":"CHA Bundang Medical Center","correspondingAuthor":false,"prefix":"","firstName":"A-Reum","middleName":"","lastName":"Han","suffix":""},{"id":141244865,"identity":"24c4b99c-c710-42dd-9da3-8e7824db0c45","order_by":3,"name":"Hee-Sun Hwang","email":"","orcid":"","institution":"Catholic University of Korea","correspondingAuthor":false,"prefix":"","firstName":"Hee-Sun","middleName":"","lastName":"Hwang","suffix":""},{"id":141244866,"identity":"f837bf19-4d47-410b-93cd-f8ed84c94639","order_by":4,"name":"Hee-Je Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIie3RIQvCQBTA8XcIWoSrbwjuK7whiB9nY6BBB0aTLGkRVs9vMRksGRTBJFiFlQ1hSUG7wRMVw9iczXD/cvDgx7vjAFSqP425BE15VgGG+JqUIC2AiiRUmgBY7pN8Jrlx0beuYhj2vOl+G1+oo0NtE7P5Mp/gwQk0nyJH7OyaIQgNt94l5qf5hNAJWSyJD3a1USc0AQbA4tV30iN+fBF+KkHkxUzC9xaUW/wCgrtzoAmKDHE4trXHWyaY0loUED51FtfZLdK5Z6V4GY11zu0kmRWQbPJ34CegUqlUqmx3hlJMy3qbGoEAAAAASUVORK5CYII=","orcid":"","institution":"Catholic University of Korea","correspondingAuthor":true,"prefix":"","firstName":"Hee-Je","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2022-09-07 10:14:25","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-2041166/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-2041166/v2","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00262-023-03385-8","type":"published","date":"2023-02-10T18:43:07+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":27336430,"identity":"b50baecf-0d3d-4895-bcd5-90fd864f2516","added_by":"auto","created_at":"2022-10-04 17:58:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1223865,"visible":true,"origin":"","legend":"\u003cp\u003eCrystal structure of the VEGF-C/FLT4 D1-2 complex and the homodimer of FLT4 D4-5. \u003cstrong\u003ea\u003c/strong\u003e The red in the cartoon depicts the designed peptide locations: the ligand binding domain for peptides 11 and 12 and the intracellular signal domain for peptides 4 and 6. \u003cstrong\u003eb\u003c/strong\u003e Detailed depiction of the peptides. T446 in the kinase domain is linked in peptide 4, and the mutation peptide 6 is from peptide 4. The K121 location is the ligand domain binding into the FLT4 region. \u003cstrong\u003ec\u003c/strong\u003e AML-MNCs were isolated and subjected to qRT-PCR. The expression level of IFN-γ was significantly increased by the peptide treatment. Genes were normalized to GAPDH. \u003cstrong\u003ed\u003c/strong\u003e FACS data showed that Cytolytic NK cells and cytokine-releasing NK cells were divided using CD56 and CD16 markers. Overall, cytokine-producing CD56\u003csup\u003e+\u003c/sup\u003e NK cells gradually increased in the peptide-treated group. \u003cstrong\u003ee\u003c/strong\u003e The expression of FLT4 was higher in AML-MNCs than in healthy donors. To perform further experiments, purity of more than 96% should be confirmed after NK cell sorting. \u003cstrong\u003ef\u003c/strong\u003e The ELISpot assay showed a high level of IFN-γ in AML-NK cells, suggesting the relevance of the peptides to IFN-γ expression. The bars represent the mean ± SE, and the asterisks depict statistically significant differences. Data shown represent the means of duplicate, independent experiments (** P \u0026lt;0.01, * P \u0026lt;0.05 vs. DMSO injected group, n=10).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2041166/v2/691d76f0b3b4e1e2bbe97937.png"},{"id":27336432,"identity":"fd0e133b-8f4f-4631-a6a7-ab67e7d27d19","added_by":"auto","created_at":"2022-10-04 17:58:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":426943,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of peptides in increasing the frequency of AML-NK cells and T cells during \u003cem\u003ein vitro\u003c/em\u003ecell proliferation. \u003cstrong\u003ea\u003c/strong\u003e Under IL-2 and IL-15 exposure, peptides 4, 6, 11, and 12 helped maintain BM and PB cell viability. On day 21, all peptides showed high frequency in proliferating PB-NK cells, with significant differences. Data from at least three independent experiments per person are shown. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 (n = 10). \u003cstrong\u003eb\u003c/strong\u003eUnlike the data for PB-NK cells, the frequency of CD3 T cells increased only in the P4-treated group, compared with that with the other peptides. Data from at least three independent experiments per person are shown. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 (n = 10).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2041166/v2/f43464836b53c081e69b71fa.png"},{"id":27337017,"identity":"56dc8aa8-b5de-46ce-8f7f-6951cc6b7fee","added_by":"auto","created_at":"2022-10-04 18:03:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":903363,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of the peptides in increasing the IFN-γ of NK and T cells in a tumor-bearing environment. \u003cstrong\u003ea\u003c/strong\u003e Schematic diagram of the \u003cem\u003ein vivo \u003c/em\u003estudy to examine the effects of the peptides in the tumor microenvironment \u003cem\u003ein vivo\u003c/em\u003e. \u003cstrong\u003eb\u003c/strong\u003eMost immune cells displayed no difference, except NK cells with P4, compared with that with other peptides. In the P4-treated group, NK cells and CD3 T cells reached their peak frequency on day 7 in the spleen. The data shown represent the means of independent experiments. Asterisks indicate statistically significant differences compared with the normal donor or leukemic control groups. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 (n = 5).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2041166/v2/f2f30ba4a9fd2782f61f3b3a.png"},{"id":27336433,"identity":"5542ffca-4802-4038-9c03-3d7b5df9171a","added_by":"auto","created_at":"2022-10-04 17:58:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":703240,"visible":true,"origin":"","legend":"\u003cp\u003eThe therapeutic potential of P4 and its accompanying high frequency of NK and T cells and low number of Treg cells in leukemic mice. \u003cstrong\u003ea\u003c/strong\u003e Schematic diagram of the \u003cem\u003ein vivo\u003c/em\u003eexperiment using ara-C and the peptide together. \u003cstrong\u003eb\u003c/strong\u003e Expressions of lymphoid lineage cells (NK, γδ T cells, IFN-γ in NK cells, and Treg cells) are shown in the PB of leukemic mice (n=4). \u003cstrong\u003ec\u003c/strong\u003e The frequency of NK, γδ T cells, IFN-γ in NK cells, and Treg cells are shown in the BM of leukemic mice (n=4). \u003cstrong\u003ed\u003c/strong\u003e The frequency of NK, γδ T cells, IFN-γ in NK cells, and Treg cells are shown in the spleens of leukemic mice (n=4). b\u003cstrong\u003e-d\u003c/strong\u003e The data shown represent the means of independent experiments. Asterisks indicate statistically significant differences compared with the normal donor or leukemic groups. **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05. The numbers for each group are as follows: 1, C1498-injected group; 2, ara-C group; 3, peptide-only group; 4, ara-C and peptide combination group; 5, \u003cem\u003eEx vivo\u003c/em\u003e non-cultured NK, ara-C, and peptide group; 6, \u003cem\u003eEx vivo\u003c/em\u003e cultured NK, ara-C, and peptide treated group.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2041166/v2/24a0481a1dbb0217267186ff.png"},{"id":44718843,"identity":"37fe086c-1e4c-4e82-b58b-a4e4266121fd","added_by":"auto","created_at":"2023-10-16 18:51:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1371219,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2041166/v2/a5cdaeb0-3442-4949-ae72-4f2de169cb04.pdf"},{"id":27336431,"identity":"b3ad5f6c-a8fe-4a0e-91f4-031f959ff7c7","added_by":"auto","created_at":"2022-10-04 17:58:12","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":712110,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarymethodsfigureandTableCII20220916.docx","url":"https://assets-eu.researchsquare.com/files/rs-2041166/v2/163779b1043f48d1ee44f308.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Therapeutic potential of FLT4-targeting peptide in acute myeloid leukemia","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAcute myeloid leukemia (AML) is defined as an increase in myeloid lineage cells with aberrant genetic changes that result in abnormal hematopoietic and immune procedures [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. To cure it, numerous trials of immune checkpoint inhibitors, small molecules, therapeutic antibodies, blocking peptides, and chimeric antigen receptor-T cells have been conducted [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Because most small molecules have a narrow therapeutic window and cause serious toxicity to normal tissues, therapeutic peptides, which have high specificity and good solubility, are regarded as promising alternatives for clinical application in treating both leukemia and solid tumors.\u003c/p\u003e \u003cp\u003ePeptide-based combinational therapy is emerging as an important strategy for treating cancer [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Whereas large proteins, including monoclonal antibodies, have some drawbacks such as difficulty being delivered into the tumor and non-specific uptake into the reticuloendothelial system, peptides have many advantages, including their small size, easy synthesis, and access to the tumor [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The biggest benefit of peptides is that they can be applied directly in clinical trials using diverse combination regimens [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Many peptide therapeutics, including various Fc-fusion peptides, mutated peptides, and extracellular domain\u0026ndash;targeting peptides, have been reported to have pros and cons in their pathophysiologic function and offer advanced opportunities to fine tune their activity spectrum [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Because synthetic peptides are small, with a 12/18-mer structure, they can penetrate cells and function as bioactive peptides that target internalized signaling pathways, making them highly efficient in regulating molecular movements. Veli-Matti L et al. reported the crystal structure of VEGF-C/Fms-related tyrosine kinase-4 (FLT4) and provided mechanistic insights about its binding and activation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Using that crystal structure information, we selected several domains as targeted binding sites: the VEGF-C binding sites into the FLT4 extracellular domain (D1-2) and FLT4 intracellular domain (D4-5). To inhibit FLT4 kinase activity, its ability to bind to its ligand should be inhibited through the extracellular domain or the homodimer structure of the intracellular kinase domain. Inhibiting FLT4 activity by blocking its phosphorylation increased the expression of IFN-γ in immune cells, suggesting that FLT4 inhibition offers the potential benefit of cancer regression in tumor microenvironments [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Despite the biological importance of the intracellular kinase domain in immune cell activation, studies about the signal domains for targeting peptides are still sparse compared with studies about the extracellular domains for targeting peptides [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFLT4 (also known as VEGFR-3) is a representative mediator of lymphangiogenesis via its ligands VEGF-C and \u0026ndash;D [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The VEGF-C/FLT4 axis is a gatekeeper signaling pathway for starting lymphangiogenesis in both hematological malignancies and solid tumors [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Receptor tyrosine kinases (RTKs) were the first kinases to be considered in cancer research, and FLT4, which is an RTK with the VEGF family, is a popular standard target for treating leukemia. Because the VEGF-C/FLT4 axis is involved in the high rate of growth and survival of leukemia cells, as well as with dysfunctional natural killer (NK) cells,[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] our group previously focused on the role of the FLT4 inhibitor MAZ51 in restoring NK cell functioning, and we demonstrated the therapeutic effects of blocking FLT4 by showing that it increased IFN-γ in NK cells in AML [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this work, we designed novel peptides targeting FLT4 by means of an intracellular kinase domain\u0026ndash;mediated effect that could replicate the effect of MAZ51 in previous studies, and we confirmed that it inhibits each downstream event, suggesting its clinical applicability.\u003c/p\u003e \u003cp\u003eTherefore, this study demonstrates that our FLT4-targeting peptide synergistically restored dysfunctional AML-NK cells when it was administered together with cytosine β-D-arabinofuranoside (ara-C), and it shows that the therapeutic potential of the peptide involves the activation of immune cells suppressed by AML.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePeptides\u003c/h2\u003e \u003cp\u003eThe FLT4 peptide ligand used in this experiment inhibits FLT4 protein activity. Its sequence is listed in Table I, and the sequences of the other tested peptides are listed in Supplementary table I. The peptides were designed by randomly cutting peptides with sizes not exceeding 18-mers based on sequences in Thr446 and Lys516 of the D5 intracellular region that were bound by strong disulfide bonds in the FLT4 region (Peptron, Daejeon, Korea). In the experiments, 20 mg/kg of the peptides were used \u003cem\u003ein vivo\u003c/em\u003e and 25 \u0026micro;M were used \u003cem\u003ein vitro\u003c/em\u003e. The FLT4 peptide was dissolved in distilled water (D.W.). NK cells expanded \u003cem\u003eex vivo\u003c/em\u003e with P4 were intravenously injected, and additional P4 was intraperitoneally injected into tumor-bearing mice.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHuman Samples\u003c/h3\u003e\n\u003cp\u003e All experiments were conducted with the approval of the Catholic Human Ethics Review Committee (KC19TESI0462). Bone marrow (BM) and PB samples were collected from 23 patients with primary AML according to the AML subtype classification designated by the World Health Organization. BM- and PB-mononuclear cells (MNCs) were fractionated by density gradient centrifugation using Ficoll-Paque\u0026reg; Plus (GE Healthcare Life Sciences, Piscataway, NJ, USA, 17-1440-03). The clinical characteristics and experimental information for the AML patients enrolled in this study are listed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The collected BM and PB MNCs (1 x 10\u003csup\u003e6\u003c/sup\u003e cells) were grown in a humidified incubator containing 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C and cultured in RPMI 1640 medium (Gibco) supplemented with 10% FBS (Gibco) for use in the peptide (Peptron, 25 \u0026micro;M stock in D.W.), IL-2 (Peprotech, 212-12-20UG, 500 U/ml), and IL-15 (Peprotech, 210-15-50UG, 50 ng/ml) treatments.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSequence of the peptides selected by binding to FLT4\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePetide ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePeptide Sequence\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP4, 440\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRQALTCTAYGVP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6, 440 mutation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRQATAYGVPLPLS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP11, 121 mutation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKSIDNEWRKGCMPREVA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP12, 121 mutation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKSIDNEWRKLGCMPREVA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eClinical characteristics and experimental information for AML patients\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatients\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell source\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAge at diagnosis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWBS count\u003c/p\u003e \u003cp\u003e[10^9/L]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ecytogenetic anomalies\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e116.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e46, XX[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e190.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e46, XY[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e46, XY, t(6;9) (p23;q34) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] /46, xy[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e46, XY[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBM, PB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e61.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e46, XY[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e208.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e46, XY, t(9;22) (q34;q11,2) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] /53, idem, +\u0026thinsp;3, +8, +\u0026thinsp;10, +10, +\u0026thinsp;15, +19, +der(22)t9;22)[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e345.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e45,X, -Y[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e46,XY, inv(16) (p13,1q22) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e406.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e46, XX, t(9;22) (q34;q11,2) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] /47, idem, +\u0026thinsp;19[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e335.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e46, XY, inv(9) (p12q13) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eFlow Cytometry And Mscs Sorting\u003c/h3\u003e\n\u003cp\u003eThe collected BM MNCs and PB MNCs were incubated together in 100 \u0026micro;l of PBS. The following human antibodies were used: APC-labeled mouse anti-human CD56 (555518; BD Biosciences), FITC-labeled mouse anti-human CD3 (555339; BD Biosciences), and PE-labeled anti-human FLT4 (356204; BioLegend, San Diego, CA, USA), along with APC mouse IgG1, κ isotype control (555751; BD Biosciences), FITC mouse IgG2a, κ isotype control (555573; BD Biosciences), PE mouse IgG1, κ isotype control (FC; 400114; BioLegend), and PE mouse IgG1, κ isotype control (559320; BD Biosciences). The collection and analysis of intracellular IFN-γ expression were performed as described above. In the \u003cem\u003ein vivo\u003c/em\u003e model, PB, spleen, and liver cells were obtained from the AML mice on day 18 post-injection with C1498 cells. BM cells were flushed from the mouse femurs, suspended in 100 \u0026micro;l of Hanks' Balanced Salt Solution (Welgene, Gyeongsangbuk-do, South Korea) and 1% FBS (Gibco), and incubated with the antibodies. After being washed, the cells were collected using a FACSCanto II flow cytometer (BD Biosciences), and the data were analyzed using FlowJo software (v10; Tree Star Inc., Ashland, OR, USA). PE-labeled mouse anti-mouse NK-1.1 (553165; BD Biosciences), perCP-conjugated mouse CD8 (ThermoFisher, 45-0081-82), APC anti-mouse CD4 antibody (100412; BioLegend) was used. For Treg cell analysis, FITC anti-mouse CD4 antibody (553729, BD Biosciences) APC rat anti-mouse CD25 (558643; BD Biosciences), FITC anti-mouse TCR γ/δ antibody (118106; BioLegend), biotin-labeled FLT4 monoclonal antibody (AFL4; 13-5988-82; Invitrogen, Carlsbad, CA, USA), APC-labeled IFN-γ monoclonal antibody (XMG1.2; 17-7311-82; Invitrogen), FOXP3 monoclonal antibody (FJK-16s; PE, eBioscience\u0026trade;; 12-5773-82; Invitrogen), PE mouse IgG2a, κ isotype control (553457; BD Biosciences), rat IgG2a κ isotype control (eBR2a), biotin (13-4321-82; Invitrogen), rat IgG1 κ isotype control (eBRG1), and APC (17-4301-82; Invitrogen) were used. To measure IFN-γ expression in the cells, we used phorbol myristate acetate (PMA; P1585; 50 ng/mL; Sigma), calcium ionophore (A23187, 500 ng/mL; Sigma), and Protein Transport Inhibitor (554724; GolgiStop\u0026trade;; 2 \u0026micro;M; BD Biosciences) to stimulate the cells for 5 hours. Then, the cells were washed with cold fixation and permeabilization solution (554722; BD Biosciences). The biotin signals were detected using the streptavidin-APC conjugate (17-4317-82; Invitrogen) as the secondary antibody. The flow cytometry data were analyzed using the appropriate isotype and unstained controls. For NK isolation, isolation of NK cells was performed by magnetic-activated cell sorting (MACS) using the NK MicroBead Kit (130-092-657, Miltenyi Biotec)\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE). The means were used to compare the data. The results were analyzed by the Kruskal-Wallis test. GraphPad Prism ver. 7 software was used for the analyses. P-values of \u0026lt;\u0026thinsp;0.05 were considered to indicate statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePeptides targeting FLT4 increased IFN-γ expressions on AML-NK cells in BM/PB\u003c/h2\u003e \u003cp\u003eWe designed functional peptides based on previous reports [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] on the crystal structure and mechanistic insights of VEGF-C/FLT4 for peptide binding and activation. Based on this, 4 peptides were selected to further examine their function in immune cells. P4 and P6 are targeting intracellular signal domain, while peptide 11 and 12 are linked to binding site of FLT4 as a competitor against VEGF-C (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Detailed loci for these peptides were depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb. To screen proper peptides in increasing the expression of IFN-γ on mononuclear cells (MNCs) in AML patients, 12 peptides were designed and tested their effects in MNCs with dose-dependent manners. The transcription level of IFN-γ was increased by P4, P6, and P11 in a dose-dependent manner, compared to that of other type of peptides (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Consistently, the expression of IFN-γ in protein level showed a concentration\u0026ndash;dependent increase in P4, P6, P11, and P12 (\u003cb\u003eFig. S1\u003c/b\u003e). In protein level, IFN-γ level was increased in CD3\u003csup\u003e\u0026minus;\u003c/sup\u003eCD56\u003csup\u003e+\u003c/sup\u003e cytokine released NK cells, compared to CD3\u003csup\u003e\u0026minus;\u003c/sup\u003eCD16\u003csup\u003e+\u003c/sup\u003e cytolytic NK cells, regardless of peptide types. It also was found that the 50uM concentration of peptide was better to be as inducer for IFN-γ expression than 25uM concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Next, to investigate the role of IFN-γ in NK cells, NK cells were isolated by MACS kit for NK cells. At least purity of 96.5% should be maintained to further study after sorting NK cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, the expression of FLT4 in MNCs was highly increased in AML patients, compared to that of normal donors. To acquire accurate data, ELISpot assay for IFN-γ was performed using isolated NK cells from leukapheresis. PMA (2.5ng/ml) and calcium ionophore (25ng/ml) group displayed that P4, P11, and P12 showed significantly increase in IFN-γ spot forming cells, compared to that of DMSO group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). A representative image for results was provided in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef \u003cb\u003eright panel\u003c/b\u003e. From present data, we found that designed 4 peptides (including 4, 6, 11, and 12) may similar or superior efficacy with FLT4 antagonist, MAZ51 in increasing the expression of IFN-γ in vitro treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEffects Of Peptides On Increasing The Frequency Of Aml-nk Cells And -t Cells\u003c/h3\u003e\n\u003cp\u003eNext, we examined that peptides have a role to support cell survival and to increase the frequency of immune cells such as NK and T cells. As adjuvant materials, IL-2 (500U/ml) and IL-15 (50ng/ml) were treated with peptides and were used in culture of AML-BM/PB cells. Data showed that BM-NK cells were dramatically increased in only P4 at 21 days after culture (13.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.4%), and P12 from 18 days (in P12, 11.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8%). However, PB-NK cells were significantly increased by culturing all peptides at day 21 (in P4, 10.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7%; in P6, 9.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9%; in P11, 6.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3%; in P12, 9.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9%)(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), suggesting the relevance of immune cells releasing IFN-γ by peptide 4 in PB and BM cells. In CD3 T cells, peptide 4 only showed significant increase in cell frequency from 12 days after culture both in IL-2/IL-15 treated group of BM, compared to non-treated group (in BM, at day 18, 49.9\u0026thinsp;\u0026plusmn;\u0026thinsp;9.7%, at day 21, 49.4\u0026thinsp;\u0026plusmn;\u0026thinsp;13.5%) Meanwhile, CD3 T of PB cells were increased in peptide 4 treated group with significant difference at day 21 only (50.0\u0026thinsp;\u0026plusmn;\u0026thinsp;6.5%)(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. Other peptides were detected with no significant difference in cells. Since AML-MNCs exhibit a rapid apoptosis when cultured in \u003cem\u003ein vitro\u003c/em\u003e due to defects in the dynamic niche, cells spontaneously result in necrosis without proliferation by cytokine [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Despite of this property, PB-, BM-NK and PB-T cells were dramatically expanded by peptide 4 under exposure of cytokines such as IL-2 and IL-15 at day 21. It suggests the role of peptide in proliferating immune cells by interfering with FLT4 kinase activity in AML [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Both in NK and T cell viability, no significant differences in all peptides treated groups were measured for 21 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and data not shown).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eThe Increase Of Nk And T Cells Using Peptides In Aml Mice\u003c/h3\u003e\n\u003cp\u003eTo examine whether peptide has an effect to increase the in vivo immune network, all P4, P6, P11, and P12 were intraperitonealy (i.p.) injected for 5 days into C57BL/6J mouse with induction of C1498 cells. At 1 week post-injection, tissues including PB, BM, and spleen from sacrificed tumor-bearing mice were collected and subjected to FACS analysis. The experimental design was presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea. FACS data from spleen showed that peptide 4 only significantly increased the frequency of IFN-γ from lymphocytes such as CD4 T, CD8 T, and NK cells at day 7, compared to that of leukemic mice. Similarly, PB-NK cells were also peaked their frequency with significance difference, compared to that of leukemic group only when peptide 4 was treated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). In case of peptide 6, the frequency of NK and CD4 T cells in spleen has increased patterns at day 7, compared to that of leukemic mice group. Due to consistency of the results by P4, we ultimately decided to use further experiments in vivo to investigate the restoration of lymphocytes using the combination of P4 as an effector and ara-C for high stimulation of immune cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eThe Effects Of Peptide With Conventional Chemotherapy On The Expression Of Ifn-γ From Leukemic Mice\u003c/h3\u003e\n\u003cp\u003eNext, to address effects of peptide with conventional chemotherapeutic agents, a syngeneic leukemic mouse model was developed using C1498 leukemic cells and ex vivo cultured NK cells with or without peptide were used. Due to the importance of functional NK production with high IFN-γ in AML, ex vivo expanded NK cells with functionality and proliferation is regarded as an essential cell source in anti-tumor therapeutic strategies [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] Thus, we applied the novel P4 in ex vivo expanded NK cells for 3 days and then NK cells were injected into tumor bearing mice with exogenous P4 and ara-C together. It was used for in vivo experiments based on the protocol shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea. The tissues including PB, BM, and spleen from each group were collected and subjected to FACS analysis. Results in PB and BM showed that IFN-γ expression was increased in NK cells in the ara-C treatment and the NK cells injected group co-cultured with the ex vivo peptide (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb-c). Also, the frequency of CD3 including CD4/CD8 in PB was high peaked in peptide only treated group and NK cells combinational treated group (\u003cb\u003eFig. S2\u003c/b\u003e). The γδ CD3 T cells in PB were significantly increased in peptide only group and NK cells without peptide stimulation injected group (\u003cb\u003eFig. S2, PB\u003c/b\u003e). However, the γδ CD8 T cells in BM were dramatically decreased in peptide and ara-C combinational treated group and the NK cells injected group co-cultured with the ex vivo peptide (\u003cb\u003eFig. S2, BM\u003c/b\u003e). In the case of the spleen, where lymphocyte activity is most expected, the frequency of lymphocytes was rather reduced by peptide treatment. These data provided that activation and proliferation of immune cells by peptide treatment can be altered depends on tissues types of AML. Although the diversity exists, we interestingly found that CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e Treg cells in PB, BM and spleen were dramatically decreased in all peptides and ara-C treated groups, compared to that of leukemic group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb-d). It implied the role for P4 functioning in increasing IFN-γ, is known as helping factor for cytolysis, from the activation of CD3 and NK cells along with regression of Treg cells in tumor microenvironments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur previous studies [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] provided evidence that blocking FLT4 enhances IFN-γ expression in dysfunctional NK cells by showing that MAZ51 treatment provided functional restoration. Based on that evidence, we developed novel peptides that can bind soluble FLT4 and replace MAZ51, and we found that P4 effectively increased IFN-γ by inhibiting FLT4 activation. Thus, these Fc targeting peptides found in tumor microenvironments are emerging as therapeutic candidates for treating tumors directly or indirectly via access to immune cells [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Chang et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] confirmed the antagonistic action of several peptides as an antibody concept that can suppress FLT4 activity by binding soluble FLT4, and they showed the therapeutic potential of peptides by inhibiting FLT4 kinase activity in tumor progression. However, therapeutic suggestions for a peptide that inhibits FLT4 activity in the dysfunctional immune cells in tumor microenvironments are still lacking, and this field remains to be developed.\u003c/p\u003e \u003cp\u003eIn 2013, the Kari A group assessed the crystal structure of human FLT4 and revealed that homotypic interactions in D5 of the Fc domain of FLT4 (Thr444, Thr446, Glu426, and Lys516) are essential for FLT4 activation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Because the signaling domain of FLT4 is susceptible to disruption by homodimers, FLT4 activation can be hampered without antibodies by continuing interactions with other molecules such as peptides that target the wild-type intracellular domain of FLT4 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Therefore, we carefully assumed that a peptide with a dominant negative action that binds to the wild-typed subunit could make an inactive oligomer that inhibits signal activation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Another possibility in activating both NK cells and T cells \u003cem\u003ein vivo\u003c/em\u003e to restore their IFN-γ expression is that the peptide binding with FLT4 is present in all cells, such as macrophages, dendritic cells, and even tumor cells, and thus accelerates their defensive activity, leading to more efficient antigen processing and presentation to CD4 and CD8 cells, which in turn confers anti-tumor effects [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Aberrant expression of FLT4 on immune cells leads to dysfunctions that can decrease abnormal signaling by blocking the FLT4 signal cascade using the instability of the homodimer structure, which ultimately results in functional immune cell restoration. That process is similar to the action of antibodies. Although potent monoclonal antibodies are significantly compromised in their ability to confer pathogen protection in \u003cem\u003ein vitro\u003c/em\u003e assays, they can produce robust immunity \u003cem\u003ein vivo\u003c/em\u003e. Thus, pathologic protection depends on the acting FcγR engagement and disparity between immunity \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e.[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] In the case of tumor-associated antigens, i.e., FLT4 in NK cells, a strong signal cascade can suppress the immune response by blocking high IFN-γ expression [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe finally showed that blocking the signal domain of aberrant FLT4 with P4 effectively increased the IFN-γ expressed by immune cells, including NK cells. Unlike MAZ51, P4 was restricted to FLT4 signaling, with no effects on other VEGFRs [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. IFN-γ also has strong, well-known anti-lymphatic and anti-angiogenic effects, consistently causing vessel regression both in murine and human tumors, and is considered a primary anti-cancer cytokine and lytic helping factor [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Despite the unraveling evidence for P4 in the \u003cem\u003ein vivo\u003c/em\u003e cascade for immune cell stimulation, increased IFN-γ in CD4 and CD8 T cells was detected, along with a low frequency of CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e Treg cells, in this study (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb-d). Consistent with H Nishikawa\u0026rsquo;s data, the \u003cem\u003eex vivo\u003c/em\u003e cultured NK cells in this study also functioned as effectors to increase IFN-γ expression and induce immunity in tumor microenvironments [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In the future, we will investigate the anti-tumor effects of P6, P11, and P12 and compare them with that of P4 in leukemic mice. Although our \u003cem\u003ein vivo\u003c/em\u003e study was incomplete because we did not test P6, P11, and P12, our present results (and our previous reports) show that the peptides we designed to target FLT4 can restore low-functioning CD56\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e\u0026minus;\u003c/sup\u003e AML-NK cells, which express high levels of the lymphatic marker FLT4. Additionally, they reveal high IFN-γ expression in the T cells and NK cells of leukemic mice treated with P4. Our experiments show that both AML-NK cells and AML leukemic cells have increased expression of the lymphatic marker FLT-4, suggesting the relevance of AML-NK cell activation via the anti-lymphatic cascade.\u003c/p\u003e \u003cp\u003eTaken together, these results suggest that the intracellular domain of a peptide targeting FLT4 can be used in combination with conventional chemotherapy to increase IFN-γ expression in NK cells and T cells. Furthermore, these results provide clues about advanced therapeutic approaches that could be used to correct the tumor microenvironment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003eJY Lee, S Park, AR Han, and HS Hwang performed the experiments and analyzed the data. JY Lee and HJ Kim wrote the manuscripts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThis work was financially supported by a grant from the National Research Foundation (NRF, 2018R1D1A1A09083557), Ministry of Education, Republic of Korea.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eapproval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were performed with authorization from the Institutional Review Board for Human Research at the Catholic University of Korea\u0026nbsp;(KC19TESI0462).\u0026nbsp;The patients/participants provided their written informed consent to participate in this study.\u0026nbsp;All protocols for testing the animals were approved by the Catholic University of Korea\u0026apos;s Institutional Animal Care and Use Committee (CUMC-2019-0135-01)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChen SJ, Shen Y, Chen Z. 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Int J Cancer.\u003cem\u003e \u003c/em\u003e2004;112(6):986-993.\u003c/li\u003e\n\u003cli\u003eNishikawa H, Kato T, Tawara I et al. IFN-gamma controls the generation/activation of CD4+ CD25+ regulatory T cells in antitumor immune response. J Immunol.\u003cem\u003e \u003c/em\u003e2005;175(7):4433-4440.\u003c/li\u003e\n\u003cli\u003eCluff E, Magdaleno CC, Fernandez E et al. Hypoxia-inducible factor-1 alpha expression is induced by IL-2 via the PI3K/mTOR pathway in hypoxic NK cells and supports effector functions in NKL cells and ex vivo expanded NK cells. Cancer. Immunol Immunother\u003cem\u003e \u003c/em\u003e2022.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Fms-related tyrosine kinase-4 (FLT4), natural killer (NK), interferon-gamma (IFN-γ), acute myeloid leukemia (AML), mononuclear cells (MNCs)","lastPublishedDoi":"10.21203/rs.3.rs-2041166/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2041166/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFms-related tyrosine kinase-4 (FLT4) is involved in tumor progression. Previously, we found that dysfunctional natural killer (NK) cells with low interferon-gamma (IFN-γ) were restored in acute myeloid leukemia (AML) by the FLT4 antagonist MAZ51. In this work, we developed 12 peptides targeting FLT4 for clinical application and ultimately selected 4 of them to examine whether they restored the frequency of lymphocytes, especially T cells and NK cells, and high IFN-γ expression, as MAZ51 treatment did in our previous study. Although clinical data from using intracellular kinase domain\u0026ndash;targeting peptides are currently available, peptides targeting FLT4 to modulate immune cells have not been fully elucidated. In this study, we focus on novel peptide 4 (P4) from the intracellular domain of FLT4 because it had dominant negative activity. Similar to MAZ51, high IFN-γ levels were expressed in AML-mononuclear cells (MNCs) exposed to P4. In addition, T and NK cell levels were restored, as were high IFN-γ levels, in a leukemic environment when P4 was co-cultured with cytosine β-D-arabinofuranoside. Interestingly, the frequency of regulatory T cells was significantly decreased by P4, implying that the peptide plays a role in modulating the tumor niche. Overall, we demonstrated the therapeutic value of functionally modulating lymphocytes using a peptide targeting FLT4 and propose the development of advanced therapeutic approaches against AML by using immune cells.\u003c/p\u003e","manuscriptTitle":"Therapeutic potential of FLT4-targeting peptide in acute myeloid leukemia","msid":"","msnumber":"","nonDraftVersions":[{"code":"","date":"2023-01-03 09:14:09","doi":"","editorialEvents":[{"type":"decision","content":"Accepted","date":"2023-01-24T22:09:18+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-01-23T08:39:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"9c5d0f78-6669-44a6-b00b-aa7386421533","date":"2023-01-15T08:36:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-01-15T01:57:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-01-04T06:38:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Immunology, Immunotherapy","date":"2023-01-03T09:00:36+00:00","index":"","fulltext":""}],"status":"private","journal":{"display":true,"email":"[email protected]","identity":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}},{"code":2,"date":"2022-10-04 17:58:10","doi":"10.21203/rs.3.rs-2041166/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-12-25T23:24:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-12-20T09:41:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-09-28T23:46:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"c0d2da9b-01f5-4d9b-9dd9-51ffbbdb2a9b","date":"2022-09-23T13:11:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"ccc6c100-3eda-4145-9255-d1249b2c9483","date":"2022-09-21T14:52:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-09-20T12:01:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-09-17T07:06:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-09-17T07:06:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Immunology, Immunotherapy","date":"2022-09-16T10:09:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"bef42f7d-18c2-4167-8a36-a1b36bc7317d","owner":[],"postedDate":"October 4th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T18:47:58+00:00","versionOfRecord":{"articleIdentity":"rs-2041166","link":"https://doi.org/10.1007/s00262-023-03385-8","journal":{"identity":"cancer-immunology-immunotherapy","isVorOnly":false,"title":"Cancer Immunology, Immunotherapy"},"publishedOn":"2023-02-10 18:43:07","publishedOnDateReadable":"February 10th, 2023"},"versionCreatedAt":"2022-10-04 17:58:10","video":"","vorDoi":"10.1007/s00262-023-03385-8","vorDoiUrl":"https://doi.org/10.1007/s00262-023-03385-8","workflowStages":[]},"version":"v2","identity":"rs-2041166","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2041166","identity":"rs-2041166","version":["v2"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}