Thrombopoietin receptor agonist antibody for treating chemotherapy-induced thrombocytopenia

doi:10.21203/rs.3.rs-2115554/v1

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Thrombopoietin receptor agonist antibody for treating chemotherapy-induced thrombocytopenia

https://doi.org/10.21203/rs.3.rs-2115554/v1

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Background: Thrombocytopenia is a common complication in cancer patients undergoing chemotherapy. Chemotherapy-induced thrombocytopenia (CIT) leads to dose reduction and treatment delays, lowering chemotherapy efficacy and survival rate. Thus, rapid recovery and continuous maintenance of platelet count during chemotherapy cycles are crucial in patients with CIT. Thrombopoietin (TPO) and its receptor, myeloid proliferative leukemia (MPL) protein, play a major role in platelet production. Although several MPL agonists have been developed to regulate thrombopoiesis, none have been approved for the management of CIT due to concerns regarding efficacy or safety. Therefore, the development of effective MPL agonists for treating CIT needs to be further expanded.

Methods: Anti-MPL antibodies were selected from the human combinatorial antibody phage libraries using phage display. We identified 2R13 as the most active clone among the binding antibodies via cell proliferation assay using BaF3/MPL cells. The effect of 2R13 on megakaryocyte differentiation was evaluated in peripheral blood CD34⁺ cells by analyzing megakaryocyte-specific differentiation markers (CD41a⁺ and CD42b⁺) and DNA ploidy using flow cytometry. The 2R13-induced platelet production was examined in 8- to 10-week-old wild-type BALB/c female mice and a thrombocytopenia mouse model established by intraperitoneal injection of 5-fluorouracil (150 mg/kg). The platelet counts were monitored twice a week over 14 days post-initiation of treatment with a single injection of 2R13, or recombinant human TPO (rhTPO) for seven consecutive days.

Results: We found that 2R13 specifically interacted with MPL and activated its signaling pathways. 2R13 stimulated megakaryocyte differentiation, evidenced by increasing the proportion of high-ploidy (≥8N) megakaryocytes in peripheral blood-CD34⁺ cells. The platelet count was increased by a single injection of 2R13 for up to 14 days. Injection of 5-fluorouracil considerably reduced the platelet count by day 4, which was recovered by 2R13. The platelets produced by 2R13 sustained a higher count than that achieved using seven consecutive injections of rhTPO.

Conclusions: Our findings suggest that 2R13 is a promising therapeutic agent for CIT treatment.

chemotherapy-induced thrombocytopenia

thrombopoietin receptor

agonist antibody

megakaryopoiesis

platelet production

Thrombocytopenia frequently occurs in patients with cancer because of chemotherapy, the malignancy itself, or infection [1]. Chemotherapy-induced thrombocytopenia (CIT, platelet count ≤ 100,000/µL after chemotherapy), occurs in approximately 16–30% of patients receiving platinum-, taxane-, or gemcitabine-based regimens [2, 3]. However, no CIT management agents have received approval from the United States Food and Drug Administration (FDA). CIT is associated with hematologic toxicity caused by hematopoiesis suppression, a significant adverse effect of chemotherapy. Since bleeding events are fatal in patients with CIT, surgical procedures, radio-, and chemotherapy are cautiously administered [4]. Therefore, the current standard management for CIT involves the postponement of chemotherapy cycles and decreasing doses to restore the platelet count to the desired level for subsequent treatments. Unfortunately, such treatment approaches lead to reduced relative dose intensity (RDI), which in turn substantially lowers the efficacy of chemotherapy and the survival rate of patients [5].

Thrombopoiesis is regulated at multiple levels by various cytokines, the most important of which is thrombopoietin (TPO). TPO and its receptor, myeloid proliferative leukemia (MPL) protein, govern the megakaryocytic lineage from hematopoietic stem cells and stimulate megakaryocyte (MK) maturation in the bone marrow (BM) niche, thereby promoting platelet production [6]. Recombinant human TPO (rhTPO) and pegylated rhTPO have been extensively studied in patients with CIT. Unfortunately, their therapeutic application ceased owing to the generation of neutralizing antibodies that cross-reacted with endogenous TPO, causing severe thrombocytopenia [7, 8]. Subsequently, the development of alternative MPL agonists has revived interest in CIT treatment. Eltrombopag and avatrombopag are oral small-molecule MPL agonists, whereas romiplostim is a subcutaneously administered peptibody. These agonists have no sequence homology with endogenous TPO, and no anti-drug antibodies have been reported [9]. Clinical studies show that small-molecule MPL agonists were less potent at increasing the platelet count than maximal doses of romiplostim in healthy subjects [10–13]. Moreover, avatrombopag failed to show efficacy in treating CIT, with insignificant results under clinical trials [14]. Although there is increasing evidence to indicate that romiplostim may be effective in CIT management, the evaluation of the safety and efficacy of romiplostim use in cancer patients with CIT has been deficient, resulting in it being consigned to off-label use [15, 16]; there have been no MPL agonists approved for CIT treatment, which suggests the need to develop effective therapeutics.

In this study, we developed and characterized the novel MPL agonist antibody, 2R13. This antibody is a fully human agonist antibody selected via a BaF3/MPL cell-based assay following phage display using human combinatorial antibody phage libraries. We found that 2R13 promoted megakaryopoiesis and platelet production both in vitro and in vivo. Furthermore, 2R13 significantly improved the platelet count in a 5-fluorouracil (5-FU)-induced thrombocytopenia mouse model, suggesting potential as a therapeutic antibody for CIT.

Cell lines and cell culture

The Expi293F^™ cell line (Gibco, Grand Island, NY, US) used for antibody production was maintained according to the manufacturer’s recommendations. The murine pre-B cell line, BaF3, was obtained from Dr. Arthur J. Sykowski (Beth Israel Deaconess Medical Center, Boston, MA, US) and cultured in Roswell Park Memorial Institute Medium (RPMI)-1640 medium (Lonza, Walkersville, MD, US) supplemented with 10% fetal bovine serum (FBS) and 5% WEHI-3B cell-conditioned medium (WEHI-CM, as a source of IL-3). BaF3 cells were stably transfected with the pCMV-human MPL plasmid (Origene, Rockville, MD, US) to establish a BaF3/MPL cell line expressing human MPL. The surface expression of human MPL was confirmed using flow cytometry with anti-CD110-APC-conjugated antibody (MiltenyiBiotec, Auburn, CA, US). The acute megakaryoblastic leukemia cell line, M07e, was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ) and grown in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% FBS and 10 ng/mL recombinant human IL-3. Normal human platelets were obtained within 1–2 days after expiration dates from the Blood Bank at Kosin University Gospel Hospital (KUGH; Busan, Republic of Korea).

Antibody selection

The naïve human combinatorial antibody phage libraries (diversity ≈ 10⁹) were obtained from Scripps Research (La Jolla, CA, US). The overall antibody panning was done as described in a previous study [17]. Briefly, MPL-binding antibodies were selected by solution-phase panning, and recombinant human MPL (rhMPL, R&D Systems, Minneapolis, MN, US) was used as the antigen. Dynabeads^™ M-270 Epoxy beads (Invitrogen, San Diego, CA, US) were coupled with rhMPL at 37°C overnight with end-over-end rotation. The beads were blocked using MPBST (2% skim milk and 0.05% Tween 20 in phosphate-buffered saline [PBS]) at room temperature for 1 h. After adding the antibody phage libraries, the blocked beads-libraries mixture was incubated at room temperature with end-over-end rotation for 1 h. Subsequently, the supernatant was removed, and the beads were rinsed three times with PBST (0.05% Tween 20 in PBS). The bound phages were eluted using 0.2 M glycine-HCl (pH 2.2) and subsequently neutralized with 2 M Tris-HCl (pH 8.0).

Enzyme-linked immunosorbent assay (ELISA)

Each well of a 96-well half-area microplate (Corning, NY, US) was coated with rhMPL (R&D Systems) or bovine serum albumin (BSA; negative control; BD Biosciences, Franklin Lakes, NJ, US) at 4°C overnight. Each well was washed with PBS and blocked with MPBS (5% skim milk in PBS) at 37°C for 1 h. Next, the phages were added to the blocked wells, incubated at 37°C for 2 h, and washed five times with PBS. The bound phages were detected using anti-M13 phage-HRP-conjugated antibody (1:2000; Sino Biological Inc, Chesterbrook, PA, US). Absorbance was measured at 450 nm using a VersaMax^™ Microplate Reader (Molecular Devices, San Jose, CA, US). The 2R13 was detected using anti-human IgG Fc-HRP-conjugated antibody (1:2000; Abcam, Cambridge, MA, US).

Antibody expression and purification

The sequences of complementarity-determining regions (CDRs) in the phagemid of selected clones were analyzed using the IMGT numbering tool web server, VBASE2. The CDR sequences of each clone were inserted into the pFuse mammalian expression vector (Invivogen, San Diego, CA, US) using the Sfi1 restriction enzyme (NEB, Ipswich, MA, US) and T4 ligase (NEB), following the manufacturer’s protocol. The antibodies were expressed using the Expi293F^™ cell expression system (Thermo Fisher Scientific, Waltham, MA, US). The supernatants were collected and filtered using a 0.22 µm filter on the fourth day after transfection. The soluble single-chain fragment variable-fragment crystallizable (scFv-Fc) antibodies in the supernatant were purified over the HiTrap protein G HP column (Cytiva, Marlborough, MA, US) using the ÄKTAprime Plus system (Cytiva). The purified antibodies were concentrated using an Amicon^® Ultra-4 Centrifugal Filter (Millipore, Burlington, MA, US) and filtered using Spin-X^® centrifuge tube filters (Corning).

Cell proliferation assay

To measure cell proliferation, each cell was incubated in the culture medium without IL-3 supplementation. For activity-based antibody selection, BaF3/MPL cells (1 × 10⁴ cells/well) were cultured in 96-well plates for 48 h in the presence of rhTPO (R&D Systems) or antibodies. Cell proliferation was measured by the CellTiter^® 96 AQueous One Solution Cell Proliferation Assay System (Promega, Madison, WI, US), and absorbance was measured at 490 nm. To validate 2R13 agonistic activity, BaF3/MPL (1 × 10⁴ cells/well), BaF3 (1 × 10⁴ cells/well), or M07e (5 × 10⁴ cells/well) cells were cultured in 96-well plates for 48 h in the presence of rhTPO (PeproTech, Rocky Hill, NJ, US) or 2R13. The Cell Titer-Glo^® Luminescent Cell Viability assay kit (Promega) was used for BaF3 and BaF3/MPL cells according to the manufacturer’s protocol. Luminescence signals were measured using a Victor3 V 1420 Multilabel Counter (Perkin Elmer Inc, Waltham, MA, US). Cell Counting Kit-8 (GlpBio, Montclair, CA, US) was used for M07e cells according to the manufacturer’s protocol, and absorbance was measured at 460 nm.

Surface plasmon resonance (SPR)

The binding affinity and rate constants of 2R13 were measured using the iMSPR mini-instrument (icluebio, Seongnam, Republic of Korea) at room temperature. The surface of the research-grade carboxylic (COOH) sensor chip (icluebio) was activated with a 1:1 mixture of 0.1 M N-hydroxysuccinimide and 0.1 M 3-(N, N-dimethylamino) propyl-N-ethyl carbodiimide with a flow rate of 50 µL/min. rhMPL (R&D Systems) or recombinant mouse MPL (rmMPL, R&D Systems) was immobilized on the chip surface by using 10 mM sodium acetate buffer (pH 4.0) at approximately 500 response units (RUs), following which the surface was blocked with 1 M ethanolamine (pH 8.0). Next, two-fold serial dilutions (256–16 nM) of 2R13 were individually injected. Data were analyzed using the iMSPR analysis software (TraceDrawer; icluebio).

Western blotting

BaF3/MPL cells (4 × 10⁵ cells/mL) and human platelets were washed with PBS and then serum-starved in RPMI1640 medium with 0.5% FBS overnight or for 3 h, respectively. The cells and platelets were stimulated with rhTPO or 2R13 and lysed in Radio Immunoprecipitation Assay (RIPA) buffer (Elpis Biotech, Daejeon, Republic of Korea) containing protease/phosphatase inhibitor cocktail (Calbiochem, San Diego, CA, US). The lysates were quantified using the bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific) and boiled with sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) sample buffer at 95°C for 5 min. The samples (10 µg/well) were separated using SDS-PAGE and transferred onto a nitrocellulose membrane (Millipore). Next, after sequential incubation with primary and secondary antibodies, chemiluminescence signals were detected using an electrochemiluminescence (ECL) kit (Thermo Fisher Scientific), which was followed by visualization with an Amersham^™ Imager 600 (GE Healthcare Life Sciences, Chicago, IL, US). Primary antibodies against the Janus family of tyrosine kinases (Jak2), p-Jak2, signal transducers and activators of transcription (Stat5), p-Stat5 (Y925), Stat3, p-Stat3 (Y705), Akt, p-Akt (S473), Erk and p-Erk1/2 (Thr202/Tyr204), were purchased from Cell Signaling Technology (Danvers, MA, US). Antibody against β‐actin was purchased from Novus Biologicals (Minneapolis, MN, US).

Reporter gene expression assay

BaF3/MPL cells (2 × 10⁶ cells/mL) were transfected with pGL4.52[luc2P/Stat5RE/Hygro] vector (Promega) using the SG Cell Line 4D-Nucleofector kit (Lonza) following the manufacturer’s protocol. After the cells were recovered in the culture medium for 48 h, the cells were selected with hygromycin (200 µg/mL; Invivogen). The selected cells (2 × 10⁴ cells/well) were incubated in 96-well plates for 6 h in the presence of rhTPO (PeproTech), control antibody, or 2R13, without WEHI-CM. Luciferase (reporter gene) expression was measured using the Bright-Glo^™ Luciferase Assay System (Promega) by an Envision^™ multimode plate reader (PerkinElmer, Waltham, MA, US).

PB-CD34⁺ cell isolation

Granulocyte colony-stimulating factor-mobilized human apheresis samples were obtained from healthy donors within the framework of standard procedures for hematopoietic stem and progenitor cell (HSPC) apheresis at KUGH after obtaining written informed consent from the donors. Peripheral blood mononuclear cells were isolated by density gradient centrifugation with Ficolle-Hypaque (Sigma, Louis, MO, US) and subjected to PB-CD34⁺ cell immunoselection using the magnetic-activated cell sorting (MACS) CD34 MicroBead UltraPure kit (Miltenyi Biotec) according to the manufacturer’s protocol. The purity of the isolated PB-CD34⁺ cells was routinely higher than 95%, as determined using CytoFLEX Flow Cytometry (Beckman, Brea, CA, US) with anti-human CD34-PE-conjugated antibody.

Megakaryopoiesis analysis

PB-CD34⁺ cells (4 × 10⁴ cells/mL) were cultured in serum-free expansion medium (SFEM, Stem Cell Technologies, Vancouver, BC, Canada) supplemented with recombinant human stem cell factor (25 ng/mL; PeproTech), recombinant human IL-6 (10 ng/mL; PeproTech), recombinant human IL-9 (10 ng/mL, PeproTech), and LDL (25 µg/mL, Stem Cell Technologies) in the presence of rhTPO or 2R13 for up to 14 days. Megakaryopoiesis was assessed by detecting MK-specific differentiation markers using flow cytometry. Cells collected at each time point (days 4, 7, 11, and 14) were washed with cold PBS and labeled with anti-CD41a-FITC-conjugated antibody (Miltenyi Biotec), anti-CD42b-PE-conjugated antibody (Miltenyi Biotec), or 7-amino-actinomycin D (AAD; eBioscience, San Diego, CA, US) at 4°C for 30 min. Subsequently, the cells were washed and analyzed using flow cytometry.

Polyploidy analysis of MKs

PB-CD34⁺ cells cultured for 13 days were labeled with anti-CD41a-FITC-conjugated antibody at 4℃ for 30 min and washed with cold PBS. The labeled cells were fixed with 1% paraformaldehyde (PFA) at room temperature for 15 min and permeabilized with 70% methanol at − 20℃ for 1 h. The cells were then treated with RNase (10 µg/mL; Roche, Basel, Switzerland), stained with propidium iodide (PI; 10 µg/mL; Sigma) at room temperature for 30 min, and analyzed using flow cytometry.

In vivo experiments

To examine the changes in platelet and white blood cell (WBC) counts induced by 2R13, 8 to 10-week-old BALB/c female mice (Nara-Biotech, Seoul, Republic of Korea) were subcutaneously injected with 0.1% BSA-PBS or rhTPO (50 ng) for seven consecutive days, or once with 2R13 (0.1, 1, 2, or 10 µg). To establish the thrombocytopenia in vivo model, the mice were intraperitoneally injected with 0.1% BSA-PBS or 5-FU (150 mg/kg; Sigma). After 1 h, they were subcutaneously injected with 0.1% BSA-PBS or rhTPO (50 ng) for seven consecutive days, or once with 2R13 (10 or 20 µg). Each mouse was anesthetized with an intraperitoneal injection of ketamine (90 mg/kg) and xylazine (10 mg/kg); then, blood samples (50 µL) were collected from the retro-orbital sinus into ethylenediaminetetraacetic acid (EDTA) capillary tubes (Marienfeld, Germany). The blood was transferred into an EDTA tube prefilled with 2.5 mM EDTA buffer. Platelets and WBCs were counted by EONE Laboratories (Incheon, Republic of Korea). To investigate a change in LSK⁺ cell count induced by 2R13, BM-derived cells were collected from the femurs and tibias. The cells were flushed with PBS and red blood cells were lysed in lysing buffer at room temperature for 1 min, followed by flow cytometric analysis.

Flow cytometry

To validate the binding activity of 2R13 against MPL, BaF3 and BaF3/MPL cells (1 × 10⁶ cells/mL) were collected and fixed with 4% PFA at 4°C for 10 min. The cells were blocked with PBS containing 0.1% Tween 20, 1% BSA, and 5% goat serum at 4°C for 1 h. Next, the blocked cells were incubated with 2R13 or control antibody at 4°C for 1 h, followed by anti-human IgG Fc-FITC-conjugated antibody (Abcam) at 4°C for 1 h. To examine the percentage of LSK⁺ cells, BM cells were resuspended in PBS and incubated with PerCP-Cy5.5-labeled lineage antibody cocktail, anti-Sca-1-FITC-conjugated antibody, and anti-c-kit-APC-conjugated antibody (BD Biosciences) at 4°C for 30 min. The samples were analyzed using the FACSCanto™ II Flow Cytometry and FlowJo software.

Statistical analysis

Data were analyzed using the GraphPad software (Prism v.9.0; San Diego, CA, US). All data are the mean ± standard deviation (SD). Between-group differences were analyzed using the unpaired t-test. Statistical significance between multiple groups was analyzed using one-way analysis of variance (ANOVA) or two-way ANOVA, followed by Dunnett’s multiple comparison test.

Selection of the human anti-MPL agonist antibody

Antibodies binding to MPL were selected by solution-phase panning from the naïve human combinatorial antibody phage libraries. We performed two rounds of panning on rhMPL as the antigen and confirmed the enrichment of binding phages (Fig. 1a). We randomly selected 12 clones from the output phage pool of the second round and individually tested their binding to rhMPL. Seven clones (2R5, 2R10, 2R11, 2R12, 2R13, 2R17, and 2R18) bound to rhMPL with an OD_{450 nm} value approximately three-fold higher than that of BSA (Fig. 1b). To identify potential agonist antibodies among these clones, we tested whether each clone in the scFv-Fc format induced the proliferation of TPO-responsive cells (BaF3/MPL). We found that three clones (2R5, 2R11, and 2R13) induced BaF3/MPL cell proliferation, with 2R13 exhibiting the most potent activity (Fig. 1c). Furthermore, we analyzed the binding activity of 2R13 to MPL. The 2R13 bound to rhMPL with an EC₅₀ of 55.28 ng/mL (Fig. 1d) and specifically interacted with BaF3/MPL cells but not BaF3 cells (Fig. 1e). The cytoplasmic domain of MPL is conserved between mice and humans, with over 80% homology noted at the amino acid level [18]. Therefore, we determined the cross-reactivity of 2R13 with rmMPL using SPR analysis. The 2R13 interacted with both rmMPL (K_D ≈ 95.6 nM) and rhMPL (K_D ≈ 6.4 nM) (Fig. 1f).

2R13 promoted cell proliferation by activating the MPL signaling pathways in mouse and human cells

To further evaluate the agonistic activity of 2R13, we examined 2R13-dependent cell proliferation in three cell types. The 2R13 showed lower potency than rhTPO with an EC₅₀ of 1.3 ng/mL but promoted BaF3/MPL cell proliferation with an EC₅₀ of 52.44 ng/mL (Fig. 2a). In contrast, the proliferation of wild-type BaF3 cells was not affected by rhTPO or 2R13; thus, MPL-specific proliferation was noted in BaF3/MPL cells (Fig. 2b). The 2R13 also induced cell proliferation in the M07e cell line, a human leukemic cell line that endogenously expresses MPL and requires TPO as a survival and proliferation factor (Fig. 2c) [19]. To determine the mechanism underlying the promotion of receptor-mediated cell proliferation by 2R13, we examined MPL-dependent signaling in BaF3/MPL cells. TPO homodimerizes MPL and activates Stat, Akt, and Erk via the receptor-associated tyrosine kinase, Jak2 [20]. The 2R13 showed patterns similar to those for rhTPO-induced protein phosphorylation in a dose-dependent manner (Fig. 2d). We further validated 2R13 signal transduction via luciferase expression mediated by Stat5 activation (Fig. 2e).

2R13 stimulated MK differentiation from PB-CD34⁺ cells

To measure the effect of 2R13 on MK differentiation from peripheral blood (PB)-derived CD34⁺ cells, we cultured PB-CD34⁺ cells for 14 days in a megakaryocytic differentiation medium supplemented with either rhTPO or 2R13. We investigated two populations, immature MKs (CD41a⁺) and mature MKs (CD41a⁺CD42b⁺), from the differentiated cells on days 4, 7, 11, and 14. On day 11, the ratio of both MK populations and the number of total nucleated cells reached a peak by rhTPO or 2R13 (Fig. 3a); 2R13 led to the production of CD41a⁺ and CD41a⁺CD42b⁺ cells in a proportion similar to that obtained with rhTPO (Fig. 3b and left panels of 3c). Analysis of the number of cells produced per input of PB-CD34⁺ cells showed that the cell counts induced by 2R13 were lower than those induced by rhTPO; The number of CD41a⁺ cells increased 5.7-fold at 50 ng/mL, 6.9-fold at 300 ng/mL and 7.9-fold at 1,000 ng/mL 2R13 vs 11.7-fold at 50 ng/mL rhTPO, compared with the control. The number of CD41a⁺CD42b⁺ cells increased 6.5-fold at 50 ng/mL, 7.7-fold at 300 ng/mL, and 8.4-fold at 1,000 ng/mL vs 12.5-fold at 50 ng/mL rhTPO, compared with the control (Fig. 3c, right panels). We obtained similar results with PB-CD34⁺ cells derived from additional donors (donor 2 and 3; Supplementary Fig. 1a, b, and Supplementary Fig. 2a, b), demonstrating the functional effectiveness of 2R13.

2R13 promoted MK maturation during MK differentiation from PB-CD34⁺ cells

During MK differentiation, committed MK progenitors initiate proliferation through a mitotic process; however, during the late phases of differentiation, the cells switch to an endomitotic process to achieve polyploid and undergo maturation [21]. Therefore, we investigated the polyploidy level achieved by 2R13 to examine the mechanism underlying the contribution of this antibody to MK differentiation. PB-CD34⁺ cells were cultured for 13 days in the megakaryocytic differentiation medium supplemented with either rhTPO or 2R13. The 2R13 led to a lesser proportion of 2N MKs and greater proportions of 4N and ≥ 8N MKs than those obtained with rhTPO (Fig. 4a, b). The ratio of high-ploidy MKs (≥ 8N) was 17.2% at 50 ng/mL, 14.5% at 300 ng/mL, and 13.6% at 1,000 ng/mL of 2R13 vs 10.8% at 50 ng/mL of rhTPO (Fig. 4c). We observed similar results for PB-CD34⁺ cells derived from donor 2 (Supplementary Fig. 3a-c), whereas cells derived from donor 3 showed a slightly different pattern of MK ploidy achieved by 2R13; the ratio of each ploidy MK was similar to that of rhTPO (Supplementary Fig. 4a-c). Furthermore, we examined the number of cells produced per input of PB-CD34⁺ cells. The number of 2R13-induced high-ploidy MKs was lower than that of rhTPO-induced high-ploidy MKs, with 2.2 at 50 ng/mL, 2.3 at 300 ng/mL, and 2.8 at 1,000 ng/mL of 2R13 vs 4.9 at 50 ng/mL of rhTPO (Fig. 4d). Similar results were obtained with PB-CD34⁺ cells derived from additional donors (Supplementary Fig. 3d and Fig. 4d). Taken together, 2R13 did not increase the number of total nucleated cells as much as rhTPO, but rather induced a greater high-ploidy MK proportion than rhTPO. Based on these findings, we conclude that 2R13 mainly supports MK maturation during MK differentiation.

2R13 stimulated the MPL signaling pathways in human platelets

The 2R13 stimulated the proliferation and the intracellular signaling pathways in BaF3/MPL cells and M07e cells, which express ectopic and endogenous MPL, respectively (see Fig. 2). We then evaluated whether 2R13 activated the MPL-dependent signaling in primary human platelets. The 2R13 dose-dependently stimulated phosphorylation of Jak2, Stat5, and Akt, similar to rhTPO (Fig. 5a). In addition, rhTPO or 2R13 led to persistently high phosphorylation levels for up to 18 h (Fig. 5b). We confirmed a similar pattern of signal activation by 2R13 in platelets derived from addition donors (donor 2 and 3; Supplementary Fig. 5a–d).

2R13 promoted platelet production in vivo

To determine whether 2R13 promoted platelet production due to MK differentiation, we monitored the platelet counts in BALB/c mice for 14 days. The mice were injected with rhTPO for seven consecutive days, or once with 2R13, and blood was collected twice a week post-initiation of treatment (Fig. 6a). The platelet counts in 2R13-injected mice gradually increased in a dose-dependent manner from day 4, reached the highest level on day 7, and then decreased. The 10 µg of 2R13 remarkably increased the platelet level on day 7, maintaining a higher count until day 14 compared with that obtained by rhTPO (Fig. 6b). These data demonstrate that 2R13 promotes platelet production following MK differentiation induction, which confirms the relevance of our in vitro observations. In contrast, the 2R13-induced WBC production did not show significant changes during this period (Fig. 6c). TPO is also involved in the regulation of hematopoietic stem cells (HSCs) and the proliferation of primitive hematopoietic progenitor cells [22]. Therefore, we examined the effect of 2R13 on HSPCs in mice. On day 7 post-initiation of treatment, we found an increased percentage of LSK⁺ cells (Lin⁻/Sca-1⁺/c-kit⁺) in BM; therefore, 2R13 did cause LSK⁺ cells proliferation (Fig. 6d and Supplementary Fig. 6).

2R13 ameliorated 5-FU-induced thrombocytopenia

We evaluated the therapeutic potential of 2R13 against CIT. The mice were injected with 5-FU 1 h before treatment of either rhTPO or 2R13 (Fig. 7a). The platelet counts were significantly reduced on day 4 (by 5-FU) and returned to control levels from day 7 onwards. The 2R13-induced platelet production peaked on day 11. The produced platelets maintained a higher count than that obtained with rhTPO until day 14. In particular, 20 µg of 2R13 rescued the platelet count to a level comparable to that in the control mice from day 4, but it did not produce more platelets than 10 µg of 2R13 from day 7 onwards (Fig. 7b). The 5-FU and 2R13 did not significantly change the WBC count during this period (Fig. 7c). Collectively, our results show that a single injection of 2R13 may help relieve CIT by effectively promoting platelet production.

MPL agonists have recently been developed and approved by the FDA for the treatment of immune thrombocytopenia (ITP), aplastic anemia, hepatitis C-associated thrombocytopenia, and perioperative thrombocytopenia. However, none have been approved for CIT treatment [23–25]. Owing to CIT, treatment delay and decrease in administered doses result in reduced RDI, which lowers chemotherapy efficacy and affects patient survival outcomes. Thus, rapid and constant recovery of the platelet count during chemotherapy cycles without impacting the ongoing cancer therapy or RDI is crucial for CIT management. Our current findings suggest that 2R13, a human agonist antibody against MPL, is a potential therapeutic agent for CIT.

Among the three known MPL-associated signaling pathways, the Akt and Erk1/2 pathways play determinant roles in MK maturation and platelet formation. Akt signaling appears to be activated throughout MK differentiation. In contrast, Erk signaling appears to be strongly activated during the early stage and turned off during the late stages of MK differentiation [26, 27]. Previous research showed that romiplostim induces greater Akt signaling than that obtained with endogenous TPO, stimulating MK proliferation without parallel stimulation of MK maturation [28]. These findings indicate that regulation of cellular signaling by endogenous TPO and MPL agonists involves different mechanisms. The 2R13 phosphorylated Stat3/5, Akt, and Erk in similar to those of rhTPO in BaF3/MPL cells, but differing patterns were observed in promoting MK differentiation from PB-CD34⁺ cells. rhTPO increased the proportion of high-ploidy MKs by simultaneously stimulating MK proliferation (increase in 2N MK ratio) and differentiation. In contrast, 2R13 induced less MK proliferation but a greater high-ploidy MK ratio than that of rhTPO in PB-CD34⁺ cells derived from two out of three donors. These results indicate that 2R13 mainly supports polyploidization and maturation rather than proliferation during MK differentiation. However, to elucidate the precise mechanism underlying the involvement of 2R13 in MK differentiation and platelet production, further studies are required for more donor-derived PB-CD34⁺ cells.

A monoclonal antibody is one of the predominantly used therapeutic modalities because of its preferred biophysical properties, including high specificity, stability, long half-life, and low immunogenicity. The long half-life ensures durable effects with less frequent administration. We found that a single injection of 2R13 potently induced platelet production in the mouse model. In particular, high-dose 2R13 (20 µg) helped ameliorate the 5-FU-induced thrombocytopenia from day 4. Treatment with 2R13 sustained a higher platelet count than that obtained using rhTPO injection for seven consecutive days during the observation period, thereby effectively relieving thrombocytopenia. These data imply that 2R13 is a potential therapeutic agent that would enable CIT management, which requires rapid and continuous improvement in platelet count during chemotherapy cycles.

TPO also plays a critical role in HSC maintenance and hematopoietic recovery. It synergizes with other early cytokines to promote HSPC survival and proliferation, supporting their expansion and differentiation into multilineage immature progenitors/precursors in the BM [29]. We found that 2R13 increased the LSK⁺ cell count, but the WBC count did not change significantly during the study period. Based on these results, 2R13 is believed to promote HSPC proliferation and strongly drive thrombopoiesis within the BM, thereby mimicking the biological role of endogenous TPO. Chemotherapy may inhibit the development of other hematopoietic cells by impairing HSC capacity for self-renewal and differentiation, leading to BM failure. The damaged BM microenvironment of patients spontaneously recovers via HSPC regeneration after chemotherapy; however, some patients receiving high-dose chemotherapy can only be restored via stem cell transplantation [30–33]. These functions of 2R13 mentioned above would aid in BM recovery and hematopoietic reconstitution, which suggests extended applicability in stem cell transplantation therapeutics.

Given that the TPO/MPL axis primes platelets, rendering them sensitive to prothrombotic and platelet-activating molecules, the use of MPL agonists could lead to venous thromboembolism, which contributes to the baseline risk associated with malignancy [34–36]. However, several studies have reported that patients receiving MPL agonists did not show significant differences in spontaneous platelet aggregation or hyperreactivity compared to healthy subjects or placebo-treated patients [37–39]. In addition, in solid tumor patients treated with romiplostim, venous thromboembolism rates were similar to those in patients who had not received it [5]. We confirmed that 2R13 activated MPL expressed in primary platelets via phosphorylation of the Jak2-Stat5 and Akt pathways, which shows the need to examine the potential for platelet activation. Further studies are required on platelet aggregation due to overactivation and changes in platelet surface markers because the effect of 2R13 on platelets is directly associated with safety as a therapeutic option for CIT.

In summary, we developed a fully human agonist antibody, 2R13, that specifically binds to and activates MPL. Our data show that 2R13 promotes polyploidization and maturation during MK differentiation, followed by effective platelet production. In particular, the amelioration of 5-FU-induced thrombocytopenia with rapid recovery and continuous maintenance of platelet count by 2R13 demonstrates its potential as a promising therapeutic agent for CIT management.

CIT

chemotherapy-induced thrombocytopenia

RDI

relative dose intensity

TPO

thrombopoietin

MPL

myeloid proliferative leukemia

bone marrow

HSC

hematopoietic stem cell

HSPC

hematopoietic stem and progenitor cell

megakaryocyte

WBC

white blood cell

peripheral blood

5-FU

5-fluorouracil

WEHI-CM

WEHI-3B cell-conditioned medium

PBS

phosphate-buffered saline

FBS

fetal bovine serum

BSA

bovine serum albumin

ANOVA

analysis of variance

Ethics approval and consent to participate

This study using human origin samples was performed in accordance with the Declaration of Helsinki and was approved by the institutional review board of Kosin University Gospel Hospital (KUGH 2016-01-002 for the use of CD34⁺ cells; KUGH 2021-03-030 for the use of platelets). Animal experiments were performed in accordance with the guidelines approved by the University College of Medicine Institutional Animal Care and Use Committee (2020-022).

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Competing interests

The authors declare that they have no competing interests.

Funding

This study was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) of Korea funded by the Korean government (MSIT) (2019M3A9H1103607 and 2020M3A9I4039539), the Basic Research Laboratory Program (2021R1A4A1031380) of the National Research Foundation (NRF) of Korea funded by the Korean government (MSIT), and the DGIST Program of the Ministry of Science and ICT (22-DGRIP-01).

Authors’ contributions

Conception and design of the study: J.S, M.K, and X.Q; Experiment and analysis of data: J.S, M.K, X.Q, and J.K; Writing-drafting the article: J.S, M.K, and X.Q; Writing-review and editing the article: S.P, J.J, and K.Y; Supervision: S.L, S.P, J.J, and K.Y. All authors read and approved the final manuscript.

Acknowledgments

Authors’ information

¹Department of New Biology, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Daegu, 42988, Republic of Korea

Jiwon Shin, Kyungmoo Yea

²Department of Biochemistry, Kosin University College of Medicine, Busan 49267, Republic of Korea

Min-Jung Kim, Jee-Yeong Jeong

³Department of Microbiology and Immunology, Inje University College of Medicine, Busan 47392, Republic of Korea

Xingguo Quan, SaeGwang Park

⁴Department of Biochemistry, Kookmin University, Seoul 02707, Republic of Korea

Ji Woong Kim, Sukmook Lee

⁵New Biology Research Center, DGIST, Daegu, 43024, Republic of Korea

Kyungmoo Yea

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Additionalfile1.pdf
Additional file 1.pdf: Fig. S1 Effect of 2R13 on MK differentiation in PB-CD34⁺ cells isolated from donor 2 a, b PB-CD34⁺ cells derived from donor 2 were stimulated with rhTPO or 2R13 at the indicated concentrations for 11 days. a Representative flow cytometric analysis of PB-CD34⁺ cell differentiation. b Percentage and number of CD41a⁺ and CD41a⁺CD42b⁺ cells. The number of cells produced per input of PB-CD34⁺ cells was calculated by multiplying the number of total nucleated cells with the percentage of CD41a⁺ or CD41a⁺CD42b⁺ cells. Data are the mean ± SD (n = 3). One-way ANOVA was used for statistical analysis. ***p < 0.001 vs control; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs rhTPO.
Additionalfile2.pdf
Additional file 2.pdf: Fig. S2 Effect of 2R13 on MK differentiation in PB-CD34⁺ cells isolated from donor 3 a, b PB-CD34⁺ cells derived from donor 3 were stimulated with rhTPO or 2R13 at the indicated concentrations for 11 days. a Representative flow cytometric analysis of PB-CD34⁺ cell differentiation. b Percentage and number of CD41a⁺ and CD41a⁺CD42b⁺ cells. The number of cells produced per input of PB-CD34⁺ cells was calculated by multiplying the number of total nucleated cells with the percentage of CD41a⁺ or CD41a⁺CD42b⁺ cells. Data are the mean ± SD (n = 3). One-way ANOVA was used for statistical analysis. ***p < 0.001 vs control; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs rhTPO.
Additionalfile3.pdf
Additional file 3.pdf: Fig. S3 Effect of 2R13 on MK polyploidization in PB-CD34⁺ cells isolated from donor 2 PB-CD34⁺ cells derived from donor 2 were stimulated with rhTPO or 2R13 at the indicated concentrations for 13 days. a Ploidy status of CD41a⁺ cells. b Representative flow cytometric profile of ploidy levels. c-d Percentage and number of high-ploidy (≥8N) MKs. The number of cells produced per input of PB-CD34⁺ cells was calculated by multiplying the number of total nucleated cells with the percentage of high-ploidy CD41a⁺ cells. Data are the mean ± SD (n = 3). One-way and two-way ANOVA were used for statistical analysis. *p < 0.05 and ***p < 0.001 vs control; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs rhTPO.
Additionalfile4.pdf
Additional file 4.pdf: Fig. S4 Effect of 2R13 on MK polyploidization in PB-CD34⁺ cells isolated from donor 3 PB-CD34⁺ cells derived from donor 3 were stimulated with rhTPO or 2R13 at the indicated concentrations for 13 days. a Ploidy status of CD41a⁺ cells. b Representative flow cytometric profile of ploidy levels. c, d Percentage and number of high-ploidy (≥8N) MKs. The number of cells produced per input of PB-CD34⁺ cells was calculated by multiplying the number of total nucleated cells with the percentage of high-ploidy CD41a⁺ cells. Data are the mean ± SD (n = 3). One-way and two-way ANOVA were used for statistical analysis. **p < 0.01 and ***p < 0.001 vs control; #p < 0.05 and ###p < 0.001 vs rhTPO.
Additionalfile5.pdf
Additional file 5.pdf: Fig. S5 MPL signal transduction induced by 2R13 in human platelets from donor 2 and donor 3 Western blot analysis of human platelets derived from donors 2 (a, b) and donor 3 (c, d), stimulated with rhTPO or 2R13, using the indicated antibodies. a, c rhTPO or 2R13 was added at the indicated concentrations for 30 min. b, d rhTPO (50 ng/mL) or 213 (300 ng/mL) was treated over the indicated period.
Additionalfile6.pdf
Additional file 6.pdf: Fig. S6 Flow cytometric analysis of LSK⁺ cells a Flow cytometry gating strategy used to isolate LSK⁺cells (Lin^-/Sca-1⁺/c-kit⁺) derived from BM. b Representative flow cytometric analysis.

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You are reading this latest preprint version

Thrombopoietin receptor agonist antibody for treating chemotherapy-induced thrombocytopenia

Status:

Version 1

Abstract

Figures

Background

Materials And Methods

Cell lines and cell culture

Antibody selection

Enzyme-linked immunosorbent assay (ELISA)

Antibody expression and purification

Cell proliferation assay

Surface plasmon resonance (SPR)

Western blotting

Reporter gene expression assay

PB-CD34⁺ cell isolation

Megakaryopoiesis analysis

Polyploidy analysis of MKs

In vivo experiments

Flow cytometry

Statistical analysis

Results

Selection of the human anti-MPL agonist antibody

2R13 promoted cell proliferation by activating the MPL signaling pathways in mouse and human cells

2R13 stimulated MK differentiation from PB-CD34⁺ cells

2R13 promoted MK maturation during MK differentiation from PB-CD34⁺ cells

2R13 stimulated the MPL signaling pathways in human platelets

2R13 promoted platelet production in vivo

2R13 ameliorated 5-FU-induced thrombocytopenia

Discussion

Conclusions

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1

Thrombopoietin receptor agonist antibody for treating chemotherapy-induced thrombocytopenia

Status:

Version 1

Abstract

Figures

Background

Materials And Methods

Cell lines and cell culture

Antibody selection

Enzyme-linked immunosorbent assay (ELISA)

Antibody expression and purification

Cell proliferation assay

Surface plasmon resonance (SPR)

Western blotting

Reporter gene expression assay

PB-CD34+ cell isolation

Megakaryopoiesis analysis

Polyploidy analysis of MKs

In vivo experiments

Flow cytometry

Statistical analysis

Results

Selection of the human anti-MPL agonist antibody

2R13 promoted cell proliferation by activating the MPL signaling pathways in mouse and human cells

2R13 stimulated MK differentiation from PB-CD34+ cells

2R13 promoted MK maturation during MK differentiation from PB-CD34+ cells

2R13 stimulated the MPL signaling pathways in human platelets

2R13 promoted platelet production in vivo

2R13 ameliorated 5-FU-induced thrombocytopenia

Discussion

Conclusions

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1

PB-CD34⁺ cell isolation

2R13 stimulated MK differentiation from PB-CD34⁺ cells

2R13 promoted MK maturation during MK differentiation from PB-CD34⁺ cells