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Neutralization of Biological Activity and Inhibition of Receptor Binding by Antibodies Against Human Thrombopoietin

Pharmaceutical Research Laboratory, Kirin Brewery Co., Ltd., Takasaki, Gunma, Japan

Key Words. Thrombopoietin • Neutralizing antibody • Peptide antibody • c-Mpl ligand • Active domain • ELISA

Dr. Takashi Kato, Pharmaceutical Research Laboratory, Kirin Brewery Co., Ltd., 3 Miyahara-cho, Takasaki, Gunma 370-12, Japan.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Thrombopoietin (TPO) is a recently isolated cytokine that primarily regulates megakaryocytopoiesis and thrombopoiesis. We recently reported the development of a variety of antibodies (Abs) to synthetic peptides of human (h)TPO and to recombinant human TPO (rhTPO). In this study, we characterized the Abs and mapped immunologically distinct areas of the molecule. Among the five different antipeptide polyclonal Abs, only one, raised against synthetic peptide D⁸ to Q²⁸, neutralized the TPO-dependent growth of FDCP-2 cells expressing human Mpl (FDCP-hMpl5 cells). One out of seven anti-rhTPO monoclonal Abs, designated as TN1, also showed neutralizing activity. TN1 was found to be specifically reactive with two proteolytic fragments, residues S¹ to R¹¹⁷ and A⁶⁰ to K¹²² of hTPO, indicating that the epitope(s) of TN1 is localized in residues A⁶⁰ to R¹¹⁷ of the molecule. These two neutralizing Abs inhibited the binding of biotinylated rhTPO to FDCP-hMpl5 cells. On the other hand, the other Abs, which reacted with five polypeptides of S⁴⁷ to D⁶², L¹⁰⁸ to A¹²⁶, N¹⁷² to A¹⁹⁰, S²⁶² to T²⁸⁴, and P³⁰⁶ to G³³² of hTPO, did not show either the neutralizing activity or the ability to inhibit the binding of biotinylated rhTPO to the cell surface hMpl. These findings indicate that two regions, residues D⁸ to Q²⁸ and A⁶⁰ to R¹¹⁷ of hTPO, may contain the domains associated with its receptor, c-Mpl. These Abs characterized here are valuable for studying the structural analysis and the biological function of hTPO mediated by its receptor.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Thrombopoietin (TPO), the c-Mpl ligand, is a major regulator of megakaryocytopoiesis and platelet production [1]. TPO stimulates the growth of committed megakaryocyte progenitors, the progressive maturation of megakaryocytes, and proplatelet formation [2-8]. In addition to its stimulating effect on megakaryocytopoiesis, TPO has also been reported to affect committed erythroid progenitors [9], hematopoietic progenitor cells [10], and leukemia cells [11].

Although human TPO (hTPO) comprises 332 amino acids [12, 13], only the N-terminal domain of the protein is sufficient for the biological activity in vitro [14] as well as in vivo [15, 16], indicating that the N-terminal domain contains the biologically active domain(s). More recently, Kenneth et al. reported the multiple receptor-binding domains of truncated hTPO in a mutagenesis study [17]. To date, however, the crystal or nuclear magnetic resonance structure of the TPO molecule has not been demonstrated. Therefore, the functional domain(s) of TPO has not been definitively identified yet.

Immunochemical methods by use of various antibodies (Abs) against cytokines have been used to measure the blood levels of the factors and analyze the molecular characteristics. We have reported blood hTPO levels in various hematopoietic disorders and liver cirrhosis by using an hTPO-specific enzyme-linked immunosorbent assay (ELISA) [18-26]. In this ELISA system, a neutralizing monoclonal Ab (mAb), termed TN1, is used as the capture Ab to detect the active molecule. Although this ELISA system is currently widely used, the precise binding site(s) of the Ab has not been clarified. In other cytokines, mapping with characterized antibodies has been performed to determine the structure of the proteins and further define the relationship between the structure and function. Such immunochemical studies using site-specific neutralizing and nonneutralizing Abs have provided valuable information about the active domains of erythropoietin, a protein which shows similarity in its amino acid sequence to a part of the N-terminal domain of TPO [27-30].

In this study, we characterized various mAbs against recombinant hTPO (rhTPO), including TN1, and the polyclonal Abs (pAbs) raised against the synthetic hTPO peptides previously reported [18, 31]. Among these Abs, only one anti-peptide pAb (anti-HT1) and one mAb (TN1) neutralized the biological activity of rhTPO in a dose-dependent fashion and inhibited the binding of the protein to its receptor. In most cases, the ability of the Abs to block binding of rhTPO correlated with their ability to neutralize biological activity. The binding regions of the mAbs were further determined by their ability to bind to C-terminal deletion muteins, synthetic peptides, and proteolytic fragments of rhTPO.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cytokines
rhTPO and recombinant mouse interleukin 3 (rmIL-3) were expressed in Chinese hamster ovary cells and in E. coli cells, respectively. The proteins were purified to homogeneity at the Production Technology Group of Kirin Brewery Co., Ltd. The protein concentrations were determined by amino acid composition analysis. The cDNAs for various deletion muteins of hTPO comprising amino acids S¹ to C¹⁵¹, S¹ to L¹⁷¹, S¹ to R¹⁹¹, S¹ to L²¹¹, or S¹ to P³¹¹ were constructed and expressed in COS1 cells. The COS1 cells were grown in Iscove's modified Dulbecco's medium (GIBCO BRL; Grand Island, NY) containing 10% heat-inactivated fetal calf serum (IMDM/FCS). The conditioned medium was harvested three days after transfection, filtrated with a 0.22 µm filter, and stored frozen until used.

Abs Against Synthetic hTPO Peptides
As previously described [31], immunogen-specific rabbit antisera could be induced with six synthetic peptides such as D⁸ to Q²⁸ (HT1), S⁴⁷ to D⁶² (HT2), L¹⁰⁸ to A¹²⁶ (HT3), N¹⁷² to A¹⁹⁰ (HT4), S²⁶² to T²⁸⁴ (HT5), and P³⁰⁶ to G³³² (HT6). All the pAbs against the peptides, which were purified from the antisera with the respective monomeric peptide-affinity columns, could bind to rhTPO on Western blots. Antigen specificity was confirmed by competitive assays using free peptides as inhibitors (data not shown).

Abs Against rhTPO
Twenty-two hybridomas producing anti-rhTPO mAbs were obtained by immunization of mice with rhTPO, as previously described [18]. Seven mAbs, termed ET1A (subclass, IgG2a), ET2A (IgG2b), ET3C (IgG3), CF6B (IgG1), CFX1 (IgG1), CF4A (IgG1), and TN1 (IgG1), were selected for their high titer to rhTPO and were used in this study. Anti-rhTPO serum, designated as PCF9, was raised in rabbits. The PCF9 polyclonal Ab was purified by use of a Protein-G column (Pharmacia Biotek; Uppsala, Sweden).

Assay for Binding of Abs to rhTPO and Synthetic hTPO Peptides
Each well of a 96-well flat-bottomed microtiter plate (Maxisorp; Nunc, Roskilde, Denmark) was coated at 4°C overnight with 100 µl of rhTPO or synthetic peptide at a concentration of 33 pmol/ml or 330 pmol /ml, respectively, in 20 mM Tris-HCl (pH 7.5) containing 0.5M NaCl and 0.1% NaN₃ (tris-buffered saline [TBS]). After preincubation of the wells with 200 µl of a blocking reagent (Super block in TBS; Pierce; Rockford, IL) for 30 min at room temperature, various concentrations of each Ab in TBS containing 20% Block Ace and 0.05% Tween 20 (BTTBS) were added to the wells and incubated for three h at room temperature. After having been washed four times with TBS containing 0.05% Tween 20 (TTBS), each well was incubated for one h at room temperature with rabbit anti-mouse IgGs (RAM) or goat anti-rabbit IgGs (GAR) conjugated with horseradish peroxidase (DAKO; Copenhagen, Denmark) in BTTBS. Four washes were performed with TTBS, followed by visualization of the binding of Abs to the polypeptide by use of a substrate color development kit (TMB, DAKO). The color intensity was measured by a 96-well plate reader (Well reader SK601; Seikagaku Kogyo; Tokyo, Japan) with a measuring filter of 450 nm and a reference filter of 570 nm.

Neutralization of Biological Activity of rhTPO by Abs
To determine whether peptide pAbs and anti-rhTPO Abs could neutralize rhTPO biological activity, we conducted a cell proliferation assay using TPO-dependent FDCP-hMpl5 cells which were genetically engineered to constitutively express human Mpl in an mIL-3-dependent FDCP-2 cell line [32]. The FDCP-hMpl5 cells were grown in IMDM/FCS containing 280 fmol/ml of rhTPO in 5% CO₂ at 37°C. The cells were washed three times with IMDM before being used in the proliferation assay. rhTPO at a concentration of 14 fmol/ml was incubated for one h at 37°C with various concentrations of each Ab in IMDM/FCS, and then these mixtures were added to the FDCP-hMpl5 (1 x 10⁴ cells/well) and the cells were incubated for 72 h in 5% CO₂ at 37°C. Thereafter, a 20-ml mixture of a tetrazolium compound (MTS) and phenazine methosulfate as a coupling reagent (CellTiter 96 AQ Assay; Promega; Madison, WI) was added to each well, and incubation was conducted for three h in 5% CO₂ at 37°C. The cell proliferation was determined by measuring the absorbance at 492 nm.

Flow Cytometric Analysis
rhTPO was biotinylated with Biotin LC-Hydrazide (Pierce). The biotinylated rhTPO stimulated the proliferation of FDCP-hMpl5 cells. FDCP-hMpl5 cells were cultured overnight in IMDM/FCS in the absence of TPO. Next, the cells were incubated for 10 min on ice with anti-mouse FcgII/III receptor antibody (FcBlock^TM; Pharmingen; San Diego, CA) in Dulbecco's phosphate buffered saline containing 1% FCS and 0.05% NaN₃. Seven pmol/ml of biotinylated rhTPO was incubated for one h at 37°C in IMDM/FCS with an excess concentration (3.3 nmol/ml) of each Ab. The Ab-rhTPO immunocomplex was reacted with the FDCP-hMpl5 cells. The binding of biotinylated rhTPO to the cells was detected by streptavidin conjugated with phycoerythrin (DAKO). Cells were analyzed by FACScan (Becton-Dickinson; San Jose, CA).

Proteolytic Fragments of rhTPO
rhTPO (280 pmol) was dissolved in 100 ml of 10 mM Tris-HCl (pH 8.0). Lysylendopeptidase (AP1 protease, Wako Pure Chemical; Osaka, Japan) was then added to the solution at an enzyme/substrate ratio of 1:20 and the mixture was incubated overnight at 37°C. The sample was reduced with dithiothreitol ([DTT], Nacalai tesque; Kyoto, Japan), and further subjected to reverse-phase HPLC using solvent A (0.1% [V/V]) trifluoroacetate in water) and solvent B (0.1% trifluoroacetate in 90% acetonitrile) as mobile phases. The digested peptide fragments were separated by a Capcell Pak C18 ( 4.6 x 150 mm; Shiseido; Tokyo, Japan), equilibrated with 5% solvent B with a gradient from 5% to 80% of mobile phase B over 50 min at a flow rate of 0.5 ml/min. Each peptide fragment was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) using Voyager^TM Elite (PerSeptive Biosystems; Framingham, CA).

Immunoblot Analysis

Western Blotting   Ten ml of rhTPO (2.8 pmol/ml) reduced with DTT (3 mg/ml) for 10 min at 96°C was subjected to SDS/PAGE using a 10%-20% precast gradient gel (Daiichi Pure Chemicals; Tokyo, Japan). The reduced rhTPO were next transferred electrophoretically from the gels to polyvinylidene difluoride (PVDF) membranes (Millipore; Marlborough, MA) with a semi-dry electroblotter (model HEP-1; Owl Scientific; Woburn, MA). After having been blocked with BTTBS containing a gelatin hydrolysate (Boeringer Mannheim; Mannheim, Germany), each membrane was incubated with 6.7 pmol/ml of each Ab for two h. After a washing with TTBS, the membranes were incubated for one h at room temperature with RAM or GAR conjugated with alkaline phosphatase (DAKO). After a washing with TTBS, the immunocomplexes were detected by alkaline phosphatase-color development kit (Bio-Rad; Hercules, CA).

Dot Blot   rhTPOs (28 fmol) and synthetic hTPO peptides (20 fmol) were transferred to PVDF membranes by use of THE CONVERTIBLE^TM filtration manifold system (GIBCO BRL). Detection of rhTPO- or peptide-Ab immunocomplexes was performed by the same method of Western blotting as described above.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Neutralization of rhTPO-Mediated Biological Activity by Abs
We examined whether six peptide-specific pAbs were capable of binding to nondenatured (intact) rhTPO in solid-phase ELISA. As shown in Table 1, all the peptide pAbs, except anti-HT3 were able to bind to intact rhTPO. The specific titers of these pAbs for rhTPO, defined here as the Abs concentrations (pmol/ml) that yielded one-half maximal binding to 3.3 pmol of rhTPO, ranged from 0.3 to 10.0 pmol/ml. In addition, the titers of the anti-rhTPO Abs ranged from 0.1 to 7.30 pmol/ml.

View larger version (13K): [in this window] [in a new window]   Figure 1. Dose-dependent inhibition of rhTPO-induced growth of FDCP-hMpl5 cells by the neutralizing Abs. rhTPO (14.1 fmol/ml) was preincubated with various concentrations of each Ab. FDCP-hMpl5 (1 x 104 cells/well) were then incubated with these mixtures. After three days, the MTS assay was performed. Symbols represent anti-HT1 (•), TN1 () and PCF9 (). Results are expressed as the mean ± SD of triplicate determinations. The absorbance of the maximal cell growth (without Ab) and the background (without factors) was 1.06 ± 0.05 and 0.47 ± 0.01, respectively.

View larger version (25K): [in this window] [in a new window]   Figure 2. Inhibition of rhTPO binding to cell-surface rhMpl by Abs. Biotinylated rhTPO (7.0 pmol/ml) was preincubated with 3.3 nmol/ml of each Ab (thick line) such as ET1A (panel B), TN1 (panel C), PCF9 (panel D), HT1 (panel E), other Abs (panel F) and control IgG (panel A to F, thin line). FDCP-hMpl5 cells were incubated with or without the Ab-rhTPO immunocomplexes (Panel A, dotted line) and then stained with PE-conjugated streptoavidin. Fluorescence was analyzed by FACScan. Results for ET2A, ET3C, CF6B, and anti-HT2 were overlayed in Panel F. Similar results were observed with CFX1, CF4A, anti-HT4, anti-HT5, and anti-HT6 (data not shown).

View larger version (37K): [in this window] [in a new window]   Figure 3. Binding of TN1 to a proteolytic fragment of rhTPO in the dot blot assay. A) AP1-digested peptides were separated with a Capcell Pak C18. Reactivities of TN1 toward AP1-peptides were tested. Specific signal of TN1 was observed in only the peptide having amino acid sequence from A60 to K122..CF6B was used as a control same isotype IgG. B) Binding activities of TN1 to three synthetic peptides that overlap the AP1-peptide A60 to K122. Three hTPO peptides, W51 to M75, A76 to N125, and L101 to N125 were synthesized with a model 431A peptide synthesizer (Perkin-Elmer; Foster City, CA) as previously reported [15]. C) Positive control of binding activities of TN1 and CF6B to the reduced rhTPO.

View larger version (8K): [in this window] [in a new window]   Figure 4. Binding regions of neutralizing and nonneutralizing Abs. The amino and carboxy termini of hTPO are indicated (black box). The binding regions (white boxes) of neutralizing Abs are shown below, and those of nonneutralizing Abs above, the hTPO molecule.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The major finding of this study is that the epitopes of two neutralizing Abs, anti-HT1 pAb and TN1 mAb, were localized in amino acid sequences D⁸ to Q²⁸ and A⁶⁰ to R¹¹⁷, respectively, in the N-terminal domain of hTPO (Table 1, Fig.3). In the neutralization assay (Table 1, Fig.1), anti-HT1 and TN1 neutralized the TPO-mediated cell growth in a dose-dependent fashion through their specific binding to rhTPO. Therefore, we predict that these regions of hTPO are involved in binding to the receptor, c-Mpl. This notion was supported by the results showing that these two neutralizing Abs could inhibit the binding of biotinylated rhTPO to its receptor (Fig.2). Contrary to this, one of the nonneutralizing Abs, ET1A mAb, specifically bound to the HT1 peptide, which was recognized by the neutralizing Ab anti-HT1 (Table 2). No neutralizing activity of ET1A was detected even at concentrations exceeding its K_d (dissociation constant) concentration by 1,000-fold (data not shown). Therefore, the lack of neutralizing activity was not apparently due to the relatively low affinity of this Ab for rhTPO. Although the differences in the neutralizing abilities of these two Abs cannot be explained at this time, ET1A mAb is likely not to recognize the receptor binding site in the hTPO region, represented by peptide HT1. On the other hand, despite their relatively high binding activity toward rhTPO, the other pAbs and mAbs recognizing specifically peptides HT2 through HT6 showed no inhibition of the biological activity of rhTPO (Tables 1 and 2). In addition, the immune complexes comprising the biotinylated rhTPO and the nonneutralizing Abs could bind to c-Mpl expressed on the cell (Fig.2). Therefore, the five regions including amino acid sequences S⁴⁷ to D⁶², L¹⁰⁸ to A¹²⁶, N¹⁷² to A¹⁹⁰, S²⁶² to T²⁸⁴, and P³⁰⁶ to G³³² in hTPO may not be involved in binding to its receptor.

In their mutagenesis study, Kenneth et al. indicated that multiple amino acid residues of hTPO, including D⁸ and K¹⁴ within helix A and K⁵² and K⁵⁹ within helix A-helix B loop, were important for its receptor binding [21]. These findings are partially consistent with our results, as the peptide HT1, which corresponds to helix A, could induce the neutralizing pAb. In contrast to their results that the two residues K⁵² and K⁵⁹ of hTPO were vital for its binding, our data showed that both pAb and mAb against peptide HT2 (S⁴⁷ to D⁶²) inhibited neither biological activity nor receptor binding. On the other hand, the binding region (A⁶⁰ to R¹¹⁷) of the other neutralizing mAb TN1 could correspond to helix B through helix C. According to previous immunochemical and in vitro mutagenesis studies on erythropoietin and IL-4, helix C of each of these proteins is one of the important domains for its bioactivity and its binding to the receptor, but helix B is not [29, 30, 33, 34]. Although our study did not show the precise epitope of TN1, these findings suggest that the predicted helix C of hTPO is one of the active domains. A definite conclusion must await extensive analysis of, for example, the crystal structures of TPO and c-Mpl.

These Abs characterized in this study should also be useful for future studies, as described below. First, anti-idiotype Abs raised against neutralizing Abs may bind to the receptor directly and provide a means to study the binding region(s) of c-Mpl. Second, the nonneutralizing Abs provide valuable probes for detection of the hTPO-huMpl complexes on the cells. Finally, although previous studies have demonstrated that there is not only the full-length form of endogenous hTPO, but also several truncated ones [31, 35], the site-specific Abs should be useful immunochemically to investigate various forms of the protein regarding the regulatory mechanisms of their generation or their secretion from hTPO-expressing tissues.

	Acknowledgments

 
We would like to thank Eiko Shimizu, Kazumi Fuju, Atsuko Kokubo and Hikaru Tsunakawa for their excellent technical support. We also wish to thank Drs. Tadashi Sudo, Katsuhiko Asano, and Jun-ichi Tanaka for their support.

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