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HYBRIDOMA Volume 24, Number 6, 2005 © Mary Ann Liebert, Inc.

Preparation and Characterization of a Monoclonal Antibody Against CKLF1 Using DNA Immunization with In Vivo Electroporation YINGYU CHEN,1 TING ZHANG,1 TING LI,2 WENLING HAN,2 YINGMEI ZHANG,2 and DALONG MA1

ABSTRACT Chemokine-like factor 1 (CKLF1) is a newly cloned human cytokine from PHA-stimulated U937 cells in our laboratory, which belongs to a novel gene family and has at least three alternative RNA splicing forms: CKLF2, CKLF3, and CKLF4. Former studies demonstrated that CKLF1 has a chemotaxis effect on different leukocytes both in vitro and in vivo. It can also stimulate the proliferation of mouse skeletal muscle cells and bone marrow cells. This study was designed to generate CKLF1-specific monoclonal antibody (MAb) for further exploration of its structure and function. Mice were immunized intramuscularly with naked plasmid DNA encoding CKLF1 gene with in vivo electroporation. Hybridomas were generated by the fusion of the spleenocytes to Sp2/0 myeloma cells. One hybridoma cell line designated as M4, which is stable in secreting antiCKLF1 MAb, was generated. It belongs to the IgG1 isotype and is specific for the unique C-terminal domain of CKLFs protein. The specificity of this antibody has been assessed by enzyme-linked immunosorbent assay (ELISA) and Western blot. Our results demonstrated that intramuscular injection of naked DNA encoding CKLF1 gene combined with in vivo electroporation is an effective and simple method to raise MAbs that can be used for basic and clinical research of CKLF1 protein.

INTRODUCTION

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HEMOKINES are potent chemoattractant proteins that display an ever-increasing spectrum of biological activity. They are small, secreted proteins that can be subdivided according to their NH2-terminal cysteine-motif into the CXC (-chemokine), CC (-chemokine), C (-chemokine), and CX3C (chemokine). Chemokine-like factor 1 (CKLF1) is a newly cloned human cytokine from PHA-stimulated U937 cells in our laboratory, whose expression is partially inhibited by IL-10.(1) It belongs to a novel gene family and has at least three alternative RNA splicing forms: CKLF2, CLKF3, and CKLF4. Among them, CKLF2 is the full-length cDNA product. All four isoforms of human chemokine-like factor contain a CC motif characteristic for the CC chemokine family. Our previous studies verified that human chemokine-like factor had multiple activities. Human CKLF1 is supposed to be a secreted protein and can attract human neutrophils, lymphocytes and monocytes in vitro. Moreover, it also stimulates the proliferation of murine skeletal muscle cells after in vivo administration of naked

CKLF1 DNA.(1) A single intramuscular injection of CKLF1 plasmid DNA into BALB/c mice could cause dramatic pathological changes in the lungs of treated mice. These changes included peribronchial leukocyte infiltration, epithelial shedding, collagen deposition, and proliferation of bronchial smooth muscle cells and fibrosis of the lung,(2) similar to the phenomena observed in chronic persistent asthma, acute respiratory distress syndrome. This implies that CKLF1 may play an important role in the pathogenesis of respiratory diseases. DNA immunization technology involves the inoculation of genetic materials into mammalian hosts, in the context of a suitable mammalian expression vector encoding the protein of interest, leading to in vivo expression and subsequently to the development of an antigen-specific antibody response.(3) Genetic immunization has been extensively tested in the past 10 years by a number of groups in vaccine research(4,5) and some reports concern production of monoclonal antibodies (MAbs).(6–8) In recent years, in vivo electroporation has attracted considerable attention because such technology significantly improved the efficiency of gene delivery.(9,10) Another advantage of DNA

1Laboratory 2Center

of Medical Immunology, School of Basic Medical Science, Peking University, Beijing, China. for Human Disease Genomics, Peking University, Beijing, China.

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immunization for antibody production is that a full spectrum of native epitopes of an antigen can be expressed in vivo, and better conformation or protein presentation can be achieved as compared with administration of recombinant protein expressed in vitro. In order to further characterize CKLF1, generation of CKLF1 MAbs is highly desirable. However, as the CKLF1 is a highly hydrophobic protein, the purification of native CKLF1 was unsuccessful. Here we report the generation of CKLF1 MAb by the DNA immunization approach instead of the conventional protein immunization strategy.

MATERIALS AND METHODS

moved from the selected mouse aseptically and was placed in 10 mL of 15% FBS–RPMI-1640 in a small Petri dish and infused with 10 mL of the same medium via a 5-mL syringe. The pooled spleen cells were washed and fused with the myeloma cell line Sp2/0 at a ratio of 5:1 with PEG1500 as previously described.(12,13) All reagents to be used for fusion were warmed to 37°C. Spleen cells and myeloma cells were mixed, centrifuged, and resuspended in 1 mL of 50% PEG1500 over 90 sec followed by serial additions of medium over 5 min. After centrifugation, fused cells were resuspended in HAT medium at a density of 1  106 cells/mL and plated on microtiter plates (100 L/well), which pre-plated mouse celiac macrophages as feeder cells. B-cell hybridoma clones secreting antibody were tested by indirect ELISA and expended for limiting dilution cloning.

Mice Eight-week-old female BALB/c mice housed under conventional conditions were used for DNA immunization and generation of MAb. Animal experiments were performed according to the Institutional Guidelines for Peking University Animal Care and Use Committee.

Plasmid construction To generate the CKLF1 eukaryotic expression vector pCDICKLF1, the coding region including the stop codon was amplified from the cDNA library of PHA-strimulated U937 cells using upstream primer (5-ATGGATAACGTGCAGCCGAAAAT-3) and downstream primer (5-TTACAAAACTTCTTTTTTTTCATG-3), then cloned into pGEM-T-easy Vector (Promega). The insert was released by cutting with EcoR1 and subcloned into the EcoR1 site of the mammalian expression vector pCDI, which replaces the Bg/II-KpnI fragment of pCDNA3 (Invitrogen) with the Bg/II-KpnI fragment of PCI.(11) The sense pCDI-CKLF1 expression vector was selected by restriction enzyme analysis and DNA sequencing. Plasmid preparations were performed by using EndoFree™ plasmid Giga Kits (Qiagen-tip 1000, Hilden, Germany), which contained a high percentage of supercoiled DNA (80%), and no RNA was detectable by gel electrophoresis.

DNA injection and electric-pulse delivery The BALB/c mouse skin overlying the anterior tibialis muscle was shaved, and a dose of 100 g of DNA in 100 L of 0.9% NaCl was injected into the anesthetized animal with a BD syringe. At defined times (1 min) after DNA injection, the twoneedle array electrodes (5 mm) were inserted into the muscle encompassing the DNA injection site to a depth of 2 mm. Four electric pulses of 20 msec in duration with 200-msec intervals at 80 V were delivered. Mice were immunized three times at 3-week intervals. The mice were bled from the tail vein, and the titers were checked by enzyme-linked immunosorbent assay (ELISA) at 2 weeks after each booster injection. The mouse with the highest antibody titer and the best sensitivity to CKLF1 was used for the fusion experiment.

Cell fusion and production of MAb Mouse myeloma cell line Sp2/0 was grown to concentrations of 4  105 to 8  105 cells/mL before use. The spleen was re-

ELISA analysis In all of the ELISA protocols, 100 L of reagents were used unless specified, and samples were assayed in triplicate. ELISA plates were coated with 2 g/mL OVA-CKLF-C27 conjugates (OVA-ALIYRKLLFNPSGPYQKKPVHEKKEVL) in coating buffer (0.1 M NaHCO3, pH 9.0). Plates were washed with PBS containing 0.05% Tween 20 (PBS-T) and blocked with 200 L of 3% BSA in PBS-T. For screening positive hybridomas, neat culture supernatants were added. After being washed three times, plates were incubated with HRP-conjugated anti-mouse IgG (Sigma) for 1 h. After washing off the secondary antibodies, OPD (Sigma) substrate was added and developed within 10 min. The detection of isotype of hybridomas was performed using antigen-mediated ELISA method (mouse MAb isotyping reagents from Sigma). Finally, optical density at 490 nm was determined on an EL-311SX ELISA Reader (Bio-Tek Instruments).

Ascites production and purification Ascites was produced in 10-week-old BALB/c mice. Each mouse was primed 1 week before i.p. injection of cells with 0.5 mL of incomplete Freund’s adjuvant (Sigma). Each mouse received 2  106 cells per 0.5 mL of serum-free medium. Ascites usually developed 7–14 days after injection. The IgG of the ascites was purified through a Protein G Sepharose™ 4 Flast Flow (Amersham Biosciences, Sweden) according to the manufacturer’s directions, and the purity was inspected by SDS-PAGE.

Western immunoblot Biosynthesis of recombinant CKLF1 lacking 17 N-terminal amino acid residues with a cell-free system by RTS500 (Roche Molecular Biochemicals, Mannheim, Germany) was carried out according to the manufacturer’s protocol and described previously.(14) Recombinant full-length CKLF1 protein containing 99 amino acids was also prepared using the Drosophila expression system (Invitrogen Life Technologies, Inc.) as described previously(15) following the protocol recommended by the manufacturer. For Western bloting, varieties of samples were separated on 15% SDS-PAGE. Then the separated proteins were transferred to nitrocellulose membranes (Hybond™, ECL™, Amersham Pharmacia, UK). After blocking in Trisbuffered saline containing 0.05% Tween-20 (TBS-T) and 5%

MAb AGAINST CKLF1 USING DNA IMMUNIZATION

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non-fat milk for 2 h at room temperature, membranes were incubated with mouse anti-CKLF1 MAb or rabbit anti-CKLF1 polypeptide antibody(16) overnight at 4°C. Blots were washed three times with TBS-T for 10 min each and then incubated with Alexa Fluor 780–labeled goat anti-mouse IgG and goat anti-rabbit secondary antibody (Odyssey) in the dark for 1 h at room temperature. Following another three washes in TBS-T for 10 min each, the IR-fluorophore on the membrane was excited at 780 nm, and the emission at 820 nm was quantitated in channel 800 of the LI-COR Infrared Imaging System (Odyssey, Lincoln, NE) and analyzed with odyssey software.

RESULTS Establishment of hybridoma cell line (clone M4) secreting anti-CKLF1 antibody Five mice received three doses of i.m. immunization with pCDI-CKLF1 naked DNA followed by in vivo electroporation. Among them, two mice produced antigen-specific antibody response with relatively high serum titers detected by indirect ELISA. Then they were killed and the spleens were removed and used for fusion with parental myeloma cells. The hybridoma superantants were first screened by ELISA for their capacity to bind CKLF-C27. One hybridoma cell line (clone M4) secreting specific antibody was isolated and subcloned by limiting dilution. This clone could stably produce MAb against CKLF1 after being cultured for 3 months in vitro. Therefore, clone M4 was selected for ascites production and used for the next experiments.

Characterization of MAb



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Indirect ELISA assay showed that the M4 MAb titers against CKLF1 in the ascite fluids reached 1:105. This antibody could specifically react with CKLF1-C27 polypeptide. In contrast, it could not cross-react with carrier protein such as OVA, BSA, and lystes of E. coli. The ELISA results were further confirmed by Western blot analysis, which revealed that MAb M4 was capable of detecting recombinant CKLF1 protein synthesized in a cell-free system and a Drosophila expression system (Fig. 1). The size of the protein band was approximately 14 kD, which is in good accordance with that of the polyclonal antibody of CKLF1.(16) M4 MAb was also found to be immunoglobulin subclass IgG1 as determined by antigen-mediated ELISA. Purification of ascites was performed by protein G affinity chromatography to 95% homogeneity as assessed by SDS-PAGE. These data showed that a strong specific response to a defined portion of a protein could be generated by DNA immunization.

DISCUSSION Several studies demonstrated that the in vivo electroporation approach drastically increased the i.m. gene transfer, resulting in a 2–4-log enhancement of gene expression.(17,18) However, the efficency of DNA immunization for antibody production varies with the nature of antigen and route of administration.(19) It has been shown that as little as 5 g of DNA encoding the

FIG. 1. Western blot analysis of recombinant CKLF1 synthesized with the cell-free system and Drosophila expression system. (A) Probed with CKLF1 MAb (clone M4). (B) Probed with rabbit anti-CKLF1 polyclonal antibody. Lanes 1 and 3 were blank plasmid as template, acting as negative control; lanes 2 and 4 were CKLF1 recombinant DNA as template. (C) Probed with CKLF1 MAb (clone M4). Lane 5 was concentrated supernatants transfected with pMT/V5-His-CKLF1; a notable band with apparent molecular mass of approximately 14 kD was observed; lane 6 was concentrated supernatants transfected with pMT/V5-His-blank, acting as negative control.

target antigen administered with gene gun could generate highaffinity MAbs successfully.(20) Recently, Moonsom et al.(21) demonstrated that intraspleenic immunization seems to be a very efficient route for the production of both polyclonal and monoclonal antibodies against cell surface proteins using plasmid DNA as the immunizing agent. However, intraspleenic immunization required surgical procedure and may cause mortality of the mice. CKLF1 is a novel gene that has been cloned in our laboratory and mapped on chromosome 16q22.1. CKLF is the fulllength cDNA product that encodes 152 amino acids and maintains four alternative RNA splicing forms: CKLF1, CKLF2, CKLF3, and CKLF4. Among them, C-terminal residue 73–99 (CKLF1-C27) of CKLF1 is identical with the other three isoforms. CKLF1 is a highly basic and hydrophobic protein consisting of 99 amino acids. It has a CC motif in its C-terminal region and exhibits potent chemotactic activity on a wide spectrum of leukocytes, both in vitro and in vivo.(1) Moreover, CKLF1 can stimulate hematopoiesis and skeletal muscle regeneration(1,22) and can also inhibit chondrocyte proliferation.(23) In this study, we demonstrated that i.m. DNA immunization with in vivo electroporation improved the efficiency of CKLF1specific antibody production mediated by CKLF1 gene immunization; subsequently, a CKLF1-specific MAb was successfully generated. Through a series of procedures such as immunization, fusion, and cloning, one hybridoma (i.e., 14 clone-producing MAb) was established, and it was shown that it performed well in a variety of assays, including ELISA and Western blot. It should be noted that M4 MAb against CKLF1 can also recognize CKLF2, CKLF3, and CKLF4. Our data indicated that our approach of using i.m. immunization of naked DNA is a relatively simple and efficient method to generate MAb targeting the CKLF-specific epitope. This MAb is a potentially useful tool for the structural and functional characterization of the CKLF1 protein.

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This work was supported by the National High Technology Research and Development Program of China (2001AA215061, 2002BA711A01) and the National Key Project for Basic Research (973, G20000569).

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Address reprint requests to: Yingyu Chen, Ph.D., M.D. Laboratory of Medical Immunology School of Basic Medical Science Peking University 38 Xueyuan Rd. Beijing 100083, P.R. China E-mail: [email protected] Received for publication July 22, 2005. Accepted for publication September 15, 2005.



ACKNOWLEDGMENTS

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CHEN AU1 Please define PHA. AU2 Please update ref. 15.