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SCIENCE IN CHINA (Series C) VOI. 40 NO. 1 February 1997 Construction of a recombinant human GM-CSF/MCAF fusion protein and study on its in vitro and in vivo antitumor effects and HUANG Cuifen (%%%) (Beijing Institute of Biotechnology, Beijing 100071, China) Received April 2, 1996 A novel cytokine fusion protein was constructed by fusing granulocyte macrophage colony stimulatAbstract ing factor (GM-CSF) with monocyte chemotactic activating factor (MCAF), which acts as a factor directing effector cells (monocytes) to a target site. The recombinant human GM-CSF/MCAF fusion protein could sustain the growth of GMCSF-dependent cell line TF1 and was chemotactic for monocytes. The in vitro antitumor effect showed that rhGM-CSF/MCAF could activate monocytes to inhibit the growth of several human tumor cell lines, including a promyelocyte leukemia cell line HL-60, a lung adenocarcinoma cell line A549, a hepatoma cell line SMMG7721 and a melanoma cell line Bowes. Furthermore, the cytotoxicity of monocytes activated by rhGM-CSF/MCAF against HL-60 and A549 was greater than that activated by GM-CSF or MCAF alone, even greater than that activated by a combination of GM-CSF and MCAF, suggesting that the fusion protein has synergistic or enhanced effects. The i n w i anti~ tumor effect indicated that rhGM-CSFIMCAF had marked antitumor effect against A549 tumor in nude mice and even completely suppressed tumor formation. rhGM-CSF/MCAF was significantly more effective in inhibiting tumor growth than rhGM-CSF. Histological analysis showed that tumor site injected with rhGM-CSF/MCAF was infiltrated by a large number of monocytes while a sparse infiltration of monocytes was observed at the tumor site injected with rhGM-CSF or normal saline, suggesting that the antitumor effect of rhGM-CSF/MCAF was mediated by the recruitment of a large number of monocytes to the tumor site. Keywords: GM-CSF, MCAF, gene cloning, fusion protein, antitumor. Studies on cytokines are becoming a hot subject in gene engineering. Researchers pay much attention to the pleiotropic biological activities of the cytokines. Cytokines with similar or complementary biological effects have been used to design cytokine fusion proteins, which exert their enhanced effects through host immune system["21. However, a common problem is that the limited number of immunoregulatory cells (effector cells) activated by the previously constructed fusion proteins to a target site may result in a low therapeutic effect and high systemic toxicities. It has been demonstrated that macrophages and granulocytes play an important role in inducing immune respose and in modulating immune response, i. e. macrophages can kill tumor cells by contact-mediated direct cytolysis and granulocytes play a major role in enhancing host immunity. To overcome the problem as mentioned above and maximize the biological effects of the cytokines, we constructed a novel fusion protein for the first time by fusing GM-CSF with MCAF, which acts as a factor directing effector cells to the target site, in an attempt to have synergistic and enhanced antitumor effects through such immune cells as macrophages. In the GM-CSF/MCAF fusion pro- * Department of Oncology, North Taiping Road Hospital, Beijing 100850, China. No. 1 A RECOMBINANT HUMAN GM-CSF/MCAF FUSION PROTEIN 19 tein, MCAF may activate monocytes, attract peripheral blood monocytes to the target site and then differentiate them into m a ~ r o ~ h a ~ e sand [ ~ ]GM-CSF , may further stimulate the proliferation and differentiation of macrophages and granulocytes[41. Therefore, it is possible to enhance host immunity and biological effects of macrophages and granulocytes. Moreover, the presence of MCAF in the GM-CSF/MCAF fusion protein is capable of enhancing the functional specificity for monocytes and of increasing the number of the effector cells at the tumor site, so the GM-CSF/ MCAF fusion protein may have an advantage over the previous cytokine fusion proteins. 1 Materials and methods 1 . 1 Materials 1 . 1 . 1 Plasmid and bacterial strain. Plasmid vector pBV220 was kindly provided by the Chinese Academy of Preventive Medical Sciences. E . coli DH5 a was maintained in our laboratory. 1 . 1 . 2 Reagents. Restriction endonucleases, T4 DNA ligase and so on were purchased from Promega and Sino-American Biotechnology Company. Primers were synthesized by our laboratory. GM-CSF antibody was a kind gift from Prof. Shen at Beijing Institute of Basic Medical Sciences. MCAF antibody was a generous gift from Dr. Rollins at Harved Medical School. Reference sample GM-CSF was purchased from GIBCO/BRL. Reference sample MCAF was purchased from Promega. Purified rhGM-CSF was a kind gift from Prof. Shen. Sephadex G-75 and CMSepharose FF were purchased from Pharmacia. Cell culture media were purchased from GIBCO/ BRL. Leukocytes were purchased from the 307th Hospital of P . L. A. 1 . 1 . 3 Cell lines. Human promyelocyte leukemia cell line HL-60 was a kind gift from Wang et a1 . at Beijing Institute of Radiation Medicine. Human lung adenocarcinoma cell line A549 was a kind gift from Prof. Li et aL. at Beijing Institute of Microbiology and Epidemiology. Human hepatoma cell line SMMC-7721 was kindly supplied by Tianjin Institute of Hygiene and Environmental Medicine. Human stomach cancer cell line Kato3 was a kind gift from Prof. Shen et al . at Beijing Institute of Basic Medical Sciences. Human melanoma cell line Bowes was a kind gift from Prof. Huang et a1 . at Beijing Institute of Biotechnology . 1 . 1 . 4 Animals. Balb/c nude mice, 4-6 weeks of age, were purchased from Institute of Oncology, Chinese Academy of Medical Sciences and maintained in laminar flow hoods under pathogenfree, temperature- and moisture-stable conditions. 1 . 2 Methods 1.2. 1 DNA recombination technology. Extraction of plasmid, digestion, isolation, ligation, transformation, identification, PCR, DNA sequencing and so on were performed as described in reference [ 51 . 1 . 2 . 2 Identification of the expressed product in E . coli . E . coli cells harboring recombinant plasmid pGMOl were grown to an OD600 of 0.4-0.5 in LB medium at 3 0 C , and then induced-at 42C for 4-5 h . The cells were harvested by centrifugation. SDS-PAGE and Western blot analyses were performed as described in reference [ 5 ] . 20 SCIENCE IN CHINA (Series C ) Vol. 40 1 . 2 . 3 Purification of rhGM-CSF/MCAF. rhGM-CSF/MCAF was expressed as inclusion bodies in E . coli. The inclusion bodies were solubilized in a solution containing 6 mol/L guanidine hydrochloride, renatured by dialysis, and purified by Sephadex G-75 gel filtration and CMSepharose FF ion exchange chromatography. The biological activity of GM-CSF was 1 . 2 . 4 Biological detection of rhGM-CSF/MCAF. examined by an M T T incorporation assay using GM-CSF-dependent cell line T F ~ ' ~ The ] . monocyte chemotactic activity of MCAF was measured by an agarose plate assayL7]. 1 x lo4 tumor cells were added to each 1 . 2 . 5 Cytotoxicity of rhGM-CSF/MCAF in vitro . well of 96-well culture plates. Serially diluted samples were then added. After the plates were incubated at 37°C in a humidified atmosphere containing 5 % C Q in air for 66 h, the cytotoxicity was determined by the MTT incorporation assayL6]. After the treat1 . 2 . 6 Monocyte tumoricidal activity by rhGM-CSF/MCAF in vitroL8]. ment of monocytes in vitro for 12 h with the serially diluted samples, tumor cells were added. The other relative procedures are the same as described in section. 1 . 2. 5 . 1 . 2 . 7 Antitumor effect of rhGM-CSF/MCAF against human lung adenocarcinoma cell line A549 in vim. Nude mice were divided into 7 groups, 4 in each group. Group A: A549 cells + normal saline ; group B : A549 cells + 1 pg rhGM-CSF/MCAF; group C: A549 cells + human leukocytes; group D: A549 cells + human leukocytes + 1 pg rhGM-CSF; group E: A549cells + human leukocytes + 0 . 1 pg rhGM-CSF/MCAF; group F: A549 cells + human leukocytes + 1 pg rhGM-CSF/MCAF; group G: A549 cells + human leukocytes + 0 . 1 pg rhGM-CSF/MCAF. 8 x lo5 A549 cells were injected subcutaneously into the right back of nude mice. Treatment was initiated on the next day after tumor cell inoculation. Mice were injected with 1 x lo7-1. 5 x lo7 human leukocytes four consecutive times per week for two weeks. The size of tumors was measured and tumor ratios were determined in two monthsLgJ. Mice were killed, and the injection sites and the other re1 . 2 . 8 Histological examination. quired sites were fixed in 10 % formalin. Standard dehydrating, paraffin-embedding,. sectioning and staining with hematoxylin and eosin were performed. 2 Results 2 . 1 Construction of recombinant plasmid directing the expression of rhGM-CSF/MCAF fusion protein We first obtained human GM-CSF gene and human MCAF gene using PCR technology. In order to clone DNA easily, endonuclease sites were introduced into primers. The upstream and downstream primers used to amplify GM-CSF gene contain EcoRI and XbaI sites, respectively. A DNA fragment containing GM-CSF gene was amplified by PCR with plasmid pcD-hGM-CSF as a template['01. The upstream and downstream primers used to amplify MCAF gene contain XbaI and BamHI sites, respectively. A DNA fragment containing MCAF gene was amplified by PCR with plasmid pMCOl as a template[111.The former fragment was digested with EcoRI and XbaI, and the latter was digested with XbaI and BamHI. Both of the digested fragments were ligated to pBV220 digested with EcoRI and BamHI. E . coli DH5a was transformed with the above con- No. 1 A RECOMBINANT HUMAN GM-CSF/MCAF FUSION PROTEIN 21 struct. Transformants were screened by in situ hybridization and DNA digestion analysis. The resulting recombinant was designated DH5a (pGMO1). With extracted pGMOl plasmid as a template, the coding sequence for rhGM-CSF/MCAF was determined by the Sanger dideoxy chain termination method. The result was the same as predicted (data not shown). For the construction of pGMO1, the coding sequence for GM-CSF was not directly ligated with the coding sequence for MCAF but with a linker sequence coding for four amino acids so as to maximize the opportunity for GM-CSF and MCAF to fold into their native three-dimensional structures and retain their biological activities respectively (The linker sequence was designed according to the respective GM-CSF and MCAF three-dimensional structures by computer-aided analysis and patented). 2.2 Expression in E . coLi , purification and immunological detection of rhGM-CSF/MCAF E . coli DH5a cells harboring recombinant plasmid pGMOl were grown at 30C and then induced at 4 2 C . The cell pellets were collected and lysed for SDS-PAGE (fig. 1 ( a ) ) . DH5a (pGMO1) expressed a 26-ku molecular weight of GM-CSF/MCAF at a level of 3 3 . 4 % of total cell protein by densitometric scanning. GM-CSF/MCAF was expressed as inclusion bodies. After the purification by Sephadex G-75 gel filtration and CM-Sephrose F F ion exchange, electrophoretically pure GM-CSF/MCAF was obtained. Western blot analysis indicated that the expressed product specifically reacted with GM - CSF and MCAF antibodies respectively ( figure l(b)). 94 ku 67ku45 ku- 21 k u - Fig. 1. SDSPAGE and Western blot analysis of rhGM-CSF/MCAF. (a) SDSPAGE; (b) Western blot with GM-CSF antibody; ( b ) ' Western blot with MCAF antibody. Lane 1 , Protein molecular weight marker; lane 2, total cellular lysate of E . coli harboring pGMO1; lane 3, inclusion bodies of rhGM-CSF/MCAF; lane 4, purified rhGM-CSF/MCAF by Sephadex G-75; lane 5, purified rhGM-CSF/MCAF by CM-Sepharose FF. 2.3 Preliminary biological detection of rhGM-CSF/MCAF Based on human GM-CSF-dependent cell line TF1, GM-CSF/MCAF and reference sample GM-CSF had specific activities of 1 . 8 x lo7 and 8 . 0 x lo6 u/mg respectively. Both of the minimum concentrations of GM-CSF/MCAF and reference sample MCAF for chemotactic activities determined by the agarose plate analysis were 40 ng/mL. These results show that GM-CSF/ MCAF retains higher biological activities. 22 2.4 SCIENCE IN CHINA (Series C) Vol. 40 Tumoricidal activity of rhGM-CSF/MCAF in vitro We first examined whether GM-CSF/MCAF had a direct cytotoxic effect on several human tumor cell lines, including a promyelocyte leukemia cell line HL-60, a lung adenocarcinoma cell line A549, a hepatoma cell line SMMC-7721, a melanoma cell line Bowes and a stomach cancer cell line Kato3. The result indicated that GM-CSF/MCAF did not have any direct cytotoxic effect on these tumor cells at concentrations ranging from 0.002 to 20 ng/mL. In view of this, we examined whether GM-CSF/MCAF stimulated human monocytes to be growth-inhibitory for these tumor cells. As shown in table 1, GM-CSF/MCAF could activate monocytes to lyse these tumor cells at concentrations ranging from 0.002 to 2 ng/mL, and the tumoricidal activity was directly proportional to the concentration. The cytotoxicity of monocytes activated by GM-CSF/MCAF against HL-60 and A549 was greater than that activated by GM-CSF or MCAF alone, even greater than that activated by a combination of GM-CSF and MCAF ( p < 0 . 0 1 ) . However, GM-CSF/ MCAF did not enhance lytic activity against SMMC - 7 7 2 1 and Bowes as mentioned above ( p > 0.05) and appeared to have no lytic activity against Kato3. The cytotoxicity of monocytes activated by GM-CSF/MCAF, GM-CSF, and the combination of GM-CSF and MCAF against some tumor cells (A549, SMMC7721) was not increased but decreased at concentrations in excess of the appropriate doses. The reason for this is not clear. Table 1 Cytotoxicity of monocytes activated by rhGM-CSF/MCAF Cytotoxicity ( $6 ) CeU line Concentration /ng-mL-' GM-CSF MCAF GM-CSF + MCAF GM-SCF/MCAF SMMC -7721 20 2 0.2 0.02 0.002 10.7i1.7 20.1 1 . 6 6.9k0.6 0 0 32.1k2.9 41.5k1.9 31.7k 1.7 43.4f 2.4 28.3+1.1 36.0k1.9 22.6k2.2 3.8k2.2 0 25.2k3.3 10.7f 1.7 0 17.0k3.9 4.4 0.6 0 0.002 0 0 9.9k1.4 11.9k1.8 + + Data are expressed as means k SE of triplicate samples. 2 . 5 Antitumor effect of rhGM-CSF/MCAF against human lung adenocarcinoma cell line A549 in vivo As described above, rhGM-CSF/MCAF could activate human monocytes to lyse A549 cells A RECOMBINANT HUMAN GM-CSF/MCAF FUSION PROTEIN No. 1 23 strongly in vitro, so we chose A549 to study the antitumor effect in nude mice. It has been reported that chemotaxis induced by human.MCAF on murine monocytes is weaker than that induced by human MCAF on human monocytes, and human GM-CSF is completely inactive on murine hematopoietic cells'49''I. T o allow GM-CSF and MCAF, especially GM-CSF in GMCSF/MCAF to exert antitumor effect, human leukocytes need to be injected. The tumor ratios in various groups of nude mice are listed in table 2. For tumor-bearing mice, the growth rates of tumors in various groups are shown in figure 2. Table 2 T u m o r ratios in various arouDs of nude mice Group No. of tumor cells injected Tumor ratio A: normal saline 5 x 10" 4/4 B: 1 pg rhGM-CSF/MCAF 5 lo8 2/4 C: human leukocytes + normal saline 5 x 10" 4/4 D: human leukocytes + 1 pg rhGM-CSF 5 x lo8 3/3 E: human leukocytes + 0 . 1 pg rhGM-CSF 5 X 10" 3/3 F: human leukocytes + 1 pg rhGM-CSF/MCAF 5 x lo8 2/4 G: human leukocytes + 0 . 1 pg rhGM-CSF/MCAF 5 lo8 4/4 A-G X X One of the mice in group D died 6 d after treatment. One of the mice in group E died 8 d after treatment. Tumors in groups appeared 17, 25, 17, 17, 17, 23 and 17 d, respectively, after inoculation. These results showed that no matter whether leukocytes were injected, 1 pg rhGM-CSF/MCAF 900, 800 - had marked antitumor effect and even completely 700 suppressed tumor formation ( p 0 . 05 ) . Although 600tumor still grew in some nude mice treated with 1 pg 500rhGM-CSF/MCAF, the tumor growth was signifi- 7 cantly suppressed as compared to the relative control ' groups ( p 0 . 0 5 ) . In the groups treated with nor- + 300200 ma1 saline, the tumors grew faster in nude mice injected with human leukocytes than in nude mice re100 ceiving no human leukocytes at the beginning ( 3 0 d, o 10 20 30 40 M 60 70 p< 0 . 0 5 ) . However, as time went on, no signifiDap after inwulation cant difference was shown between the above two groups ( 6 0 d, p 0 . 0 5 ) . The reason for this may be Fig. 2 . Effect of rhGM-CSF/MCAF on the growth of A549 tumors in nude mice. A , Normal saline; that the immune system was changed after leukocytes 0,pg rhGM-CSF/MCAF; leukocytes + were injected into nude mice. In addition, there was no,al ,,line; a,leukocytes + lpgr h ~ ~ - ~ ~ leukono significant difference whether leukocytes were in- r ,leukocytes + 0. 1 pg rhGM-CSF; jected into nude mice treated with 1 pg rhGM-CSF/ 'yteS pg rhGM-CSF/MCAF; leukocytes + 0 . 1 pg rhGM-CSF/MCAF. MCAF. rhGM-CSF showed no marked antitumor effect in nude mice injected with human leukocytes ( p > O . 0 5 ) . < E %8 < - < .* +, + 2.6 Histological examination Mice were killed two months later. Histological analysis showed that no salivary gland, lung SCIENCE IN CHINA (Series C) Vd. 40 and other required tissue metastases were observed both in the control groups and in the experimental groups. The A549 tumor cells were columnar with large nucleus and in adenomatous array in the control groups (data not shown), and no tumor cells were observed under a microscope in the experimental groups which did not show visible tumors. To examine whether rhGM - CSF / MCAF can attract monocytes to the tumor site in v i m , 1 x lo6 A549 tumor cells were injected into the right back of nude mice 6 h before treatment with normal saline, 1 pg rhGM-CSF or 1 pg rhGM-CSF/MCAF around the tumor site. Mice were killed 24 h after the treatment and the injection sites were processed for histological examination. As shown in fig. 3, a sparse infiltration of monocytes and neutrophils was observed in tumor and loose connective tissues in the group treated with normal saline. The infiltration in the group treated with 1 pg rhGM-CSF was similar in composition to that in the group treated with normal saline. In contrast, a predominantly monocytic infiltration was present in tumor and loose connective tissues in the group treated with 1pg rhGM-CSF/MCAF. Fig. 3. Histological examination of A549 tumors in nude mce. (a) A sparse infitration of monocytea and neutmphils is present in the tumor and loose connective tissues. Larger hyperchmmic cells in the right of the f& are A549 cdb (normal saline, x 490) ; (b) similar to (a) ( 1 pg rhGM-CSF, x 490) ; (c) an abundant monocytic infiltration is pnsent in the tumor and loose connective tissues (1 pg rhGM-CSF/MCAF, x 490); (d) the monocytic infiltration of C at higher magnification ( x 980). 3 Discussion Recently, researchers have paid much attention to cancer immunotherapy because tumor is No. 1 A RECOMBINANT HUMAN GM-CSF/MCAF FUSION PROTEIN 25 eventually eradicated through host immune system, in which cellular immune response is the major host defense and killing system. Macrophages and granulocytes play an important role in inducing immune response and in modulating immune response, so the GM-CSF/MCAF fusion protein was constructed in an attempt to exert its effects such as antitumor effect by increasing the number of immunoregulatory cells at the target site. Our results show that rhGM-CSF/MCAF retains higher biological activities of GM-CSF and MCAF respectively. Namely, rhGM-CSF/MCAF can sustain the growth of GM-CSF-dependent cell line TF1 and is chemotactic for monocytes, suggesting that the biological activities of GMCSF and MCAF are compatible. Grabstein et aL. demonstrated that rhGM-CSF could activate monocytes to inhibit the growth of human melanoma cells and bladder carcinoma cells[131. Matsushima et a l . demonstrated that MCAF did not have any direct growth inhibitory activity on tumor cells, but MCAF could stimulate monocytes to be growth-inhibitory for several human tumor cell lines, including a melanoma cell line A375, a mammary cell line MCF7, a colon carcinoma cell line HT29, a rhabdomyosarcoma cell line HTB82 and a leiomyosarcoma cell line H T B ~ ~ ' ~ ] . Our results show that rhGM-CSF/MCAF does not have any direct growth inhibitory activity on tumor cells either, but it can stimulate monocytes to inhibit the growth of human promyelocyte leukemia cell line HL-60, human lung adenocarcinoma cell line A549, human hepatoma cell line SMMC-7721 and human melanoma cell line Bowes. Furthermore, the cytotoxicities of monocytes activated by rhGM-CSF/MCAF against HL-60 and A549 were greater than that activated by GM-CSF or MCAF alone, even greater than that activated by the combination of GM-CSF and MCAF, suggesting that the fusion protein has synergistic or enhanced effects. Because rhGMCSF/MCAF could activate monocytes to lyse A549 strongly in vitro, we chose A549 to study its antitumor effect in pude mice. The result showed that rhGM-CSF/MCAF had marked antitumor effect and even completely suppressed tumor formation. The antitumor effect was dose-dependent. The more the dose, the stronger the inhibitory effect. rhGM-CSF/MCAF was significantly more effective in inhibiting tumor growth than rhGM-CSF. T o observe whether rhGM-CSF/MCAF can stimulate and attract monocytes in nude mice, we injected rhGM-CSF/MCAF alone but not human leukocytes. Histological analysis showed that the tumor site injected with rhGM-CSF/MCAF was infiltrated by a large number of monocytes, but a sparse infiltration of monocytes was observed at the tumor site injected with rhGM-CSF or normal saline, suggesting that the antitumor effect of rhGM-CSF/MCAF was mediated by the recruitment of a large number of monocytes to the tumor site. Phase I clinical trial of GM-CSF indicated that, in cancer patients, GM-CSF could activate monocytes and enhance monocyte cytotoxicity[14]. In addition, GM-CSF can stimulate the proliferation of granulocytes. Although the role that granulocytes play in the inhibition of tumor growth remains unclear, granulocytes may have indirect inhibitory effect on Because human GM-CSF and human MCAF on human hematopoietic cells are more effective than those on murine hematopoietic cells, we predict that rhGM-CSF/MCAF will exert stronger effects such as antitumor, antiinfection effects with animals that are more relative to human as a model. These results suggest that it is possible to kill target cells by effector cells that are recruited to the target site by chemokines and that rhGMCSF/MCAF may become a candidate drug for cancer immunotherapy. 26 Vol. 40 SCIENCE IN CHINA (Series C) AeknowI-t The authors are very grateful to Duan Haiqing, Sui Yongiun and Wang Hengliang for their assistance in biological detection. References . 1 Curtis, B. M. Williams, D. E., Broxmeyer, H. 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