ANTICANCER RESEARCH 26: 1057-1064 (2006)

IgG Isotype Conversion of a Novel Human Anti-carcinoembryonic Antigen Antibody to Increase its Biological Activity

JIAN HUANG1, HIROTOMO SHIBAGUCHI1,2, JUN ZHAO2, NAIXIANG LUO1, MOTOMU KUROKI1,2, TETSUSHI KINUGASA1, YUMIKO HIROSE2, HIROMI YAMADA1 and MASAHIDE KUROKI1,2

1Department of Biochemistry, Faculty of Medicine and 2Molecular Oncology Center, Fukuoka University, Fukuoka, Japan

Abstract. Background: The IgG isotype of antibodies is very apoptosis induction by the blockade of receptor/ligand important for their biological functions such as complement- interactions, the targeted delivery of prodrugs, toxins, dependent cytotoxicity (CDC) and antibody-dependent cellular radioisotopes and chemotherapeutics to the engagement of cytotoxicity (ADCC). To increase the biological activity of a immune effector mechanisms, such as complement- novel human monoclonal antibody (C2-45) against dependent cytotoxicity (CDC) and antibody-dependent carcinoembryonic antigen (CEA), we tried to genetically convert cellular cytotoxicity (ADCC) (5, 6). its isotype from IgG4 to IgG1. Materials and Methods: VH and Tumor antigens are molecules which occur in or on VL genes were cloned from the parental antibody C2-45(IgG4) tumor cells and are either not demonstrable or are and inserted into the pAc-κ-CH3 expression vector which significantly less concentrated in normal tissues (7). There contained the constant region gene of human IgG1. The are many tumor antigens against which monoclonal recombinant gene was transfected into Sf9 insect cells to antibodies (mAbs) are used for both immunotherapy and produce recombinant protein. The resulting recombinant gene therapy of cancer (6). One of the major antibody- protein, designated C2-45(cIgG1), in the culture medium was recognized tumor antigens, used as the target of cancer purified by affinity chromatography and characterized for its therapy for various cancers including gastrointestinal cancer CEA binding activity and biological activity. Results: The (8), is the carcinoembryonic antigen (CEA). converted C2-45(cIgG1) retained the original antigen-binding For the efficient antibody therapy of cancer, fully human activity and showed significantly higher CDC and ADCC mAbs (HmAbs) are superior to mouse, mouse/human activities against CEA-expressing tumor cells than did the chimeric or humanized mAbs, because of their minimum original C2-45(IgG4). Conclusion: C2-45(cIgG1) may be immunogenicity to humans and their efficient collaboration useful for antibody-based immunotherapy of human CEA- with human effector cells (9). Recently, we generated fully expressing tumors. HmAbs to CEA using the KM mouse, which lacks the endogeneous mouse antibody genes instead carrying the The last decade has seen the successful development of human antibody genes (10). Among them, 22 HmAbs novel antibody-based therapeutics, either in the form of reacted with CEA but did not react with 3 other CEA- native antibodies or as conjugates with radionuclides and related cell adhesion molecules (CEACAMs), namely toxins (1-4). Antibody-based therapies can be designed to CEACAM1, CEACAM6 and CEACAM8. In addition, the specifically target tumor cells via the recognition of antigens affinity constants for the CEA of these HmAbs were overexpressed on their surface. The mechanisms of comparable to those of the previously prepared mouse antibody-based therapies range from antiproliferation and mAbs to CEA. The IgG isotype of antibodies, however, is very important for their biological functions such as CDC or ADCC. Human antibodies of the IgG1 and IgG3 isotypes can potentially support the effector functions of Correspondence to: Dr. Masahide Kuroki, Department of CDC and ADCC (2). In the 12 HmAbs examined, 8 were Biochemistry, Faculty of Medicine, Fukuoka University, 7-45-1 IgG4, 2 were IgG3, 1 was IgG2, and the other was IgG1. Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan. Tel: +81-92-801- The IgG1 antibody, designated C2-74(IgG1), exhibited 1011, Ext. 3240, Fax: +81-92-801-3600, e-mail: kurokima@fukuoka- u.ac.jp more potent biological activity against CEA-expressing tumor cells, compared to those obtained by the other IgG- Key Words: Isotype conversion, CEA, HmAb, CDC, ADCC, isotype HmAbs (10). In the present study, to increase the immunotherapy. biological activity of an anti-CEA HmAb of IgG4,

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Figure 1. Construction of the recombinant C2-45(cIgG1) gene.

designated C2-45(IgG4), with the highest affinity constant Prism Big Dye terminator cycle sequencing ready reaction kit (PE to CEA, an attempt was made to genetically convert its Applied Biosystems, Foster City, CA, USA) (12). isotype from IgG4 to IgG1. Gene transfection and protein expression. The recombinant pAc-κ- CH3 expression vector was co-transfected into Sf9 insect cells Materials and Methods (Invitrogen, OR, USA) with AcNPV baculovirus DNA using the BD BaculoGoldì Transfection Kit (Pharmingen, NJ, USA), and Gene cloning and construction of recombinant gene. The Vκ and VH the final infected cells were cultured in a spinner flask (13). The genes of C2-45 were cloned from the 45kHscFv gene, the cDNA for resulting IgG-isotype converted antibody, designated C2-45(cIgG1), the single-chain variable fragment of C2-45 (11), by polymerase chain was secreted into the cultured medium. reaction (PCR). Briefly, the Vκ gene was amplified using a forward primer (5’-AAGAGCTCATTGTGTTGACGCAGTCTCCA-3’) Cell culture. The transfected Sf9 insect cells were grown at 27ÆC flanking the Sac I cloning site and a reverse primer (5’-AGAAGCTT under air conditioning in Grace’s insect medium (Invitrogen) TGGCGCGCCTTTGATCTC-3’) flanking the Hind III site. The VH supplemented with 10% fetal calf serum (FCS), 100 U/ml of gene was amplified using a forward primer (5’-AACTCGAGCAG penicillin and 100 Ìg/ml of streptomycin. Then the cells were TGTGAGGTGCAGCT-3’) flanking the Xhol I site and a reverse adapted to Sf-900 II SFM (serum-free medium) for the production primer (5’-AAGCTAGCTGAGGAGACGGTGACCAG-3’) flanking of C2-45(cIgG1). Three human gastric cancer cell lines, MKN-45 the Nhe I site (Figure 1). After digestion with the respective (CEA-expressing), KATO III (CEA-expressing) and MKN-74 restriction enzymes, the Vκ and VH cDNAs were inserted into the (CEA-nonexpressing), were obtained from the Japanese Cancer corresponding sites of the pAc-κ-CH3 baculovirus expression vector Research Resources Bank (Tokyo, Japan) and maintained in (Progen Biotechnik GmbH, Heidelberg, Germany), which contains D-MEM (Sigma Chemical, St. Louis, MO, USA) supplemented the human Cκ and CÁ1 (CH1+CH2+CH3) genes, by using the T4 with 10% FCS and the antibiotics, at 37ÆC in a humidified ligation kit (Promega Co., Madison, WI, USA) (Figure 1). The atmosphere with 5% CO2 and 95% air. Chinese hamster ovary recombinant gene was characterized by DNA sequencing with an (CHO) cells expressing CEA (CHO-CEA) or CHO cells expressing Applied Biosystems Model 373A DNA sequencer using the ABI MK-1 (CHO-MK-1) were cultured in ·-MEM (Sigma Chemical)

1058 Huang et al: IgG Isotype Conversion of a Human Anti-CEA Antibody

Figure 2. Western blot analysis of the converted IgG1 antibody C2-45(cIgG1) and an original IgG1 antibody C2-74(IgG1). Both the antibodies were electrophoresed on 5-20% gel without reduction (A) and with reduction by 2-mercaptoethanol (2ME) (B). After blocking, the membrane was reacted successively with biotinylated goat anti-human IgG (H+L) and horseradish peroxidase-avidin D. The proteins on the membrane were visualized with ECL Western blotting detection reagents.

supplemented with 10% FCS and the antibiotics. All the cells were Flow cytometry. To confirm antibody binding to the tumor cells, harvested as previously described (14) and used in flow cytometry, flow cytometry was also performed, as described elsewhere (17). CDC or ADCC assays, respectively. Human peripheral blood lymphocytes were isolated from the blood of healthy volunteer CDC assay. The CDC activity of the purified antibodies was donors and cultured with IL-2 in RPMI1640 (Sigma Chemical) determined, as described in detail elsewhere (10). Briefly, target supplemented with 10% FCS and the antibiotics for 3 days to tumor cells (1x104/well) were seeded in wells of 96-well plates generate human lymphokine-activated killer (LAK) cells, as (Corning Inc., Corning, NY, USA). After being cultured for described previously (10). 48 hours, the cells were incubated in 100 Ìl of medium containing diluted antibodies and 20% fresh human serum for 2 hours at Antibody purification by affinity chromatography. The recombinant 37ÆC. Following washing, the remaining living cells were quantified antibody C2-45(cIgG1), its original antibody C2-45(IgG4) and using the colorimetric reagent Cell Counting Kit-8 (Dojindo C2-74(IgG1) were purified from the culture medium by affinity Laboratories, Kumamoto, Japan). The % cytotoxicity was chromatography using the column of CNBr-activated Sepharose 4 calculated as follows: % cytotoxicity = [(OD490 of target cells – Fast Flow (Amersham Biosciences AB, Uppsala, Sweden) OD490 of experiment)/(OD490 of target cells – OD490 of conjugated with goat anti-human IgG (Vector Laboratories, Inc., background)] x 100. Burlingame, CA, USA). ADCC assay. The ADCC activity of purified antibodies in the Western blotting. The purified antibodies were analyzed by SDS- presence of LAK cells was determined by the method of PAGE, followed by electroblotting onto nitrocellulose membranes, Naundorf et al. (18) with slight modifications, using the double as described previously (14). staining kit Cellstain (Dojindo Laboratories) containing calcein- AM and propidium iodide solutions, which stain viable and dead Human IgG activity. Quantitation of the human antibodies was cells, respectively. Briefly, target tumor cells (5x106) were labelled carried out by solid-phase enzyme immunoassay (SPEIA) using with 0.2 M calcein-AM for 15 minutes at 37ÆC in D-MEM/10% 96-well plates (Microlon 96K, Greiner, Frickenhausen, Germany), FCS. After washing with PBS, the cells were resuspended at a as reported previously (15). A purified human IgG1(κ) myeloma density of 5x106 cells/ml in RPMI1640/10% FCS. The LAK cells protein was used as the standard. were washed in PBS followed by RPMI1640/10% FCS and mixed with calcein-AM-labelled target cells at an effector/target (E/T) Reactivity with purified CEA in SPEIA. The reactivity of the ratio of 10:1. This was followed by the addition of 20 Ìl of serially antibodies with CEA was estimated by another SPEIA using purified diluted antibody solutions, resulting in the indicated CEA immobilized on 96-well plates, as reported elsewhere (16). concentrations. After reaction for 4 hours at 37ÆC, the cells were

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Reactivity of C2-45(cIgG1) to CEA-expressing cells. The binding of C2-45(cIgG1) to CEA-expressing cells was investigated by flow cytometric analysis (Figure 4). As expected from the reactivity in SPEIA, C2-45(cIgG1) kept almost the same binding activity to CEA-expressing MKN-45, KATO III and CHO-CEA cells as that of C2-45(IgG4), as shown by the high fluorescence intensity compared with the background level obtained with control human IgG. However, neither antibody showed any binding activity to CEA-non-expressing MKN-74 cells.

CDC activity of C2-45(cIgG1). To investigate the antitumor activity of C2-45(cIgG1), first its CDC activity was analyzed against CEA-expressing MKN-45 and CEA-non-expressing Figure 3. Reactivity of C2-45(cIgG1) with purified CEA in ELISA. MKN-74 cells in vitro. As shown in Figure 5A, C2-45(cIgG1) C2-45(cIgG1), C2-74(IgG1), C2-45(IgG4) or control human IgG was and C2-74 (IgG1) showed substantial and dose-dependent incubated with immobilized CEA on a 96-well plate for 1 hour at 37ÆC. lyses of the MKN-45 cells in the presence of human fresh After washing, the antibodies bound to CEA were detected with serum, but C2-45(IgG4) did not. These HmAbs did not biotinylated goat anti-human IgG. reveal any significant cell lysis against the MKN-74 cells (Figure 5B). collected by centrifugation and resuspended in FACS buffer ADCC activity of C2-45(cIgG1). The ADCC activity of containing 1 Ìg/ml propidium iodide. The cells were further human LAK cells against CEA-expressing MKN-45 cells incubated for another 15 minutes at 37ÆC and 2x104 cells were was compared among C2-45(cIgG1), C2-45(IgG4) and then analyzed by flow cytometry. Quantitation of cytotoxicity was C2-74(IgG1) at different antibody concentrations. As shown based on the number of live and dead target cells in the control in Figure 6A, the activity of C2-45(cIgG1) was significantly reaction without antibody. higher than that of both C2-45(IgG4) and C2-74(IgG1). The ADCC activity of the 3 antibodies against CEA-non- Results expressing MKN-74 cells was extremely low (Figure 6B).

Production of C2-45(cIgG1). The recombinant pAc-κ-CH3 Discussion expression vector was amplified through several rounds of infection into Sf9 cells to produce high-titer virus stocks that The IgG isotype of human antibodies is very important for were used to infect growing cultures for protein production. their biological functions such as CDC or ADCC (2). To The amount of C2-45(cIgG1) in the serum-free culture increase the biological activity of a novel human anti-CEA supernatant of the final infected cells was about 4,000 ng/ml antibody C2-45(IgG4) with the highest affinity constant to after a 3-day culture of 2x106 cells. CEA, its isotype was genetically converted from IgG4 to IgG1. The converted C2-45(cIgG1) antibody retained the Western blotting. The purified antibodies were analyzed by original antigen-binding activity and exhibited significantly SDS-PAGE followed by Western blotting. Under non- higher CDC and ADCC activity against CEA-expressing reducing conditions, C2-45(cIgG1) and C2-74(IgG1) gave a tumor cells than did the original C2-45(IgG4). band at a portion corresponding to the assembled protein CDC is initiated by complement component C1q binding of H2L2 and the molecular mass (150 kDa) of C2-45(cIgG1) to the Fc region of IgG, which is bound to the surface of a was similar to that of C2-74(IgG1) (Figure 2A). Under tumor cell. Subsequent target-cell killing can occur in a cell- reducing conditions, 2 bands appeared at positions dependent or cell-independent manner (2). The initiation corresponding to the H- and L-chains. The H- and L-chain of the complement system is one of the most important migration patterns of C2-45(cIgG1) and C2-74(IgG1) were effector mechanisms of IgG1 and IgG3 molecules (2, 19). almost the same (Figure 2B). However, IgG2 does not appear to initiate the complement system effectively, while IgG4 is also ineffective (19). In the Reactivity of C2-45(cIgG1) with purified CEA. When tested by present experiments in combination with human sera, SPEIA using purified CEA immobilized on 96-well plates, C2- C2-45(cIgG1) exhibited a very high CDC activity against 45(cIgG1), as well as its original antibody C2-45(IgG4) and CEA-expressing tumor cells, C2-74(IgG1) showed a C2-74(IgG1), retained their reactivity with CEA (Figure 3). moderate activity, but C2-45(IgG4) did not. This finding is

1060 Huang et al: IgG Isotype Conversion of a Human Anti-CEA Antibody

Figure 4. Reactivity of C2-45(cIgG1) with CEA-expressing cells in flow cytometric analysis. The cells used were incubated with C2-45(cIgG1), C2-45(IgG4) or control human IgG for 1 hour on ice. After washing, the cells were stained with fluorescein-conjugated rabbit anti-human IgG.

noteworthy because antibodies to CEA, including mouse antibody in vivo came with the development of mice that mAbs, have not been considered to be very effective for lack FcÁRI and FcÁRIII (1, 21). The activation of ADCC is CDC activity against CEA-expressing tumor cells (20). This also one of the important effector mechanisms of IgG1 and result also implies that the injection of HmAb alone, such IgG3 molecules (19). The importance of the Fc–Fc receptor as C2-45(cIgG1), might be effective for antibody therapy of interaction for antitumour activity was subsequently shown CEA-expressing tumors since the human complement for the clinically important antibodies Herceptin and system is always available in our bodies (10). Rituxan (1). The antitumour activities of Herceptin and Tumor cell killing by ADCC is triggered by the Rituxan were greatly reduced in mice that lacked the interaction between the Fc region of an antibody bound to activation receptors FcRI and FcRIII, whereas disruption a tumor cell and the FcÁ receptors on immune effector cells of the gene that encodes the inhibitory receptor FcRIIB such as neutrophils, macrophages and natural killer cells substantially enhanced the antitumour activity (1). The (2). The first demonstration that the Fc-FcÁ receptor antitumour activity of Herceptin was also attenuated by a interaction is important for the antitumor activity of an mutation (D265A) that impairs binding to FcRIII and

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Figure 5. CDC activity of C2-45(cIgG1) against CEA-expressing tumor cells. CEA-expressing MKN-45 cells (A) and CEA-non-expressing MKN- Figure 6. ADCC activity of C2-45(cIgG1) against CEA-expressing tumor 74 cells (B) were incubated with different concentrations of C2-45(cIgG1), cells. CEA-expressing MKN-45 cells (A) and CEA-non-expressing MKN- C2-74(IgG1), C2-45(IgG4) or control human IgG in the presence of 20% 74 cells (B) were incubated with LAK cells at an E/T ratio of 10:1 for 4 human fresh serum for 2 hours at 37ÆC. Cell lysis was determined by a hours in the presence of different concentrations of C2-45(cIgG1), C2- colorimetric assay, as described in the text. The values shown are the mean 74(IgG1), C2-45(IgG4) or control human IgG. Cell lysis was determined of triplicate samples ± SD. by flow cytometry, as described in the text.

FcRIIB. These studies indicated the potential for increasing In conclusion, the results indicated that C2-45(cIgG1) the antitumour activity of an antibody by manipulating the may be useful for the antibody-based immunotherapy of Fc region to increase its affinity for the activation human CEA-expressing tumors. receptor(s) and/or by abrogating its ability to bind to the inhibitory receptor. In this study, when tested in References combination with human LAK cells, C2-45(cIgG1) revealed 1 Clynes RA, Towers TL, Presta LG and Ravetch JV: Inhibitory a very strong ADCC activity against CEA-expressing tumor Fc receptors modulate in vivo cytotoxicity against tumor targets. cells, and C2-74(IgG1) also showed a fairly high activity. In Nat Med 6: 443-446, 2000. this context, it is of interest to note that mouse mAbs, even 2 Carter P: Improving the efficacy of antibody-based cancer if they have the highest affinity, showed only a slight ADCC therapies. Nat Rev Cancer 1: 118-129, 2001. activity with human LAK cells (22). The results of the 3 Wels W, Biburger M, Muller T, Dalken B, Giesubel U, Tonn T and Uherek C: Recombinant immunotoxins and retargeted ADCC analysis thus indicated that C2-45(cIgG1) may killer cells: employing engineered antibody fragments for exhibit an additive effect on LAK cell-mediated adoptive tumor-specific targeting of cytotoxic effectors. Cancer Immunol immunotherapy of CEA-expressing tumors. Immunother 53: 217-226, 2004.

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