Highly Effective Recombinant Format of a Humanized IgG-like Bispecific Antibody for Cancer Immunotherapy with Retargeting of Lymphocytes to Tumor Cells

Abstract

We previously reported the marked in vitro and in vivo antitumor activity of hEx3, a humanized diabody (small recombinant bispecific antibody) with epidermal growth factor receptor (EGFR) and CD3 retargeting. Here, we fabricated a tetravalent IgG-like bispecific antibody with two kinds of single-chain Fv (scFv), i.e. humanized anti-EGFR scFv and anti-CD3 scFv, that contains the same four variable domains as hEx3, on the platform of human IgG1 (hEx3-scFv-Fc). hEx3-scFv-Fc prepared from mammalian cells showed specific binding to both EGFR and CD3 target antigens. At one-thousandth (0.1–100 fmol/ml) of the dose of normal hEx3, hEx3-scFv-Fc showed intense cytotoxicity to an EGFR-positive cell line in a growth-inhibition assay using lymphokine-activated killer cells with the T-cell phenotype (T-LAK cells). The enhanced antitumor effect was more clearly observed when peripheral blood mononuclear cells (PBMCs) were used as effector cells, indicating the utility of IgG-like fabrication. These results suggested that the intense antitumor activity is attributable to the multivalency and the presence of the fused human Fc, a hypothesis that was supported by the results of flow cytometry, PBMC proliferation assay, and protein kinase inhibition assay. Furthermore, the growth inhibition effects of hEx3-scFv-Fc were considerably superior to those of the approved therapeutic antibody, cetuximab, which recognizes the same EGFR antigen even when using PBMCs as effector cells. The high potency of hEx3-scFv-Fc may translate into improved antitumor therapy and lower costs of production because of the smaller doses needed. We previously reported the marked in vitro and in vivo antitumor activity of hEx3, a humanized diabody (small recombinant bispecific antibody) with epidermal growth factor receptor (EGFR) and CD3 retargeting. Here, we fabricated a tetravalent IgG-like bispecific antibody with two kinds of single-chain Fv (scFv), i.e. humanized anti-EGFR scFv and anti-CD3 scFv, that contains the same four variable domains as hEx3, on the platform of human IgG1 (hEx3-scFv-Fc). hEx3-scFv-Fc prepared from mammalian cells showed specific binding to both EGFR and CD3 target antigens. At one-thousandth (0.1–100 fmol/ml) of the dose of normal hEx3, hEx3-scFv-Fc showed intense cytotoxicity to an EGFR-positive cell line in a growth-inhibition assay using lymphokine-activated killer cells with the T-cell phenotype (T-LAK cells). The enhanced antitumor effect was more clearly observed when peripheral blood mononuclear cells (PBMCs) were used as effector cells, indicating the utility of IgG-like fabrication. These results suggested that the intense antitumor activity is attributable to the multivalency and the presence of the fused human Fc, a hypothesis that was supported by the results of flow cytometry, PBMC proliferation assay, and protein kinase inhibition assay. Furthermore, the growth inhibition effects of hEx3-scFv-Fc were considerably superior to those of the approved therapeutic antibody, cetuximab, which recognizes the same EGFR antigen even when using PBMCs as effector cells. The high potency of hEx3-scFv-Fc may translate into improved antitumor therapy and lower costs of production because of the smaller doses needed. Therapeutic antibodies have great potential in cancer immunotherapy. Approximately 20 therapeutic antibodies have been approved by the United States Food and Drug Administration and there are more than 150 additional antibodies in clinical development (1Reichert J.M. Rosensweig C.J. Faden L.B. Dewitz M.C. Nat. Biotechnol. 2005; 23: 1073-1078Crossref PubMed Scopus (631) Google Scholar). In attempts to accelerate the development of such drugs for use in cancer immunotherapy, problems with limited efficacy and high production costs have become so serious that attempts are now being made to construct highly effective and potent recombinant antibodies from human and/or humanized protein fragments (2Wu A.M. Senter P.D. Nat. Biotechnol. 2005; 23: 1137-1146Crossref PubMed Scopus (900) Google Scholar). These recombinant antibodies also may provide low production costs. Bispecific antibodies (BsAbs) 2The abbreviations used are: BsAbbispecific antibodyEGFepidermal growth factorEGFRepidermal growth factor receptorscFvsingle-chain FvT-LAKlymphokine-activated killer cells with T-cell phenotypePBMCperipheral blood mononuclear cellsADCCantibody-dependent cellular cytotoxicityCHOChinese hamster ovaryPBSphosphate-buffered salineMTS3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner saltmAbmonoclonal antibodyMAPKmitogen-activated protein kinaseFITCfluorescein isothiocyanate. 2The abbreviations used are: BsAbbispecific antibodyEGFepidermal growth factorEGFRepidermal growth factor receptorscFvsingle-chain FvT-LAKlymphokine-activated killer cells with T-cell phenotypePBMCperipheral blood mononuclear cellsADCCantibody-dependent cellular cytotoxicityCHOChinese hamster ovaryPBSphosphate-buffered salineMTS3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner saltmAbmonoclonal antibodyMAPKmitogen-activated protein kinaseFITCfluorescein isothiocyanate. are attractive formats for recombinant antibodies, because they can be designed to redirect T-cells toward tumor cells without the involvement of major histocompatibility complex, by cross-linking the antigens on the tumor cells and the CD3-T-cell receptor complex on the T-cells. In particular, BsAbs can redirect cytotoxic T-lymphocytes, which are the most potent killer cells in the immune system but cannot be engaged by monoclonal antibodies (mAbs) because T-lymphocytes lack Fcγ receptors (3Kufer P. Lutterbuse R. Baeuerle P.A. Trends Biotechnol. 2004; 22: 238-244Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 4Cao Y. Lam L. Adv. Drug Delivery Rev. 2003; 55: 171-197Crossref PubMed Scopus (77) Google Scholar). bispecific antibody epidermal growth factor epidermal growth factor receptor single-chain Fv lymphokine-activated killer cells with T-cell phenotype peripheral blood mononuclear cells antibody-dependent cellular cytotoxicity Chinese hamster ovary phosphate-buffered saline 3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt monoclonal antibody mitogen-activated protein kinase fluorescein isothiocyanate. bispecific antibody epidermal growth factor epidermal growth factor receptor single-chain Fv lymphokine-activated killer cells with T-cell phenotype peripheral blood mononuclear cells antibody-dependent cellular cytotoxicity Chinese hamster ovary phosphate-buffered saline 3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt monoclonal antibody mitogen-activated protein kinase fluorescein isothiocyanate. Epidermal growth factor receptor (EGFR) is overexpressed in a wide range of human malignancies, and its expression level is correlated with poor clinical outcome in patients with any of several cancers (5Fischer-Colbrie J. Witt A. Heinzl H. Speiser P. Czerwenka K. Sevelda P. Zeillinger R. Anticancer Res. 1997; 17: 613-619PubMed Google Scholar). BsAbs targeting CD3 and EGFR have been generated, and their efficacy in T-cell retargeting toward these antigens has been documented in several tumor models in vitro and in vivo (6Tosi E. Valota O. Negri D.R. Adobati E. Mazzoni A. Meazza R. Ferrini S. Colnaghi M.I. Canevari S. Int. J. Cancer. 1995; 62: 643-650Crossref PubMed Scopus (25) Google Scholar, 7Jung G. Brandl M. Eisner W. Fraunberger P. Reifenberger G. Schlegel U. Wiestler O.D. Reulen H.J. Wilmanns W. Int. J. Cancer. 2001; 91: 225-230Crossref PubMed Scopus (39) Google Scholar, 8Pfosser A. Brandl M. Salih H. Grosse-Hovest L. Jung G. Int. J. Cancer. 1999; 80: 612-616Crossref PubMed Scopus (25) Google Scholar, 9Reusch U. Sundaram M. Davol P.A. Olson S.D. Davis J.B. Demel K. Nissim J. Rathore R. Liu P.Y. Lum L.G. Clin. Cancer Res. 2006; 12: 183-190Crossref PubMed Scopus (79) Google Scholar). Therapeutically potent BsAbs have been prepared by fusion of hybrid hybridoma and by chemical conjugation; however, such classical methods have led to the formation of several nonfunctional analogs (10Bohlen H. Manzke O. Patel B. Moldenhauer G. Dorken B. Cancer Res. 1993; 53: 4310-4314PubMed Google Scholar). Advances in recombinant technology have made it feasible to generate smaller BsAbs, such as minibodies, tandem singlechain Fv (scFv), and diabodies, which generally consist of two VH and two VL domains from two different antibodies (3Kufer P. Lutterbuse R. Baeuerle P.A. Trends Biotechnol. 2004; 22: 238-244Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 11Kontermann R.E. Acta Pharmacol. Sin. 2005; 26: 1-9Crossref PubMed Scopus (114) Google Scholar, 12Shahied L.S. Tang Y. Alpaugh R.K. Somer R. Greenspon D. Weiner L.M. J. Biol. Chem. 2004; 279: 53907-53914Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). In comparison with classical BsAbs (e.g. in the F(ab′)2 form), small BsAbs are expected to have low immunogenicity, high tumor penetration ability, and high efficiency because of their physical proximity between lymphocytes and targeted tumor cells (13Wu A.M. Chen W. Raubitschek A. Williams L.E. Neumaier M. Fischer R. Hu S.Z. Odom-Maryon T. Wong J.Y. Shively J.E. Immunotechnology. 1996; 2: 21-36Crossref PubMed Scopus (194) Google Scholar). In fact, we previously determined the effectiveness of the VH and VL domains from EGFR monoclonal antibody 528 in cancer immunotherapy, and we constructed a humanized functional bispecific diabody, hEx3, retargeting lymphokine-activated killer cells with the T-cell phenotype (T-LAK cells) against EGFR-positive cell lines (14Hayashi H. Asano R. Tsumoto K. Katayose Y. Suzuki M. Unno M. Kodama H. Takemura S. Yoshida H. Makabe K. Imai K. Matsuno S. Kumagai I. Kudo T. Cancer Immunol. Immunother. 2004; 53: 497-509Crossref PubMed Scopus (37) Google Scholar, 15Asano R. Sone Y. Makabe K. Tsumoto K. Hayashi H. Katayose Y. Unno M. Kudo T. Kumagai I. Clin. Cancer Res. 2006; 12: 4036-4042Crossref PubMed Scopus (63) Google Scholar). However, the small molecular form, the removal of the Fc region, and the monovalent binding to two different antigens result in rapid clearance from the circulation, a lack of induction of antibody-dependent cellular cytotoxicity (ADCC), and low affinity, respectively. The steady overcoming of these three problems is advancing the usefulness of BsAbs as clinical reagents. To date, several kinds of IgG-like BsAbs with the human Fc portion have been constructed. Evaluation of their functions has revealed that BsAbs with the Fc portion is an attractive molecule as a therapeutic reagent because it can overcome the above-mentioned problems of diabodies (16Carter P. J. Immunol. Methods. 2001; 248: 7-15Crossref PubMed Scopus (144) Google Scholar, 17Marvin J.S. Zhu Z. Acta Pharmacol. Sin. 2005; 26: 649-658Crossref PubMed Scopus (80) Google Scholar, 18Lu D. Jimenez X. Zhang H. Atkins A. Brennan L. Balderes P. Bohlen P. Witte L. Zhu Z. J. Immunol. Methods. 2003; 279: 219-232Crossref PubMed Scopus (42) Google Scholar). One of the most applicable molecules is the scFv-IgG form, in which two different scFv are fused at the N termini of the CL and the CH1 domains of an IgG, respectively: (scFvA)-CL and (scFvB)-CH1-CH2-CH3. Coexpression of the two polypeptide chains in a single host results in the formation of a tetravalent bispecific molecule, Bs(scFv)4-IgG. Only homogeneous BsAbs are generated as a result of the natural heterodimerization between the CL and the CH1 domains (17Marvin J.S. Zhu Z. Acta Pharmacol. Sin. 2005; 26: 649-658Crossref PubMed Scopus (80) Google Scholar, 19Zuo Z. Jimenez X. Witte L. Zhu Z. Protein Eng. 2000; 13: 361-367Crossref PubMed Scopus (38) Google Scholar). Therefore, we selected the scFv-IgG form as an IgG-like bispecific antibody for efficient retargeting of cytotoxic T-lymphocytes to malignant cells. Here, we describe the construction of a novel Bs(scFv)4-IgG molecule, designated hEx3-scFv-Fc, from humanized anti-EGFR and anti-CD3 scFv (Fig. 1A). hEx3-scFv-Fc prepared from the supernatant of stably transfected Chinese hamster ovary (CHO) cells showed specific binding to both target cells and remarkably higher cytotoxicity to EGFR-positive cell lines than that of the diabody form of hEx3. This efficacy was well characterized by flow cytometry, peripheral blood mononuclear cell (PBMC) proliferation assay, and protein kinase inhibition assay. To our knowledge, this is the first report of the construction and potency of a Bs(scFv)4-IgG molecule retargeted to surface antigens on T-lymphocytes and tumor cells; this molecule may prove to be a highly attractive recombinant therapeutic antibody with the possibility of low cost production. Construction of Expression Vector for hEx3-scFv-Fc-The gene encoding the humanized anti-human CD3 antibody OKT3 scFv with VH-VL orientation, referred to as hOHL, was amplified from bacterial expression vector by the first PCR (15Asano R. Sone Y. Makabe K. Tsumoto K. Hayashi H. Katayose Y. Unno M. Kudo T. Kumagai I. Clin. Cancer Res. 2006; 12: 4036-4042Crossref PubMed Scopus (63) Google Scholar, 20Adair J.R. Athwal D.S. Bodmer M.W. Bright S.M. Collins A.M. Pulito V.L. Rao P.E. Reedman R. Rothermel A.L. Xu D. Hum. Antibodies Hybridomas. 1994; 5: 41-47Crossref PubMed Scopus (49) Google Scholar). The leader peptide for protein in mammalian cells was to the of the by the the gene encoding the humanized anti-human EGFR antibody 528 scFv with VH-VL orientation, referred to as was amplified from bacterial expression vector by the first PCR by a PCR to the leader peptide (15Asano R. Sone Y. Makabe K. Tsumoto K. Hayashi H. Katayose Y. Unno M. Kudo T. Kumagai I. Clin. Cancer Res. 2006; 12: 4036-4042Crossref PubMed Scopus (63) Google Scholar). expression for the and chains of hEx3-scFv-Fc were constructed (Fig. The expression vector of hEx3-scFv-Fc was constructed by the VH gene of humanized OKT3 expression vector on with by with and expression vector for the hEx3-scFv-Fc was constructed by the VL gene of the humanized OKT3 expression vector on with the by with and of and hEx3-scFv-Fc diabody was prepared from in as previously (14Hayashi H. Asano R. Tsumoto K. Katayose Y. Suzuki M. Unno M. Kodama H. Takemura S. Yoshida H. Makabe K. Imai K. Matsuno S. Kumagai I. Kudo T. Cancer Immunol. Immunother. 2004; 53: 497-509Crossref PubMed Scopus (37) Google Scholar, 15Asano R. Sone Y. Makabe K. Tsumoto K. Hayashi H. Katayose Y. Unno M. Kudo T. Kumagai I. Clin. Cancer Res. 2006; 12: 4036-4042Crossref PubMed Scopus (63) Google Scholar, R. Kudo T. Y. Makabe K. Hayashi H. Suzuki M. Tsumoto K. Kumagai I. J. PubMed Scopus Google Scholar, S. Asano R. Tsumoto K. S. Katayose Y. Kodama H. Yoshida H. Suzuki M. Imai K. Matsuno S. Kudo T. Kumagai I. Protein Eng. 2000; 13: PubMed Scopus Google Scholar). hEx3-scFv-Fc, cells were with of from vector and stably hEx3-scFv-Fc was by and The cells were to at in by The supernatant was and hEx3-scFv-Fc was from the supernatant by in a protein in with the by the The were into phosphate-buffered saline and with The of the antibodies was by and the was a and in at of PBMCs and were by of from a for the induction of cells, PBMCs were for at a cell of in with recombinant human by in a with OKT3 The cells were to and for in as reported previously H. Suzuki M. Katayose Y. M. Takemura S. Yoshida H. H. Asano R. M. Imai K. Y. Matsuno S. Kudo T. Immunol. PubMed Scopus Google Scholar). cells were first on with of of hEx3-scFv-Fc for being with they were to fluorescein antibody, for hEx3, anti-human Fc for hEx3-scFv-Fc, Fc for for on The cells were by flow (15Asano R. Sone Y. Makabe K. Tsumoto K. Hayashi H. Katayose Y. Unno M. Kudo T. Kumagai I. Clin. Cancer Res. 2006; 12: 4036-4042Crossref PubMed Scopus (63) Google Scholar). In vitro growth inhibition of cell lines was with a 3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt assay cell proliferation The target cells cells in of were on were to well removal of the by of cells cells) of recombinant antibodies were to a cell of of the cells for at well was with three to effector cells and target cells, and of of a of was to The were for at on a at a of inhibition of target cells was as R. Kudo T. Makabe K. Tsumoto K. Kumagai I. PubMed Scopus Google growth inhibition of target cells of of of of with was also removal of the used for the target cells, of cells hEx3-scFv-Fc and of were to for at with was as (14Hayashi H. Asano R. Tsumoto K. Katayose Y. Suzuki M. Unno M. Kodama H. Takemura S. Yoshida H. Makabe K. Imai K. Matsuno S. Kumagai I. Kudo T. Cancer Immunol. Immunother. 2004; 53: 497-509Crossref PubMed Scopus (37) Google Scholar). of recombinant antibodies was also by using a assay. In target cells were to at an cell of of recombinant antibodies were of at of supernatant were and for in a The cytotoxicity to cancer cells was as (14Hayashi H. Asano R. Tsumoto K. Katayose Y. Suzuki M. Unno M. Kodama H. Takemura S. Yoshida H. Makabe K. Imai K. Matsuno S. Kumagai I. Kudo T. Cancer Immunol. Immunother. 2004; 53: 497-509Crossref PubMed Scopus (37) Google of of PBMCs was in a cell proliferation assay system PBMCs in of were into well of in the presence of of recombinant for at reagent was and the cells for an additional The was to the assay for cells in was with a at a of S. Kudo T. Asano R. Suzuki M. Tsumoto K. Katayose Y. Kodama H. Yoshida H. S. H. Imai K. Matsuno S. Kumagai I. Cancer Immunol. Immunother. PubMed Scopus Google Scholar). of the of protein kinase was with a in with the was with without antibodies for and with at for cell were by and to by The mitogen-activated protein kinase was by using binding was by using antibody and the system E. L. W. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). of BsAbs from of mammalian expression and were into cells. The and in growth and these were with high in by protein (Fig. in clearly the of hEx3-scFv-Fc in the expression system (Fig. The of hEx3-scFv-Fc was a of of BsAbs in of BsAbs to targeted antigens was by flow The of hEx3-scFv-Fc for and EGFR-positive was than that of but also those of the (Fig. and Furthermore, hEx3-scFv-Fc to the cells transfected with EGFR in the same but to normal (Fig. and the binding of hEx3-scFv-Fc to and that hEx3-scFv-Fc recognizes the same as the of Cancer with and the of BsAbs with and the human Fc region, cancer growth inhibition was by assay. hEx3-scFv-Fc growth of the cell even at a dose than This was more lower than that by hEx3, indicating a marked in cytotoxicity by binding with for targeted antigen (Fig. PBMCs were as effector cells, hEx3-scFv-Fc also cancer cell growth more than hEx3. was cytotoxic at high because of the presence of T-cells in the cytotoxicity of hEx3-scFv-Fc the of PBMC by the Fc in hEx3-scFv-Fc and the potential of hEx3-scFv-Fc without molecules in (Fig. OKT3 and 528 can the Fc from the same human IgG1 as The growth inhibition by these was to at the low of at their were than that of hEx3, which (Fig. and In hEx3-scFv-Fc, with both and the human Fc region, showed even at a of because of the fusion of the two molecules and the Fc (Fig. To the of in the cytotoxicity of hEx3-scFv-Fc, we the effects of anti-CD3 and anti-EGFR in the presence of hEx3-scFv-Fc (Fig. and the of anti-CD3 anti-EGFR showed inhibition of antitumor The cytotoxicity was also by assay of cells with a low of (Fig. hEx3-scFv-Fc at showed cytotoxicity in an and the effect to a higher of hEx3, i.e. the diabody These results that hEx3-scFv-Fc cytotoxicity in an and that the of Fc and is for its of PBMCs by assay was to the proliferation of PBMCs in to recombinant antibodies (Fig. even at a high of because of its lack of the Fc region, humanized OKT3 and 528 IgG, with the Fc region, proliferation of PBMCs at doses of in to was also observed at the low of at this effect on the PBMCs because of anti-CD3 In the of hEx3-scFv-Fc we observed effective of PBMCs that was than the proliferation by OKT3 These results that the construction of hEx3-scFv-Fc led to in proliferation and that the antibody proliferation of PBMCs that was with that by of Protein by Bispecific IgG-like and and in the of cell growth and and are by a wide of such as growth and and Here, the inhibition effect of hEx3-scFv-Fc on of protein kinase was by with antibody (Fig. between hEx3, hEx3-scFv-Fc, and 528 was observed at a high of epidermal growth factor at low and 528 showed inhibition of of with those in T. A. H. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). at any of hEx3-scFv-Fc at and These results suggested that the of hEx3-scFv-Fc was for its inhibition of protein of hEx3-scFv-Fc with Therapeutic growth inhibition effects of hEx3-scFv-Fc and the approved therapeutic antibody were by assay in two kinds of cancer cell lines by using cells and PBMCs as effector cells. is a monoclonal antibody with for human EGFR as well as is in therapeutic use in the United States and is effective in EGFR-positive cancer G. J. R. 2005; PubMed Scopus Google Scholar, I. A. T. Biol. 2000; PubMed Scopus Google Scholar). In and for cancer showed activity (Fig. in the presence of however, it effect in the presence of cells. This is because cells there are with Fc (e.g. and Fc cells are to efficient In hEx3-scFv-Fc was highly effective in that its cytotoxicity was of the of effector This is because hEx3-scFv-Fc can cancer cells and lymphocytes its and can also its human Fc hEx3-scFv-Fc, with its and to immune functions such as and is a novel as a reagent in and/or BsAbs a human Fc region, as are antibody with high therapeutic potency in cytotoxic T-cells against tumor cells and Furthermore, they can be by using protein and have than small BsAbs such as however, the classical of IgG-like BsAbs by the fusion of two by in vitro chemical cross-linking from two antibodies to production. The BsAbs that are can be but is because the have such as molecular (10Bohlen H. Manzke O. Patel B. Moldenhauer G. Dorken B. Cancer Res. 1993; 53: 4310-4314PubMed Google Scholar). In have the of attractive IgG-like molecules human Fc and with for targeted antigen (17Marvin J.S. Zhu Z. Acta Pharmacol. Sin. 2005; 26: 649-658Crossref PubMed Scopus (80) Google Scholar, J.B. L.G. P. Protein Eng. 1996; PubMed Scopus Google Scholar, A.M. Zhu Z. A. L.G. P. Nat. Biotechnol. PubMed Scopus Google Scholar, Z. M. Hu M. B. J. Immunol. Methods. 2005; PubMed Scopus Google Scholar). The tetravalent BsAbs of the scFv-IgG form, selected from reported IgG-like BsAbs, was generated by of the two polypeptide i.e. (scFvA)-CL and in a single The natural heterodimerization between the CL and CH1 domains in vivo can the for of IgG-like BsAbs, which is a serious in classical methods Z. Jimenez X. Witte L. Zhu Z. Protein Eng. 2000; 13: 361-367Crossref PubMed Scopus (38) Google Scholar, D. Zhang H. D. A. Jimenez X. D. Balderes P. Liu M. Bohlen P. Witte L. Zhu Z. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). In construction of recombinant antibodies with IgG-like can by the of scFv and the Fc this the for antigen and bispecific binding to become J. Immunol. 1997; Google Scholar). with IgG, the scFv-IgG constructed from humanized anti-EGFR and anti-CD3 scFv, designated as hEx3-scFv-Fc, showed in and to targeted antigen by flow (Fig. and This that the constructed have that a in the activity of the binding and by and surface methods are in The more cytotoxicity of hEx3-scFv-Fc as with the diabody at doses in the of is to the of high and activity the presence of human this was supported by the results of PBMC proliferation assay and protein kinase inhibition assay. the of protein in may its lower we have that has to that of prepared from mammalian expression system In comparison with humanized OKT3 and 528 IgG, hEx3-scFv-Fc also more cytotoxicity than the These that the cross-linking of effector and target cells is more than T-cell by the anti-CD3 antibody and/or growth inhibition by the anti-EGFR hEx3-scFv-Fc cytotoxicity in an EGFR and and the of Fc and is in the molecule with These results are also supported by the comparison it its cytotoxicity with that of the approved therapeutic anti-EGFR antibody, We have reported the high antitumor activity of the diabody and humanized in vivo (14Hayashi H. Asano R. Tsumoto K. Katayose Y. Suzuki M. Unno M. Kodama H. Takemura S. Yoshida H. Makabe K. Imai K. Matsuno S. Kumagai I. Kudo T. Cancer Immunol. Immunother. 2004; 53: 497-509Crossref PubMed Scopus (37) Google Scholar, 15Asano R. Sone Y. Makabe K. Tsumoto K. Hayashi H. Katayose Y. Unno M. Kudo T. Kumagai I. Clin. Cancer Res. 2006; 12: 4036-4042Crossref PubMed Scopus (63) Google Scholar). In vivo of hEx3-scFv-Fc are now there was a report of a clinical of which is an that and CD3 and has an Fc portion binding to Fc This has been as a there is the development of human antibody and human antibody because of and by we have into the of and peptide hEx3-scFv-Fc with its human and humanized can be expected to the possibility of such to In we constructed and characterized a tetravalent IgG-like bispecific antibody, designated as hEx3-scFv-Fc, that has for targeted antigen and a human Fc that the induction of of the Fc also the of recombinant antibody by the use of a protein and is expected to a than that of the To our knowledge, this report is the first of the possibility of cancer by the use of a tetravalent to cells to cancer cells with high