ISSN: 0250-7005
`
`
`ANTICANCER RESEARCH
`
`International Journal of Cancer Research and Treatment
`
`Volume 26, Number 6A, November-December 2006
`Proceedings of the 9th Annual Meeting of the
`Society of Biotherapeutic Approaches,
`December3, 2005, Tokyo, Japan
`Special Issue Edited by
`H. Hori, T. Fukushima, M. Kuroki, G.-I. Soma
`
`Contents
`
`* The Therapeutic Potential of Immuno-cell Therapy of Cancer in Combination with Aminobisphosphonates.
`§. GOTO, A. NOGUCHI, H. JINGUJI, M. TAKAHARA (Tokyo, Japan) ........c.cccccesessssesesessesesesessssesseneesesees
`
`Identification and Characterization of Lipopolysaccharide in Acetic Acid Bacteria. Y. TANIGUCHI, T.
`NISHIZAWA, C. KOUHCHI, H. INAGAWA, T. YAMAGUCHI, S. NAGAI, A. TAMURA, G.-I. SOMA
`(Tokushima-shi; Noda-shi; Shimonoseki-shi; Himeji; Ito-gun; Takamatsu-shi, Japan) ...0..0.0.+0+0.s0s0-sessese00e0eeneseeee
`
`A Soluble 17 kDa Tumour Necrosis Factor (TNF) Mutein, TNF-SAM2, with Membrane-bound TNF-like
`Biological Characteristics. A. YOSHIDA, C. KOHCHI, H. INAGAWA,T. NISHIZAWA,H. HORI, G.-I.
`SOMA (Tokushima-shi; Noda-shi; Shimonoseki-shi, Japan) ..........c.cscssssssssssesssossesesseresesnsvesesesvsveseasavevensassssueneacs
`
`* Unique Molecular Characteristics of the Environmental Responses of Mucosal Macrophages. K. NAKATA,
`H.
`INAGAWA, T. NISHIZAWA, C. KOHCHI, Y. TANIGUCHI, N. YOSHIOKA, G.-I. SOMA
`NEMEC rRPOO UTERECAM, CGINEINNE, IIPIUEE ch ss odes on cs agow sae vuny pa aaestova taeaadveLsiaictcecby dicaedanienueacenpcaousiereas
`
`A Case of Recurrent Ovarian Cancer Successfully Treated with Adoptive Immunotherapy and Lentinan.
`Peel, M. TOMONAGA,S. GOTO (Fishtiokit, Japan) .o.cccccasesisssssanscssscesesdansonsevavtcaonsarsvencessensnsoiexene
`
`3989
`
`3997
`
`4003
`
`4009
`
`4015
`
`* Possible Applications of Antibodies or their Genes in Cancer Therapy. M. KUROKI, J. HUANG, H.
`SHIBAGUCHI, T. TANAKA, J. ZHAO, N. LUO, K. HACHIMINE, T. KINUGASA,S.-I. MAEKAWA,
`4019
`5. ENATSU, W. HAMANAKA,T. FUKAMI, M. KUROKI (Fukuoka, Japan) .....cccccccsssessssesesesesssssseseesesesseee
`Contents continued on the back cover
`
`74516)| Fae Page 1
`
`
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`i
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`Miltenyi Ex. 1019 Page 1
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`

`

`Contents continued
`
`Evaluation of Intratumoral Administration of Tumor Necrosis Factor-alpha in Patients with Malignant
`Glioma. $. OSHIRO, H. TSUGU, F. KOMATSU, H. OHNISHI, Y. UENO, S. SAKAMOTO,
`T. FUKUSHIMA, G.-I. SOMA (Fukuoka; Tokushima, Japan) ........cssssssssessesesseosssesessesssssssssesecsessesssssssseseesesecs
`
`Mechanisms of Small Globular Protein-induced Plasma Membrane Permeability and Cytotoxicity in
`U87-MG Human Malignant Glioblastoma Cells. H. ONISHI, S. JIMI, H. TSUGU,S. LEE, T. FUKUSHIMA
`DPRPRRRBMLL OREMEMEN 2cac5 ctsucincssu-csvusssuteedecscecdtsatesa ers esnsandagncoesestensatoonan ctaceren apaceteemermeesteers cetanneasier oevesstieneestaiic
`
`Anti-tumor Activity of De Novo Designed Small Globular Protein (SGP) In Vivo. H. TSUGU, H. ONISHI,
`TRS EeeRte, LE (PURO SONG) coresccscccccsssecsacsniszectoacscusecssessdueesexsoatininaniessates aoe entaversesetransescivonce>
`
`Expression of Tn and Sialyl-Tn Antigens in Endometrial Cancer: Its Relationship with Tumor-produced
`Cyclooxygenase-2, Tumor-infiltrated Lymphocytes and Patient Prognosis. S. OHNO, Y. OHNO, H. NAKADA,
`N. SUZUKI, G.-I. SOMA, M. INOUE(Ishikawa; Kyoto; Tokushima; Chiba, Japan) ....1....:0..ssssssssssesssvesssvessesesees
`
`Molecular Dynamics of Human-specific Cytolysin: Analysis of Membrane Binding Motif for Therapeutic
`Application. K. OHKURA, H. HORI, H. NAGAMUNE (Aichi; Tokushima, Japan) ........:.cccsescsssesssseseseeveoes
`
`A Bio-defensive Function Induced by Low-dose Lipopolysaccharide Pre-treatment Against CCl,-induced
`Fulminant Hepatic Failure in Rats. Y. UEDA, K. YABE, Z. TAIRA (Tokushima, Japan)...........0.0.000-000000-
`
`A Fully Human Chimeric Immune Receptor for Retargeting T-cells to CEA-expressing TumorCells.
`H. SHIBAGUCHI, N.-X. LUO, M. KUROKI, J. ZHAO, J. HUANG, K. HACHIMINE, T. KINUGASA,
`PB RTOSIRD (PRR, DONATED cncccnsnn cmesenrnssvenacoevnsnveacnsensnsesetaasteauanciezalttCuts past lenmanausiadasensesandcsgsesisansedeeniuenne
`
`The Role of Ge Protein Oligosaccharide Structure as a Risk Factor for COPD. K. OHKURA, H. NAGASAWA,
`Y. UTO, N. OKAMURA, A. MURAKAMI, H. HORI(Aichi; Tokushima, Japan) .......s0sccssssssescsesssssesssessveensesesaneens
`
`4027
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`4033
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`4043
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`4047
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`4055
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`4063
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`4067
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`4073
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`* Reviews (pages 3989, 4009, 4019)
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`Miltenyi Ex. 1019 Page 2
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`Miltenyi Ex. 1019 Page 2
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`

`

`ANTICANCER RESEARCH26: 4067-4072 (2006)
`
`A Fully Human Chimeric ImmuneReceptor for Retargeting
`T-cells to CEA-expressing TumorCells
`
`HIROTOMO SHIBAGUCHI!?, NAI-XIANG LUO!, MOTOMU KUROKI!”, JUN ZHAO?,
`JIAN HUANG!, KEN HACHIMINE!, TETSUSHI KINUGASA! and MASAHIDE KUROKI!
`
`'Department of Biochemistry, Faculty of Medicine and
`?Molecular Oncology Center, Fukuoka University, Fukuoka, Japan
`
`Miltenyi Ex. 1019 Page 3
`
`Abstract. Background: Recombinant chimeric immune
`receptors (CIRs) with anti-CEA specificity can retarget grafted
`T-cells to CEA-expressing tumors in an HLA-independent
`manner. To reduce the immunogenicity of conventional CIR in
`humans, an attempt was made to generate a CIR encodedbyall
`human genes. Materials and Methods; A single-chain variable
`fragmented (scFv) antibody gene was prepared from variable
`region genes of the C2-45 human mAb clone specific for CEA.
`The scFv gene was connected to a gene construct comprised of
`the cDNAs for the human CD8&a hingeregion, the human CD28
`transmembrane and cytoplasmic domains, and the human
`CD3€
`intracellular domain. The resulting human CIR gene,
`designated L45scFv-CIR, was inserted into the pcDNA3.1
`expression vector and transfected into human primary T-cells.
`Results: Flow cytometric analysis using allophycocyanin-labeled
`CEA demonstrated the expression of the L45scFv-CIR protein
`on the T-cells and its specific antigen binding activity.
`Conclusion: This L45scFv-CIR gene, consisting offour human
`genes, may be a useful tool for eradication of CEA-expressing
`but HLA-downregulated tumorcells.
`
`Most immunotherapeutic strategies targeting tumor cells
`aim to induce and enhance the number of tumor-specific T-
`lymphocytes in patients by peptide vaccination. Transfer of
`such tumor specific T-lymphocytes into patients can have
`clinical significance (1). However, the isolation of tumor-
`specific cytotoxic T-lymphocytes (CTLs) and their expansion
`to significant numbers for clinical application on an
`
`Correspondence to: Hirotomo Shibaguchi, Ph.D., Department of
`Biochemistry, Faculty of Medicine, Fukuoka University, 7-45-1
`Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan. Tel: +81-92-801-
`1011, Ext. 3247, Fax: +81-801-3600, e-mail: shiba-h@cis.fukuoka-
`u.ac.jp
`
`Key Words: Chimeric immune receptor, scFv, T-lymphocytes,
`electroporation,
`antibody-based
`therapy,
`carcinoembryonic
`antigen, CEA.
`
`0250-7005/2006 $2.00+.40
`
`
`
`individual
`
`basis
`
`is
`
`cumbersome
`
`and
`
`the
`
`outcome
`
`unpredictable (2-4). Furthermore, HLA class I molecules on
`tumorcells, essential for target recognition of CTL, are
`frequently
`down-regulated
`and/or
`lost
`during
`the
`development of malignancies (5, 6).
`Theclinical application of the chimeric immune receptor
`(CIR), as a mean to redirect the specificity of T-cells toward
`the cellular target of interest, holds great promise for the
`adoptive immunotherapy of cancer and infectious disease
`(7-9). The CIR is based on an artificial immune receptor
`composed of an extracellular antigen-binding domain linked
`through
`a
`transmembrane motif
`to
`a
`cytoplasmic
`lymphocyte-signaling moiety. The CIR approach using
`antibody-derived single-chain variable fragment (scFv) as a
`recognition unit, is especially suited to immunotherapy of
`cancer, being able to bypass many of the mechanisms by
`which tumors avoid immunorecognition, such as HLA
`down-regulation,
`lack of expression of co-stimulatory
`molecules, CTL resistance
`and induction of T-cell
`suppression, and where the use of both CD8* CTL and
`CD4* T-cells are best combined for optimum anti-tumor
`efficacy (6, 10, 11).
`To date, the majority of CIR genes are reconstructed
`with murine scFv specific for the tumor antigen (12).
`However, murine scFv is immunogenic in humans (13).
`Therefore, we have developed a novel CIR with a human
`scFv portion that
`recognizes CEA, one of the most
`important tumor-associated antigens (TAAs). Recently, we
`generated fully human monoclonal antibodies (mAbs)
`specific for CEA using KM mouse™ that
`lacks
`the
`endogenous genes for immunoglobulin and instead carries
`human immunoglobulin genes (14). In a recent study, we
`also generated a human scFv antibody, designated
`45kKHscFv, after cloning the variable region cDNAsofthe
`C2-45 human mAb(15).
`the scFv gene was fused to a
`In the present study,
`construct containing the human CD8a hinge region, human
`CD28 transmembrane and cytoplasmic domain and human
`CD3¢ intracellular domain genes. After transfection of this ,
`
`4067
`
`Miltenyi Ex. 1019 Page 3
`
`

`

`
`
`
`Vy
`Linker
`
`
`Leader sequence
`Bg!/\l Nhel
`\ua
`
`—
`
` 45kHscFv/
`TOPO
`=
`ge
`L45scFv-CiR
` Sacl
`
`
`
`ANTICANCER RESEARCH26: 4067-4072 (2006)
`
`C2-45 hybridoma
`
`Cloning of V region genes
`
`45xHscFv
`
`F39scFv/CIR-2
`
`Sac|
`BamH |
`
` CD3é
`
`CD8a_
`CD28
`——
`
`
`
` F39scFv/
`CIR-2/
`pcDNA3.1
`
`
`Bg/ll Nhel
`
`BamH |
`
`ee
`
`
`L45scFv-
`
`CIR/
`pcDNA3.1
`
`
`Figure 1. Schematic illustration ofthe construction ofthe L45scFv-CIR expression vector.
`
`fully human gene into primary human T-lymphocytes, their
`©xpression of the resulting CIR and binding to CEA-
`POSitive cells was investigated.
`
`Materials and Methods
`
`Celts. A CEA-expressing gastric cancer cell line, MKN-45, was
`maintained in DMEM supplemented with 10% FBS, 100 U/mlof
`Penicillin and 100 ug/ml of streptomycin. Peripheral blood
`Mononuclear cells (PBMCs) were obtained from healthy volunteers
`using LSM® lymphocyte separation medium according to the
`Manufacturer's protocol. After 1 h incubation in 10-cm diameter
`culture dishes, non-adherent PBMCs were collected and cultured
`im AIM-V® medium (Invitrogen, Carlsbad, CA, USA).
`Chimeric immune receptor construction and expression vector
`Production. CDNA encoding the anti-CEA chimeric immune receptor,
`
`designated L45sceFv-CIR, was generated with specific primers
`including unique restriction enzymesites using the human 45kHscFv
`gene and another mouse/human CIR, F39scFv/CIR-2, with mouse
`scFv (14-16). The specific primers were as follows: L45scFv-CIR
`forward, 5’-aatgagctccagtctecaggcaccctetetttgtct-3"; LASscFv reverse,
`5'-ccatggctcgagaaatgaggeccaaceggcca-3’; CIR forward, 5’-ageggccg
`cectgagcaaatacatcatg-3’ and L4S5scFv-CIR reverse, 5’-taagettggatcege
`sgectaageepecageecctgcatgt-3’. As schematically represented in Figure
`1, the L45scFy-CIR with a leader sequence was inserted into a
`pcDNA3.1 expression vectorattherestriction enzymesites, Bgill and
`BamH] with T4 ligase, and transformed into TOP10 competentcells.
`The plasmid DNA was cloned and sequenced with a DNA sequencer
`using BigDye™ terminator (PE Applied Biosystems, Foster City, CA,
`USA)as described previously (15).
`
`Gene transfection. The plasmid DNA was purified using the
`PureLink™ HQ Mini Plasmid Purification kit (Invitrogen) for
`transfection. The L45scFv-CIR or pmaxGFP (Amaxa Biosystems,
`
`4068
`
`Miltenyi Ex. 1019 Page 4
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`Miltenyi Ex. 1019 Page 4
`
`

`

`Rosetting test. MKN-45 cells were cultured to about 50%
`confluence in a 4-well LAB-TEK® ChamberSlide™ (Nalge Nunc
`International, Naperville, IL, USA), and were incubated with
`transfected or non-transfected T-cells (1x10® cells/ml) at 37°C for
`1 h. Cells were then washed three times with PBS to remove
`nonspecific binding T-cells.
`
`Results
`
`Detection of L45scFv-CIR gene in T-cells. Twenty-four h
`after transfection of the L45scFv-CIR gene into primary
`T-cells, total RNA wasextracted from the transfectants and
`non-transfectants, and analyzed by RT-PCR using specific
`primers described above to detect the gene transcription. As
`shown in Figure 2,
`the RT-PCR products from the
`transfectants and the plasmid DNAincluding L45scFv-CIR
`showed a major band of about1.4 kbp, but no specific band
`was detected in the non-transfectants, indicating that the
`mRNA for L45scFv-CIR gene was transcripted in the
`transfectants.
`
`Expression of L45scFv-CIR in T-cells. Flow cytometric
`analysis was employed to detect the functional expression of
`L45scFv-CIR on the surface of
`transfectants. CEA
`molecules labeled with APC were specifically bound to
`transfectants, but not to non-transfectants (Figure 3A). In
`contrast, APC-BSA was bound to neither L45scFv-CIR
`transfectants nor non-transfectants, as shown in Figure 3B.
`The efficacy of L45scFv-CIR gene transfection to primary
`human T-cells was about 80%.
`
`Targeting of T-cells expressing L45scFv-CIR to CEA-positive
`twnor cells. The binding capability of L45scFv-CIR
`transfected T-cells was tested by rosette formation. MKN-45
`target cells were incubated with transfectants or non-
`transfectants. Nonspecifically bound T-cells were removed by
`washing. The L45scFv-CIRtransfected T-cells were bound to
`MKN-45 cells, whereas the non-transfected T-cells did not
`react with MKN-45 cells (Figure 4).
`
`Discussion
`
`In the presentstudy, a novel CIR gene, L45scFv-CIR, with
`four human genes was successfully generated. Primary
`human T-cells expressing L45scFv-CIR on their surface
`specifically recognized APC-labeled CEA and bound to
`
`Flow cytometry. CEA and BSA_were labeled with the
`
`MKN-45cells in vitro.
`Allophycocyanin Labeling kit (Dojindo Molecular Technologies
`CIRs expressed on T-cells have usually been generated
`Inc., Kumamoto, Japan). T-cells expressing L45scFv-CIR were
`using variable region genes of mouse mAbs or humanized
`identified by immunofluoresence with allophycocyanin (APC)-
`labeled CEA or BSA 24 h after transfection. Briefly, 1x105
`mAbs(12). The reports of phase I/II trials using mAbs of
`transfectants were incubated with 5 g/ml of APC-CEA or -BSA
`mouse origin, however, indicated that the production of
`for 1 h at 4°C. After washing twice with PBS, cells were analyzed
`human
`anti-mouse
`antibody
`(HAMA) was
`still
`a
`by a flow cytometer (FACSCalibur, BD Biosciences, San Jose, CA,
`considerable problem (17-19),
`suggesting that CIRs
`USA)
`using CellQuest
`software
`(BD Biosciences). The
`containing variable regions of mouse origin are possibly
`electroporated cells without gene were used as a control.
`
`4069
`
`Shibaguchiet al: Human CIR Specific for CEA
`
`0.3
`
`“4 PCR product
`
`Figure 2. Detection of L45scFv-CIR gene mRNA on transfected primary
`T-cells by RT-PCR with specific primers. Total RNA (1 ug) obtained from
`the parental cells and transfectants was used as template RNA. The
`plasmid DNA containing L45scFv-CIR was used as a positive control.
`
`Cologne, Germany) genes were transfected into freshly isolated
`PBMCs using Nucleofector™ (Amaxa Biosystems) with the
`human T-cell Nucleofector® kit according to the manufacturer's
`protocol with minor modification. Briefly, 3 ug of DNA was
`added to 5x10° PBMCsresuspended in 100 ul of human T-cell
`Nucleofector® Solution, electroporated using the U-14 program
`of Nucleofector™ device, and immediately transferred into the
`prewarmed AIM-V medium.
`
`RT-PCR. Total RNA was extracted from the transfectants 24 h
`after transfection with ISOGEN (Nippon Gene, Toyama, Japan)
`according to the manufacture’s protocol. The RT-PCR was
`performed using the AccessQuick™ RT-PCR system (Promega,
`Madison, WI, USA)with L45scFv-CIR specific primers described
`above (L45scFv-CIR forward and L45scFv-CIR reverse). Thefinal
`products were analyzed by electrophoresis on 1.5% agarose gel
`followed by staining with ethidium bromide.
`
`Miltenyi Ex. 1019 Page 5
`
`Miltenyi Ex. 1019 Page 5
`
`

`

`B BSA
`
`Transfectant
`
`count
`count
`Transfectant
`
`
`
`ANTICANCER RESEARCH 26: 4067-4072 (2006)EErereeT)
`
`160=200
`Cell
`Cell
`
`120
`
`80
`
`Control
`
`Transfectant
`
`
`102
`io!
`10°
`104
`10!
`102
`Fluorescenceintensity
`Fluorescenceintensity
`Figure 3. The L45scFv-CIR expression on human T-cells. Primary human T-cells (5x10 cells) were transfected with 2 yg ofthe L45scFv-CIR gene.
`Transfectants (1x109 cells) were stained with 1ugiml ofAPC-labeledBSA or CEA for I h at 4°C 24 hrs afternucleofection. The expression ofL45scFv-
`CIR on human T-cells was then determined byflow cytometry. Non-transfected T-cells were also stained as control
`
`A
`
`Figure 4. Binding ofL45scFv-CIR transfectedprimary human T-cells to CEA-expressing tumor cells. The CEA expressing MKN-45 cells were mixed
`with parental T-cells or transfectants at the ratio of 1:5 and incubatedfor rossetteformationfor 1 h at 37°C.
`
`the
`immunogenic. Therefore, our L45scFv-CIR has
`advantage of avoiding the HAMA problem becauseofits
`construction design with all genes of human origin.
`To date, it has been widely accepted that CD28 is one
`the most
`important co-stimulatory molecules on
`of
`T-lymphocytes. An
`early
`study
`showed
`that
`the
`
`simultaneous expression of recombinant scFv-CD3¢ and
`scFy-CD28 receptors in a T-cell line resulted in signaling via
`both receptors and enhanced cellular activation (20).
`Signaling via CD28is required for optimum IL-2 production,
`cell cycle progression and survival (21-23). In addition to
`signaling through the TCR/CD3 complex, a co-stimulatory
`
`4070
`
`Miltenyi Ex. 1019 Page 6
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`Miltenyi Ex. 1019 Page 6
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`

`

`Shibaguchiet al: Human CIR Specific for CEA
`
`signal is required for full activation and proliferation of T-
`
`cells, especially unprimedorresting cells (24-26). Therefore,
`the signaling domain of CD28 was addedto the cytoplasmic
`domain of L45scFv-CIR.
`
`“It is quite difficult to efficiently introduce and express
`genes in resting lymphocytes with viral vectors (27, 28). In
`
`present study, the L45scFv-C/R wastransfected into
`
`ing human primary T-cells using the Nucleofector™
`
`ce. The percentage of viable cells transfected with the
`
`scFv-CIR constructs was between 60-80%. This
`
`ficiency compares favorably with the 20-40% obtained by
`transduction of prestimulated human primary T-cells
`
`ith similar constructs (27-30).
`
`The cytokine production and cell cytotoxicity by primary
`
`T-cells expressing L45scFv-CIR remainto be investigated.
`
`ing into consideration the results using the same three
`
`‘nes in the cytoplasmic region of another CIR with
`
`iouse anti-CEA scFv (31), the primary human T-cells
`
`pressing the L45scFv-CIR constructed in this study
`
`ight work functionally as well.
`In conclusion,
`the
`
`L45scFv-CIR with four genes of human origin might be a
`
`ful tool to enhance effector function to resting human
`-cells and to eradicate CEA-positive tumorcells in an
`
`HLA-independent manner.
`
`Acknowledgements
`
`
`
`
`work was supported in part by Grant-in-Aids for High-
`
`echnology Research Center andfor Scientific Research® from the
`
`Ministry of Education, Culture, Sports, Science and Technology,
`
`
`
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`Received May 23, 2006
`Accepted July 3, 2006
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