Volume 34, Number 16/17, November/December 1997
`
`ISSN 0161-5890
`
`L
`
`Board of Regional Editors
`
`Hidde Ploegh, Boston, USA
`
`CHAIRMAN
`
`J. Donald Capra, Oklahoma City, USA
`Michael Carroll, Boston, USA
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`Steve Gerondakis, M elbourne, Australia
`V. Horejsi, Prague, Czech Republic
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`Ed Palmer, Basel, Switzerland
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`!) PERGAMON
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`Miltenyi Ex. 1004 Page 1
`
`

`

`Molecular Immunology
`
`Board of Regional Editors
`Dr HIDDE PLOEGH (Chairman of the Board), Department of Pathology, Harvard Medical School, 200 Longwood Ave., 02-137,
`Boston, MA 02115, U.S.A.
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`Dr STEVE GERONDAKIS, The Walter and Eliza Ha/I Institute of Medical Research, Royal Melbourne Hospital, Victoria 3050, Australia
`Dr V. HOREJSI, Institute of Molecular Genetics, Academy of Sciences of Czech Republic, Videnska 1083, 142 20 Praha, Czech
`Republic
`Dr ED PALMER, Basel Institute for Immunology, Grenzacherstrasse 487, CH-4005, Basel, Switzerland
`Founding Editor
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`Toronto, Canada
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`Miltenyi Ex. 1004 Page 2
`
`

`

`Volume 34, No. 16-17
`
`November-December 1997
`
`Molecular
`Immunology
`
`Contents
`
`C. D. Partidos and C. Kanse
`
`1105 Specificity of the T-cell responses in covalently linked
`peptides each comprising of a T helper epitope
`
`I. Dalum, M. R. Jensen,
`K. Gregorius, C. M. Thomasen,
`H. I. Elsner and S. Mouritsen
`
`1113
`
`Induction of cross-reactive antibodies against a self
`protein by immunization with a modified self protein
`containing a foreign T helper epitope
`
`K. B. Vu, M. A. Ghahroudi, L. Wyns
`and S. Muyldermans
`
`1121 Comparison of llama VH sequences from conventional
`and heavy chain antibodies
`
`N. G. Saito and V. Paterson
`
`K. Tominaga, T. Kirikae and
`M. Nakano
`
`I. C. Nicholson, K. A. Lenton,
`D. J. Little, T. Decorso, F. T. Lee,
`A. M. Scott, H. Zola and
`A. W. Hohmann
`
`E. Lunde, B. Bogen and I. Sandlie
`
`B. B. Jrad and E. Bahraoui
`
`1133 Contribution of peptide backbone atoms to binding of
`an antigenic peptide to class I major histocompatibility
`complex molecule
`
`1147 Lipqpolysacc~9ri~e (LPS)-induced IL-6 production by
`efh{orypnic fitrfoblasts isolated and cloned from LPS-(cid:173)
`responsive and LPS-hyporesponsive mice
`
`,;r
`
`1157 Cbnstructiofr and characterisation of a functional corn
`specific single chain Fv fragment for immunotherapy
`of B lineag~ •eukaemia and lymphoma
`
`1167
`lrnmunoglobulin as a vehicle for foreign antigenic
`•",, peptides immunogenic to T cells
`
`1177 Linear and cyclic peptides mimicking the disulfide loops
`in HIV-2 envelope glycoprotein induced antibodies with
`different specificity
`
`E. Virts, D. Barritt, E. Siden and
`W. C. Raschke
`
`1191 Murine mast cells and monocytes express distinctive
`sets of CD45 isoforms
`
`J.C. Almagro, I. Hernandez,
`M. del Carmen Ramirez and
`E. Vargas-Madrazo
`
`1199 The differences between the structural repertoires of
`VH germ-line gene segments of mice and humans:
`implication for the molecular mechanism of the immurn:~
`response
`
`INDEXED/ABSTRACTED IN: Excerp Med, Ind Med, MEDL/NE, CABS, Biosis Data,
`PASCAL-CNRS Data, ACSA, CAB Inter, Cam Sci Abstr, Chem Abstr, Life Sci,
`ISI/BIOMED Database, Sci Cit Ind and SC/SEARCH Data
`
`-continued on inside back cover
`
`(I) Pergamon
`
`ISSN 0161-5890
`IMCHAZ 34(16/17) 1105-1236 (1997)
`
`Printed in Great Britain by BPC-AUP Aberdeen Ltd.
`
`253
`
`111111 II IIIIIII IIIIIIIIIIIIHlll 11
`
`0161-5890(1997)34:16-17;1-0
`
`cdhelp@elsevier.co.uk
`
`Miltenyi Ex. 1004 Page 3
`
`

`

`Molecular
`Immunology
`
`Contents -
`
`continued from outside back cover
`1215 Structural analysis of an anti-estradiol antibody
`
`U. Lamminmaki, B. 0. Villoutreix,
`P. Jauria, P. Saviranta, M. Vihinen,
`L. Nilsson, 0. Teleman and T. Lovgren
`M. Salmi, D. J. Smith, P. Bono,
`T. Leu, J. Hellman, M.-T. Matikainen
`and S. Jalkanen
`
`1227 A mouse molecular mimic of human vascular adhesion
`protein-1
`
`Forthcoming papers
`
`~ Pergamon
`
`ISSN 0161-5890
`IMCHAZ 34(16/17) 1105-1236 (1997)
`
`Miltenyi Ex. 1004 Page 4
`
`

`

`~Pergamon ~ PH: S0161-5890(97)00144-2
`
`Molecular Immunology, Vol. 34, No. 16-17, pp. 1157-1165, 1997
`© 1997 Elsevier Science Ltd. All rights reserved
`Printed in Great Britain
`0161-5890/97 $17.00 + 0.00
`
`CONSTRUCTION AND CHARACTERISATION OF A
`FUNCTIONAL CD19 SPECIFIC SINGLE CHAIN Fv
`FRAGMENT FOR IMMUNOTHERAPY OF B LINEAGE
`LEUKAEMIA AND LYMPHOMA
`
`IAN C. NICHOLSON,*§ KELLY A. LENTON,t DEBBIE J. LITTLE,* TINA
`DECORSO,t FOOK THEAN LEE,i ANDREW M. SCOTT,i HEDDY ZOLA*~
`and ARTHUR W. HOHMANN*
`* Child Health Research Institute, Women's and Children's Hospital, Adelaide, South Australia;
`t Flinders University of South Australia; and tLudwig Institute for Cancer Research, Austin and
`Repatriation Medical Centre, Studley Road, Heidelberg, Vic 3084 Australia
`
`(First received 20 July 1997; accepted in revised form 7 November 1997)
`
`Abstract-The B cell specific antigen CD19 is a target for the immunotherapy of B lineage leukaemias
`and lymphomas. We have engineered a single chain Fv (scFv) fragment from the mouse hybridoma
`cell line FMC63 which produces monoclonal antibody specific for CD19. The genes encoding the
`FMC63 heavy and light chain variable regions were amplified from cDNA and a scFv was constructed
`by splice overlap extension PCR. Analysis of staining of lymphoblastoid cell lines, peripheral blood
`lymphocytes and tonsil sections demonstrated that the monovalent scFv fragment has the same
`cellular specificity as the parent hybridoma antibody. Kinetic studies with radiolabelled material
`, compared with 4.2 x 10- 9 for the
`showed that the scFv binds target cells with a Ka of 2.3 x 10- 9
`parent antibody. This CD19 scFv will be used in experimental models to test its therapeutic efficacy
`and immunogenicity, with a view to application in the diagnosis and treatment of human B cell
`cancers. © 1997 Elsevier Science Ltd. All rights reserved.
`
`Key words: scFv, CD19, antibody therapy, leukaemia, lymphoma.
`
`INTRODUCTION
`imaging and immunotherapy of
`Antibody directed
`tumours relies on targeting tumour-associated antigens.
`CD 19 is expressed on most B lineage malignancies,
`including acute lymphoblastic leukaemia, chronic lym(cid:173)
`lymphoma.
`leukaemia and non-Hodgkin's
`phocytic
`Because CD19 is absent from bone marrow progenitor
`cells it is a potential target for immunotherapy of these
`malignancies (Uckun et al., 1988). Antibodies against
`CD19 inhibit the growth of tumour cells (Ghetie et al.,
`1994). CD19 is not readily shed from cells (Uckun et al.,
`1988) and is internalised with bound antibody, allowing
`delivery of anti-CD 19-linked toxins (Uckun et al., 1988).
`Animal models have indicated the potential value of anti(cid:173)
`bodies to CD19 (Jansen et al., 1992; Pietersz et al., 1995).
`Antibody alone (Hekman et al., 1991), with IL-2 (Vlas-
`
`§ Present Address: Department of Development and Genetics,
`Babraham Institute, Babraham, Cambridge, CB2 4AT,
`U.K.
`,-i Author to whom correspondence should be addressed: Prof.
`Heddy Zola, Child Health Research Institute, 72 King
`William Road, North Adelaide, South Australia, 5006,
`Australia
`
`veld et al., 1995), or conjugated to toxin (Grossbard et
`al., 1993; Stone et al., 1996) have been used in clinical
`trials for therapy of leukaemia and lymphoma and CD 19
`scFv have been described (Bejcek et al., 1995).
`Some of the limitations of therapeutic monoclonal
`antibodies can be overcome by engineering smaller and
`more effective antibody fragments (Winter et al., 1994).
`scFv are single gene fusions of the antibody heavy and
`light chain variable regions joined by a peptide linker.
`Because they are smaller than whole antibodies, scFv
`show improved penetration into poorly vascularised
`tumours (Yokota et al., 1992) and in clinical trials have
`shown negligible immunogenicity (Begent et al., 1996).
`Functional moieties such as toxins, enzymes, or sites for
`binding drugs or radioisotopes can be incorporated
`(Ghetie and Vitetta 1994; Pietersz et al., 1992). Engi(cid:173)
`neered antibody fragments can be produced on a large
`scale in bacterial or mammalian expression systems (Pack
`et al., 1993; Bebbington, 1995).
`We describe the production and characterisation of a
`CD19 scFv, CHRI-19Fvl. Staining of lymphoblastoid
`cell lines, peripheral blood lymphocytes and tonsil sec(cid:173)
`tions indicates that CHRI-19Fvl has the same cellular
`specificity as the parent antibody and has retained a high
`level of binding albeit with a 2-fold reduction in affinity.
`1157
`
`Miltenyi Ex. 1004 Page 5
`
`

`

`1158
`
`I. C. NICHOLSON et al.
`
`MATERIALS AND METHODS
`
`Cloning of antibody variable region genes
`FMC63 (CD19; IgG2a/K; Zola et al., 1991) hybridoma
`cells were grown in RPMI 1640 with 10% fetal calf serum.
`Total RNA was isolated using RNAzol (Biotecx Lab(cid:173)
`oratories, Houston, TX, U.S.A.). cDNA was synthesised
`using reverse transcriptase (Promega, Madison, WI,
`U.S.A.). Heavy chain variable region (VH) cDNA was
`amplified using a degenerate primer to the first eight
`codons (MVH.FWR) (Orlandi et al., 1989) and a primer
`to the y constant region (MVH.y) (Gavilondo et al.,
`1990). Light chain variable region cDNA (V d was ampli(cid:173)
`fied using a mixture of primers to the first eight codons
`of framework 1 (MVK.B5-l-B5-5) (Leung et al., 1993)
`and a primer to the K constant region (MVK.K) (Gav(cid:173)
`ilondo et al., 1990) (Table 1 ). PCR products were purified
`using the Wizard PCR Prep System (Promega), cloned
`into pGem-T (Promega) and transformed into E. coli
`TG 1 cells. Recombinant colonies were selected by blue(cid:173)
`white screening and insert size estimated using heavy
`chain primers MVH.FWR and MVH.JOIN, and light
`chain primers MVK.B5 and MVK.FOR2 (Table 1 ).
`Inserts from positive colonies were amplified using M 13
`sequencing primers and sequenced.
`
`Assembly of scFv fragment
`V H and V L DNA templates were extended at the 3'
`and 5' ends by PCR using primers encoding the linker
`sequence (Gly4Ser)3 (Huston et al., 1988). VH DNA was
`amplified with the primers Vector.V H and H.SOE.anti;
`VL with B5-2.SOE.Sense and VL.Vector.2 (Table 1).
`Products were purified using the Wizard PCR Prep Sys(cid:173)
`tem and the extended V H and V L products were used for
`assembly of the scFv by SOE-PCR (Clackson et al.,
`1992). Cycling began (in the absence of primers) with
`denaturation at 95°C for 5 min followed by five cycles of
`74°C for 1 min, 95°C for 1 min then a final extension at
`74° for 10 min. For amplification of the scFv template,
`Tli DNA polymerase, dNTPs and the primers Vector.VH
`and VL.Vector2 were added directly to the PCR mix. The
`pooled products was amplified for 10 cycles using Taq
`polymerase to add an overhanging "A" residue for lig(cid:173)
`ation into pGem-T. The ligation was transformed into
`competent E. coli TG 1 cells. Recombinant colonies were
`screened for scFv by PCR using the MVH.FWR and
`MVK.FOR2 primers. The construct was re-amplified
`the Vector.VH and
`from positive colonies using
`VL.Vector2 primers, digested with NcoI and NotI and
`cloned into the pHEN-1 phagemid (Hoogenboom et al.,
`1991 ). The ligation was digested with XhoI to reduce the
`
`Table 1. Primers used for isolation of immunoglobulin heavy chain and light chain variable region genes and the assembly of the
`scFv construct
`
`Primer Name
`
`Sequence (5' to 3'
`
`Region
`
`Strand
`
`MVH.FWR
`MVH.y
`MVH.Join
`
`MVK.B5-1
`MVK.B5-2
`MVK.B5-3
`MVK.B5-4
`MVK.B5-5
`MVK.K
`MVK.For.2
`
`Vector.VH
`
`V L.Vector.2
`
`TTCSAGGTSMARCTGCAGSAGTCT
`ACACACAGGRRCAGTGGATAGAC
`TGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAG
`
`GAAATTGTKCTCACMCARTCTCC
`GACATCCAGATGACMCAGWCTMC
`GATATTGTGATGACMCAGGMC
`GATGTTGTGATGACCCAAACTCC
`ARYATTGTGATGACCCAGWCTC
`ACTGGATGGTGGGAAGATGGA
`GTTAGATCTCCAGCTTGGTCCC
`
`CCATGACTCGCGGCCCAGCCGGCCATGGCCSAGGTS
`MARCTGCAGSAGTCT
`TGCGGCCGCCCGTTTGATCTCCAGCTTGGTCCC
`
`H.SOE.Anti
`
`ACCCGACCCACCACCGCCCGAGCCACCGCCACCTGAGGA
`GACGGTGACCGTGGTCCCTTGGCCCCAG
`B5-2.SOE.Sense TCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGACA
`TCCAGATGACACAGACTACA
`
`MB.USP
`M13.RSP
`pHEN.USP
`pHEN.RSP
`
`GTAAAACGACGGCCAGT
`CACACAGGAAACAGCTATGACCATG
`CAGTCATAATGAAATACCTATTGCCTAC
`CGTTAGTAAATGAATTTTCTGTATGAGG
`
`VH FWRl
`CHI
`VH FWR4
`
`VKFWRl
`VKFWRl
`VKFWRl
`VK FWRl
`CKl
`VKFWR4
`
`pHENl-VH
`FWRI
`pHENl-VK
`FWR4
`
`sense
`antisense
`anti sense
`
`sense
`sense
`sense
`sense
`antisense
`antisense
`
`sense
`
`antisense
`
`antisense
`
`sense
`
`sense
`
`sense
`anti sense
`
`Miltenyi Ex. 1004 Page 6
`
`

`

`CD 19-specific single-chain Fv antibody construct
`
`1159
`
`non-recombinant background and was transformed into
`competent E.coli HB2151 (Carter et al., 1985) for soluble
`expression of the scFv protein. Ampicillin-resistant col(cid:173)
`onies were screened for scFv using the MVH.FWR and
`MVK.FOR2 primers and positive clones were tested for
`production of soluble scFv protein.
`
`Expression, purification and size exclusion chromato(cid:173)
`graphy
`scFv was prepared in 2YT medium containing 100
`µg/ml ampicillin, grown to log phase, induced with 1 mM
`ITPG and incubated with shaking for 24 hat 22°C. A
`monoclonal antibody against c-myc (encoded as a tag in
`pHEN), 9E10 (Evan et al., 1985; ATCC, Rockville, MD,
`U.S.A.), was used to detect scFv expression by slot-blot
`analysis on nitrocellulose. scFv was purified from con(cid:173)
`centrated culture supernatant using an affinity column
`made with 9El0 antibody. The purified scFv was ana(cid:173)
`lysed on a Superose12 FPLC column (Pharmacia); scFv
`was detected by slot-blot using 9E10 antibody.
`
`Flow cytometry and immunohistology
`Binding of scFv to Mann, JVM13, HRIK, KM3 (all
`CD19+) and Jurkatt (CD19-) cell lines to peripheral
`blood and tonsil cells was detected using 9El0 antibody,
`biotinylated horse anti-mouse IgG (Vector Laboratories,
`Burlingame, CA, U.S.A.) and streptavidin-conjugated
`phycoerythrin (Caltag Laboratories, San Francisco, CA,
`U.S.A.). FMC63 antibody was used as a positive control,
`detected with biotinylated horse anti-mouse IgG and
`streptavidin-conjugated phycoerythrin. The ability of
`CHRI-19Fvl and several CD19 antibodies to inhibit the
`binding of FMC63 and two other labelled CD19 anti(cid:173)
`bodies was tested using JVM13 and PE labelled FMC63
`(AMRAD, Melbourne, Australia), FITC labelled B43
`and CyChrome-labelled HIB 19 (Pharmingen, San Diego,
`U.S.A.). Cells (5 x 105 in 50 µl) were incubated with
`blocking antibody for 30 min at 4°C, washed, mixed with
`labelled antibody, incubated for 30 min at 4°C, washed
`and analysed. Samples were analysed using a F ACScan
`(Becton Dickinson, San Jose, CA, U.S.A.) or an EPICS
`Elite cytometer (Coulter, Hialeah, FL, U.S.A.).
`Frozen sections of human tonsil were examined using
`a high-sensitivity immunofluorescence staining method
`(Zola et al., 1995).
`
`immunoreactivities and affinities of the labelled anti(cid:173)
`bodies were determined as described by Lindmo et al.
`(1984). Briefly, 10 ng of labelled antibodies were mixed
`with concentrations of Daudi cells from 0-12 x 106 cells
`in 1.0 ml of medium at 4°C. After constant mixing by
`inversion for 45 min at 4 °C the cells were washed three
`times with medium. Antibody binding was determined
`by comparison to standards. Immunoreactivity, expre(cid:173)
`ssed as percentage binding, was derived by double
`reciprocal plot of percentage binding against cell number.
`Affinity was determined by Scatchard analysis (Lindmo
`et al., 1984); aliquots of unlabelled antibody from 0-5 µg
`were mixed with 10 ng of labelled antibody and 6 x 106
`cells in 1.0 ml medium.
`
`Radiochemical purity
`Radiochemical purity of labelled antibody was ana(cid:173)
`lysed using thin-layer chromatography developed with
`10% trichloroacetic acid. Labelled antibody (1 ng) was
`spotted on the TLC strip and developed until the front
`reached the top of the strip. Strips were divided in two
`and counted separately. Over 90% of radioactivity
`remained at the origin, bound to the antibody.
`
`RESULTS
`
`Isolation of antibody variable region genes
`DNA sequences of the heavy and light chain variable
`region genes used to construct the scFv are shown in Fig.
`1. The sequences are in frame and contain no stop codons.
`Alignment to GeneBank confirmed that both sequences
`are unique mouse immunoglobulin genes. Two other light
`chain sequences were isolated; one derived from the
`fusion partner (Carroll et al., 1988) while the second
`contained four termination codons.
`
`Assembly of scFv fragment
`The SOE-PCR generated one major band at 740 bp
`(the expected size for the scFv fragment) and two minor
`bands at about 500 bp and 350 bp. Following cloning
`into pGem-T recombinant colonies containing an insert
`of 740 bp were amplified and cloned into pHen-1. Clone
`CD 19 .105 was used in further studies. Nucleotide sequ(cid:173)
`encing confirmed that the insert was a correctly assembled
`scFv construct.
`
`Binding kinetics
`Purified scFv (1.8 mg/ml) and intact FMC63 (3.0
`mg/ml) in PBS were transferred to 0.1 M sodium borate
`buffer pH 8.6 by centrifugal desalting (Lee et al., 1993)
`using Bio-gel P6Dg (Biorad, Australia). Aliquots (300
`µCi) of 1251-Bolton Hunter Reagent (1 251-BHR; Du
`Pont-AMRAD, Australia) (Bolton and Hunter, 1973)
`dissolved in dry benzene were dried down in V-bottom
`vials using a gentle stream of nitrogen. The vials were
`cooled on ice and the proteins added and incubated on
`ice for 2 h with occasional mixing. The labelled antibodies
`were purified by centrifugal buffer exchange to PBS. The
`
`Expression of scFv fragment
`Slot blot analysis indicated that more scFv was secreted
`into the culture supernatant at lower temperatures and
`we routinely used a temperature of 22°C for 24 h after
`induction with IPTG. Yields were approximately 200 µg
`per litre in the supernantant. scFv derived from culture
`supernatant was used for the experiments described.
`Approximately 40 µg per litre of culture was recovered
`from the cell pellet by freeze-thaw extraction and
`approximately 1 mg/1 was obtained from disrupted cells
`following 6 h cultures in media containing 10 mM
`MgS04.
`
`Miltenyi Ex. 1004 Page 7
`
`

`

`1160
`
`I. C. NICHOLSON et al.
`
`60
`20
`10
`50
`40
`30
`*
`*
`*
`*
`*
`*
`GAG GTG AAA CTG CAG GAG TCA GGA CCT GGC CTG GTG GCG CCC TCA CAG AGC CTG TCC GTC
`Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val>
`
`120
`110
`100
`70
`90
`80
`*
`*
`*
`*
`*
`*
`ACA TGC ACT GTC TCA GGG GTC TCA TTA CCC GAC TAT GGT GTA AGC TGG ATT CGC CAG CCT
`Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro>
`
`180
`170
`140
`160
`150
`130
`*
`*
`*
`*
`*
`*
`CCA CGA AAG GGT CTG GAG TGG CTG GGA GTA ATA TGG GGT AGT GAA ACC ACA TAC TAT AAT
`Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn>
`
`240
`230
`200
`190
`220
`210
`*
`*
`*
`*
`*
`*
`TCA GCT CTC AAA TCC AGA CTG ACC ATC ATC AAG GAC AAC TCC AAG AGC CAA GTT TTC TTA
`Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu>
`
`300
`290
`270
`260
`250
`280
`*
`*
`*
`*
`*
`*
`AAA ATG AAC AGT CTG CAA ACT GAT GAC ACA GCC ATT TAC TAC TGT GCC AAA CAT TAT TAC
`Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr>
`
`360
`310
`350
`340
`330
`320
`*
`*
`*
`*
`*
`*
`TAC GGT GGT AGC TAT GCT ATG GAC TAC TGG GGT CAA GGA ACC TCA GTC ACC GTC TCC TCA
`Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser>
`
`CHRI-19Fvl H chain
`
`60
`50
`20
`10
`40
`30
`*
`*
`*
`*
`*
`*
`GAC ATC CAG ATG ACA CAG ACT ACA TCC TCC CTG TCT GCC TCT CTG GGA GAC AGA GTC ACC
`Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr>
`
`120
`110
`70
`100
`90
`80
`*
`*
`*
`*
`*
`*
`ATC AGT TGC AGG GCA AGT CAG GAC ATT AGT AAA TAT TTA AAT TGG TAT CAG CAG AAA CCA
`Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro>
`
`180
`170
`160
`150
`140
`130
`*
`*
`*
`*
`*
`*
`GAT GGA ACT GTT AAA CTC CTG ATC TAC CAT ACA TCA AGA TTA CAC TCA GGA GTC CCA TCA
`Asp Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser>
`
`240
`230
`220
`210
`190
`200
`*
`*
`*
`*
`*
`*
`AGG TTC AGT GGC AGT GGG TCT GGA ACA GAT TAT TCT CTC ACC ATT AGC AAC CTG GAG CAA
`Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln>
`
`290
`300
`280
`270
`260
`250
`*
`*
`*
`*
`*
`*
`GAA GAT ATT GCC ACT TAC TTT TGC CAA CAG GGT AAT ACG CTT CCG TAC ACG TTC GGA GGG
`Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly>
`
`350
`360
`320
`340
`330
`310
`*
`*
`*
`*
`*
`*
`GGG ACT AAG TTG GAA ATA ACA CGG GCT GAT GCT GCA CCA ACT GTA TCC ATC TTC CCA CCA
`Gly Thr Lys Leu Glu Ile Thr Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro>
`
`369
`*
`TCC AGT AAT
`Ser Ser Asn>
`
`CHRI-19Fvl L chain
`Fig. 1. DNA sequence of FMC63V H and FMC63V L· Sequences have been submitted to EMBL
`Nucleotide Sequence Database.
`
`Miltenyi Ex. 1004 Page 8
`
`

`

`CD 19-specific single-chain Fv antibody construct
`
`1161
`
`Affinity purification and molecular size
`The scFv was eluted from the 9El0 column as a single
`peak. Polyacrylamide gel analysis showed a single band
`of approximately 30 kDa. Size exclusion chro(cid:173)
`matography confirmed a molecular weight of 30 kDa
`under non-dissociating conditions; virtually no dimer was
`detected.
`
`Target cell binding demonstrated by flow cytometry
`The scFv and the parent FMC63 monoclonal antibody
`both stained co19+ B and pre-B lymphoblastoid lines
`Mann, JVM 13 (Fig. 2), KM3 and HRIK, but showed no
`reactivity with the Jurkatt T cell line (data not shown).
`About 10% of peripheral blood lymphocytes and 50%
`of tonsil lymphocytes reacted with the scFv (Fig. 2). The
`intensity of staining of the scFv was less than that of the
`parent monoclonal antibody . Pre-mixing the scFv with
`9E 10 anti-c-myc antibody to make a divalent complex
`increased the staining intensity, but not to the level
`obtained with parent FMC63 (Fig. 2).
`
`Competition with parent and other CDl9 monoclonal anti(cid:173)
`bodies
`CHRI-19Fvl was able to inhibit by 85% the binding
`of parent FMC63 monoclonal antibody (Table 2). The
`scFv also strongly inhibited binding of the HIB 19 and
`B43 CD19 antibodies (Table 2).
`
`Binding kinetics
`The labelling efficiencies for the intact FMC 63 and
`scFv were 41 % and 48.4% and specific activities were 2.5
`and 1.8 mCi/mg respectively. Binding to Daudi cells was
`measured in the cold to minimise internalisation of the
`radioconjugates. The immunoreactivities, as determined
`through double reciprocal plots shown in Fig. 3, were
`54.3% for 125I-BHR-FMC 63 and 13.1 % for 125I-BHR(cid:173)
`scFv. The Ka and number of antibody binding sites,
`derived from Scatchard plots (Fig. 4), were 4.17 x 109
`M- 1 and 32,000 sites per cell for the whole antibody and
`2.32 x 109 M- 1 and 30,000 sites per cell for CHRI-19Fvl.
`
`Staining of lymphoid tissue
`The pattern of staining observed with the CD19 scFv
`was similar to that seen with the monoclonal FMC63
`antibody, with germinal centres stained strongly and scat(cid:173)
`tered staining in the interfollicular area (data not shown).
`Staining intensities were lower with the scFv than with
`the parent antibody.
`
`Potential immunogenicity
`The amino acids present at the solvent exposed residues
`in the FMC63 heavy chain and light chain proteins are
`indicated in Table 3. For the light chain, 13 of the 15
`amino acids present in the FMC63 sequence were com(cid:173)
`mon in human sequences. For the heavy chain, 20 of the
`26 residues were common in human sequences.
`
`-Whole antibody
`.. Negative control
`- - scFv 4µg/ml
`
`d
`
`Blood
`
`Tonsil
`
`..
`
`-Whole antibody
`· Negative control
`--scFv
`scFv complex
`• - - -
`Fig. 2. a: Staining of the B lymphoblastoid cell line Mann with FMC63 scFv (bold trace) and parent
`antibody (normal trace). b: Staining of the B prolymphocytic leukaemia derived cell line JVM13 with
`FMC63 scFv (bold trace) and parent antibody (normal trace). The bold dotted trace shows scFv pre(cid:173)
`complexed with anti-c-myc antibody. c: Staining of peripheral blood and tonsil d: cells with FMC63
`scFv (bold trace) and parent antibody (normal trace). In each panel, the negative control X63 is
`shown as a dotted trace.
`
`_
`
`Whole antibody
`Negative control
`- - scFv 4µ8'ml
`
`Miltenyi Ex. 1004 Page 9
`
`

`

`1162
`
`I. C. NICHOLSON et al.
`
`Table 2. Blocking of binding of directly-conjugated CD19 antibodies B43, FMC63 and HIB19 by
`CD19 antibodies and the CD19scFv
`
`Blocking
`antibody
`
`X63
`FMC63
`CD19 scFv
`AB-I
`B4
`4G7 (Leu12)
`HD37
`HB13d
`
`Reference
`
`Negative control
`Zola et al., 1991
`This study
`Dorken et al., 1989
`Nadler et al., 1983
`Meeker et al., 1984
`Pezzutto et al., 1987
`CD20 control
`
`Staining antibody*
`
`B43
`(Pharmingen)
`
`FMC63
`(AMRAD)
`
`HIB19
`(Pharmingen)
`
`100
`16
`30
`73
`19
`21
`31
`126
`
`100
`3
`15
`30
`6
`2
`10
`93
`
`100
`8
`16
`55
`11
`8
`10
`111
`
`* Numbers represent mean fluorescence intensity, normalised to give a value of 100 when the
`blocking antibody is X63, a non-binding lgG 1.
`
`60
`
`50
`
`'c?-
`"O 40
`C
`:::,
`0 co
`>,
`"O
`0 30
`.0
`:g
`<(
`"O
`~
`c6 20
`.0 co
`
`_J
`
`10
`
`0
`
`2.00
`
`1.75
`
`(I) 1.50
`~
`LI..
`(I)
`>
`
`1.25
`
`u ro
`
`(I)
`0::
`0) 1.00
`C
`i5
`C co 0.75
`u
`<+= ·u
`(I)
`Q. 0.50
`CJ)
`
`0.25
`
`0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45
`
`Specific Binding / nM
`Fig. 4. Scatchard plots of binding of 1251-FMC 63 (•)and 1251-
`scFv ( O) antibodies to CD 19 expressing Daudi cells. From
`the slopes of the lines, the association constants for the two
`conjugates were Ka = 4.2 x 109 M- 1 and 2.3 x 109 M- 1 respec(cid:173)
`tively. From the intercept values at the abscissa, the binding
`capacity per cell were determined to be 32,000 and 30,000
`molecules.
`
`8
`
`10
`
`0
`
`2
`
`4
`6
`Cell Concentration x 106
`/ ml
`Fig. 3. Binding of 1251-FMC 63 (circles) and 1251-scFv (squares)
`to Daudi cells. Double inverse plots Lindmo et al. (1984) pro(cid:173)
`vided immunoreactive fraction values of 54.3% and 13.1 % for
`the whole antibody and scFv respectively.
`
`DISCUSSION
`
`Functional immunoglobulin variable region gene
`sequences isolated from the CD19 hybridoma FMC63
`were joined and used to encode a 30 kDa protein, CHRI(cid:173)
`l 9Fvl. The scFv retained the specificity of the parent
`monoclonal antibody when tested against cell lines, blood
`and tonsil lymphocytes and frozen sections of human
`tonsil and was able to compete with parent antibody.
`Improved binding seen with a preformed divalent com(cid:173)
`plex suggests that the reduction in binding affinity is at
`least in part due to the loss of valency. The linkage of
`the two V region domains using a peptide sequence, in
`
`contrast to the natural antibody structure, may reduce
`affinity further. Scatchard analysis of the affinity of
`CHRI-19Fvl and the parent antibody showed a 2-fold
`reduction in affinity. This reduction compares to 8-10-
`fold reductions reported for other scFvs (Milenic et al.,
`1991; Wels et al., 1992). It may be possible to increase
`the affinity by in vitro mutation and selection using phage
`display techniques (Winter et al., 1994; Irving and
`Hudson, 1995) or by constructing a divalent form of the
`single chain antibody, which may be expected to show an
`
`Miltenyi Ex. 1004 Page 10
`
`

`

`CD 19-specific single-chain Fv antibody construct
`
`1163
`
`Table 3. Comparison of FMC63 surface exposed residues with accessible residues in human and mouse immunoglobulin variable
`regions. Numbering of residues is according to the Kabat system (Kabat et al., 1990). np: FMC63 residue not listed as found at
`a frequency of 5% or greater.
`
`Heavy Chain
`
`Light Chain
`
`Residue
`Number
`
`Mouse
`Amino
`Acids
`
`Human Amino
`Acids
`
`FMC63 VH
`Amino Acid
`
`Human
`Frequency
`
`Residue
`Number
`
`Mouse
`Amino
`Acids
`
`Human
`Amino
`Acids
`
`Human
`FMC63 VL
`Amino Acids Frequency
`
`D,E,A,S
`D,Q,E
`V,Q,S, Y
`V,Q,L
`T,L
`T
`S,A, L, P P, S, G, A, L
`P,V,L
`L,P,V,A
`R, K, S, Q, T R, S, T, P
`p
`