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`an XhoI restriction site in the hinge region in frame
`
`prior to the CH2 and CH3 domains of human IgG1. Using a
`
`Her—2 cDNA clone as a template PCR fragments is generated
`
`using standard molecular biology techniques known to a
`
`person skilled in the art. These fragments consist of a
`
`unique 5’ restriction site, a start codon followed by a
`
`eukaryotic leader sequence that is linked in frame to
`
`either the total extracellular (EC) domain of Her—2 or to
`
`a part of the EC domain of Her—2 that is followed in
`
`frame by an XhoI restriction site. These PCR fragments
`
`are subsequently cloned in frame with the CH2—CH3 IgGl
`
`region into the pCDNA3.lzeo expression vector.
`
`In
`
`addition to the fusion protein containing the total EC
`
`domain of Her—2, several smaller fusion proteins are
`
`generated containing non—overlapping fragments of the
`
`Her—2 EC domain. These constructs encoding the Her—Z—Ig
`
`fusionproteins are used for transient transfection of
`
`293T cells using the lipofectamine reagent
`
`(Gibco). Five
`
`days after transfection the supernatants of the 293T
`
`cells are harvested and Her—Z—Ig fusion proteins are
`
`purified using protein A affinity chromatography
`
`according to standard procedures.
`
`Her—Z—Ig fusion proteins containing non—overlapping
`
`fragments of the Her—2 EC domain are coated for 2 hours
`
`at 37° onto the surface of Maxisorpm‘plastic tubes (Nunc)
`
`at a saturating concentration (0.5 — 5 ug/ml). The tubes
`
`are blocked for l h in 2% fat free milk powder dissolved
`
`in PBS (MPBS). Simultaneously, 500 pl
`
`(approximately 1012
`
`Cfu) of a semi—synthetic phage display library (a sub—
`
`library according to the terminology used above)
`
`in which
`
`only one Vkappal light chain is represented prepared as
`
`described by De Kruif et al
`
`(l995a,b) and references
`
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`
`therein,
`
`is added to two volumes of 4% MPBS.
`
`In addition,
`
`human serum is added to a final concentration of 15% and
`
`blocking is allowed to proceed for 30—60 min. The Her—2—
`
`Ig—coated tubes are emptied and the blocked phage library
`
`is added. The tube is sealed and rotated slowly for l h,
`
`followed by 2‘h of incubation without rotation. The tubes
`
`are emptied and washed 10 times in PBS containing 0.1%
`
`Tween—20,
`
`followed by washing 5 times in PBS.
`
`1 ml
`
`glycine—HCL, 0.05 M, pH 2.2 is added, and the tube is
`
`rotated slowly for 10 min. The eluted phages are added to
`
`500 pl
`
`lM Tris—HCl pH 7.4. To this mixture, 3.5 ml of
`
`exponentially growing XL—l blue bacterial culture is
`
`added. The tubes are incubated for 30 min at 37°C without
`
`shaking. Subsequently,
`
`the bacteria are plated on 2TY
`
`agar plates containing ampicillin,
`
`tetracycline and
`
`glucose. After overnight incubation of the plates at
`
`37°C,
`
`the colonies are scraped from the plates and used
`
`to prepare an enriched phage library, essentially as
`
`described by De Kruif et al.
`
`(1995a). Briefly, scraped
`
`bacteria are used to inoculate ZTY medium containing
`
`ampicillin,
`
`tetracycline and glucose and are grown at
`
`37°C to an ODmmm of ~0.3. Helper phages are added and
`
`allOWed to infect the bacteria after which the medium is
`
`changed to ZTY containing ampicillin,
`
`tetracycline and
`
`kanamycin.
`
`Incubation is continued overnight at 30°C. The
`
`next day,
`
`the bacteria are removed from the 2TY medium by
`
`centrifugation after which the phages are precipitated
`
`using polyethylene glycol 6000/NaCl. Finally,
`
`the phages
`
`are dissolved in a small volume of PBS—1% BSA, filter—
`
`10
`
`15
`
`20
`
`25
`
`30
`
`sterilized and used for a next round of selection. The
`
`selection/re—infection procedure is performed twice.
`
`After the second round of selection,
`
`individual E.coli
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`colonies are used to prepare monoclonal phage antibodies.
`
`Essentially,
`
`individual colonies are grown to log—phase
`
`and infected with helper phages after which phage
`
`antibody production is allowed to proceed overnight.
`
`Phage antibody containing supernatants are tested in
`
`ELISA for binding activity to Her—Z—total EC—Ig coated 96
`
`wells plates.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`Selected phage antibodies that are obtained in the screen
`
`described above, are validated by ELISA for specificity.
`
`For this purpose, Her—Z—Ig fusion proteins containing
`
`non—overlapping fragments of the Her—2 EC domain are
`
`coated to Maxisorp ELISA plates. After coating,
`
`the
`
`plates are blocked in 2% MPBS. The selected phage
`
`antibodies are incubated in an equal volume of 4% MPBS.
`
`The plates are emptied, washed once in PBS, after which
`
`the blocked phages are added.
`
`Incubation is allowed to
`
`proceed for l h,
`
`the plates are washed in PBS 0.1% Tween—
`
`20 and bound phages are detected using an anti—M13
`
`antibody conjugated to peroxidase. The procedure is
`
`performed simultaneously using a control phage antibody
`
`directed against thyroglobulin (De Kruif et al.
`
`l995a,b),
`
`which serves as a negative control.
`
`In another assay the selected phage antibodies are
`
`analyzed for their ability to bind BT474 human breast
`
`cancer cells that express Her-2. For flow cytometry
`
`analysis, phage antibodies are first blocked in an equal
`
`volume of 4% MPBS for 15 min at 4°C prior to the staining
`
`of the BT474 cells. The binding of the phage antibodies
`
`to the cells is visualized using a biotinylated anti—M13
`
`antibody (Santa Cruz Biotechnology)
`
`followed by
`
`streptavidin—phycoerythrin (Caltag).
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`Alternatively, phage antibodies recognizing multiple
`
`epitopes on Her—2 are selected using a method based upon
`
`competition of phage binding to Her—2 with binding of the
`
`well characterized murine anti—Her—Z antibodies HERSO,
`
`HER66 and HER70 (Spiridon et a1, 2002). To this purpose
`
`2x106 BT474 cells are incubated at 4°C with
`
`approximately 1
`
`013 cfu (0.5 ml) of a semi—synthetic phage
`
`display library in which only one Vkappal light chain is
`
`represented prepared as described supra and blocked with
`
`2 volumes of medium containing 10% of FBS. The mixture is
`
`slowly rotated at 4°C for 2 hours in a sealed tube.
`
`Subsequently, non—bound phages are removed by two washes
`
`with 50 ml of cold medium containing 10% FBS. Hereafter,
`
`phages recognizing multiple epitopes on Her—2 are eluted
`
`by resuspending the BT474 cells in 1 ml of cold medium
`
`containing saturating concentrations (5—20 pg/ml) of the
`
`HERSO, HER66 and HER70 murine anti—Her—Z antibodies. The
`
`cells are left on ice for 10 min, spun down and the
`
`supernatant containing the anti—Her—Z phage antibodies is
`
`used to reinfect XLl—Blue cells as described supra.
`
`From the panel of Her—Z—specific phage antibodies
`
`generated by de screens described above,
`
`three phage
`
`antibodies are selected that are recognizing three
`
`different non—overlapping epitopes on the Her—2 protein.
`
`The VH sequences and the unique Vkappal light chain
`
`sequence of these clones, provisionally designated
`
`VKlHERZ—l, VKlHERZ—Z and VKlHER2—3, are cloned behind the
`
`HAVTZO leader sequences of expression plasmid pCRU—KOl
`
`(ECACC deposit 03041601), or a similar expression
`
`plasmid,
`
`to obtain plasmids encoding a full length human
`
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`IgGl—kappa with binding specificities for Her~2. These
`
`plasmids are provisionally designated as pCRU—VKlHERZ—l,
`
`pCRU—VKlHERZ—Z and pCRU—VKlHERZ—B, respectively.
`
`Stable PER.C6TM derived cell lines are generated,
`
`according to methods known to the person skilled in the
`
`art,
`
`the cell lines expressing antibodies encoded by
`
`genetic information on either pCRU—VKlHERZ—l, pCRU~
`
`VKlHER2—2 or pCRU—VKlHER2—3 and a cell line expressing
`
`antibodies encoded by all three plasmids. Therefore,
`
`PER.C6TM cells are seeded in DMEM plus 10% FBS in tissue
`
`culture dishes (10 cm diameter) or T80 flasks with
`
`approximately 2.5 x 106 cells per dish and kept overnight
`
`under their normal culture conditions (10% C02
`
`concentration and 37°C). The next day,
`
`transfections are
`
`performed in separate dishes at 37°C using Lipofectamine
`
`(Invitrogen Life Technologies) according to standard
`
`protocols provided by the manufacturer, with either 1~2
`
`ug pCRU—VKlHERZ—l,
`
`1—2 ug pCRU—VKlHERZ—Z,
`
`1—2 ug pCRU—
`
`VKlHER2~3 or 1 ug of a mixture of pCRU—VKlHERZ—l,
`
`pCRU—
`
`VKlHERZ—Z and pCRU—VKlHERZ—B. As a control for
`
`transfection efficiency, a few dishes are transfected
`
`with a LacZ control vector, while a few dishes are not
`
`transfected and serve as negative controls.
`
`After 5 hours cells are washed twice with DMEM and refed
`
`with fresh medium without selection. The next day, medium
`
`is replaced with fresh medium containing 500 ug/ml G418.
`
`Cells are refreshed every 2 or 3 days with medium
`
`containing the same concentrations of G418. About 20—22
`
`days after seeding, a large number of colonies are
`
`visible and from each transfection at least 300 are
`
`picked and grown via 96—well and/or 24—well via 6—well
`
`plates to T25 flasks. At this stage, cells are frozen (at
`
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`
`least 1, but usually 4 vials per sub—cultured colony) and
`
`production levels of recombinant human IgG antibody are
`
`determined in the supernatant using an ELISA specific for
`
`human IgGl..Also, at this stage G418 is removed from the
`
`culture medium and never re—applied again. For a
`
`representative number of colonies larger volumes are
`
`cultured to purify the recombinant human IgG1 fraction
`
`from the conditioned supernatant using Protein A affinity
`
`chromatography according to standard procedures. Purified
`
`human IgG1 from the various clones is analyzed on SDS—
`
`PAGE,
`
`ISO-electric focusing (IEF), assayed binding to
`
`Her—2—Ig fusion proteins by ELISA, and analyzed for
`
`binding to Her—2 on the surface of BT474 cells by flow
`
`cytometry.
`
`Clones obtained from the co—transfection of pCRU-VKlHERZ—
`
`l, pCRU—VKlHERZ—Z and pCRU—VKlHER2—3 are screened by PCR
`
`on genomic DNA for the presence or absence of each of the
`
`three constructs. The identity of the PCR products is
`
`further confirmed by DNA sequencing.
`
`Next, it is demonstrated that a clonal cell line accounts
`
`for the production of each of the three binding
`
`specificities. Therefore, a limited number of colonies,
`
`which screened positive for the production of each of the
`
`three binding specificities (both by PCR at the DNA level
`
`as well as in the specified binding assays against Her—
`
`2), are subjected to single cell sorting using a
`
`fluorescence activated cell sorter (FACS)
`
`(Becton &
`
`10
`
`15
`
`20
`
`25
`
`Dickinson FACS VANTAGE SE). Alternatively, colonies are
`
`seeded at 0.3 cellS/well to guarantee clonal outgrowth.
`
`30
`
`Clonal cell populations, hereafter designated as sub—
`
`clones, are refreshed once a week with fresh medium. Sub—
`
`clones are grown and transferred from 96—wells via 24—
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`-81—
`
`and 6—wells plates to T25 flasks. At this stage,
`
`sub—
`
`clones are frozen (at least 1, but usually 4 vials per
`
`sub—clone) and production levels of recombinant human IgG1
`
`antibody are determined.in the supernatant using a human
`
`IgG1 specific ELISA. For a representative number of sub—
`
`clones,
`
`larger volumes are cultured to purify the
`
`recombinant human IgG1 fraction from the conditioned
`
`supernatant using Protein A affinity chromatography
`
`according to standard procedures.
`
`lO
`
`l5
`
`Purified human IgGl from the various sub—clones is
`
`subsequently analyzed as described above for human IgG1
`
`obtained from the parental clones, i.e. by SDS—PAGE,
`
`Iso—
`
`electric focusing (IEF) and binding to Her—2. Sub—clones
`
`will also be screened by PCR on genomic DNA for the
`
`presence or absence of each of the three constructs pCRU—
`
`VKlHERZ—l, pCRU-VKlHERZ—Z and pCRU—VKlHER2~3. The
`
`identity of the PCR products is further confirmed by DNA
`
`sequencing.
`
`Other methods such as Southern blot and/or FISH can also
`
`20
`
`be used to determine whether each of the three constructs
`
`are present in the clonal cell line.
`
`Sub—clones that are proven to be transgenic for each of
`
`the three constructs are brought into culture for an
`
`extensive period to determine whether the presence of the
`
`transgenes is stable and whether expression of the
`
`antibody mixture remains the same, not only in terms of
`
`expression levels, but also for the ratio between the
`
`various antibodies that are secreted from the cell.
`
`Therefore,
`
`the sub—clone culture is maintained for at
`
`least 25 population doubling times either as an adherent
`
`culture or as a suspension culture. At every 4—6
`
`population doublings, a specific production test is
`
`25
`
`30
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`
`performed using the human IgG specific ELISA and larger
`
`volumes are cultured to obtain the cell pellet and the
`
`supernatant. The cell pellet is used to assess the
`
`presence of the three constructs in the genomic DNA,
`
`either via PCR, Southern blot and/or FISH. The
`
`supernatant is used to purify the recombinant human IgG1
`
`fraction as described supra. Purified human IgG1 obtained
`
`at the various population doublings is analyzed as
`
`described, i.e. by SDS—PAGE, Iso—electric focusing (IEF)
`
`and binding to Her—2 by ELISA and by flow cytometry using
`
`BT474 cells.
`
`Functionality of the antibody mixture of anti-Her—Z
`
`antibodies is analyzed in cell—based assays to determine
`
`whether the human IgG1 mixture inhibits proliferation
`
`and/or induces apoptosis of BT474 cells.
`
`In addition the
`
`antibody mixtures are analyzed for their potential to
`
`induce antibody dependent cellular toxicity and
`
`complement dependent cytotoxicity of BT474 cells.
`
`In each of the experiments described below the
`
`functionality of the antibody mixture recognizing Her—2
`
`can be analyzed and compared to each of the individual
`
`IgG1 antibodies and to an equimolar combination of the
`
`three individual monospecific IgGl molecules.
`
`To assess the ability of the antibody mixtures to inhibit
`
`the proliferation of BT474 cells,
`
`these cells are allowed
`
`to adhere overnight in 96—well plates (1.5 x lOs/well) and
`
`are subsequently incubated with several concentrations (5
`
`— 20 ug/ml) of the antibody mixtures against Her—2 for 72
`
`hours. The proliferation of the cells is measured by 3H~
`
`10
`
`15
`
`2O
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`
`thymidine incorporation during the last 6 hours of
`
`culture. Inhibition of growth is determined by plotting
`
`the percentage of 3H—thymidine incorporation compared with
`
`untreated cells (taken as 100% reference value).
`
`10
`
`15
`
`2O
`
`25
`
`To analyze apoptosis induction of BT474 cells,
`
`these
`
`cells are allowed to adhere overnight
`
`in 48—well plates
`
`(2.5 X 105/well in lml) and are subsequently incubated
`
`with several concentrations (5 — 20 ug/ml) of the
`
`antibody mixtures against Her—2 for 4 hours. Hereafter
`
`the cells are harvested by trypsinization, are washed
`
`twice with PBS and are incubated at RT for 10 min with
`
`100 pl FITC—labelled annexin V (Caltag) diluted 1:25 in
`
`annexin V binding buffer (Caltag). Prior to the analysis
`
`of the samples by flow cytometry (FACSCalibur, Becton
`
`Dickinson, San Jose, CA) propidium iodide (PI)(Sigma)
`
`is
`
`added to a final concentration of 5 ug/ml to distinguish
`
`necrotic cells (annexin V’/PI+)
`
`from apoptotic cells
`
`(annexin V+/PI", early apoptotic cells; annexin V+/PI+,
`
`late apoptotic cells).
`
`Antibody Dependent Cellular Cytotoxicity of the antibody
`
`mixtures is analyzed using peripheral blood mononuclear
`
`cells as effector cells and BT474 cells as target cells
`
`in a standard 51Cr release assay as descibed supra (Huls
`
`et al, 1999). Complement dependent cytotoxicity is
`
`determined in a similar assay. Instead of the effector
`
`cells, now 50 pl human serum is added to the target
`
`cells. Subsequently,
`
`the assay is performed as described
`
`30
`
`supra.
`
`Alternatively, ADCC and CDC of the antibody mixtures is
`
`determined using a Europium release assay (Patel and
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`-g4_
`
`Boyd,
`
`l995)or using an LDH release assay (Shields et al,
`
`2001).
`
`The functionality of the antibody mixtures against Her—2
`
`is also tested using in vivo animal models, such as for
`
`instance described in Spiridon et al, 2002.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`Example 5
`
`Expression of different functional human IgGs in the milk
`
`of transgenic animals
`
`The V3 and Vh sequences of phages against proteins present
`
`on human B—cells, i.e. CD22 clone B28), CD72
`
`(clone II—2)
`
`and HLA—DR (clone I—2)
`
`(Fig. 7) are cloned into
`
`expression plasmid pBCl
`
`(as provided in the pBCl Mouse
`
`Milk Expression System, Invitrogen Life Technologies)
`
`to
`
`obtain mammary—gland and lactation—specific expression of
`
`these human IgG molecules in transgenic animals,
`
`according to the manufacturers instructions. These
`
`mammary—gland specific expression vectors, encoding the
`
`antibody sequences for anti—CD22, anti—CD72 and anti—HLA—
`
`DR are introduced into the murine germline according to
`
`the manufacturers instructions. Obtained pups are
`
`screened for the presence of each of the three constructs
`
`by PCR on DNA isolated from the tail. Pups, either male
`
`or female, confirmed for being transgenic for each of the
`
`three antibodies, are weaned and matured. Female
`
`transgenic mice are fertilized at the age of 6—8 weeks
`
`and milk samples are obtained at several time points
`
`after gestation. Male transgenic mice are mated with non—
`
`transgenic females and female transgenic offspring (as
`
`determined with PCR as described above)
`
`is mated and
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`
`milked as described above for the female transgenic
`
`founders. Whenever needed,
`
`female or male transgenic
`
`founders are mated for another generation to be able to
`
`obtain sufficient amounts of transgenic milk for each
`
`founder line. Transgenic milk is analyzed for the
`
`presence of human IgG with a human IgG specific ELISA,
`
`which does not cross—react with mouse IgG or other mouse
`
`milk components. Human IgG is purified from transgenic
`
`mouse milk using Protein A affinity chromatography
`
`according to standard procedures. Purified human IgG is
`
`analyzed on SDS—PAGE, Isoelectric focusing and binding on
`
`the targets CD22, CD72 and HLA—DR. Functionality of the
`
`antibody mixture is analyzed as described supra.
`
`Example 6
`
`Production of an IgA/IgG mixture against a
`
`predefined target in a PER.C6'm derived clone.
`
`The Vfi—VL sequences of the phage UBS—54 directed against
`
`the homotypic adhesion molecule EP—CAM (Huls et al.,
`
`1999) was not only cloned into a vector encoding the
`
`constant domains of a human IgG1 with Kappa light chain
`
`(expression vector pUBS3000Neo), but also into an
`
`expression vector encoding the constant domains of a
`
`human IgAl with Kappa light chain (expression vector
`
`pUBSS4—IgA, Fig. 8). Hence, antibodies derived from
`pUBS3000Neo and pUBSS4—IgA do bind to the same epitope on
`
`EPCAM. The only differences antibodies derived from
`
`pUBS3000Neo and pUBSS4—IgA are in the sequences encoding
`
`the constant domains of the heavy chain, resulting in
`
`either an IgG1 or IgAl isotype. The Kappa light chain
`
`sequences of these two vectors are identical.
`
`10
`
`15
`
`2.0
`
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`30
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`
`Stable PER.C6TM derived cell lines expressing antibodies
`
`encoded by genetic information on pUBS3000Neo and pUBSS4—
`
`IgA are generated by procedures well known to persons
`
`skilled in the art. Therefore, PER.C6TM cells are seeded
`
`in DMEM plus 10% FBS in tissue culture dishes (10 cm
`
`diameter) or T80 flasks with approximately 2.5 x 106 cells
`
`per dish and kept overnight under their normal culture
`
`conditions (10% C02 concentration and 37°C). The next day,
`
`transfections are performed in separate dishes at 37°C
`
`using Lipofectamine (Invitrogen Life Technologies)
`
`according to standard protocols provided by the
`
`manufacturer, with either 1—2 ug pUBS3000Neo and pUBS54—
`
`IgA. As a control for transfection efficiency, a few
`
`dishes are transfected with a LacZ control vector, while
`
`a few dishes are not transfected and serve as negative
`
`controls.
`
`After 4—5 hours, cells are washed twice with DMEM and
`
`refed with fresh medium without selection. The next day,
`
`medium is replaced with fresh medium containing 500 ug/ml
`
`G418. Cells are refreshed every 2 or 3 days with medium
`
`containing the same concentrations of G418. About 20—22
`
`days after seeding, a large number of colonies is visible
`
`and from each transfection at least 300 are picked and
`
`grown via 96—well and/or 24—well via 6—well plates to T25
`
`flasks. At this stage, cells are frozen (at least 1, but
`
`usually 4 vials per sub—cultured colony) and production
`
`levels of recombinant human IgG and human IgA antibody
`
`are determined in the supernatant using an ELISA specific
`
`for human IgG1 as well as an ELISA specific for human
`
`IgA. Also, at this stage G418 is removed from the culture
`
`medium and never re—applied again. For a representative
`
`number of colonies larger volumes are cultured to purify
`
`10
`
`15
`
`20
`
`25
`
`30
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`
`the recombinant human IgG1 and human IgA fraction from the
`
`conditioned supernatant using for instance (a combination
`
`of) Protein L or LA affinity chromatography, cation
`
`exchange chromatography, hydrophobic interaction
`
`chromatography and gel filtration. Purified human
`
`immunoglobulins from the various clones are analyzed on
`
`SDS—PAGE, ISO-electric focusing (IEF) and binding to the
`target EPCAM using cell lines having a high expression of
`
`this molecule. The clones will also be screened by PCR on
`
`genomic DNA for the presence or absence of pUBSBOOONeo
`
`and pUBSS4—IgA. The identity of the PCR products is
`
`further confirmed by DNA sequencing.
`
`A limited number of clones, which are screened positive
`
`for the production of both EPCAM IgG1 and EPCAM IgA, are
`
`subjected to single cell sorting using a fluorescence
`
`activated cell sorter (FACS)
`
`(Becton Dickinson FACS
`
`VANTAGE SE). Alternatively, colonies are seeded at 0.3
`
`cells/well to guarantee clonal outgrowth. Clonal cell
`
`populations, hereafter designated as sub-clones, are
`
`refreshed once a week with fresh medium. Sub—clones are
`
`grown and transferred from 96~wells via 24— and 6—wells
`
`plates to T25 flasks. At this stage,
`
`sub—clones are
`
`frozen (at least 1, but usually 4 vials per sub-clone)
`
`and production levels of recombinant human IgG1 and IgA
`
`antibody are determined in the supernatant using a human
`
`IgG1 specific ELISA and a human IgA specific ELISA. For a
`
`representative number of sub—clones,
`
`larger volumes are
`
`cultured to purify the recombinant human IgG1 and human
`
`IgAl fraction from the conditioned supernatant using for
`
`instance (a combination of) Protein L or LA affinity
`
`chromatography, cation exchange chromatography,
`
`hydrophobic interaction chromatography and gel
`
`10
`
`15
`
`20
`
`25
`
`30
`
`

`

`WO 2004/009618
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`PCT/EP2003/007690
`
`-8g_
`
`filtration. Purified human immunoglobulins from the
`
`various clones are analyzed on SDS—PAGE,
`
`Iso—electric
`
`focusing (IEF) and binding to the target EPCAM using cell
`
`lines having a high expression of this molecule.
`
`Sub—clones will also be screened by PCR on genomic DNA
`
`for the presence or absence of pUBSBOOONeo and pUB854—
`
`IgA. The identity of the PCR products is further
`
`confirmed by DNA sequencing.
`
`Other methods such as Southern blot and/or FISH may also
`
`be used to determine whether both constructs are present
`
`in the clonal cell line.
`
`Example 7
`
`—
`
`Production of a human IgGl/IgGg mixture
`
`against multiple targets in a clonal PER.C6“'M cell line
`
`Phage clone UBS—54 and Clone K53
`
`(Fig. 3) were obtained
`
`as described in Example 1. The VH and VL of clone UBS—54
`
`was inserted into an expression vector containing the
`
`HAVTZO leader sequence and all the coding sequences for
`
`the constant domains of a human IgG1 with a Kappa light
`
`chain by a method essentially as described (Boel et al,
`
`2000). The resulting plasmid was designated as
`
`pUBS3000Neo (Fig. 4). It will be clear that expression
`
`vectors containing heavy chain constant domains of any
`
`desired isotype can be constructed by routine methods of
`
`molecular biology, using the sequences of these regions
`
`that are all available in the art. The VH and VL sequences
`
`10
`
`15
`
`20
`
`25
`
`'of Phage clone K53 are cloned into an expression vector
`
`containing the HAVTZO leader sequence and all the coding
`
`30
`
`sequences for the constant domains of a heavy chain of a
`
`human IgGgWith a Kappa light chain by a method
`
`

`

`WO 2004/009618
`
`PCT/EP2003/007690
`
`-39_
`
`essentially as described (Boel et al, 2000). This
`
`expression vector is designated as pK53IgG3.
`
`These plasmids are transiently expressed either alone or
`
`in combination in PER.C6TM cells.
`
`In brief, each 80 cm2
`
`flask is transfected by incubation for 4 hours with 140
`
`pl lipofectamine + 10 pg DNA (either pUBS3000Neo,
`
`pK53IgG3 or 10 ug of both)
`
`in serum—free DMEM medium at
`
`37°C. After 4 hours,
`
`this is replaced with DMEM + 10%
`
`FBS, and the cells are grown overnight at 37°C. Cells are
`
`then washed with PBS and the medium is replaced with
`
`Excell 525 medium (JRH Bioscience). The cells are allowed
`
`to grow at 37°C for 6 days, after which the cell culture
`
`supernatant is harvested. Human IgG specific ELISA
`
`analysis, i.e. measuring all IgG sub—types,
`
`is done to
`
`determine the IgG concentration in transfected and non—
`
`transfected PER.C6"PM cells. Human IgG from each
`
`supernatant is subsequently purified using Protein A
`
`affinity chromatography (Hightrap Protein A HP, cat.no.
`
`1—040203) according to standard procedures,
`
`following
`
`recommendations of the manufacturer
`
`(Amersham
`
`Biosciences). After elution, samples are concentrated in
`
`a Microcon YM3O concentrator (Amicon) and buffer
`
`exchanged to 10 mM sodium phosphate, pH 6.7. Samples are
`
`analysed for binding to the targets EPCAM and CD46 using
`
`cell lines having a high expression of these molecules
`
`such as LSl74T cells. Twelve ug of purified IgG, either
`
`transiently expressed UBS—54 IgGl, K53 IgG3 or IgG from
`
`the cells in which both antibodies were co—transfected,
`
`is subsequently analyzed on Isoelectric focusing gels
`
`(Serva Pre—cast
`
`IEF gels, pH range 3—10, cat.no. 42866).
`
`Samples are loaded on the low pH side and after focussing
`
`stained with colloidal blue. The pI values of the major
`
`10
`
`15
`
`20
`
`25
`
`30
`
`

`

`WO 2004/009618
`
`PCT/EP2003/007690
`
`-90_
`
`isoforms for each sample are determined to illustrate
`
`whether there has been expression of UBS—54 IgGl, K53
`
`IgG3 or bispecific heterodimers, depending on how the
`
`cells were transfected. The identification of
`
`heterodimers would indicate that single cells have
`
`translated both the IgG3 heavy Chain of K53 and the IgGl
`
`heavy chain of UBS—54 and assembled these into a full
`
`length IgG molecule together with the common light chain.
`
`The absence of bispecific heterodimers indicates that it
`
`is possible to translate both the IgG3 heavy chain of K53
`
`and the IgGl heavy chain of UBS—54 in single cells, but
`
`that these do not assemble into a full length IgG
`
`molecule together with the common light chain, i.e.
`
`there
`
`is preferential binding of IgG1 and IgG3 heavy chains.
`
`This could however also be explained by the lack of co—
`
`expression of UBS—54 IgGl and K53 IgG3. Therefore, stable
`
`clonal cell lines expressing both pUBSSOOONeo and
`
`pK53IgG3 are generated by procedures as such well known
`
`to persons skilled in the art. PER.C6PM cells are seeded
`
`in DMEM plus 10% FBS in tissue culture dishes (10 cm
`
`diameter) or T80 flasks with approximately 2.5 x 106 cells
`
`per dish and kept overnight under their normal culture
`
`conditions (10% 002 concentration and 37°C). The next day,
`
`transfections are performed in separate dishes at 37°C
`
`using Lipofectamine (Invitrogen Life Technologies)
`
`according to standard protocols provided by the
`
`10
`
`15
`
`20
`
`25
`
`manufacturer, with either 1—2 ug pUBS3000Neo, pK53IgG3 or
`
`both. As a control for transfection efficiency, a few
`
`dishes are transfected with a LacZ control vector, while
`
`30
`
`a few dishes will be not transfected and serve as
`
`negative controls.
`
`

`

`WO 2004/009618
`
`PCT/EP2003/007690
`
`-91_.
`
`After 4—5 hours, cells are washed twice with DMEM and
`
`refed with fresh medium without selection. The next day,
`
`medium is replaced with fresh medium containing 500 ug/ml
`
`G418. Cells are refreshed every 2 or 3 days with medium
`
`containing the same concentrations of G418. About 20—22
`
`days after seeding, a large number of colonies is visible
`
`and from each transfection at least 300 are picked and
`
`grown via 96—well and/or 24—well via 6—well plates to T25
`
`flasks. At this stage, cells are frozen (at least 1, but
`
`usually 4 vials per sub—cultured colony) and production
`
`levels of recombinant human IgG antibody are determined
`
`in the supernatant using an ELISA specific for all sub—
`
`types of human IgG. Also, at this stage G418 is removed
`
`from the culture medium and never re—applied again. For a
`
`representative number of colonies larger volumes are
`
`cultured to purify the recombinant human IgG from the
`
`conditioned supernatant using Protein A affinity
`
`chromatography (Hightrap Protein A HP, cat.no. 1—040203)
`
`according to standard procedures,
`
`following
`
`recommendations of the manufacturer
`
`(Amersham
`
`Biosciences). Purified human immunoglobulins from the
`
`various clones are analyzed on SDS—PAGE, Iso-electric
`
`focusing (IEF) and binding to the targets EPCAM and CD46
`
`using cell lines having a high expression of these
`
`molecules such as LSl74T cells. The clones are also
`
`screened by PCR on genomic DNA for the presence or
`
`absence of pUBS3000Neo and pK53IgG3. The identity of the
`
`PCR products is further confirmed by DNA sequencing.
`
`A limited number of clones, which are screened positive
`
`for the production of both EPCAM IgG1 and K53 IgG3, are
`
`subjected to single cell sorting using a fluorescence
`
`activated cell sorter (FACS)
`
`(Becton Dickinson FACS
`
`10
`
`15
`
`20
`
`25
`
`30
`
`

`

`WO 2004/009618
`
`PCT/EP2003/007690
`
`-92_
`
`VANTAGE SE). Alternatively, colonies are seeded at 0.3
`
`cells/well to guarantee clonal outgrowth. Clonal cell
`
`populations, hereafter designated as sub—clones, are
`
`refreshed once a week with fresh medium. Sub—clones are
`
`grown and transferred from 96—wells via 24— and 6—wells
`
`plates to T25 flasks. At this stage,
`
`sub—clones are
`
`frozen (at least 1, but usually 4 vials per sub—clone)
`
`and production levels of recombinant human IgG antibody
`
`are determined in the supernatant using a human IgG
`
`specific ELISA. For a representative number of sub—
`
`clones,
`
`larger volumes are cultured to purify the
`
`recombinant human IgG fraction from the conditioned
`
`supernatant using Protein A affinity chromatography
`
`(Hightrap Protein A HP, cat.no. 1—040203) according to
`
`standard procedures,
`
`following recommendations of the
`
`manufacturer (Amersham Biosciences). Purified human
`
`immunoglobulins from the various clones are analyzed on
`
`SDS—PAGE, Iso~electric focusing (IEF) and binding to the
`
`targets EPCAM and CD46 using cell lines having a high
`
`expression of this molecules, such as for instance LSl74T
`
`cells, or transfectants expressing these molecules.
`
`Sub-clones are also screened by PCR on genomic DNA for
`
`the presence or absence of pUBS3000Neo and pK53IgG3. The
`
`identity of the PCR products is further confirmed by DNA
`
`10
`
`15
`
`20
`
`25
`
`sequencing.
`
`Other methods such as Southern blot and/or FISH may also
`
`be used to determine whether both constructs are present
`
`in the clonal cell line.
`
`Once the clonal sub—clones are available and confirmed
`
`30
`
`positive for the expression of both UBs—54 IgG1 and K53
`
`IgG3,
`
`the presence of functional K53 and UBS-54 shows
`
`that it is possible to generate a mixture of functional
`
`

`

`WO 2004/009618
`
`PCT/EP2003/007690
`
`-93_
`
`IgG’s with different isotypes with the common light chain
`
`in a single cell. Analysis of the expression of
`
`bispecific antibodies binding both EpCAM and CD46 will
`
`reveal to what extent the different heavy Chains having a
`
`different subtype will pair, which will influence the
`
`amount of bispecific antibodies produced. It is expected
`
`that no or very low levels of bispecific antibodies will
`
`be found in this case.
`
`10
`
`Example 8. Selection of phage carrying single chain Fv
`
`fragments specifically recognizing rabies virus glyco
`
`protein (RVGP) using RVGP—Ig fusion protein, and
`
`expression of mixtures of antibodies against the rabies
`
`15
`
`virus.
`
`This example describes the production of mixtures of
`
`antibodies against the rabies virus, as another potential
`
`target. As an antigen,
`
`the Rabies Virus Glycoprotein
`
`(RVGP)
`
`is chosen, but other rabies antigens may be chosen
`
`or included as well for this purpose. Several monoclonal
`
`antibodies recognizing RVGP have already been described
`
`in the art, and polyclonal antibodies have been
`
`recognized to be useful in treatment of rabies infections
`
`as well
`
`(e.g. EPO402029; EPO445625).
`
`

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