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`Eurapilaohoa Patamarnt _
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`Europaan Patent Oflioa
`Offioa curopéan dos hrovata
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`EUROPEAN PATENT APPUCATION
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`®mu‘:c 12 N 5100
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`A 51 K 39/395
`//C1231/91, GU1N33f54
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`® Priority: 1a.o1.ao us rmss
`
`
`
`E) Appuuni: 11-IEBDARDDFTRU-ETEEBOFTHELELAND
`STANFORD Jumon umvansmr
`End:-u Man, Stnnhrd umersny
`SI'.uIfdI'd.Ca!lfon-da54305lU3l
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`ZTJNR Iulladn HM
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`ATIECHDEFHGBITULUHLSE
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`® Inventor: I1|l|llI.HanryB.
`3‘ Cohrillo
`Stanford Caiflornio. 3||3fl5{U5I
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`@ nepnmnmm: Il.an-BoII.DIvIIICh|-Initphar It :1.
`MEWBU'FWEl..I.|S&OO'III&72I‘.ha.r|aryl.m1o
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`® Human hyhrldon-III. |IraI:urIon and product.
`
`® Human rnonodonal andbodv compositions. human-
`hurnan monoclonal hybridorna calla. hurnan non-viral trans-
`fo-rmad parduslariy non-Epslein-Barr virua'trana'lorI'I1ed. neo-
`plastic iyrnphaid calls. human antibody genes and their usaa.
`Human neoplastic oalin are developed for fusing with
`In-irnunhad lvrnphoid can: In provido atahla human-human
`hybridoma an-aim producing complete monoclonal anti-
`hodiaa for a oradefinod antigen. From a myaioma cell line,
`rapidly growing 8-azaguanino resistant HAT aanaitiva nails
`are coloured. The aalactad myoldrna calls an aoaaad with
`immunized lymphoid nails and the rasuhlng all mbrl-ura
`grown under controlled selective conditions. Lymphoma
`calls rnav be mhatituud for ma n-nrolorna oalla. Ahar
`expansion of the desired hvhridorna calls. the monoclonal
`antlhadiaa may he harvoaiai The hvhridornaa sum as a
`noun: for rnnsaonger am {or light and heavy chains which
`may he uaod for produuion of light and heavy chain
`Irnrnunuglobulin prouaina through hybrid DNA techniques.
`U-28-B-AR. call line has been deposited at Call Distribution
`Cantor. The Salk Inaflnna on July 17. 1530. and tho £LT.l'.‘..C.
`on Scplarnbar I1, 1380.
`
`1
`
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`
`,'
`
`
`oruuauuau-nsngouno-«Lu.
`
`Merck Ex. 1066, pg 1504
`
`
`
`
`
`r.-A-.a'_1.0:-d.KI$fl.-£>&h-..v-...'.n.‘na.n.
`
`EP0044722A1
`
`Merck Ex. 1066, pg 1504
`
`

`
`0044722’
`
`HUMAN EYBRIDDMAS, PRECURSORS AND PRODUCTS
`
`The mamalian capacity for producing immunoglobu-
`lins has found application in medicine and industry.
`The
`ability of immunoglobulins to distinguish specifically
`between chemical compounds of slightly differing structure
`has found broad application in the detection and measurement
`of a wide variety of compounds.
`in therapeutic applications,
`immunoglobulins can be administered to provide passive
`immunity against diseases. Major stumbling blocks in the
`wide application of immunoglobulin therapy were the hetero-
`geneity of antisera and the limited availability of human
`antisera for a specific antigen.
`The seminal discovery-by Kohler and Milstein of
`mouse "hybridomas“ capable of secreting specific monoclonal
`antibodies against predefined antigens ushered in a new era
`in experimental
`immunology. Many of the problems associated
`with heteroantisera are circumvented;
`the clonal selection
`and immortality of such hybridoma cell lines assure the
`monoclonality, monospecificity and permanent availability of
`their antibody products.‘ At the clinical level.
`the use of
`such antibodies is clearly limited by the fact that they are
`foreign proteins and would act as antigens to humans.
`Human cells have only been difficultly cultured in
`mztro. Efforts to achieve a human hybridoma which is a cross
`.e1«een a lymphoid cell and a myeloma cell have heretofore
`
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`Merck Ex.
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`Merck Ex. 1066, pg 1505
`
`

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`0044722
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`been unsuccessful. The problems of maintaining a stable
`culture of human cells have inhibited the ready production of
`human—human hybridomas.
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`-..-u--u-—.—o-u.-._-.
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`The production of mouse hybridomas is described by
`Kohler. G. and Hilstein. K.
`(1975) Nature §§§: 495~7;
`(1976)
`Euro. J.
`Immunol §: 511-519. Chimeric hybridomas generated
`by fusing mouse myeloma cells with huan imunoglohulin-
`producing cells were described by Levy, R. and Dilley, I.
`(1978) PNAS USA 1§: 4211-2415.
`Permanent cultures of
`specific antibody-producing human B-lymphocytes obtained by
`transformation with Epstein-Barr virus is described by
`steinitz, H.
`(1977) Nature gig: 42a—422.
`'
`SUMMARY OF THE INVENTION
`Non-viral transformed. particularly non-Epstein»
`Barr virus transformed, neoplastic lymphoid cells are grown
`under conditions to provide strains having HAT sensitivity
`for use as fusion partners. The neoplastic lymphoid cells
`may then be fused with lymphocytes to provide hybridomas
`capable of stably producing immunoglobulins to a predeter-
`mined ligand.
`
`In accordance with the subject invention. novel
`human neoplastic lymphoid cell strains are provided, which
`are employed for fusion with lymphoid cells to produce
`hybridomas capable of producing complete monoclonal anti-
`bodies having a unique specificity and homogeneous composi-
`tion. The invention therefore involves the development of
`the neoplastic lymphoid cell strains; the preparation of
`lymphoid cells producing antibodies to a specific antigen;
`the fusion of the immunized lymphoid cells and neoplastic
`lymphoid cells to produce hybridoma cells; the selective
`culturing of the hybridoma cells; and the production of
`monoclonal antibodies. The antibodies may be produced to a
`wide variety of haptens and antigens and may find use in
`immunoassays, passive imunization.
`treatment against infec-
`tion, diagnosis and treatment of cancer, and the like.
`In
`addition to the production of IgG, human-human hybridomas
`
`Merck Ex. 1066, pg 1506
`
`Merck Ex. 1066, pg 1506
`
`

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`oo44722‘
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`3
`
`offer opportunities for the production of complete human
`monoclonal Ign,
`Igm. and IgE.
`The human—human hybridomas can also serve as a
`useful source of cans for the heavy and light chains of
`antibodies for specific antigens. By known molecular biology
`techniques.
`the means may he used for the generation of genes
`which when inserted into the appropriate vector can serve as
`a source of the proteins. Upon assembling of the light and
`heavy chains, antibodies are produced.
`*
`Nonsviral transformed, particularly non-Epstein-
`Barr virus transformed. neoplastic lymphoid cells may be
`employed as fusion partners. The fusion partners are
`characterized by being differentiated. EAT medium sensitive.
`and unable to metabolize hyperxanthine.
`Illustrative of
`neoplastic lymphoid cells are cells obtained from a host with
`a lymphoma and cells obtained from a host with a myeloma.
`The lymphocytes are the principal cell type of lymph tissue.
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`5
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`The lymphoma line may then be employed in the same way as the
`myeloma line to provide hybridomas for the production of
`immunoglobulins specific for a predetermined determinant.
`a
`Human gyeloma Cell Line
`The human myeloma cell line is chosen to provide a
`stable cell line which is EAT medium sensitive and unable to
`metabolize hypoxanthine.
`The particular cell line chosen was
`U—266 which was originally described by Nilsson. K. et a1.,
`(1970) clin. Exp.
`Immunol 1: "477-439.
`' EAT sensitivity is achieved by culturing cells in a
`medium containing a purine analog such as B—azaguanine.
`Cells remaining viable under these conditions are mutants
`lacking an alternative biosynthetic pathway for the produc-
`tion of purines.
`'
`Specifically.
`the cells are first cultured at a
`high B-azaguanine concentration,
`then at a low a—azaguanine
`concentration, followed by cultivation at intermediate con-
`centration levels.
`In each instance.
`incubation times are
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`25
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`30 .
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`35
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`Merck ‘Ex. 1066, pg 1507
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`.--L.
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`n.._
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`—-._.-u.-nh....I....‘4.-..-....'.u
`
`Merck Ex. 1066, pg 1507
`
`

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`0044722
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`about one week, with the viable cells being isolated prior to
`the next incubation.
`The 8-azaguanine concentration varies
`in the range of about 3 to 2Spg/ml, usually in the range of
`about 5 to zopg/ml. At each stage the number of cells being
`incubated should be sufficient to ensure the isolation of
`viable cells at the end of the incubation. There should be
`at least 1 x 103, preferably 5 x 103 cells per microwell.
`Alternatively. a sngle stage may be employed with a semisolid
`
`medium e.g. agarose.
`The number of successive incubations with nutrient
`media containing B-asaguanine will be at least two and not
`more than about eight.
`I
`Selection is further made of the fastest growing
`8-azaguanine resistant HAT sensitive clones and it is these
`clones that are expanded. Rapidly growing clones normally
`
`double in about 24 to 36 hours.
`Except for the 8-azaguanine and HAT, the nutrient
`Prior to fusion the selected
`, media employed are conventional.
`cells are expanded in non-selective nutrient medium to enhance
`the number of cells.
`I
`
`Human Lymphoid cells
`_
`” The human lymphoid cells are cells immunised against
`a hapten or antigen. Various sources of lymphoid cells may
`be emloyed.
`one source is spleen specimens. which specimens
`are devoid of malignancies. The host should be immunized at
`least once. and at least about two weeks prior to the
`splenectomy. After freeing a single cell suspension of the
`spleen tissue of red blood cells and granulccytes,
`the viable
`mononuclear cells are suspended in an appropriate nutrient
`medium, and non—adherent cells separated from adherent cells.
`Desirably,
`the cells are grown in the presence of a mitogen
`for‘about 5-7 days to enhance fusibility. The lymphoid cell
`culture may then be fused with the myeloma cell line.
`Instead of 33 gigg immunization. spleen cells can
`be isolated and imunized in vitro.
`A single cell suspension
`of spleen cells is prepared. viable cells are isolated and
`seeded in nutrient medium with the appropriate antigen at an
`appropriate concentration. After sufficient time for immuni-
`zation. viable cells are isolated and used for fusion.
`
`Merck Ex. 1066, pg 1508
`
`Merck Ex. 1066, pg 1508
`
`

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`-1_n.u.u...-.._.-.-—-¢v-ZI-
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`0044722
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`An alternative to spleen lymhoid cells are
`
`lymphocytes isolated from peripheral blood. which are then
`combined in an appropriate nutrient medium containing
`'
`
`macrophages and a sufficient amount of an antigen to prime
`
`_
`
`the lymphocytes. After a sufficient time for priming. gen-
`
`erally from about two to four days.
`
`the viable cells may be
`
`separated from the dead cells and employed for fusion. The
`
`lymphocyte cells can be isolated by Ficoll-Eypaque gadient
`
`centrifugation and viable cells grown in nutrient medium,
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`10
`
`containing about 15% FCS. about 40ug/ml antigen. and about
`105 macrophages/ml and the cells incubated for three days to
`prime the cells and produce blast cells.
`The viable cells
`may then be used for fusion.
`_
`Fusion
`
`15
`
`The fusion is carried.out by combining the neo-
`
`plastic cells and lymphoid cells in an appropriate non-ionic
`
`detergent containing medium, normally polyethylene glycol of
`from about 1000 to eooodaltons. The period for the fusion is
`
`generally under about 3min. and the resulting cells are
`washed free of the nonrionic detergent. While ratios-other
`
`20
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`25
`
`than 1:1 of the two cell lines may be employed,
`
`the_best
`
`results have been obtained with a 1:1 ratio. Therefore, for
`
`enhanced probability of success in the fusion and isolation
`of desired hybridoma cells, an approximately 1:1 ratio of
`cells should be employed. The individual cell concentration
`will generally be from about 106 to 103, preferably about 1-2
`x 107 cells/ml. The cells are then seeded at relatively high
`concentrations in microplates in nutrient media.
`there being
`at least about 104-105 cells per well. preferably about 1-2 x
`105 cells per well. After a sufficient time for expansion,
`generally 1-4 days, usually about 2 days,
`the cells are then
`selected by incubation in BAT medium. While normally EAT
`resistant hybrids grow out within about one to two weeks, it
`is desirable that the culture be expanded in EAT medium for
`
`from about three to four weeks.
`
`The BAT medium which is employed is described in
`
`Littlefield, Science leg. 709 (1964) and contains a combina-
`tion of hypoxanthine. aminopterin or methotrexate, and
`
`thymidine.
`
`Merck Ex. 1066, pg 1509
`
`Merck Ex. 1066, pg 1509
`
`

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`0044722
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`Atter the initial incubation with the HA! medium,
`the supernatant fluid of each culture microwell is tested £or
`immunoglobulin production.
`conveniently, staph. protein
`I A-binding can be employed for detection of IgG and Iga (oz).
`If detection of other imunoglobulins is of interest, radio-
`labeled heterologous antisera to specific types of heavy
`chains can be used for the detection of each of the other
`types of immunoglobulins._ conveniently, any immunoassay may
`be used which can distinguish the various immunoglobulins,
`such as radioimmunoassays.
`the cells in the
`Once positive wells are detected,
`positive wells may be cloned under limiting dilution condi-
`tions. The resulting clones are then expanded and the mono-
`clonal antibodies are'then harvested in accordance with known
`procedures. The monoclonal antibodies may be freed of other
`proteins in accordance with known techniques, such as elec-
`trophoresis, chromatography, or the like.
`Monoclonal Antibodies
`the monoclonal human
`By appropriate immunization,
`antibodies may be prepared against any hapten or antigen.
`.By
`antibodies is intended-to include not only IgG, but also Igu,
`IgE and Iga. Particularly“ antibodies may be produced
`against.drugs, both naturally occurring and synthetic, such
`as opioids, amphetamines, barbiturates, steroids,
`catecholamines, dilantin,
`theophylline, histamine, PCP,-
`cannabinoids, or the like.
`Antigens of interest include bistocompatability and
`other cell membrane antigens, pathogen surface antigens,
`viral antigens,
`toxins, allergens, and the like.
`For a more complete list of ligands of interest,
`"see u.s. Patent No. 4,193,983 particularly columns 7-11
`inclusive, which disclosure is incorporated herein by
`
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`
`reference.
`
`.
`the subject invention
`as indicated previously,
`provides for production of the various immunoglobulins IgG,
`Igfl, Iga and IgE.
`‘As compared to previous immunoglobulin
`compositions,
`the subject compositions are homogeneous in
`composition. That is, greater than 90 weight 2, usually
`
`Merck Ex. 1066, pg 1510
`
`Merck Ex. 1066, pg 1510
`
`

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`0044722
`
`7
`
`greater than about 95 weight Z. more usually greater than
`
`about 99 weight 2 will have the same composition.
`
`By referring to.the same composition it is intended
`that the chemical composition and amino acid sequence of the I
`chains be the same;
`the chains be of substantially the same
`.chain length, normally the same chain length; and the folding
`of the molecules be substantially the same to define the same
`specificity.
`In effect. the primary, secondary and tertiary t
`structures of the immunoglobulin molecules in the composition’
`are substantially the same.
`'
`‘
`By having a uniform comosition of imunoglobulins A
`many advantages ensue. First. one is ensured of freedom fromi
`immuoglobulins specific for other than the predefined antigen.
`The presence of undesired immunoglobulins is disadvantageous
`for analytical work as well as for therapeutic purposes.
`
`_
`
`Secondly, one is assured of a single binding site, as compared
`to antibody compositions obtained from myeloma patients.
`-
`Third. one can obtain an exact titer for a specific
`
`determinant site, rather than averaging over the entire
`I_
`composition. with analytes, better control of cross-
`_
`reactivities can be achieved with a homogeneous composition.
`The subject monoclonal human antibodies find use in"
`
`conventional applications for antibodies, such as immuno-
`
`assays, cell sorting, electrophoretic analysis, histology,
`
`cytology and the_like. Besides the conventional uses. the
`subject monoclonal human antibodies have additional uses
`
`since they are not xenogeneic (foreign) proteins for other
`humans.
`I
`‘
`
`Because the human monoclonal antibodies will be
`accepted by the human imune system,
`the monoclonal human
`antibodies can be used for induction of passive immunity.
`Among immune sera which are presently available are antisera'
`
`for tetanus, hepatitis. vaccinia, mumps, rabies, pertussis,
`
`botulism, gas gangrene, varicella, as well as other diseases.
`The antisera are normally administered parenterally
`or by ingestion in dosages varying from 10d to 20,000 units,_
`_or in amounts based on immune serum of 0.005 to lml/kg of the
`host.
`(Medical Pharmacology 6th ed. Edited by Meyers,
`
`Merck Ex. 1066, pg 1511
`
`Merck Ex. 1066, pg 1511
`
`

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`0044722
`
`Jaivetz and Goldfien, Lange Medical Publications, 1978. pages
`612-615.) Particular dosages will vary depending upon the
`manner of adinistration. various carriers or media can be
`used, such as physiological saline, capsules, plasma, or the
`like. other additives may also be included, such as stabil-
`izers, drugs. proteins, and the like.
`-
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`35
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`for site directed therapy. By preparing antibodies recog-
`nizing determinant sites of an organ, abnormal cell e.g.
`‘tumor, or infectious cell, the antibody can serve to direct a
`drug or other therapeutic means to such site and maintain
`such drug or therapeutic means at such site. For example.
`the antibodies can be attached to slow release particles
`containing a particular drug for treatment of an infection.
`The antibodies would bind to the infected site. maintaining a
`high localized concentration of the drug in the infected
`area.
`_
`other uses include diagnosis, where the antibodies
`would be radioactively labeled, providing for localization of
`the radioactive label at a particular site, permitting
`I
`scintigraphy of a particular organ or other internal site.
`The hybridomas can also serve as a concentrated
`source of messenger RNA or as a source of the genes for the
`light and heavy chains of IgG.
`The desired messenger RNAs may be obtained as
`follows. The hybridoma cells are swollen on ice, ruptured,
`the nuclei removed by centrifugation, the supernatant iso-
`lated and centrifuged to produce a pellet containing the
`membrane-bound polysomes. The pellet is resuspended in
`appropriate medium. deproteinized by conventional means and I
`the RNA precipitated by adding buffer and ethanol.
`The poly A-rich mRNA can be concentrated with an
`oligo d1-cellulose or poly dU-Sepharose chromatographic
`column. The mRNA mixture is then resolved employing density
`gradient centrifugation and/or gel electrophoresis and the
`fractions collected.
`'
`l
`The mRNA fractions may then be assayed_for in a
`number of ways. The mRNA from the parent myeloma cell may be
`S
`
`Merck Ex. 1066, pg 1512
`
`
`
`.-I\.
`
`
`
`
`.n...—..-u-.—n__n‘-.g.—..—am":aIh.'.n
`_._.__n..-.m...a..‘....‘....a....._......n. '_
`
`
`
`Merck Ex. 1066, pg 1512
`
`

`
`yoo44722
`
`9
`
`treated in the same way and common bands between the mRHA
`
`mRRA
`from the hybridomas and the myeloma cells discarded.
`molecules of the appropriate molecular weights for the light
`and heavy chains can be employed under the same conditions of
`density gradient centrifugation to further narrow the number
`of bands.
`
`10
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`15
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`25
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`3o_
`
`35
`
`For further elimination of mRNA molecules other
`than those expressing the desired light and heavy chains,
`probes can be prepared of RNA or ssDHA.' The probes are
`synthesized from nucleotides corresponding to the codon
`sequence coding for a portion of the polypeptide light and
`heavy chains respectively.
`.the probe will usually have at
`least 20 bases, preferably at least about 30 bases.
`A 32?
`
`marker is employed for autoradiographic visualization.
`The probe is hybridized with the electrophoretic ‘
`fractions under conditions where only mRNA substantially
`homologous with the probe will hybridize.
`(See, Southern, J.
`Hol. Biol. gg, 503 (1975)). where the probe is based on the
`variable portions of the light and.heavy chains,-only the
`desired mnnas will be isolated. or highly concentrated frac-
`tions thereof.
`
`It is not necessary, however,
`
`to isolate the mRHAs
`
`expressing the desired light and heavy chains. Purification
`can be achieved subsequently by isolation of transformants
`
`producing the desired light and heavy chains, emloying
`antisera to the chains for detecting the desired clones.
`After isolating the mamas substantially pure or as
`a mixture, coma may be prepared by employing reverse tran-
`scriptase in accordance with conventional techniques (nuell,
`et al. J. Biol. Chem. £21:
`2471 (19?8)). The dsDNA is
`generated using DNA polymerase and S1 nuclease (wickens, at
`al., ibig ggg:
`2483 (1978)). Sequencing of the 5'-ends will
`determine the sites of initiation of the light and‘heavy
`chains. The DNA sequence preceding the fhmet codhn may be
`removed employing an exonuclease and replaced with a short
`
`_sequence providing cohesive ends, a host ribosomal start site
`
`or other appropriate coding.
`
`Merck'E'>'<. 1066, pg 1513
`
`
`
`_..n.—.__.——H'
`
`Merck Ex. 1066, pg 1513
`
`

`
`0044722
`
`10
`
`The dsDNA for the light and heavy chains may be
`joined to any conventional vector by conventional means.
`vectors will normally have a marker. conveniently antibiotic
`resistance, for selection of transformants.
`Illustrative
`vectors include psc1o1. Aplac. pBR322. Tips. and the like
`which may be used for transformation of bacteria and yeast.
`The dsnnh may be joined to the vector by means of blunt end
`ligation, for example, with T-4 ligase; or the termini modi-
`fied. by ligation of a short dsnna having a staggered end and
`a blut end to provide for cohesive ends; or by adding on
`complementary sequences employing deoxynucleotidyl trans-
`ferase. As indicated previously, modification of the termini
`can be used for introducing particular signals, providing for
`binding to the vector. as well as providing restriction
`sites. The dsDNA is joined to the replicon to provide a
`ribosomal start site near the femet codon. various
`techniques are available for either introducing a ribosomal
`start site on the dsDHA adjacent to £¥met codon or joining
`the gene adjacent the ribosomal start site of the vector.
`The vector and dsDHA are joined under hybridizing
`and ligating conditions to produce circular DNA or plasmids
`and'host cells transformed under transforming conditions e.g.
`calcium shock. The cells are then grown under selective
`conditions to kill any untransformed host cells.
`The re-
`maining viable cells are streaked on selective media and
`individual clones grown and tested for production of the
`desired light and heavy chains. The light and heavy chains
`are isolated from the clones. by rupturing the cells and then
`employing conventional separation techniques, such as density
`gradient centrifugation, electrophoresis, chromatography, and
`the like. The purified light and heavy chains are then -
`combined under mildly oxidizing conditions to provide for
`folding of the chains together and disulfide formation.
`As an alternative to emloying the mamas,
`the DNA
`may be synthesized based on the mana sequence.
`See European
`Patent Application 0 001 929. Oligodeoxyribonucleotides can
`be prepared and joined together to provide ssDNA. The coding
`strand of ssDNA can be synthesized with appropriate host
`
`10
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`35
`
`Merck Ex. 1066, pg 1514
`
`J}
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`‘:3
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`-._—:......._——n.-u-:u._-——L.-..'
`
`Merck Ex. 1066, pg 1514
`
`

`
`11
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`004472
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`5
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`signals. e.g. ribosomal start and stop, promoter and operate:
`, sigals. Also. appropriate restriction sites are provided at
`the termini for joining_to the vector and retrieving the
`genes after cloning. Once the gene has been synthesized, it
`may be inserted into a replicon as described above.
`The following examples are offered by way of illus-
`tration and not by way of limitation.
`'
`EXPERIMENTAL
`
`10
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`15
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`20
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`30
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`In order to demonstrate the subject invention with
`_
`neoplastic granulocytes,
`the following preparation was
`carried out.
`A EAT medium sensitive mutant cell line was
`selected from the U-266 human myeloma cell line originally:
`described by Nilsson et al. supra, U-266 cells were
`incubated for one week in RPMI-1640 medium containing 15% PC
`and zopg/a1 B—azaguanine: dead cells were then removed by"
`Ficoll—Hypaque gradient centrifugation and viable cells were
`incubated in RPMI-1640 plus 15x FCS plus Spg/ml 8-azaguanine
`for three days followed by isolation of viable cells using a
`Ficoll-Hypaque gradient. The viable-cells were then seeded
`in one well
`.5 x 103 cells). The cells are then grown at
`gradually increasing concentrations of B-azaguanine going
`from 5 to Zppg/ml at Spg/ml increments for 1 week at each
`concentration. The viable cells are then maintained in
`RPMI-1640 plus 15% FCS plus Zopg/ml B—azaguanine.
`cultures
`of the fastest growing 8-azaguanine resistant clone were
`expanded, after verifying that they were HAT sensitive." find
`mutant cell line. U—266-'-AR1
`maintained in am-11-1540 plus
`15% PCS plus Spg/ml 8—azaguanine. The cells are seeded at a
`concentration of 105/ml 3-5 days before fusion. On the day
`of fusion. the cell concentration is about o'.a-1.0 x 105
`cell/ml. The viability is above 90%.
`Fresh spleen specimens were obtained from untreab
`patients with Hodgkin's disease undergoing staging laparotm
`with splenectomy. The spleens were devoid of involvement h
`Hodgkins disease. At least two weeks prior to surgery, sud
`patients were‘sensitized and later challenged with
`2-dinitrochlorobenzene.
`'
`'
`'
`A single cell suspension prepared from the spleen
`tissue was freed of red blood cells and granulocytes by
`Merck Ex. 1066, pg 1515
`
`
`
`is...
`
`Merck Ex. 1066, pg 1515
`
`

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`Ficoll-Hypaque gradient centrifhgation and the viable mono-
`nuclear cells suspended in RPHI-1640 medium. Adherent cells
`were removed by incubation of the mononuclear cells in
`plastic dishes three times for zonins. each at 37°C and
`removal of the non-adherent cells after each incubation. The
`lymphocyte-enriched mononuclear cell suspensions thus
`obtained were then fused with U-266-aR;#hnman_myeloma cell
`line.
`'3‘ FiTc.c.
`cm. 803,3 .'s-eaosi-re-,5
`II set‘-*r 16160
`Fusion was achieved by mixing 2 x 107 myeloma cells
`and 2 x 107 lymphoid cells, washing twice in RPMI—l640 and
`then fusing in 2.0ml 38% wyv polyethylene glycol
`(‘1400mw) at
`37°C. After the last wash, the supernatant is removed as
`quickly as possible and the polyethylene glycol added drop-
`wise over a minute at 37°C. The cell pellet is carefully
`stirred for 1min. in polyethylene glycol.
`then gently resus-
`pended with a lml pipette. The cells are centrifuged at
`doorpm for dmins. and B00rpm for émins. The polyethylene
`glycol supernatant is discarded, the pellet resuspended in
`warm (37°C) serum-free RPMI-1640 and washed twice with warm
`(37°c) RPMI-1640 medium, and then suspended at a concentra-
`tion of 10° cells/ml in RPM!-1640 plus 1574 res. ' The cells
`are seeded in 0.2ml aliquots in microtiter plates with flat-
`bottom wells in RPM!-1640 plus 20% FCS and then incubated in
`the same medium for 48hrs. After 48hrs., the medium is
`changed to EAT medium and the cells incubated in HAT medium
`for eight days. The EAT medium is 1O'4§ hypoxanthine; 6.3 x
`10'BH methotrexate: 1.5 x 10-63 thymidine; 40i.u./ml insulin
`and 13.2mg/looml oxaloacetate.
`‘
`The supernatant fluid of each cu1ture'microwel1 is
`then tested for imunoglobulin production by employing a
`solid-phase radioimunoassay using 1251 labeled staph. pro-
`tein A as the detector. This test is only diagnostic of IgG
`(11, 12, 14) and IgA(u2). Therefore, production of other
`immunoglobulins such as Ig and IgE would go undetected. By
`employing appropriate antibodies,
`the other_types of immuno-
`globulins could also be detected.
`'
`Cultures containing immunoglobulin producers were
`expanded for two days in RPMI-1640 plus 20% FCS plus
`Merck Ex. 1066, pg 1516
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`15
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`20
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`25
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`
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`Merck Ex. 1066, pg 1516
`
`

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`13
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`0044722
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`the culture was grown in
`40i.u./ml insulin. After two days,
`HA! medium for another 1-2 weeks. wells that showed immuno-
`globulin production were then tested for production of anti-
`bodies binding specifically to dinitropheny1—BSA. Several
`anti—dinitropheny1 antibody-producing cultures were detected.
`Cells from such wells were cloned by limiting dilution pro-
`cedure and cultures of the clone producing the'highest level
`of specific anti-dinitrophenyl antibody were expanded.
`A hybridoma cell clone producing a high level of
`anti-dinitrophenyl antibody was incubated overnight in medium
`containing 14¢-leucine. The immunoglobulins in the super-
`natant were imunoprecipitated with rabbit anti-Fc and anti-
`light chain antisera and the precipitate analyzed sequen-
`tially by sodium dodecylsulfate-polyacrylamide gel electro-
`phoresis and by isoelectric focusing.
`
`In a second experiment a human spleen was isolated
`and treated as previously described. After cutting into
`pieces and forming a single cell suspension. red cells are
`
`removed employing a Ficoll-Eypague gradient centrifugation.
`The viable cells are seeded at 2 x 106 cells/ml in tissue
`culture flasks in urn:-1540 + 15x rcs + 1o'5§
`2-mercaptoethanol to which was added sheep red blood cells to
`a final concentration of one percent. After 4 days, the
`non-adherent cells were isolated and dead cells removed
`
`The
`employing a Ficoll—Hypaque gradient centrifugeion.
`buffy-coat was isolated.and used for fusion under the same
`
`conditions as described previously. After incubation in HA!
`medium as described above clones were screened for IgG using
`1251-labeled staph. protein A. The production of Igc-was not
`detected. The clones were then screened for IgM production
`using a test analogous to the Jerne Plague Forming Assay.
`The test employs superimposed layers of agar, with SRBC and
`complement in one layer and the hybridoma cells in the other
`layer. Production of Ig results in lysis of the SRBC with
`formation of a plaque. Production of Ign was observed by
`plaque formation with at least one clone§
`
`lymphoma
`Following the procedure described above,
`cells can be obtained which are HAT srusitive and may be used
`as fusion partners for humanrhuman hybridomas.
`
`Merck Ex. 1066, pg 1517
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`10
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`15
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`25
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`-|
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`I i12
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`3! 1 !
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`Merck Ex. 1066, pg 1517
`
`

`
`oo44722
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`14
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`5
`
`10
`
`In accordance with the subject invention, a novel
`myeloma strain is provided which can be used for fusion with
`lymphoid cells to produce hybridomas. The hybridomas which
`are produced can be stably cultured in vitro and provide for
`a continuous source of monospecific monoclonal antibodies.
`In this manner, a wide variety of antibody compositions can
`be produced which are free of xenogeneic proteins. The
`complete human monoclonal antibodies can find wide uses,
`since they will be accepted by humans and are a homogenous
`composition having a unique binding site.
`‘
`Although the foregoing invention has been described
`in some detail by way of illustration and example for pur-
`poses of clarity of understanding. it will be obvious that
`certain changes and modifications may be practiced_
`
`
`
`iI
`
`3 IT
`
`Merck Ex. 1066, pg 1518
`
`Merck Ex. 1066, pg 1518
`
`

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`..........—'....
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`
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`'oo44722
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`20
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`cells in each well;
`
`growing the cells in HAT medium to produce clones
`
`free of HAT sensitive cells;
`
`selecting for clones producing monoclonal antibodies
`
`for said predefined determinant site.
`
`‘
`
`2. A method according to Claim 1, wherein said neoplastic
`
`&. Human monoclonal antibodies derived from a human-human
`
`25
`
`hybridoma.
`
`5. Antibodies according to Claim h, wherein said hasn-
`
`Merck Ex. 1066, pg 1519
`
`Merck Ex. 1066, pg 1519
`
`

`
`
`
`human hybridoma is derived from a myeloma cell.
`7.
`.Hnman monoclonal antibodies according to any of Claims
`A to 6, wherein said antibodies are IgM.
`8. Human monoclonal antibodies accordin
`
`g to any of Claims
`
`4 to 6, wherein said antibodies are Igs.
`Myeloma cells of the U-266-AR1 strain or cells derived
`
`9.
`
`to
`
`therefrom.
`10. Human hybridoma cells
`11.
`A method for treating a human host subject to an
`cal response from contact with a
`
`undesirable physiologi
`
`surface antigen of a pathogeni
`
`o cell; which comprises:
`
`15
`
`administering to said human ho
`antibodies specific for said antigen in an amount sufficient
`
`st human monoclonal
`
`to diminish said udesirable response.
`12.
`A composition of matter consisting essentially of
`
`20
`
`human IgG molecules characterized by being:
`a)
`substantially free of incomplete IgG molecules;
`b)
`homogeneous in chemical composition;
`and
`c)
`specifically binding to a predefined determinant
`
`site on an antigen.
`
`Merck Ex. 1066, pg 1520
`
`Merck Ex. 1066, pg 1520
`
`

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`_
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`.
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`0
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`Empm_,3m PARTIAL EUROPEAN semen REPORT
`which uncle! Rule 45 of Ihe European Patent convention
`shafl be considered. forthe purposes of subsequani
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`0044722
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`EP 31 3° 3235
`
`DOCUMENTS CONSIDERED TO BE REL