`European Patent Office
`Office européen des brevets
`
`7 Publication number:
`
`0 044- 722
`A1
`
`®
`
`EUROPEAN PATENT APPLICATION
`
`® Application number: 813032863
`__
`@ Dateoffllmg: 16.07.81
`
`@ lnt..Cl.3: C 12 N 5/00
`C 12 N 15/00, C12 F 1/00
`A 61 K 39/395
`//C1 2R1/91, G01 N33/54
`
`
`
`Priority: 13.07.30 us 170255
`
`
`@ Applicant: THE some OFTRUSTEES or THE LELAND
`STANFORD JUNIOR UNIVERSITY
`Encina 6-930, Stanford University
`
`
`
`@ Date of publication of application:
`27.01.82 Bulletin B2/4
`
`Designated Contracting States:
`ATBECH DEFRGBITLILU NLSE
`
`Stanford, California 94305(USl
`
`® Inventor: Kaplan, Henry S.
`631 Cabrillo
`Stanford California, 54305lUS)
`
`® inventor: Olsson,Lennart
`20A Ibstrupvei
`DK-2820 GentoftelDKl
`
`Representative: Harrison, David Christopher at al.
`MEWBURN ELLIS & CO 70 8:72 Chancery Lane
`London WC2A 1AD(GB)
`
`7,»
`
`@ Human hybridomas, precursors and products.
`
`@ Human monoclonal antibody compositions, human-
`human monoclonal hybridoma cells, human non-viral trans- ‘
`formed particularly non-Epstein-Barr virus transformed, neo-
`plastic lymphoid cells. human antibody genes and their uses.
`Human neoplastic cells are developed for fusing with
`immunized lymphoid cells to provide stable human-human
`hybridoma strains producing complete monoclonal anti-
`bodies for a predefined antigen. From a myeloma cell line,
`rapidly growing B-azaguanine resistant HAT sensitive cells
`are selected. The selected myeloma cells are crossed with
`immunized lymphoid cells and the resulting cell mixture
`grown under controlled selective conditions. Lymphoma
`cells may be substituted for
`the myeloma cells. After
`expansion of the desired hybridoma cells. the monoclonal
`antibodies may be harvested. The hybridomas serve as a
`source for messenger RNA for light and heavy chains which
`may be used for production of
`light and heavy chain
`immunoglobulin proteins through hybrid DNA techniques.
`U-266-AR1 cell line has been deposited at Cell Distribution
`Center. The Salk Institute on July 17, 1980, and the A.T.C.C.
`on September 11, 1980.
`
`EP0044722A1
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`.,
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`Croydon Printing Company Ltd.
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`Sanofi/Regeneron Ex. 1032, pg 920
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`Mylan Ex. 1032, pg 920
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`0044722"
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`-HUMAN HYBRIDOMAS, PRECURSORS AND PRODUCTS
`
`The mammalian capacity for producing immunoglobu-
`lins has found application in medicine and industry.
`The
`ability of immunoglobulins to distinguish specifically
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`5
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`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-
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`10
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`geneity of antisera and the limited availability of human
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`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
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`15
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`in experimental immunology. Many of the problems associated
`with heteroantisera are circumvented;
`the clonal selection
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`20
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`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
`iitrg. Efforts to achieve a human hybridoma which is a cross
`Aetieen a lymphoid cell and a myeloma cell have heretofore
`
`
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`'4, 2’i
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`'1‘
`}
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`a BAD onueungr
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`Sanofi/Regeneron Ex. 1032, pg 921
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`Mylan Ex. 1032, pg 921
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`The problems of maintaining a stable
`been unsuccessful.
`culture of human cells have inhibited the ready production of
`human-human hybridomas.
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`10
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`The production of mouse hybridomas is described by
`5 Kohler, o. and Milstein, K.
`(1975) Nature ;5__e_: 495-7;
`(1976)
`Euro. J.
`Immunol Q: 511-519. Chimeric hybridomas generated
`by fusing mouse myeloma cells with human immunoglobulin-
`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, M.
`(1977) Nature 2_§g: 420,-422.
`SUMMARY OF THE INVENTION
`Non-viral transformed, particularly non-Epstein-
`l5 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-
`
`;
`
`’
`
` 20 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 immunization,
`treatment against infec-
`tion, diagnosis and treatment of cancer, and the like.
`In
`addition to the production of IgG, human-human hybridomas
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`25
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`Sanofi/Regeneron Ex. 1032 pg 922
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`Mylan Ex. 1032, pg 922
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`0044722
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`offer opportunities for the production of complete human
`
`monoclonal
`
`IgA,
`
`IgM, and IgE.
`
`The human-human hybridomas can also serve as a
`
`useful source of mRNA for the heavy and light chains of
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`5
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`antibodies for specific antigens.
`
`By known molecular biology
`
`the mRNAs may be used for the generation of genes
`techniques,
`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.
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`10
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`'
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`Non-viral transformed, particularly non-Epstein-
`
`Barr virus transformed, neoplastic lymphoid cells may be
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`15
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`20
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`The fusion partners are
`employed as fusion partners.
`characterized by being differentiated, HAT 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.
`Human Lymphoma Cell Line.
`A human lymphoma may be modified as described below
`
`for a myeloma line to provide a HAT sensitive fusion partner.
`The lymphoma line may then be employed in the same way as the
`myeloma line to provide hybridomas for the production of
`s
`immunoglobulins specific for a predetermined determinant.
`
`Human Myeloma Cell Line
`The human myeloma cell line is chosen to provide a
`
`25
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`stable cell line which is HAT medium sensitive and unable to
`metabolize hypoxanthine.
`The particular cell line chosen was
`U-266 which was originally described by Nilsson, K. et al.,
`
`(1970) Clin. Exp. Imunol 2: 477-489.
`' HAT sensitivity is achieved by culturing cells in a
`30 _
`' medium containing a purine analog such as 8-azaguanine.
`
`Cells remaining viable under these conditions are mutants
`
`lacking an alternative biosynthetic pathway for the produc-
`
`tion of purines.
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`'
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`35
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`the cells are first cultured at a
`Specifically,
`high 8=azaguanine concentration,
`than at a low 8—azaguanine
`concentration, followed by cultivation at intermediate cone
`
`centration levels.
`
`In each instance,
`
`incubation times are
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`Sanofi/Regeneron Ex. 1032, pg 923
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`Mylan Ex. 1032, pg 923
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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 25pg/ml, usually in the range of
`about 5 to 20pg/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 8-azaguanine will be at least two and not
`more than about eight.
`'
`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-
`the nutrient
`Except for the 8-azaguanine and HAT,
`_ media employed are conventional. Prior to fusion the selected
`cells are expanded in non-selective nutrient medium to enhance
`the number of cells.
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`Human Lymphoid Cells
`” The human lymphoid cells are cells immunized against
`a hapten or antigen. Various sources of lymphoid cells may
`be employed.
`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 granulocytes,
`the viable
`mononuclear cells are suspended in an appropriate nutrient
`30 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 lg yiyg immunization, spleen cells can
`be isolated and immunized in yitgg.
`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.
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`35
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`Sanofi/Regeneron Ex. 1032, pg 924
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`Mylan Ex. 1032, pg 924
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`An alternative to spleen lymphoid cells are
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`[lymphocytes isolated from peripheral blood, which are then
`
`combined in an appropriate nutrient medium containing
`
`'
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`;
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`macrophages and a sufficient amount of an antigen to prime
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`5
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`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-Hypaque gradient
`
`centrifugation and viable cells grown in nutrient medium,
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`10
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`containing about 15% FCS, about 40pg/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
`
`
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`p
`4
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`é
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`15
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`The fusion is carried out by combining the neo-
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`plastic cells and lymphoid cells in an appropriate non-ionic
`
`detergent containing medium, normally polyethylene glycol of
`
`from about 1000 to 4000daltons.
`
`The period for the fusion is
`
`generally under about 3min. and the resulting cells are
`20 washed free of the non—ionic detergent. While ratios-other
`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
`25 H cells should be employed.
`The individual cell concentration
`will generally be from about 106 to 108, preferably about 1-2
`x 107 cells/ml.
`The cells are then seeded at relatively high
`
`there being
`concentrations in microplates in nutrient media,
`at least about lO4—l06 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
`
`30
`'
`
`selected by incubation in HAT medium. While normally HAT
`
`resistant hybrids grow out within about one to two weeks, it
`
`is desirable that the culture be expanded in HAT medium for
`from about three to four weeks.
`
`35
`
`The EAT medium which is employed is described in
`
`Littlefield, Science lgg, 709 (1964) and contains a combina-
`
`tion of hypoxanthine, aminopterin or methotrexate, and
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`thymidine.
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`Sanofi/Regeneron Ex. 1032, pg 925
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`After the initial incubation with the BAT medium,
`the supernatant fluid of each culture microwell is tested for
`imunoglobulin production. Conveniently, Staph. protein
`A-binding can be employed for detection of IgG and IgA (a2).
`If detection of other immunoglobulins 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
`By appropriate immunization, the monoclonal human
`antibodies may be prepared against any hapten or antigen. By
`antibodies is intended to include not only IgG, but also IgM,
`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 histocompatability 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-ll
`inclusive, which disclosure is incorporated herein by
`reference.
`
`35
`
`the subject invention
`As indicated previously,
`provides for production of the various immunoglobulins IgG,
`IgM,
`IgA and IgE.
`‘As compared to previous immunoglobulin
`compositions,
`the subject compositions are homogeneous in
`composition. That is, greater than 90 weight %, usually
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`Sanofi/Regeneron Ex. 1032 pg 926
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`greater than about 95 weight %, more usually greater than
`.about 99 weight 2 will have the same composition.
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`5
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`10
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`15
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`By referring to the same composition it is intended
`that the chemical composition and amino acid sequence of the 0
`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 1
`structures of the immunoglobulin molecules in the composition"
`are substantially the same.
`.
`By having a uniform composition of immunoglobulins
`many advantages ensue. First, one is ensured of freedom fromi
`immunoglobulins 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.
`0
`Third, one can obtain an exact titer for a specific
`determinant site, rather than averaging over the entire
`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 immune»
`
`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.
`T
`'
`
`Because the human monoclonal antibodies will be
`accepted by the human immune 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 100 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,
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`3
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`Sanofi/Regeneron Ex. 1032, pg 927
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`%‘
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`0044722
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`5
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`Jaivetz and Goldfien, Lange Medical Publications, 1978, pages
`612-615.) Particular dosages will vary depending upon the
`manner of administration. 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.
`The human monoclonal antibodies can also be used
`for site directed therapy.
`By preparing antibodies recog-
`nizing determinant sites of an organ, abnormal cell e.g.
`10 ‘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
`.
`scintigraphy of a particular organ or other internal site.
`T
`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
`the RNA precipitated by adding buffer and ethanol.
`The poly A-rich mRNA can be concentrated with an
`oligo dT-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.
`'
`1
`i
`The mRNA fractions may then be assayed for in a
`number of ways.
`The mRNA from the parent myeloma cell may be
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`15
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`20
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`25
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`30‘
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`35
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`t
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`
`f
`E
`Q
`3
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`"Q
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`
`
`
`
`n..4.....*-.'......l.............
`
`.
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`Sanofi/Regeneron Ex. 1032, pg 928
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`treated in the same way and common bands between the mRNA
`
`mRNA
`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
`
`5
`
`density gradient centrifugation to further narrow the number
`of bands.
`
`For further elimination of mRNA molecules other
`
`than those expressing the desired light and heavy chains,
`probes can be prepared of RNA or ssDNA.
`The probes are
`
`10
`
`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 32P
`
`marker is employed for autoradiographic visualization.
`
`15
`
`The probe is hybridized with the electrophoretic
`
`fractions under conditions where only mRNA substantially
`
`homologous with the probe will hybridize.
`
`(See, southern, J.
`
`Mol. Biol. gg, 503 (1975)). Where the probe is based on the
`
`20
`
`variable portions of the light and heavy chains, only the
`desired mRNAs will be isolated, or highly concentrated frac-
`tions thereof.
`
`It is not necessary, however,
`
`to isolate the mRNAs
`
`expressing the desired light and heavy chains. Purification
`can be achieved subsequently by isolation of transformants
`
`25
`
`producing the desired light and heavy chains, employing
`antisera to the chains for detecting the desired clones.
`
`After isolating the mRNAs substantially pure or as
`a mixture, CDNA may be prepared by employing reverse tran-
`scriptase in accordance with conventional techniques (Buell,
`
`30
`
`The dsDNA is
`2471 (1978)).
`et al. J. Biol. Chem. ggz
`' generated using DNA polymerase and S1 nuclease (Wickens, et
`
`al.,
`ibid gggz
`2483 (1978)).
`Sequencing of the 5'-ends will
`
`determine the sites of initiation of the light and heavy
`chains. The DNA sequence preceding the f-met codon may be
`
`35
`
`removed employing an exonuclease and replaced with a short
`
`_sequence providing cohesive ends, a host ribosomal start site
`
`or other appropriate coding.
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`Sanofi/Regeneron Ex. 1032, pg 929
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`i i
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`T
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`3;
`ii?
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`0044722
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`5
`
`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, kplac, pBR322, Y1p5, and the like
`which may be used for transformation of bacteria and yeast.
`The dsDNA 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 dsDNA having a staggered end and
`a blunt 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 f-met codon. Various
`techniques are available for either introducing a ribosomal
`start site on the dsDNA adjacent to f-met codon or joining
`the gene adjacent the ribosomal start site of the vector.
`The vector and dsDNA 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-
`25 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 employing the mRNAs,
`the DNA
`35 may be synthesized based on the mRNA 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
`
`15
`
`20
`
`30
`
`
`
`7?
`
`i.....‘_~‘.'......_.;;4...
`
`
`
`
`
`..—-H-Iu.‘.'..~.-....a.a..a.....L...._.......4.....’.
`
`Sanofi/Regeneron Ex. 1032, pg 930
`
`Mylan Ex. 1032, pg 930
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`
`
`0044722
`
`11
`
`signals, e.g. ribosomal start and stop, promoter and operator
`
`..signals. 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
`
`5 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.
`EXPER IMENTAL
`
`_
`
`In order to demonstrate the subject invention with
`
`10
`
`the following preparation was
`neoplastic granulocytes,
`carried out.
`A HAT 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% FCS
`
`15
`
`and 20pg/ml 8—azaguanine; dead cells were then removed by
`
`20
`
`25
`
`30
`
`Ficoll-Eypaque gradient centrifugation and viable cells were
`
`incubated in RPMI-1640 plus 15% 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 8-azaguanine going
`from 5 to fopg/ml at Spg/ml increments for 1 week at each
`concentration.i The viable cells are then maintained in
`
`RPMI-1640 plus 15% FCS plus 20pg/ml 8-azaguanine. Cultures
`of the fastest growing 8-azaguanine resistant clone were
`expanded, after verifying that they were HAT sensitive.i This
`mutant cell line, U-2664AR1 was maintained in RPMI-1640 plus
`15% FCS 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 0.8-1.0 x 106
`cell/ml. The viability is above 90%._
`7
`Fresh spleen specimens were obtained from untreated
`patients with Hodgkin's disease undergoing staging laparotomy
`with splenectomy.
`The spleens were devoid of involvement by
`
`35 Hodgkins disease. At least two weeks prior to surgery, such
`patients were sensitized and later challenged with
`2-dinitrochlorobenzene.
`A"
`'
`A
`A single cell suspension prepared from the spleen
`tissue was freed of red blood cells and granulocytes by
`
`
`
`.‘
`-§
`§
`‘
`
`j
`
`’
`
`Qi
`“E
`
`A
`
`‘
`
`Sanofi/Regeneron Ex. 1032, pg 931
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`Mylan Ex. 1032, pg 931
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`jfiueuc/uzwfi
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`12
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`0044722
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`Iii?! __
`
`311% 33% 7579/
`
`Ficoll-Hypaque gradient centrifugation and the viable mono-
`nuclear cells suspended in RPMI-1640 medium. Adherent cells
`were removed by incubation of the mononuclear cells in
`plastic dishes three times for 20mins. 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£*human myeloma cell
`line.
`it GTCE.
`cru. @032» .‘3ePosi1‘.e_'>
`H SEPT I%0
`7 myeloma cells
`Fusion was achieved by mixing 2 x 10
`and 2 x 107 lymphoid cells, washing twice in_REMI-1640 and
`then fusing in 2.0ml 38% w/v polyethylene glycol
`("l400mw) 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
`400rpm for 4mins. and 800rpm for 4mins. 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 RPMI-1640 plus 15% res. ' The cells
`are seeded in 0.2ml aliquots in microtiter plates with flat-
`bottom wells in RPMI-1640 plus 20% FCS and.then incubated in
`the same medium for 48hrs. After 48hrs.,
`the medium is
`changed to HAT medium and the cells incubated in HAT medium
`for eight days. The HAT medium is 10'4g hypoxanthine; 6.3 x
`10'8§ methotrexate; 1.5 x l0'6§ thymidine; 40i.u./ml insulin
`and l3.2mg/100ml oxaloacetate.
`'
`The supernatant fluid of each culture microwell is
`then tested for immunoglobulin production by employing a
`solid—phase radioimmunoassay using 1251 labeled fitgph. pro-
`tein A as the detector. This test is only diagnostic of IgG
`(11, V2, v4) and IgA(u2). Therefore, production of other
`immunoglobulins such as IgM 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
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
` i i 1
`
`.‘._..-_.;._.....-.
`
`
`
`oiz
`3
`
`Sanofi/Regeneron Ex. 1032, pg 932
`
`Mylan Ex. 1032, pg 932
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`
`
`é1
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`,4
`
`
`
`E
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`13
`
`0044722
`
`5
`
`10
`
`15
`
`20
`
`the culture was grown in
`40i.u./ml insulin. After two days,
`HAT medium for another 1-2 weeks. Wells that showed immuno-
`globulin production were then tested for production of anti-
`bodies binding specifically to dinitrophenyl-BSA. Several
`anti-dinitrophenyl 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 immunoprecipitated 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-Hypaque gradient centrifugation.
`The viable cells are seeded at 2 x 106 cells/ml in tissue
`culture flasks in RPMI-1640 + 15% FCS + 1o"5g
`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
`
`25
`
`employing a Ficoll-Hypaque gradient centrifugaion.
`
`The
`
`buffy-coat was isolated and used for fusion under the same
`
`30
`
`35
`
`conditions as described previously. After incubation in HAT
`medium as described above clones were screened for IgG using
`125I-labeled §§§ph. protein A.
`The production of IgG was not
`detected.
`The clones were then screened for IgM production
`using a test analogous to the Jerne Plaque 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 IgM results in lysis of the SRBC with
`formation of a plaque. Production of Igm was observed by
`plaque formation with at least one clone§
`
`lymphoma
`Following the procedure described above,
`cells can be obtained which are HAT s?1sitive and may be used
`as fusion partners for human—human hybridomas.
`
`Sanofi/Regeneron Ex. 1032, pg 933
`
`Mylan Ex. 1032, pg 933
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`
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`0044722
`
`14
`
`S
`
`10
`
`In accordance with the subject invention, a novel
`provided which can be used for fusion with
`myeloma strain is
`The hybridomas which
`lymphoid cells to produce hybridomas.
`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
`d which are free of xenogeneic proteins. The
`be produce
`nd wide uses,
`lete human monoclonal antibodies can fi
`comp
`humans and are a homogenous
`since they will be accepted by
`composition having a unique binding site.
`Although the foregoing invention has been described
`ample for pur-
`ome detail by way of illustration and ex
`tanding, it will be obvious that
`poses of clarity of unders
`certain changes and modifications may be practiced‘
`
`in s
`
`
`
`3.2I-.
`J‘.
`
`
`
`......_._...-........‘...:.4.s.-»....._.........4.,.--i_,__,
`
`Sanofi/Regeneron Ex. 1032 pg 934
`
`Mylan Ex. 1032, pg 934
`
`
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`15
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`’oo44722
`
`CLAIMS
`
`1.
`
`A method for producing human—human hybridomas
`
`producing specific human monoclonal antibodies against
`a predefined determinant site which comprises:
`
`fusing lymphoid cells immunized against a pre-
`
`defined determinant site with rapidly growing HAT
`‘sensitive non-Epstein—Barr virus transformed neoplastic
`
`lymphoid cell in a fusing medium at an approximately
`
`equivalent cell ratio to produce a cell mixture;
`
`1o
`
`dividing into each of a plurality of wells a suf-
`
`ficient number of cells of said cell mixture to encourage
`growth and incubating said cells in a nutrient medium
`
`for a sufficient time to expand the number of viable
`
`cells in each well;
`
`growing the cells in HAT medium to produce clones
`free of HAT sensitive cells;
`and
`
`15
`
`selecting for clones producing monoclonal antibodies
`for said predefined determinant site.l
`2.
`A method according to Claim 1, wherein said neoplastic
`lymphoid cells are HAT sensitive myeloma cells which are
`
`20
`
`grown in the presen