Europaisches Patentamt
`European Patent Office
`Office europeen des brevets
`
`@ Publication number:
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`0 044 722
`A1
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`@
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`EUROPEAN PATENT APPLICATION
`
`@ Application number: 81303286.9
`@ Date offiling: 16.07.81
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`@ lnt.CI.3: C 12 N 5/00
`C 12 N 15/00, C 12 P 1/00
`A 61 K 39/395
`//C12R1/91, G01 N33/54
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`@ Priority: 18.07.80 US 170255
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`@ Date of publication of application:
`27.01.82 Bulletin B:l/4
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`@ Designated Contracting States:
`AT BE CH DE FR GB IT U LU NL SE
`
`@Applicant: THEBOARDOFTRUSTEESOFTHELELAND
`STANFORD JUNIOR UNIVERSITY
`Encina 6-930, Stanford University
`Stanford, California 94305(US)
`@ Inventor: Kaplan, HenryS.
`631 Cabrillo
`Stanford California, 94305(USI
`@ Inventor: Olsson, Lennart
`20A lbstrupvej
`DK-2820 GentofteiDKI
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`@ Representative: Harrison, David Christopher et al,
`MEWBURN ELLIS & CO 70 & 72 Chancery Lane
`London WC2A 1AD(GBI
`
`'
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`1
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`@ Human hybridomas, precursors and products.
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`@ Human monoclonal antibody compositions, human(cid:173)
`human monoclonal hybridoma cells, human non-viral trans·
`formed particularly non-Epstein-Barr virus transformed, neo(cid:173)
`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
`,... celis may be substituted for the myeloma cells. After
`<t expansion of the desired hybridoma cells, the monoclonal
`antibodies may be harvested. The hybridomas serve as a
`N source for messenger RNA for light and heavy chains which
`N may be used for production of light and heavy chain
`r"'o
`immunoglobulin proteins through hybrid DNA techniques.
`U-266-AR, 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.
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`Croydon Printing C<>mpony Lld.
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`Sanofi/Regeneron Ex. 1032, pg 920
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`Merck Ex. 1032, pg 946
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`HUMAN HYBRIDOMAS, PRECURSORS AND PRODUCTS
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`The mammalian capacity for producing immunoglobu(cid:173)
`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 app.lication 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 11 hybridomas 11 capable of secreting specific monoclonal
`antibodies against predefined antigens ushered in a new era
`in experimental immunology. Many of the problems associated
`w1th heteroantisera are circumvented; the clonal selection
`and immortality of such hybridoma cell lines assure the
`monoclonality, monospecificity and permanent availability of
`thei.r antibody products. At the clinical level, the use of
`sucL 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
`·;:tro. Efforts to achieve a human hybridoma which is a cross
`.,,,_ 1een a lymphoid cell and a myeloma cell have heretofore
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`Sanofi/Regeneron Ex. 1032, pg 921
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`Merck Ex. 1032, pg 947
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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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`The production of mouse hybridomas is described by
`5 Kohler, G. and Milstein, K. {1975) Nature 356: 495-7; (1976)
`Euro. J. Immunol £: 511-519. Chimeric hybridomas generated
`by fusing mouse myeloma cells with human immunoglobulin(cid:173)
`producing cells were described by Levy, R. and Dilley, I.
`{1978) PNAS USA 75: 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 269: 42Q-422.
`SUMMARY OF THE INVENTION
`Non-viral transformed, particularly non-Epstein(cid:173)
`Barr virus transformed, neoplastic ~ymphoid 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(cid:173)
`mined ligand.
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`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(cid:173)
`tion. The invention therefore involves the development of
`the neoplastic lymphoid cell strains; the preparation of
`lymphoid cells producing antibodies to a specific antigen;
`~e fusion of the immunized lymphoid cells and neoplastic
`lymphoid cells to produce hybridoma cells; the selective
`culturing of the hybridoma c,ells; 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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`Sanofi/Regeneron Ex. 1032, pg 922
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`Merck Ex. 1032, pg 948
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`offer opport1mities 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
`antibodies for specific antigens. By known molecular biology
`techniques, the mRNAs may be 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.
`Non-viral transformed, particul.arly non-Epstein-
`Barr virus transformed, neoplastic lymphoid cells may be
`employed as fusion partners. The fusion partners are
`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 des~ribed 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
`immunoglobulins specific for a predetermined determinant.
`Human Myeloma Cell Line
`The human myeloma cell line is chosen to provide a
`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. Immunol 1: 477-489.
`- HAT sensitivity is achieved by culturing cells in a
`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(cid:173)
`tion of purines.
`Specifically, the cells are first cultured at a
`high s~azaguanine concentration, then at a low 8-azaguanine
`concentration, followed by cultivation at intermediate con(cid:173)
`centration levels.
`In each instance, incubation times are
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`Sanofi/Regeneron Ex. 1032, pg 923
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`Merck Ex. 1032, pg 949
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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 25~g/ml, usually in the range of
`about 5 to 20~g/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.
`Except for the 8-azaguanine and HAT, the nutrient
`media employed are conventional. Prior to fusion the selected
`cells are expanded in non-selective nutrient medium to enhance
`the number of cells.
`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
`medium, and non-adherent cells separated from adherent cells.
`.
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`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 In vivo immunization, spleen cells can
`be isolated and immunized in vitro. A single cell suspension
`of spleen cell~ is prepared, viable cells are isolated and
`seeded in nutrient medium with the appropriate antigen at an
`appropriate concentration. After sufficient time for immuni(cid:173)
`zation, viable cells are isolated and used for fusion.
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`Sanofi/Regeneron Ex. 1032, pg 924
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`Merck Ex. 1032, pg 950
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`An alternative to spleen lymphoid 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 ljmphocytes. After a sufficient time for priming, gen(cid:173)
`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,
`containing about 15% FCS, about 40~g/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 he used for fusion.
`Fusion
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`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 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
`cells should he 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
`concentrations in microplates in nutrient media, there being
`at least about 104-106 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 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.
`The HAT medium which is employed is described in
`Littlefield, Science 145, 709 (1964) and contains a combina(cid:173)
`tion of hypoxanthine, aminopterin or methotrexate, and
`thymidine.
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`Sanofi/Regeneron Ex. 1032, pg 925
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`Merck Ex. 1032, pg 951
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`After the initial incubation with the HAT medium,
`the supernatant fluid of each culture microwell is tested for
`immunoglobulin 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(cid:173)
`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.
`Once positive w~lls are detected, the cells in the
`positive wells may be cloned under limiting dilution condi(cid:173)
`tions. The resulting clones are then expanded and the mono(cid:173)
`clonal ~ntibodies are· then harvested in accordance wi~~ known
`procedures. The monoclonal antibodies may be freed of other
`proteins in accordance with known techniques, such as elec(cid:173)
`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 intendedto include not only IgG, but also IgM,
`IgE and IgA. Particularly,_ antibodies may be produced
`against ~ugs, 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 7-11
`inclusive, which disclosure is incorporated herein by
`reference.
`As indicated previously, the subject invention
`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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`Merck Ex. 1032, pg 952
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`greater than about 95 weight %, more usually gre~ter than
`about 99 weight % will have the same CClmposition.
`By referring to.the same composition it is intended
`that the chemical composition and amino acid sequence of the
`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 ,
`structures of the immunoglobulin molecuies in the composition-
`are substantially the same.
`By having a uniform composition of immunoglobulins
`many advantages ensue. First, one is ensured of freedom from
`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.
`Third, one can obtain an exact titer for a specific
`determinant site, rather than averaging over the entir~
`composition. With analytes, better control of cross(cid:173)
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`reactivities can be achieved with a homogeneous composition.
`The subject monoclonal human antibodies find use in
`conventional applications for antibodies, such as immune(cid:173)
`assays, ce~l 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.
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`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 tp 20,000 uni~s,
`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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`Sanofi/Regeneron Ex. 1032, pg 927
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`Merck Ex. 1032, pg 953
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`Jaivetz and Goldfien, Lange Medical Publications, 1978, pages
`612-615. } Particular dosages will vary dep.mding upon the
`manner of administration. Various carriers or media can be
`used, such as physiological sali~e, capsules, plasma, or the
`like. Other additives may also be included, such as stabil(cid:173)
`izers, drugs, proteins, and the like.
`The human monoclonal antibodies can also be used
`for site directed therapy. By preparing antibodies recog(cid:173)
`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.
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`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.
`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(cid:173)
`lated and centrifuged to produce a pellet containing the
`membrane-bound polysomes. The pellet is resuspended in
`30· 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.
`The rnRNA fractions may then be assayed for in a
`number of ways. The mRNA from the parent myeloma cell may be
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`Sanofi/Regeneron Ex. 1032, pg 928
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`Merck Ex. 1032, pg 954
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`treated in the same way and common bands between the mRNA
`from the hybridomas and the myeloma cells discarded.
`rnRNA
`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.
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`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
`synthesized from nucleotides corresponding to the codon
`.
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`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.
`The probe is hybridized with the electrophoretic
`fractions under conditions where only mRNA substantially
`(See, Southern, J.
`homologous with the probe will hybridize.
`Mol. Biol. 98, 503 (1975)). Where the probe is based on the
`variable portions of the light and heavy chains,.only the
`desired mRNAs will be isolated, or highly concentrated frac(cid:173)
`tions thereof.
`It is not necessary, however, to i~olate the mRNAs
`expressing the desired light and heavy chains. Purification
`can be achieved subsequently by isolation of transforrnants
`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, eDNA may be prepared by employing reverse tran(cid:173)
`scriptase in accordance with conventional techniques (Buell,
`et al, J. Biol, Chern. 253: 2471 (1978)). The dsDNA is
`generated using DNA polymerase and Sl nuclease (Wickens, et
`al., ibid 253: 2483 (1978)). Sequencing of the 5 1 -ends will
`determine the sites of initiation of the light and heavy
`chains. The DNA sequence preceding the f-met codon may be
`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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`Merck Ex. 1032, pg 955
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`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
`5 vectors include pSClOl, ~plac, pBR322, Ylp5, 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-~ ligase; or the termi.ni modi(cid:173)
`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(cid:173)
`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 s~;nt~esized with appropriate host
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`Sanofi/Regeneron Ex. 1 032, pg 930
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`Merck Ex. 1032, pg 956
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`signals, e.g. ribosomal start and stop, promoter and operator
`signals. Also, appropriate restriction sites are provided at
`the termini for joinin~ 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(cid:173)
`tration and not by way of .limitation.
`EXPERIMENTAL
`In order to demonstrate the subject invention with
`neoplastic granulocytes, the following preparation was
`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
`and 20~g/ml 8-azaguanine; dead cells were then removed by
`Ficoll-Hypaque gradient centrifugation and viable cells were
`incubated in RPMI-1640 plus 15% FCS plus S~g/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 2_0~g/ml at S~g/ml increments for 1 week at each
`concentration. The viable cells are then maintained in
`RPMI-1640 plus 15% FCS plus 20~g/ml 8-azaguanine. Cultures
`of the fastest growing 8-azaguanine resistant clone were
`expanded, after verifying that they were HAT sensitive. This
`mutant cell line, U-266-AR1 was maintained in RPMI-1640 plus
`15% FCS plus S!Jg/ml 8-azaguan:ine. The cells are seeded at a
`concentration of 105/ml 3-5 days before fusion. On the day
`c;>f fusion, the cell concentration is about o·.S-1.0 x 106
`cell/ml. The viability is ·above 90%.
`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 single ~ell suspension prepared from the spleen
`tissue was freed of red blood cells and granulocytes by
`
`20
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`25
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`30
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`- !
`
`;
`• A
`..
`
`Sanofi/Regeneron Ex. 1032, pg 931
`
`Merck Ex. 1032, pg 957
`
`

`
`Ficoll-Hypaque gradient centrifugation and the viable mono(cid:173)
`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
`rernoval.of the non-adherent cells after each incubation. The
`lymphocyte-enriched mononuclear cell suspensions thus
`line. * ATc.c_
`obtained were then fused with U-266-AR1*hurnan myeloma cell
`II .S:EPT ,qU>
`c:: ftL &.a~> ~c.Po.sir-e~
`Fusion was achieved by mixing 2 x 107 myeloma cells
`and 2 x 107 lymphoid cells, washing twice in RPMI-1640 and
`then fusing in 2.0ml 38% wjv polyethylene glycol (-1400mw) at
`37°C. After the last wash, the supernatant is removed as
`quickly as possible and the polyethylene glycol added drop(cid:173)
`wise over a minute at 37°C. The cell pellet is carefully
`stirred for 1min. in polyethylene glycol, then gently resus(cid:173)
`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(cid:173)
`tion of 106 cells/ml in RPMI-1640 plus 15% FCS. · The cells
`are seeded in 0.2ml aliquots in microtiter plates with flat(cid:173)
`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-~ hypoxanthine; 6.3 x
`10-a~ methotrexate; 1.5 x 10- 6~ thymidi~e; 40i.u.jml insulin
`and 13.2mg/100ml oxaloacetate.
`The supernatant fluid of each culture·microwell is
`then tested for immunoglobulin production by employing a
`solid-phase radioimmunoassay using 125r labeled Staph. pro(cid:173)
`tein A as the detector. This test is only diagnostic of IgG
`(y1 , y2 , y4 ) and IgA(a 2). Therefore, production of other
`immunoglobulins such as IgM and IgE would go undetected. By
`employing appropriate antibodies, the other types of immuno(cid:173)
`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
`
`Sanofi!Regeneron Ex. 1 032, pg 932
`
`i ~ ..
`
`A
`
`' ,
`
`l
`
`Merck Ex. 1032, pg 958
`
`

`
`13
`
`0044722
`
`5
`
`10
`
`~
`
`~
`
`20
`
`l
`' 'I
`' ~
`l
`l
`-I-
`
`25
`
`1 l ' l
`
`40i.u./ml insulin. After two days, the culture was grown in
`HAT medium for another 1-2 weeks. Wells that showed immuno(cid:173)
`globulin production were then tested for production of anti(cid:173)
`bodies binding specifically to dinitrophenyl-BSA. Several
`anti-dinitrophenyl antibody-producing cultures were detected.
`Cells from such wells were cloned by limiting dilution pro(cid:173)
`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 14c-leucine. The immunoglobulins in the super(cid:173)
`natant were immunoprecipitated with rabbit anti-Fc ~nd anti(cid:173)
`light chain antisera and the precipitate analyzed sequen(cid:173)
`tially by sodium-dodecylsulfate-polyacrylamide gel electro-
`IS 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 RPMl-1640 + 15% FCS + 10-5!1
`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
`employing a Ficoll-Hypaque gradient centrifugaion. The
`buffy-coat was isolated and used for fusion under the same
`conditions as described previously. After incubation in BAT
`medium as described above clones were screened for IgG using
`125r-labeled staph. 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.
`Following the procedure described above, lymphoma
`cells can be obtained which are HAT s'<sitive and may be used
`as fusion partners for human-human hybridomas.
`
`30
`
`35
`
`Sanofi/Regeneron Ex. 1032, pg 933
`
`Merck Ex. 1032, pg 959
`
`

`
`14
`
`0044722
`
`5
`
`10
`
`In accordance with the subject invention, a novel
`myeloma strain is provided which can be used for fusion wjth
`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 mannex, 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 hav~ng a unique binding site.
`Although the foregoing invention has been described
`in some detail by way of illustration and example for pur(cid:173)
`poses of clarity of understanding, it will be obvious that
`certain changes and modifications may be practiced •
`
`Sanofi/Regeneron Ex. 1032, pg 934
`
`. j
`
`_J
`
`i
`.l
`1
`
`j
`'
`
`'
`1
`
`' I
`j
`i .,
`
`!
`
`·1
`!
`j
`I
`
`Merck Ex. 1032, pg 960
`
`

`
`15
`
`0044722
`
`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-
`
`5
`
`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;
`
`dividing into each of a plurality of wells a suf(cid:173)
`
`10
`
`ficient number of cells of said cell mixture to encourage
`
`gro\~h and in