United States Patent [19]
`Morrison et al.
`
`US005807715A
`Patent Number:
`Date of Patent:
`
`[11]
`[45]
`
`5,807,715
`Sep. 15, 1998
`
`[54]
`
`METHODS AND TRANSFORMED
`MAMMALIAN LYMPHOCYTE CELLS FOR
`PRODUCING FUNCTIONAL ANTIGEN
`BINDING PROTEIN INCLUDING CHIMERIC
`IMMUNOGLOBULIN
`
`Inventors: Sherie L. Morrison, Scarsdale, NY;
`Leonard A. Herzenberg, Stanford;
`Vernon T. Oi, Menlo Park, both of
`Calif.
`
`[73]
`
`Assignee:
`
`The Board of Trustees of The Leland
`Stanford Junior University, Stanford,
`Calif.
`
`Appl. No.: 266,154
`
`Filed:
`
`Jun. 27, 1994
`
`Related US. Application Data
`
`Continuation of Ser. No. 893,610, Jun. 3, 1992, abandoned,
`which is a continuation of Ser. No. 675,106, Mar. 25, 1991,
`abandoned, which is a continuation of Ser. No. 441,189,
`Nov. 22, 1989, abandoned, which is a continuation of Ser.
`No. 90,669, Aug. 28, 1987, abandoned, which is a continu
`ation-in-part of Ser. No. 644,473, Aug. 27, 1984, abandoned.
`
`Int. Cl.6 ......................... .. C12N 15/00; C12N 15/13;
`C07K 16/00
`U.S. Cl. ................... .. 435/69.6; 435/172.3; 435/326;
`530/387.1; 530/387.3; 536/23.53
`Field of Search ............................ .. 435/69.6, 246.27,
`435/320.1, 172.3, 326; 530/3873, 387.1;
`935/15; 536/2353
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`4,399,216
`4,816,397
`4,816,567
`
`8/1983 Axel et a1. ................................ .. 435/6
`
`3/1989 Boss . . . . . . . . .
`
`. . . . .. 435/68
`
`3/1989 Cabilly .................................. .. 530/387
`
`FOREIGN PATENT DOCUMENTS
`
`0125023 11/1984 European Pat. Off. .
`8303971 11/1983 WIPO.
`
`OTHER PUBLICATIONS
`
`Falkner et al Nature vol. 298:286—288, Jul. 1982.
`Gillies et al Nucleic Acid Res vol. 11 No. 22:7981—7997,
`1983.
`Stafford et al Nature vol. 306:77—79, Nov. 1983.
`Rice et al., “Regulated expression of an immunoglobulin k
`gene introduced into a mouse lymphoid cell line”, Proc.
`Natl. Acad. Sci. USA, vol. 79, 7862—65 (1982).
`Ochi et al., “Functional immunoglobulin M production after
`transfection of cloned immunoglobulin heavy and light
`chain genes into lymphoid cells”, Proc. Natl. Acad. Sci.
`USA, vol. 80, 6351—55 (1983).
`Sharon, et al., “Expression of a VHCK chimaeric protein in
`mouse myeloma cells”, Nature, vol. 309, 364—367 (1984).
`Cabilly S. et al 1984 (Jun.) PNAS, USA 81:3273—3277
`Generation of Antibody Activity from Immunoglobulin
`Polypeptide Chains Produced in Esherichia coli.
`Gillies S.D. et al 1983 Cell 33: 717—728.
`Seno et al 1983 Nucleic Acid Research 11(3);719—726.
`Dolby et al 1980 PNAS 77(10) 6027—6031 Oct. 1980.
`Stedman’s Medical Dictionary 25th edition, p. 902, 1992.
`
`Primary Examiner—Lila Feisee
`Assistant Examiner—Julie E. Reeves
`Attorney, Agent, or Firm—Fish & Neave; Vicki S. Veenker;
`EdWard F. MulloWney
`
`[57]
`
`ABSTRACT
`
`Methods for producing functional immunoglobulin are pro
`vided. The methods involve transfecting and expressing
`exogenous DNA coding for the heavy and light chains of
`immunoglobulin. In some embodiments, chimeric immuno
`globulins are provided having variable regions from one
`species and constant regions from another species by linking
`DNA sequences encoding for the variable regions of the
`light and heavy chains from one species to the constant
`regions of the light and heavy chains respectively from a
`different species. Introduction of the resulting genes into
`mammalian host cells under conditions for expression pro
`vides for production of chimeric immunoglobulins having
`the speci?city of the variable region derived from a ?rst
`species and the physiological functions of the constant
`region from a different species.
`
`62 Claims, 2 Drawing Sheets
`
`Blvl-ll
`
`l
`
`Ell/"7
`l
`
`pSVZAH-S707 HuGZ
`
`Mylan v. Genentech
`IPR2016-00710
`Merck Ex. 1150, Pg. 1
`
`

`

`U.S. Patent
`
`Sep. 15, 1998
`
`Sheet 1 of2
`
`5,807,715
`
`Bl’ H/ (@Ui/NMKH”)
`
`Bl’ H/
`
`pSVZAH-S 107 HuGI
`Mouse VDJ
`pSVZAH-S707 Hu62
`
`Human constant region
`
`5%; H/ M ///
`
`,oSVZAH-S707 Huk
`
`'
`
`V///i
`
`Mouse VJ
`
`Hum an kappa
`
`pBRJZZ on’
`
`Pvu/l
`
`.
`SV40 on
`
`t
`co-gp or
`Eco_neO
`
`FIG. 1B
`
`Merck Ex. 1150, Pg. 2
`
`

`

`U.S. Patent
`
`Sep. 15, 1998
`
`Sheet 2 of2
`
`5,807,715
`
`E. Coli gpl‘
`
`FIG. 2
`
`Merck Ex. 1150, Pg. 3
`
`

`

`1
`METHODS AND TRANSFORMED
`MAMMALIAN LYMPHOCYTE CELLS FOR
`PRODUCING FUNCTIONAL ANTIGEN
`BINDING PROTEIN INCLUDING CHIMERIC
`IMMUNOGLOBULIN
`
`This is a continuation of application Ser. No. 07/893,610,
`?led Jun. 3, 1992, noW abandoned, Which is a continuation
`of application Ser. No. 07/675,106, ?led Mar. 25, 1991, noW
`abandoned, Which is a continuation of application Ser. No.
`07/441,189, ?led Nov. 22, 1989, noW abandoned, Which is
`a continuation of application Ser. No. 07/090,669, ?led Aug.
`28, 1987, noW abandoned, Which is a continuation-in-part of
`application Ser. No. 06/644,473, ?led Aug. 27, 1984 noW
`abandoned.
`
`10
`
`15
`
`BACKGROUND OF THE INVENTION
`
`1. Field of the Invention
`Naturally occurring receptors, such as immunoglobulins,
`enzymes, and membrane proteins have seen an extraordi
`nary expansion in commercial applications over the last
`decade. With the advent of monoclonal antibodies, the
`usefulness of immunoglobulins has been greatly expanded
`and in many situations has greatly extended prior uses
`employing polyclonal antibodies. HoWever, in many
`applications, the use of monoclonal antibodies is severely
`restricted Where the monoclonal antibodies are to be used in
`a physiological (in vivo) environment. Since, for the most
`part, monoclonal antibodies are produced in rodents, e.g.,
`mice, the monoclonal antibodies are immunogenic to other
`species.
`While the constant regions of immunoglobulins are not
`involved in ligand binding, the constant regions do have a
`number of speci?c functions, such as complement binding,
`immunogenicity, cell receptor binding, and the like. There
`Will, therefore, be situations Where it Will be desirable to
`have constant regions Which bind to cells or proteins from a
`particular species having binding regions for a particular
`ligand.
`2. Relevant Literature
`KWan et al., J. Exp. Mod. (1981) 153:1366—1370 and
`Clarke et al., Nucl. Acids Res. (1982) 10:7731—7749
`describe VH and VK exons from the mouse phosphocholine
`binding antibody-producing S107 myeloma cell line. Oi et
`al., Proc. Natl. Acad. Sci. USA (1983) 80:825—829, report
`that the mouse light chain gene is not expressed ef?ciently
`in a rat myeloma cell.
`
`20
`
`25
`
`30
`
`35
`
`40
`
`SUMMARY OF THE INVENTION
`
`Chimeric multi-subunit receptors are provided, Where
`each of the subunits is an expression product of a fused gene.
`Each fused gene comprises a DNA sequence from one host
`species encoding the region involved With ligand binding
`joined to a DNA sequence from a different source, either the
`same or a different host species, encoding a “constant”
`region providing a structural framework and biological
`properties. Introduction of the fused genes into an appro
`priate eukaryotic host cell under conditions for expression
`and processing provides for a functional assembled multi
`subunit receptor product.
`
`55
`
`60
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1A is a schematic diagram of the chimeric mouse
`:human heavy chain gene vector; and FIG. 1B is the chi
`meric light chain vector.
`
`65
`
`5,807,715
`
`2
`FIG. 2 is a schematic diagram of chimeric human IgG
`anti-DNS expression vectors.
`
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`
`Novel methods and compositions are provided, for pro
`duction of polypeptide products having speci?c binding
`af?nities for a predetermined ligand and predetermined
`biological, particularly physiological, properties, each of
`Which are not normally associated With the binding region
`peptide sequences. Particularly, multi-subunit chimeric
`receptors are provided Which result from fused genes having
`the portion of the polypeptide involved With binding of a
`predetermined ligand having an amino acid sequence sub
`stantially the same (>90% conserved) as an amino acid
`sequence having the same function from one host, While the
`portion involved With providing structural stability, as Well
`as other biological functions, being analogously derived
`from a different host. The resulting composition can be
`either an inter- or intraspecies chimera. At least tWo fused
`genes are involved, Which genes are introduced into an
`appropriate eukaryotic host under conditions for expression
`and processing, Whereby the fused genes are expressed and
`the resulting subunits bound together, resulting in an
`assembled chimeric receptor.
`The receptors prepared in accordance With the subject
`invention Will be multi-subunit, Where the units are held
`together either by non-covalent binding or a combination of
`non-covalent and covalent binding, particularly disul?de
`linkages through cysteine, and having at least one binding
`site, usually at least tWo binding sites, and not more than
`about ten binding sites. Receptors of interest include both
`B-cell and T-cell receptors, more particularly,
`immunoglobulins, such as IgM, IgG, IgA, IgD and IgE, as
`Well as the various subtypes of the individual groups. The
`light chain may be K or )t. The heavy chains are referred to
`as p, y, 0t, 6, and e.
`In discussing the tWo regions of each subunit, the tWo
`regions Will be referred to as “variable” and “constant” by
`analogy to immunoglobulins. The variable region is the
`region involved With ligand binding and, therefore, Will vary
`in conformation and amino acid sequence depending upon
`the ligand. The region Will usually be composed of a
`plurality of smaller regions (hypervariable or complemen
`tary determining regions), involving a region having as its
`primary function binding to the ligand (V) and a region
`associated With joining the V region to the constant region,
`the joining region
`There may also be a hypervariable
`
`region joining the V and J regions, the diversity region These regions are related to gene segments observed in the
`
`genes encoding immunoglobulin variable regions.
`The constant region Will not be associated With ligand
`binding and Will be relatively limited in the variations in its
`conformation and amino acid sequence Within any one
`species and Within any one class, each class generally having
`from 1 to 4 subclasses. Each constant region is speci?c for
`a species. Within the classes there Will be allotypes, indi
`vidual polymorphisms Within a class Within a species.
`The varible region of the immunoglobulins Will be
`derived from a convenient mammalian source, Which may
`be a rodent, e.g., mouse or rat, rabbit, or other vertebrate,
`mammalian or otherWise, capable of producing immunoglo
`bulins. The constant region of the immunoglobulin, as Well
`as the J chain for IgM and IgA (not the same as the J region
`of the heavy or light immunoglobulin chain), Will be derived
`from a vertebrate source different from the source of the
`
`Merck Ex. 1150, Pg. 4
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`

`5,807,715
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`3
`variable region, particularly a mammalian source, more
`particularly primate or domestic animal, e.g., bovine,
`porcine, equine, canine, feline, or the like, and particularly,
`humans. The different source of the constant region can be
`either from a different species or from the same species as
`the mammalian source utiliZed to provide the variable
`region. Thus, the constant region of the receptor Will nor
`mally be chosen in accordance With the purpose of the
`receptor. For example, Where the receptor is to be introduced
`into the host, the constant portion Will be selected so as to
`minimiZe the immune response of the host to the receptor
`and to optimiZe biological efficiency, such as complement
`?xation or physiological half-life (catabolism). Where the
`receptor is to bind to particular cell membrane surface
`receptors, the constant region Will be chosen in accordance
`With the host of the receptor recognition site.
`The fused gene derived from the tWo host sources Will be
`prepared by joining the 5‘-end of a sequence encoding the
`constant region in reading frame to the 3‘-end of a sequence
`encoding the variable region. (In referring to 5‘ or 3‘ for a
`double strand, the direction of transcription is With 5‘ being
`upstream from 3‘.) With immunoglobulins, tWo fused genes
`Will be prepared, one for the light chain and one for the
`heavy chain. With T-cell receptors, the tWo fused genes Will
`be for each of the tWo chains involved in the formation of the
`T-cell receptor. The DNA sequences employed for prepar
`tion of the fused gene may be derived from a variety of
`sources. These sources include genomic DNA, cDNA, syn
`thetic DNA, and combinations thereof. The genomic DNA
`may or may not include naturally occurring introns.
`The DNA obtained from natural sources, namely the
`genomic DNA or CDNA, may be obtained in a variety of
`Ways. Host cells coding for the desired sequence may be
`isolated, the genomic DNA may be fragmented, conve
`niently by one or more restriction endonucleases, and the
`resulting fragments may be cloned and screened With a
`probe for the presence of the DNA sequence coding for the
`polypeptide sequence of interest. For the variable region, the
`rearranged germline heavy chain DNA Will include V, D,
`and J regions, including the leader sequence, Which may be
`subsequently removed as Well as any introns. The rearranged
`germline light chain coding DNA Will include the V and J
`regions including the leader sequence, as Well as any introns
`Which may be subsequently removed. The particular source
`of the exons de?ning the domains and the manner of
`splicing, Where introns are present, is not germane to this
`invention. Once the cloned fragment has been identi?ed
`Which contains the desired DNA sequence, this fragment
`may be further manipulated to remove super?uous DNA,
`modify one or both termini, remove all or a portion of
`intervening sequences (introns), or the like.
`In providing a fragment encoding the variable region, it
`Will usually be desirable to include all or a portion of the
`intron doWnstream from the J region. Where the intron is
`retained, it Will be necessary that there be functional splice
`acceptor and donor sequences at the intron termini. The gene
`sequence betWeen the J (joining region) and the constant
`region of the fused gene may be primarily the intron
`sequence associated With (1) the constant region, (2) the J
`region, or (3) portions of each. The last may be a matter of
`convenience Where there is a convenient restriction site in
`the introns from the tWo sources. In some instances, all or a
`portion of the intron may be modi?ed by deletion, nucleotide
`substitution(s) or insertion, to enhance ease of manipulation,
`expression, or the like. When the variable region is chosen
`to be syngeneic With the host cells employed for expression,
`all or at least about 80% of the intron sequence can be
`
`45
`
`55
`
`65
`
`4
`selected from the naturally occurring intron sequence asso
`ciated With the J region. In some instances it Will be
`necessary to provide adapters to join the intron or truncated
`intron to the constant region. By cleaving Within the intron,
`the variable region Will be separated from its natural con
`stant region.
`Alternatively, it may be desirable to have the fused gene
`free of the intron betWeen the variable and constant regions.
`Thus, the 3‘ terminus Will be at or in the joining region.
`Normally all or a portion of the J region Will be associated
`With the host providing the variable region. By restriction
`enZyme analysis or sequencing of the J region, one can
`select for a particular site for the 3‘ terminus of the variable
`region.
`Alternatively, one can use an exonuclease and by employ
`ing varying periods of digestion, one can provide for varying
`3‘-termini, Which can then be used for linking to the constant
`region and selection made for a functional product in a
`variety of Ways. For example, Where joining of the variable
`region to the constant region results in a unique restriction
`site, the fused DNA fragments may be screened for the
`presence of the restriction site.
`Alternatively, it may be found desirable to include an
`adapter or linker to join the variable region to the constant
`region, Where the adapter or linker may have the same or
`substantially the same sequence, usually at least substan
`tially the same sequence, of the DNA sequence of the tWo
`fragments adjacent the juncture. The adapter or linker Will
`be selected so as to provide for the tWo sequences to be in
`common reading frame. Furthermore, by employing
`adapters, one could add an additional degree of variability in
`the binding af?nity of the chimeric receptor, by providing for
`the expression of different amino acids in the J region.
`The joining of the various fragments is performed in
`accordance With conventional techniques, employing blunt
`ended or staggered-ended termini for ligation, restriction
`enZyme digestion to provide for appropriate termini, ?lling
`in of cohesive ends as appropriate, alkaline phosphatase
`treatment to avoid undesirable joining, and ligation With
`appropriate ligases.
`For cDNA, the cDNA may be cloned and the resulting
`clone screened With an appropriate probe for cDNA coding
`for the desired variable or constant region. Once the desired
`clone has been isolated, the CDNA may be manipulated in
`substantially the same manner as the genomic DNA.
`HoWever, With cDNA there Will be no introns or intervening
`sequences. The cDNA is cleaved at or near the juncture of
`the variable region With the constant region so that the
`variable region is separated from the constant region and the
`desired region retained. Where a convenient restriction site
`exists, the CDNA may be digested to provide for a fragment
`having the appropriate terminus. The restriction site may
`provide a satisfactory site or be extended With an adapter.
`Alternatively, primer repair may be employed, Where for the
`variable region a complementary sequence to the site of
`cleavage and successive nucleotides in the 3‘ direction of the
`complementary sequence is hybridiZed to the sense strand of
`the CDNA and the nonsense strand replicated beginning
`With the primer and removal of the single-stranded DNA of
`the sense strand 3‘ from the primer. The reverse is true for
`the constant region. Other techniques may also suggest
`themselves. Once the fragment has been obtained having the
`predetermined 3‘ or 5‘ terminus, as appropriate, it may then
`be employed for Joining to the other region.
`Finally, one or both of the regions may be synthesiZed and
`cloned for use in preparing the fused gene. For the most part,
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`Merck Ex. 1150, Pg. 5
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`

`

`15
`
`5
`the same or substantially the same constant region can be
`repetitively used, so that a library of constant regions may be
`established Which can be selected for joining to variable
`regions. Thus, the constant regions Would have an appro
`priate 5‘ terminus for joining directly or through an adapter
`to a variable region.
`In order for expression of the fused gene, it Will be
`necessary to have transcriptional and translational signals
`recogniZed by an appropriate eukaryotic host. For the most
`part, desirable eukaryotic hosts Will be mammalian cells
`capable of culture in vitro, particularly leukocytes, more
`particularly myeloma cells, or other transformed or onco
`genic lymphocyte, e.g., EBV transformed cells.
`Alternatively, non-mammalian cells may be employed, such
`as fungi, e.g., yeast, ?lamentous fungi, or the like.
`The DNA sequence coding for the variable region may be
`obtained in association With the promoter region from
`genomic DNA. To the extent that the host cells recogniZe the
`transcriptional regulatory and translational initiation signals
`associated With the variable region, then the region 5‘ of the
`variable region coding sequence may be retained With the
`variable region coding sequence and employed for transcrip
`tional and translational initiation regulation.
`The contiguous non-coding region 5‘ to the variable
`region Will normally include those sequences involved With
`initiation of transcription and translation, such as the TATA
`25
`box, capping sequence, CAAT sequence, and the like. Usu
`ally the 5 ‘-non-coding sequence Will be at least 150 bp, more
`usually at least 200 bp, usually not exceeding about 2 kbp,
`more usually not exceeding about 1 kbp.
`The non-coding region 3‘ to the constant region may be
`retained for its transcriptional termination regulatory
`sequences, such as termination and polyadenylation. Thus,
`by retaining the 3‘-region naturally contiguous to the DNA
`sequence coding for the constant region, the transcriptional
`termination signals may be provided for the fused gene.
`Where the transcriptional termination signals are not satis
`factorily functional in the expression host cell, then a 3‘
`region functional in the host cell may be substituted.
`Conveniently, the non-coding 3‘ region may be obtained
`from a non-coding contiguous 3‘ region of a constant region
`from the expression host. The 3‘-non-coding region may be
`joined to the constant region by any of the means described
`previously for manipulation and ligation of DNA fragments.
`This region could then be used as a building block in
`preparing the fused gene.
`The fused gene for the most part may be depicted by the
`folloWing formula:
`
`35
`
`45
`
`Wherein:
`TIR intends the transcriptional regulatory and transla
`tional initiation region and is generally of at least about 150
`bp and not more than about 2 kbp, Which may be in Whole
`or in part the sequence naturally joined to the V coding
`region;
`LS refers to a DNA sequence encoding a leader sequence
`and processing signal functional in the expression host for
`secretion and processing for removal of the sequence; this
`leader sequence can contain an intron, as is knoWn in the art
`to occur;
`e is 0 or 1;
`V is a segment coding for the variable domain in reading
`frame With LS, When LS is present;
`f is 0 or 1;
`D is a segment coding for the diversity domain and is
`present for the heavy chain (b=1) and is absent for the light
`chain (b=0);
`
`55
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`65
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`5,807,715
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`6
`J is a segment coding for the joining region;
`Vi and Di are introns associated With the letter-indicated
`coding segments having functional donor and acceptor splic
`ing sites;
`a, b and c are the same or different and are 0 or 1, Wherein
`When b is 0, c is 0; a, b, and c are all preferably 0;
`I is an intron Which may be naturally contiguous to the J
`segment or naturally contiguous to the C domain or a
`combination of fragments from both, or a fragment thereof,
`desirably including an enhancer sequence functional in said
`expression host, or I may be foreign in Whole or in part to
`the J and C segments;
`d is 0 or 1 (preferably 0);
`C is the constant domain and may code for a p, y, 6, 0t or
`6 chain, preferably p, y, or 0t, usually including at least 80%
`of the constant region sequence, and may be the same as or
`a modi?ed naturally occurring allotype or an altered con
`stant region encoding an improved protein sequence; and
`TTR is the transcriptional termination region providing
`for transcriptional termination and polyadenylation Which
`may be naturally associated With C or may be joined to C,
`being functional in the expression host; usually being at least
`about 100 bp and may be 1 kbp or more.
`Fused genes lacking, or containing modi?cations in, the
`hinge region or other immunoglobulin constant region
`domains can also be prepared, in like manner to the modi
`?cations described above, in Which case the formula Will be
`as shoWn above but With the hinge region of the constant
`chain being absent or modi?ed.
`The constructs for each of the different subunits may be
`joined together to form a single DNA segment or may be
`maintained as separate segments, by themselves or in con
`junction With vectors.
`The subunit constructs may be introduced into a cell by
`transformation in conjunction With a gene alloWing for
`selection Where the construct Will become integrated into the
`host genome.
`A large number of vectors are available or can be readily
`prepared Which provide for expression in a host, either by
`maintenance as an extrachromosomal element or by inte
`gration into the host genome. For a mammalian host, a Wide
`variety of vectors are knoWn based on viral replication
`systems, such as Simian virus, bovine papilloma virus,
`adenovirus and the like. These vectors can be used as
`expression vectors Where transcriptional and translational
`initiation and termination signals are present and one or
`more restriction sites are available for insertion of a struc
`tural gene. In addition, the vectors normally have one or
`more markers Which alloW for selection of host cells Which
`contain the expression vector. The marker may provide for
`prototrophy to an auxotrophic host; biocide resistance, e.g.,
`resistance to antibiotics, such as G418, or heavy metals, such
`as copper; or the like. If desired, expression vectors can be
`prepared by joining the various components, such as the
`replication system, markers, and transcriptional and trans
`lational regulatory initiation and termination signals in con
`junction With the fused gene. Frequently, a vector Will
`include a prokaryotic replication system, Which alloWs for
`cloning, manipulation, puri?cation, and expansion of the
`desired DNA sequence.
`A Wide variety of transcriptional and translational regu
`latory sequences may be employed, depending upon the
`nature of the host. The transcriptional and translational
`regulatory signals may be derived from viral sources, such
`as adenovirus, bovine papilloma virus, Simian virus, or the
`like, Where the regulatory signals are associated With a
`particular gene Which has a high level of expression.
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`Merck Ex. 1150, Pg. 6
`
`

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`5,807,715
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`7
`Alternatively, promoters from mammalian expression
`products, such as actin, collagen, myosin, etc., may be
`employed. Transcriptional initiation regulatory signals may
`be selected Which alloW for repression or activation, so that
`expression of the fused genes can be modulated. Of interest
`are regulatory signals Which are temperature-sensitive so
`that by varying the temperature, expression can be repressed
`or initiated, or are subject to chemical regulation, e.g.,
`metabolite.
`Once the vector DNA sequence containing the fused gene
`has been prepared for expression, the DNA construct may be
`introduced into an appropriate host. Various techniques may
`be employed, such as protoplast fusion, calcium phosphate
`precipitation, or other conventional technique. After the
`fusion, the cells are groWn in a selective medium or are
`phenotypically selected leaving only cells transformed With
`the DNA construct. Expression of the fused gene results in
`assembly to form the receptor. To date, expression has been
`accomplished in lymphocytes.
`The host cells Will for the most part be immortaliZed cells,
`particularly myeloma or lymphoma cells. These cells may be
`groWn in an appropriate nutrient medium in culture ?asks or
`injected into a syngeneic host, e.g., mouse or rat, or immu
`node?cient host or host site, e.g., nude mouse or hamster
`pouch. Particularly, the cells may be introduced into the
`abdominal cavity for production of ascites ?uid and har
`vesting of the chimeric receptor. Alternatively, the cells may
`be injected subcutaneously and the antibodies harvested
`from the blood of the host. The cells may be used in the same
`manner as hybridoma cells. See Diamond et al., N. Eng. J.
`Med. (1981) 3034:1344 and Kennatt, McKearn and Bechtol
`(eds.), Monoclonal Antibodies: Hybridomas—A New
`Dimension in Biologic Analysis, Plenum, 1980, Which are
`incorporated herein by reference.
`Where a leader is present With a processing signal for
`secretion and selective cleavage of the leader (signal)
`sequence, the resulting assembled receptor Will be secreted
`into the nutrient medium of the transformed cells and may
`be harvested. Where secretion does not occur, after suf?cient
`time for the receptor to be expressed in reasonable amounts,
`the cells may be killed, lysed, and the receptors isolated and
`puri?ed. Where transcriptional initiation can be modulated,
`the cells may be groWn to high density under non-permissive
`conditions, folloWed by groWth under permissive conditions
`Where the receptor is expressed.
`The receptors may be naturally glycosylated, unnaturally
`glycosylated or be free of glycosyl groups, depending on the
`host, conditions of cellular groWth and subsequent treat
`ment. Where a mammalian host cell is employed for
`expression, usually natural glycosylation Will occur. Glyco
`sylation can be prevented by an appropriate inhibitor, e.g.,
`tunicamycin. Alternatively, glycosyl groups may be
`removed by hydrolysis, e.g., enZymatic hydrolysis using
`hydrolases. In expression hosts other than mammalian cells,
`unglycosylated or unnatural glycosylated receptors may be
`obtained.
`The receptor may be isolated and puri?ed in accordance
`With conventional conditions, such as extraction,
`precipitation, chromatography, af?nity chromatography,
`electrophoresis, or the like. By employing antibodies spe
`ci?c for the constant region(s), af?nity chromatography Will
`alloW for concentration and puri?cation of the chimeric
`receptor.
`The chimeric receptors can be used in the same manner as
`other receptors for binding to speci?c ligands in diagnostic
`assays, af?nity chromatography or the like. In addition,
`because a chimeric receptor of substantially reduced immu
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`55
`
`60
`
`65
`
`8
`nogenicity can be produced, the chimeric receptors can ?nd
`use in therapy, for passive immuniZation for in vivo
`imaging, for speci?c treatment of diseased cells, or the like.
`For in vivo imaging, the chimeric antibody Will normally be
`conjugated to a radionuclide, e.g., technetium, rhenium, or
`the like. For biocidal activity, the antibody may be joined to
`the A-portion of toxins, liposomes containing biocidal
`reagents, radionuclides, or other biocidal agent.
`Alternatively, the antibodies can be used in combination
`With the host immune system, e.g., complement, due to the
`presence of the native constant region. In vitro, the subject
`chimeric antibodies can be used in conjunction With comple
`ment to remove particular cells from a mixture of cells,
`Where the target cells have a ligand complementary to the
`binding site of the chimeric antibody.
`The folloWing examples are offered by Way of illustration
`and not by Way of limitation.
`EXAMPLE 1
`Materials and Methods
`
`Chimeric Genes
`The cloned S107 variable region (heavy) (VH) and S107
`VK variable region (light, kappa) genes Were obtained from
`Dr. MattheW Scharff (Dept. of Cell Biology, Albert Einstein
`College of Medicine, Bronx, NY. 10641). The S107 VH
`gene Was spliced to human IgG1 and IgG2 constant region
`genes using SalI linkers as shoWn in FIG. 1A. Both con
`structs Were inserted into the vector pSVZAH-gpt (Oi et al.,
`Proc. Natl. Acad. Sci. USA (1983) 80:825—829; Mulligan
`and Berg, Science (1980) 209:1422—1427). The S107 VK
`gene Was spliced to the human K gene at a unique HindIII
`site located in the large intron betWeen the JK and CK exons
`as shoWn in FIG. 1B. This chimeric light chain gene
`construct Was inserted into both pSVZAH-gpt and pSVZ-neo
`plasmid vectors (Mulligan and Berg, Proc. Natl. Acad. Sci.
`USA (1981) 78:2072—2076) and pSV184AH-neo plasmid
`vectors (Oi and Morrison, Biotechniques (1986)
`4:214—221).
`Transfection
`Protoplast fusion and calcium-phosphate (CaPO4) pre
`cipitation techniques (Oi et al., (1983) supra; Sandri-Goldin
`et al., Mol. Cell. Biol. (1981) 1:743—752; Chu and Sharp,
`Gene (1980) 13:197—202) Were used to transfect these
`chimeric immunoglobulin genes into the J558L myeloma
`cell line (a lambda (9») light chain producing mouse
`myeloma cell line) and the non-immunoglobulin-producing
`An derivative of the P3 myeloma cell line. Mycophenolic
`acid (Gibco Laboratories, Santa Clara, Calif. 95050) Was
`used for selection of cells transfected With pSVZAH-gpt
`vectors as described previously (Oi et al., (1983) supra; Ochi
`et al., Proc. Natl. Acad. Sci. USA (1983) 80:6351—6355).
`G418 (Gibco Laboratories) at 1.0 mg/ml Was used for
`selection of cells transfected With pSV2-neo vectors
`(Mulligan and Berg, (1980) supra).
`When both light and heavy chimeric genes Were trans
`fected into the J558L cell line using protoplast fusion
`techniques, light and heavy chimeric immunoglobulin genes
`Were transfected sequentially using G418 selection for the
`chimeric light chain gene vector and mycophenolic acid for
`the chimeric he