`Moore et al.
`
`US005840545A
`[11] Patent Number:
`[45] Date of Patent:
`
`5,840,545
`Nov. 24, 1998
`
`[54] HYBRID DNA PREPARED BINDING
`COMPOSITION
`
`[75] Inventors: Kevin W. Moore, San Bruno;
`Alejandro Za?'aroni, Atherton, both of
`Calif.
`
`[73] Assignee: Schering Corporation
`
`[21] Appl. No.: 461,071
`[22] Filed:
`Jun. 5, 1995
`
`Related US. Application Data
`
`[62] Division of Ser. No. 394,923, Feb. 23, 1995, abandoned,
`which is a continuation of Ser. No. 210,540, Mar. 17, 1994,
`abandoned, which is a continuation of Ser. No. 61,760, May
`13, 1993, abandoned, which is a continuation of Ser. No.
`928,526, Aug. 11, 1992, abandoned, which is a continuation
`of Ser. No. 740,862, Jul. 31, 1991, abandoned, which is a
`continuation of Ser. No. 235,835, Aug. 18, 1988, abandoned,
`which is a continuation of Ser. No. 558,551, Dec. 5, 1983,
`Pat. No. 4,642,334, which is a continuation of Ser. No.
`358,414, Mar. 15, 1982, abandoned.
`
`[51]
`
`Int. Cl.6 .......................... .. C12P 21/02; C12P 21/08;
`C12N 1/21; C12N 15/13
`[52] US. Cl. ................ .. 435/696; 435/172.3; 435/252.33
`[58] Field of Search ............................ .. 735/2402, 172.3,
`735/691, 69.6; 514/2; 530/387.1, 387.3;
`435/691, 69.6, 1721, 172.3, 3201, 252.33
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`4,036,945
`4,642,334
`
`7/1977 Haber et a1. ......................... .. 424/1.49
`2/1987 Moore et al. ......................... .. 530/388
`
`FOREIGN PATENT DOCUMENTS
`
`A 0 035 265 9/1981
`
`European Pat. Off. .
`
`OTHER PUBLICATIONS
`
`Bodey et al., “Human Cancer Detection and Immunotherapy
`With Conjugated and Non—conjugated Monoclonal Antibod
`ies”, Anticancer Res., vol. 16, pp. 661—674 (1996).
`Jain, “Vascular and Interstitral Barners to Delivery of Thera
`peutic Agents in Tumors”, Cancer Metastasis Rev. vol. 9(3),
`pp. 253—266, 1990 (Abstract only).
`Baumgartner et al., “Immunotherapy of EndothoXemia and
`Septicemia”, Immunobiol, vol. 187, pp. 464—477, (1993).
`Givol, 1974, Essays in Biochemistry 10:73—104.
`Gough, Aug., 1981, TIBS 203—205.
`Early et al., 1981, Genetic Engineering: Principles and
`Methods 3:157—183.
`Leder, 1982, Scienti?c American 19:2702—2710.
`
`Natl. Acad. Sci. USA
`
`Gough et al., 1980, Biochemistry 19: 2702—2710.
`Adams et al., 1980, Biochemistry 19: 2711—2719.
`Inbar et al., Proc. Nat. Acad. Sci. USA 69(9):2659—2662.
`Hochman et al., 1973, Biochemistry 12(6):1130—1135.
`Sharon et al., 1976, Biochemistry 15(7):1591—1594.
`Kooistra et al., 1978, Biochemistry 17(2):345—351.
`Accolla et al., 1980, Proc. Nat. Acad. Sci., USA
`77(1):563—566.
`O’Sullivan et al., 1979, Annals of Clinical Biochemistry
`16:221—240.
`MioZZari et al., 1978, J. Bacteriol 133(3):1457—1466.
`Pliickthun, 1991, Bio/technology 9:545—551.
`Kakimoto et al., 1974, The Journal of Immunology
`112(4):1373—1382.
`Lin et
`al.,
`1978, Proc.
`75(6):2649—2653.
`Rosemblatt et al., 1978, Biochemistry 17:3877—3882.
`Cunningham, Understanding Immunology, pp. 38—39.
`Alberts, 1989, Molecular biology ofthe cell, pp. 1025—1026.
`Francis et al., 1974, Proc. Nat. Acad. Sci., 71:1123—1127.
`Raso
`et
`al.,
`1980,
`Journal
`of Immunology
`125(6):2610—2616.
`Hales et al., 1980, Methods in enzymology 70:334—355.
`O’Sullivan et al., 1981, Methods in enzymology 73: 147—166.
`Boss et al., 1984, Nucleic Acids Research 12:3791—3806.
`Wood et al., 1985, Nature 314:446—448.
`Cabilly et al., 1984, Proc. Natl. Acad. Sci. USA
`8 1 : 3273—3277.
`Kenton et al., 1984, Proc. Natl. Acad. Sci. USA
`81:2955—2599.
`Liu et al., 1984, Proc. Natl. Acad. Sci. USA 81:5369—5373.
`Gherna et al., 1985, American Type Culture Collection
`Sixteenth edition, p. 243.
`Gubler et al., 1986, J. Immunol. 136:2492—2497.
`SaXena et al., 1970, Biochemistry 9:5015—5023.
`
`Primary Examiner—David GuZo
`Attorney, Agent, or Firm—Cynthia L. Foulke; EdWin P.
`Ching
`[57]
`
`ABSTRACT
`
`Proteinaceous binding compositions are prepared employing
`hybrid DNA technology, Where the variable region polypep
`tides of immunoglobulins are substantially reproduced to
`provide relatively small protein molecules having binding
`speci?city and lacking the undesirable aspects of the heavy
`regions of immunoglobulins. The compositions ?nd a Wide
`range of use, particularly for physiological purposes for
`diagnosis and therapy. The binding compositions may be
`modi?ed by labeling With radioisotopes, ?uorescers, and
`toxins for speci?c applications in diagnosis or therapy.
`
`2 Claims, No Drawings
`
`Merck Ex. 1065, pg 1490
`
`
`
`1
`HYBRID DNA PREPARED BINDING
`COMPOSITION
`
`5,840,545
`
`This application is a division of application Ser. No.
`08/394,923, ?led Feb. 23, 1995, noW abandoned, Which is a
`continuation of application Ser. No. 08/210,540, ?led Mar.
`17, 1994, noW abandoned, Which is a continuation of appli
`cation Ser. No. 08/061,760, ?led May 13, 1993, noW
`abandoned, Which is a continuation of application Ser. No.
`07/928,526, ?led Aug. 11, 1992, noW abandoned, Which is
`a continuation of application Ser. No. 07/740,862, ?led Jul.
`31, 1991, noW abandoned, Which is a continuation of appli
`cation Ser. No. 07/235,835, ?led Aug. 18, 1988, noW
`abandoned, Which is a continuation of application Ser. No.
`06/558,551, ?led Dec. 5, 1983, now US. Pat. No. 4,642,334,
`Which is a continuation of application Ser. No. 06/358,414,
`?led Mar. 15, 1982, noW abandoned.
`
`10
`
`15
`
`BACKGROUND OF THE INVENTION
`
`1. Field of the Invention
`The mammalian immunological system is unique in its
`broad ability to produce protein compounds having
`extremely high speci?city for a particular molecular struc
`ture. That is, the proteins or immunoglobulins Which are
`produced have a conformation Which is speci?cally able to
`complement a particular structure, so that binding occurs
`With high affinity. In this manner, the mammalian immune
`system is able to respond to invasions of foreign molecules,
`particularly proteins in surface membranes of
`microorganisms, and toxins, resulting in detoxi?cation or
`destruction of the invader, Without adverse effects on the
`host.
`The primary immunoglobulin involved in the defensive
`mechanism is gamma-globulin (IgG). This immunoglobulin,
`Which is a glycoprotein of about 150,000 daltons, has four
`chains, tWo heavy chains and tWo light chains. Each of the
`chains has a variable region and a constant region. The
`variable regions are concerned With the binding speci?city
`of the immunoglobulin, While the constant regions have a
`number of other functions Which do not directly relate to the
`antibody af?nity.
`In many situations it Would be desirable to have mol
`ecules Which are substantially smaller than the
`immunoglobulins, While still providing the speci?city and
`af?nity Which the immunoglobulins afford. Smaller mol
`ecules can provide for shorter residence times in a mamma
`lian host. In addition, Where the immunoglobulin has to be
`bound to another molecule, it Will be frequently desirable to
`minimiZe the siZe of the ?nal product. Also there are many
`economies in being able to produce a smaller molecule
`Which ful?lls the function of a larger molecule.
`There are situations Where it Will be desirable to be able
`to have a large number of molecules compactly held
`together. By having smaller molecules, a greater number can
`be brought together into a smaller space. Furthermore,
`Where the binding molecule can be prepared by hybrid DNA
`technology, one has the opportunity to bind the binding
`portion of the molecule to a Wide variety of other
`polypeptides, so that one can have the binding molecule
`covalently bonded at one or both ends to a polypeptide
`chain.
`Where immunoglobulins are used in in vivo diagnosis or
`therapy, antisera from an allogenic host or from a mono
`clonal antibody may be immunogenic. Furthermore, When
`conjugates of other molecules to the antibody are employed,
`the resulting conjugate may become immunogenic and elicit
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`55
`
`60
`
`65
`
`2
`host antibodies against the constant region of the immuno
`globulin or against any other part of the molecule.
`It is therefore important that methods be developed Which
`permit the preparation of homogeneous compositions hav
`ing high speci?city for a particular ligand, While avoiding
`the shortcomings of complete immunoglobulins, and pro
`viding the many advantages of loWer molecular Weight.
`2. Description of the Prior Art
`Discussions concerning variable regions of heavy and
`light chains of immunoglobulins may be found in Sharon
`and Givol, Biochem. (1976) 15:1591—1594; Rosemblatt and
`Haber, Biochem. (1978) 17:3877—3882; and Early and
`Hood, Genetic Engineering (1981) 3:157—188. Synthesis of
`part of a mouse immunoglobulin light chain in a bacterial
`clone is described by Amster et al., Nucleic Acids Res.
`(1980) 8:2055—2065. See also the references cited through
`out the speci?cation concerning particular methodologies
`and compositions.
`
`SUMMARY OF THE INVENTION
`Novel protein complexes are provided by producing
`homogeneous compositions de?ning the variable regions of
`the light and heavy chains of an immunoglobulin, Which
`individually or together form a speci?c binding complex to
`a predetermined haptenic or determinant site. Employing
`hybrid DNA technology, cDNA is tailored to remove nucle
`otides extraneous to the variable regions of the light and
`heavy chains. The resulting tailored ds cDNA is inserted into
`an appropriate expression vector Which is then introduced
`into a host for transcription and translation. The resulting
`truncated light and heavy chains de?ne at least a major
`portion of the variable regions and are combined to form a
`complex capable of speci?cally binding to a predetermined
`haptenic site With high af?nity.
`
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`
`The subject invention concerns a hybrid DNA strategy for
`the preparation of speci?c binding polypeptides, normally
`comprised of tWo different polypeptide chains, Which
`together assume a conformation having high binding affinity
`to a predetermined ligand or haptenic site thereof. The
`polypeptide chains form binding sites Which speci?cally
`bind to a predetermined ligand to form a complex having
`strong binding betWeen the ligand and the binding site. The
`binding constant or avidity Will generally be greater than
`105, more usually greater than 106, and preferably greater
`than 108. The haptenic binding site or determinant binding
`site of the polypeptide chain may be associated With a hapten
`or antigen.
`One or both of the different polypeptide chains derived
`from the variable region of the light and heavy chains of an
`immunoglobulin may be used to provide speci?c binding to
`a ligand. For the most part each of the polypeptide chains of
`the light and heavy variable regions Would be employed
`together for binding to the ligand. In describing this
`invention, it Will be understood that While the tWo different
`chains are indicated as forming a complex, either of the
`chains could be used individually, Where feasible due to
`suf?cient binding af?nity of the particular chain to the
`reciprocal ligand.
`The tWo polypeptide chains Which, individually or
`together, provide the compositions of this invention Will
`form a receptor site, analogous to the binding site of an
`immunoglobulin. The composition Will be referred to as an
`
`Merck Ex. 1065, pg 1491
`
`
`
`5,840,545
`
`10
`
`15
`
`35
`
`25
`
`3
`rFv With the individual chains referred to as L-rFv or H-rFv.
`The L- and H- designations Will normally mean light and
`heavy respectively, but in some instances the tWo chains
`may be the same and derived from either the light or heavy
`chain sequences. The polypeptide chains of the rFv Will
`generally have feWer than 125 amino acids, more usually
`feWer than about 120 amino acids, While normally having
`greater than 60 amino acids, usually greater than about 95
`amino acids, more usually greater than about 100 amino
`acids. Desirably, the H-rFv Will be from about 110 to 125
`amino acids While the L-rFv Will be from about 95 to 115
`amino acids.
`The amino acid compositions Will vary Widely, depending
`upon the particular idiotype involved. Usually there Will be
`at least tWo cysteines separated by from about 60 to 75
`amino acids and joined by a disul?de bond to form cystine.
`The tWo chains Will normally be substantial copies of
`idiotypes of the variable regions of the light and heavy
`chains of immunoglobulins, but in some situations it may be
`sufficient to have combinations of either the light or the
`heavy variable region chains.
`In many instances, it Will be desirable to have one or both
`of the rFv chains labeled or bound to a support. Various
`labels may be employed, such as radioisotopes, ?uorescers,
`or toxins. In other situations, one or both of the chains may
`be bound to an inert physiologically acceptable support,
`such as synthetic organic polymers, polysaccharides, natu
`rally occurring proteins, or other non-immunogenic sub
`stances.
`In some situations, it may be desirable to provide for
`covalent crosslinking of the tWo chains, Which could involve
`providing for cysteine residues at the carboxyl termini. The
`chains Will normally be prepared free of the constant
`regions, including or being free of all or a portion of the J
`region. The D region Will normally be included in the
`transcript of the H-rFv.
`For the most part only a relatively small percent of the
`total amino acids Will vary from idiotype to idiotype in the
`rFv. Therefore, there Will be areas providing a relatively
`constant framework and areas that Will vary, namely, the
`hypervariable regions.
`The C-terminus region of the rFv Will have a greater
`variety of sequences than the N-terminus and, based on the
`present strategy, can be further modi?ed to permit variation
`45
`from the naturally occurring heavy and light chains. A
`synthetic oligonucleotide can be employed to vary one or
`more amino acids in a hypervariable region.
`The preparation of the rFv employing hybrid DNA tech
`nology Will noW be described in greater detail.
`The preparation of the rFv Will be divided into three parts:
`(1) isolation of appropriate DNA sequences; (2) introduction
`of the DNA sequences coding for the members of the rFv
`into an appropriate expression vector; and (3) expression
`and isolation of the mimetic variable regions of the light
`(L-rFv) and heavy (H-rFv) chains to provide the rFv.
`I. Isolation of Appropriate DNA Sequences.
`In preparing the DNA sequences, a source of the genes
`encoding the variable region Will be required. The variable
`regions may be derived from IgA, IgD, IgE, IgG or IgM,
`most commonly, from IgM and IgG. This can be achieved by
`immuniZing an appropriate vertebrate, normally a domestic
`animal, and most conveniently a mouse. The immuniZation
`may be carried out conventionally With one or more repeated
`injections of the immunogen into the host mammal, nor
`mally at tWo to three Week intervals. Usually three days after
`
`55
`
`65
`
`4
`the last challenge, the spleen is removed and dissociated into
`single cells to be used for cell fusion to provide hybridomas.
`The immunogen Will be the antigen of interest, or Where
`a hapten, an antigenic conjugate of the hapten to an antigen.
`In order to prepare the hybridomas, the spleen cells are
`fused under conventional conditions employing a fusing
`agent, e.g. PEG6000, to a variety of inter- or intra- species
`myeloma cells, particularly mouse cells such as SP-2/0,
`NS-1, etc. and then suspended in HAT selective media. The
`surviving cells are then groWn in microtiter Wells and
`immunologically assayed for production of antibodies to the
`determinant site(s) of interest.
`Assays for antibodies are Well knoWn in the art and may
`employ a variety of labeled antigens or haptens, Where the
`labels are conveniently radioisotopes, ?uorescers, enZymes,
`or the like. Other techniques may also be employed, such as
`sandWich techniques involving tWo antibodies, one bound to
`a support and the other being labeled. The cells from
`microtiter Wells scored as positive are cloned either by
`limiting dilution or cloning in soft agar. The resulting cloned
`cell lines are then propagated in an appropriate nutrient
`medium and, if necessary, may be stored froZen in liquid
`nitrogen.
`After selection of a particular cell line providing a mono
`clonal antibody of interest, the cells are expanded.
`Conveniently, the cells may be groWn to a density of about
`1><106 cells/ml in a 1 L culture. The cells are then harvested
`by centrifugation and lysed.
`In order to obtain the desired DNA sequence, one can look
`to either the gene expressing the variable region or the
`messenger RNA, Which expresses the variable region. The
`dif?culty With employing genomic DNA is in juxtaposing
`the sequences coding for the variable region, Where the
`sequences are separated by introns. One must isolate the
`DNA fragment(s) containing the proper exons, excise the
`introns and then splice the exons in the proper order and
`orientation. For the most part, this Will be difficult, so that
`the alternative technique employing the messenger RNA
`Will be the method of choice.
`Where the messenger RNA is to be employed, the cells
`Will be lysed under RNase inhibiting conditions. The mes
`senger RNA has the advantage that the mature messenger is
`free of introns, so that the sequence is continuous for the
`entire variable region. Dif?culties With messenger RNA
`have been encountered, due to incomplete reverse transcrip
`tion but these dif?culties can be minimiZed. The ?rst step is
`to isolate the messenger RNA. Conveniently, messenger
`RNA can be separated from other RNA because of its
`polyadenylation, employing an oligo-(dT) cellulose column.
`The mixture of messenger RNAs Will be obtained free of
`other RNA. The presence of messenger RNAs coding for the
`heavy and light chain polypeptides of the immunoglobulins
`may then be assayed by hybridiZation With DNA single
`strands of the appropriate genes. Conveniently, the
`sequences coding for the constant portion of the light and
`heavy chains may be used as probes, Which sequences may
`be obtained from available sources (see, for example, Early
`and Hood, Genetic Engineering, SetloW and Hollaender eds.
`Vol. 3, Plenum Publishing Corp., NeW York (1981), pages
`157—188.)
`Whether the messenger RNA codes for the correct immu
`noglobulin may be determined by in vitro translation
`employing a rabbit reticulocyte cell-free extract (Pelham and
`Jackson, Eurp. J. Biochem. (1976) 66:247—256). The result
`ing translation product may then be isolated by employing
`antibodies speci?c for one or more of the regions of the
`
`Merck Ex. 1065, pg 1492
`
`
`
`5,840,545
`
`5
`chain of interest, for example, using rabbit anti(mouse IgG)
`Where the chains are derived from mouse immunoglobulin.
`The immunoprecipitate may be further analyzed by poly
`acrylamide gel electrophoresis, and the presence of com
`plexes determined by using radiotagged receptors for
`antigen-antibody complexes, such as S. aureus protein A, Rf
`factor, or the like. In addition, RNA blot hybridiZation can
`be employed to further insure that the correct messenger
`RNA is present.
`The crude mixture of mRNA sequences containing the
`desired mRNA sequences Will be treated as folloWs. In order
`to enhance the probability that full length cDNA is obtained,
`the method of Okayama and Berg, Mol. Cell. Biol. (1982)
`may be employed. Alternatively, the methods described by
`Efstradiadis and Villa-Komaroff (1979) in Genetic Engi
`neering: Principles and Methods 1, SetloW and Hollaender,
`eds., NeW York, Plenum Press, pages 15—36, or Steinmetz et
`al. (1981) Cell 24:125—134, may be employed. The ?rst
`strand of cDNA is prepared employing a virus reverse
`transcriptase in the presence of primer. Asecond strand may
`then be prepared employing reverse transcriptase, the Kle
`noW fragment of DNA polymerase I or T4 polymerase. If
`necessary, the resulting ds cDNA may then be treated With
`a single-strand-speci?c nuclease, such as S1 nuclease for
`removal of single stranded portions to result in ds cDNA,
`Which may then be cloned.
`
`1O
`
`15
`
`25
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`6
`The host colonies, usually bacterial, Which have DNA
`Which hybridiZes to either the light or heavy chain probes are
`picked and then groWn in culture under selective pressure. In
`order to maintain selective pressure, it is desirable that the
`vector Which is employed have biocidal, particularly
`antibiotic, resistance. After suf?cient time for expansion of
`the host, the host cells are harvested, conveniently by
`centrifugation. The hybrid plasmid DNA may then be iso
`lated by knoWn procedures. (Gunsalus et al., J. Bacteriol.
`(1979) 140:106—133).
`The isolated plasmid DNA is then characteriZed by
`restriction enZyme digestion and DNA sequence analysis.
`These analyses insure that the isolated cDNA clones com
`pletely encode the variable region and, optionally, the leader
`sequences for the light or heavy chain of the desired immu
`noglobulin. Furthermore, by having a restriction map of the
`variable regions and leader sequences, as Well as the ?ank
`ing sequences, one can determine the appropriate restriction
`sites for excising a DNA fragment Which Will alloW for
`appropriate modi?cation of the DNA sequence for insertion
`into a vector and expression of the polypeptide of interest.
`Where no unique restriction site is available at an appropri
`ate position in the ?anking regions, partial digestion may be
`employed, With selection of fragments having the variable
`region and, optionally, the leader sequence intact. Where the
`5‘ and 3‘ ?anking regions are too extended, these can be
`cheWed back using Bal 31 to varying degrees by varying the
`period of digestion.
`Furthermore, by knoWing the DNA sequence of the cod
`ing strand in the region of the C-terminus of the heavy and
`light chain variable regions, a stop codon may be introduced
`at the C-terminus by the folloWing procedure of in vitro
`mutagenesis. The cDNA is restricted With the appropriate
`enZyme(s) to provide a variable region coding segment With
`additional 5‘ and 3‘ ?anking sequences. This segment is
`puri?ed, for example, by gel electrophoresis, gradient den
`sity centrifugation, etc. After isolating the desired segment,
`the tWo strands of the segment are dissociated, conveniently
`by boiling. Alternatively, the undesired strand of the intact
`cDNA-plasmid clone may be nicked and digested.
`A synthetic, single-stranded DNA oligomer is prepared,
`conveniently by synthesis, Which Will have at least about 12
`nucleotides, more usually about 15 nucleotides, and Will
`generally have feWer than about 50 nucleotides, usually
`feWer than 30 nucleotides, since a more extended oligomer
`is not required.
`Where heteroduplexing is involved, the non
`complementary nucleotides Will usually be ?anked by at
`least about three, more usually at least about six nucleotides
`complementary to the hybridiZed strand. The heteroduplex
`ing oligonudleotide Will be complementary to the sequence
`at or about a signi?cant juncture ie between the leader
`sequence and the variable region or the variable region and
`the constant region. The synthetic DNA oligomer Will be
`complementary to the coding (“sense”) strand of the
`variable-region sequence, but altered to encode a termina
`tion codon at the C-terminus of the variable region. That is,
`the oligomer Will be complementary to the coding strand
`except at or about the amino acid Which is involved at the
`juncture of the variable region and the D-, J- or C-regions of
`the light and heavy chains, particularly at or intermediate the
`D- or J-regions or intermediate the J-region, or at the J
`region and C-region juncture. It is intended that there Will be
`some variation in the polypeptides Which are prepared, so far
`as extending beyond the variable domains or not including
`all of the amino acids at the C-terminus of the variable
`region.
`
`II. Preparation of Genes Coding For L-rFv and H
`rFv and Introduction into an Expression Vector For
`Ampli?cation
`A Wide variety of vectors may be employed for ampli?
`cation or expression of the ds cDNA to produce the light and
`heavy chains of the immunoglobulin. A vector having an
`appropriate restriction site is digested With the appropriate
`endonuclease. The ds cDNA obtained from the reverse
`transcription of the mRNA may be modi?ed by ligating
`linkers, treatment With terminal transferase or other tech
`niques to provide staggered (complementary) or blunt ended
`termini. The vectors may have one, tWo or more markers for
`selection of transformants. Desirably, the vector Will have a
`unique restriction site in one of multiple markers, so that the
`transformants may be selected by the expression of one
`marker and the absence of expression of the other marker.
`Various markers may be employed, such as biocide
`resistance, complementation of an auxotroph, viral
`immunity, or the like.
`After transforming an appropriate host With the ds cDNA
`prepared from the mRNA, e.g. E. coli, B. subtilis, S.
`cerevisiae, etc., in accordance With conventional Ways, the
`transformants are plated and selected in accordance With the
`particular markers. The resulting colonies are screened, by
`restriction electrophoretic pattern, hybridiZation to a labeled
`probe or by any other conventional means. See, for example,
`Hanahan and Meselson (1980), Gene 10:63—67. One proce
`dure employs colony hybridiZation, Where the transformants
`are groWn on a solid medium to produce colonies. Cells
`from the colonies are transferred to a nitrocellulose replica
`?lter, the transferred cells incubated for further groWth,
`lysed, dried and baked. The replica ?lter is then hybridiZed
`With appropriate radio-isotope labeled probes. conveniently,
`there are readily available probes for the determinant sites
`present in the constant regions of a variety of mammalian
`immunoglobulins. The colonies may be probed based on the
`nature of the particular immunoglobulin, as Well as the
`different determinant sites, Which may be present With the
`particular immunoglobulin.
`
`35
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`45
`
`55
`
`65
`
`Merck Ex. 1065, pg 1493
`
`
`
`5,840,545
`
`7
`An excess amount of the oligomer is combined With the
`denatured strands of the restriction fragment under suffi
`ciently stringent hybridization conditions. Thus, the oligo
`mer speci?cally heteroduplexes to the complementary por
`tions of the coding strand, While providing one or more stop
`and/or nonsense codons to insure the termination of expres
`sion at the desired amino acid at the C-terminus.
`After suf?cient time for hybridization at the desired level
`of stringency, suf?cient amounts of the four deoxynucle
`otides are added in conjunction With the KlenoW fragment of
`DNA polymerase I. A strand complementary to the coding
`sequence of the variable-region and any 5‘-?anking
`sequence is synthesiZed by enZymatic elongation of the
`primer resulting in a sequence complementary to the strand
`to Which the oligonucleotide is bound. The single-stranded
`DNA sequence on the coding strand located 3‘ to the region
`hybridiZed to the synthetic oligonucleotide is degraded by
`the 3‘—5‘ exonuclease activity of the DNA polymerase. In
`this manner, ds cDNA is obtained Which speci?cally codes
`for the variable-region and upstream ?anking regions asso
`ciated With the light and heavy chains. Each of the heavy and
`light chains is encoded to terminate expression at a prede
`termined codon in the V, D or J region.
`The resulting heteroduplexed blunt-ended ds CDNA frag
`ments are then employed for preparation of homoduplexed
`ds CDNA coding for the light and heavy variable regions
`With the stop codons at the desired sites. Conveniently, the
`blunt ended fragments are modi?ed as described previously,
`e.g. joined to linkers Which code for restriction sites Which
`are absent in the variable region sequences, or may be tailed
`e.g. polyG or polyC, or used directly for insertion. With
`restriction site linkers, after insertion of the fragment into an
`appropriate vector having complementary termini, the frag
`ment can be recovered by restriction at the linker sites. The
`linkers are joined to the coding sequences With an appro
`priate ligase, e.g. T4 ligase, the resulting fragment restricted
`to provide cohesive ends, and the product annealed to the
`complementary ends of a vector.
`At this stage, the vector Which is employed provides for
`ampli?cation and convenient isolation of transformants hav
`ing the variable region coding sequence insert. Numerous
`vectors for ampli?cation in bacteria or other hosts exist such
`as pBR322, pSClOl, pRK290, 2 p-plasmid, etc. The hybrid
`plasmid containing the mismatched sequences Will replicate
`in the host to generate tWo different plasmid molecules, one
`With the original sequence and one With the “tailored” or
`“site mutated” sequence derived from the synthetic oligo
`nucleotide. Therefore, each transformant colony is groWn in
`small (approximately 2 ml) culture for plasmid isolation.
`The transformants are groWn, the plasmid DNA isolated
`in accordance With knoWn procedures, and used for a second
`cycle of transformation to provide individual clones repli
`cating the tailored sequence. The clones may be screened by
`?lter blot hybridiZation, probing With a labeled synthetic
`oligonucleotide Which Will include the synthetic oligonucle
`otide employed in tailoring the variable region sequence, or
`other convenient technique. Thus, plasmids are obtained
`having ds cDNA ?anked by appropriate restriction sites and
`having a stop codon at a predetermined site.
`Having noW de?ned the 3‘-terminus of the coding strand
`or, alternatively, the C-terminus amino acid, the 5‘-region or
`N-terminus of the polypeptide is noW de?ned. Of course, the
`particular order in Which the tWo termini are modi?ed is
`primarily one of convenience, and can even be done
`simultaneously, Where primer repair is used at the 5‘-end of
`the coding strand in conjunction With site mutation at the
`3‘-end.
`
`15
`
`25
`
`35
`
`45
`
`55
`
`65
`
`8
`Different strategies may be evolved, depending upon the
`nature of the host in Which expression is to be obtained, and
`Whether such host recogniZes the leader sequence as a
`secretory signal for secretion of the polypeptide With con
`comitant removal of the leader sequence polypeptide. Where
`this opportunity is not available, the strategy Will involve
`removal of the leader sequence to provide a start codon at
`the 5‘-terminus of the sequence of the coding strand coding
`for the variable region, Which sequence can be inserted into
`an expression vector, so as to be under the control of a
`predetermined promoter and ribosomal start site.
`Based on the sequence of the leader region or the
`sequence coding for the N-terminus of the variable region,
`different oligonucleotides for homo- or heteroduplexing can
`be prepared.
`Where the leader sequence is retained, primer repair is
`employed to remove the 5‘-?anking sequence of the coding
`strand. When the primer repair of the N-terminus is per
`formed simultaneously With the C-terminus mutagenesis,
`after treatment With the DNA polymerase, the resulting
`partial double stranded DNA Will be treated With a 5‘-3‘
`single strand exonuclease to remove the 5‘-?anking region as
`Well as a ligase to provide for covalent linking of the
`replicated strand to the N-terminus oligonucleotide.
`Where the leader sequence is to be removed, in vitro
`mutagenesis is employed to introduce an f-met codon at the
`N-terminus of the DNA sequence coding for the variable
`region.
`Alternative strategies may be employed for recovering the
`desired ds cDNA and performing the in vitro mutagenesis.
`If useful restriction sites are distant from the coding regions,
`the plasmid may be digested With the appropriate restriction
`endonuclease, folloWed by digestion With a double-strand
`exonuclease e.g. Bal 31. The resulting ds cDNA may be
`cloned and the proper sequence selected and modi?ed, as
`appropriate, as described above. If the non-coding ?anking
`region at the 5‘-terminus of the coding strand is too long, it
`may be digested With an endonuclease, Where a convenient
`restriction site is available or by digestion With an exonu
`clease e.g. Bal 31.
`By repeating the above described procedure for modify
`ing the 3‘-terminus, except that the oligonucleotide is noW
`complementary to the non-coding (nonsense) strand, and
`includes an initiation codon at the 5‘-end (primer repair) or
`Within the oligonucleotide (in vitro muta-genesis), the
`5‘-terminus of DNA sequence encoding the variable regions
`may be tailored. Normally, the oligonucleotide homodu
`plexes for p