United States Patent
`Cabilly et al.
`
`[19]
`
`[11] Patent Number:
`
`[45] Date of Patent:
`
`4,816,567
`
`Mar. 28, 1989
`
`Nisonoff, A. et al., Arch. Biochem. Biophys., vol. 93, pp.
`460-462, 1960.
`Glennie, M. J. et al., Nature, vol. 295, pp. 712-714, 1982.
`Eisen, H. N., Immunology, Harper & Row, Publishers,
`pp. 415 and 428-436, 1974.
`Hozumi, N. et al. Nuc Acids Res. vol. 5(6) pp.
`1779-1799, Chem. Abst. 89:87928t, 1978.
`Wetzel, R. et al. Gene, vol. 16, pp. 63-71, 1981.
`Williams et al., Science, vol. 215, pp. 687-689, 1982.
`Falkner, F. G. et al., Nature, vol. 298, pp. 286-288,
`1982.
`
`Boss et al. Hamer et al., Ed. “Gene Expressions-Proc.
`Cetus-UCLA Symposium .
`.
`. Mar. 26-Apr. 1, 1983”
`pp. 513-522.
`Amster et al., “Nucleic Acid Research” 8(9): 2055-2065
`(1980).
`DeBoer et al., Rodriguez ct al., Ed. “Promoters”
`462-481 (1982).
`Gougb, “Tibs” 6(8): 203-205 (Aug. 1981).
`Morrison, “J. of Immunology” 123(2): 793-800 (Aug.
`1979).
`Kohler “P.N.A.S. USA” 77(4): 2197-2199 (Apr. 1980).
`Thomas M. Roberts, Promoters, Rodriguez et al., Eds.
`(1982), pp. 452-461.
`Kemp et al., “Proc. Natl. Acad. Sci. USA” 78(7):
`4520-4524 (Jul. 1981).
`Valle et al., “Nature” 300271-74 (4 Nov. 1982).
`Microbiology 3rd ed., Harper Int. Ed. 338-379 (1980).
`Hitzeman et al. Science 219: 620-625 (1983).
`Mercereau-Puijalon et al., in “Expression of Eukary-
`otic Viral and Cellular Genes” Pettersson et al. (ED.)
`295-303 (1981) Academic Pr.
`Primary Examt'ner—Jayme Huleatt
`
`[57]
`
`(List continued on next page.)
`ABSTRACT‘
`
`including con-
`Altered and native immunoglobulins,
`stant-variable region chimeras, are prepared in recombi-
`nant cell culture. The immunoglobulins contain variable
`regions which are immunologically capable of binding
`predetermined antigens. Methods are provided for re-
`folding directly expressed immunoglobulins into immu-
`nologically active form.
`
`7 Claims, 15 Drawing Sheets
`
`Sanofi/Regeneron Ex. 1007, pg 161
`
`Mylan Ex. 1007, pg 161
`
`[54] RECOMBINANT IMMUNOGLOBIN
`PREPARATIONS
`
`[75]
`
`Inventors: Shmuel Cabilly, Monrovia; Herbert
`L. Heyneker, Burlingame; William E.
`Holmes, Pacifica; Arthur D. Riggs,
`La Verne; Ronald B. Wetzel, San
`Francisco, all of Calif.
`
`[73] Assignec:
`
`Genentech, Inc., South San
`Francisco, Calif.
`
`[21] App1.No.: 483,457
`[22] Filed:
`Apr. 8, 1983
`
`[51]
`
`[56]
`
`C07K 15/14; C07K 15/05;
`Int. c1.4 ................
`C12P 21/00; C12N 15/00; C12N 1/20
`[52] U.S.Cl. .................................... .. 530/337; 435/68;
`435/172.3; 435/320; 435/252.3; 435/252.31;
`435/252.33; 435/252.34; 935/10; 935/15;
`935/29; 935/73; 530/388
`[58] Field of Search .................... .. 435/68, 172.3, 240,
`435/253, 172.2, 317, 320; 260/112 B; 536/27;
`935/11, 15, 27, 29, 73; 530/387, 388
`References Cited
`U.S. PATENT DOCUMENTS
`4,444,878 4/1984 Paulus ............................. 435/188
`4,512,922 4/1985 Jones et al.
`.
`435/68 X
`4,518,584
`5/1985 Mark et al.
`.
`435/172.3
`4,704,362 11/1987 Itakura et al.
`.. 435/ 172.3 X
`
`
`
`FOREIGN PATENT DOCUMENTS
`0057107
`8/1982 European Pat. Off.
`.
`0073656
`8/1982 European Pat. Off.
`......... 435/ 172.3
`0068763
`1/1983 European Pat. Off.
`.
`0120694 10/1984 European Pat. Off.
`.
`OTHER PUBLICATIONS
`
`Dolby, T. W. et al. Proc. Natl. Acad. Sci., vol. 77, (10)
`pp. 6027-6031, 1980.
`Rice, D. et al. Proc. Natl. Acad. Sci. vol. 79 pp.
`7862-7865, 1982.
`
`Accolla, R. S. et al., Prac. Natl. Acad. Sci., vol. 77, (1)
`pp. 563-566, 1980.
`Raso, V. et al., Cancer Res, vol. 41, pp. 2073-2078,
`1981.
`
`

`
` Page 2
`
`4,816,567
`
`OTHER PUBLICATIONS
`1
`.
`.
`Keshet et a1. Nuclexc Ac1ds Res. 9(1): 19-30 (1981).
`Taniguchi et al., Proc. Natl. Acad. Sci. USA, 77(9):
`5230-5233 (1930).
`Ohsuye et al" Nucleic Acids ReS' 11(5): 12834295
`(1983)
`
`Kadonaga et al., J. Biol. Chem. 259(4): 2149-2154
`(1934).
`.
`.
`H
`.
`,,
`11:ff.I.l'::3]S; Ti N:t°le;u1a“rNC111:1:::g Ap'.:'33l{Se1,D,' 11918 .
`35§1_3591 (E83)
`' ‘
`C‘
`5
`°3~
`(
`)-
`Roberts T M in“‘Promoters Structures and Function”
`Rodriguez, R. L. (ED.) 452-461 (1982).
`
`Sanofi/Regeneron Ex. 1007, pg 162
`
`Mylan Ex. 1007, pg 162
`
`

`
`US. Patent Mar. 23, 1989
`
`Sheet 1 of4
`
`4,816,567
`
`Sanofi/Regeneron Ex. 1007, pg 163
`
`Mylan Ex. 1007, pg 163
`
`

`
`U.S. Patent Mar. 23, 1939
`
`Sheet 2 of4
`
`4,816,567
`
`6Fig.88.
`
`Sanofi/Regeneron Ex. 1007, pg 164
`
`Mylan Ex. 1007, pg 164
`
`

`
`U.S. Patent Mar. 23, 1939
`
`Sheet 3 of4
`
`4,816,567
`
`Sanofi/Regeneron Ex. 1007, pg 165
`
`Mylan Ex. 1007, pg 165
`
`

`
`U.S. Patent Mar. 23, 1989
`
`Sheet 4 of 4
`
`4,816,567
`
`O.|0ug anti-CEA
`
`O.30pg anti-CEA
`I.OOug anti-CEA
`K cells (|Op|)
`
`ycells (|Oul)
`K, cells (noun)
`RW57-K3000
`
`RW57—K3000
`
`::-:;::}
`K7 cells (20p|)
`0, Kcells (zom)
`
`
`
`Sanofi/Regeneron Ex. 1007, pg 166
`
`Mylan Ex. 1007, pg 166
`
`

`
`1
`
`RECOMBINANT IMMUNOGLOBIN
`PREPARATIONS
`
`BACKGROUND OF THE INVENTION
`
`This invention relates to the field of immunoglobulin
`production and to modification of naturally occuring
`immunoglobulin amino acid sequences. Specifically, the
`invention relates to using recombinant techniques to
`produce both immunoglobulins which are analogous to
`those normally found in vertebrate systems and to take
`advantage of these gene modification techniques to
`construct chimeric or other modified forms.
`A. Immunoglobulins and Antibodies
`Antibodies are specific immunoglobulin polypeptides
`produced by the vertebrate immune system in response
`to challenge by foreign proteins glycoproteins, cells, or
`other antigenic foreign substances. The sequence of
`events which permits the organism to overcome inva-
`sion by foreign cells or to rid the system of foreign
`substances is at least partially understood. An important
`part of this process is the manufacture of antibodies
`which bind specifically to a particular foreign sub-
`stance. The binding specificity of such polypeptides to a
`particular antigen is highly refined, and the multitude of
`specificities capable of being generated by the individ-
`ual vertebrate is remarkable in its complexity and vari-
`ability. Thousands of antigens are capable of eliciting
`responses, each almost exclusively directed to the par-
`ticular antigen which elicted it.
`Immunoglobulins include both antibodies, as above
`described, and analogous protein substances which lack
`antigen specificity. The latter are produced at low lev-
`els by the lymph system and in increased levels by mye-
`lomas.
`A.l Source and Utility
`Two major sources of vertebrate antibodies are pres-
`ently utilized——generation in situ by the mammalian B
`lymphocytes and in cell culture by B-cell hybrids. Anti-
`bodies are made in situ as a result of the differentiation
`of immature B lymphocytes into plasma cells, which
`occurs in response to stimulation by specific antigens. In
`the undifferentiated B cell, the portions of DNA coding
`for the various regions on the immunoglobulin chains
`are separated in the genomic DNA. The sequences are
`reassembled sequentially prior to transcription. A re-
`view of this process has been given by Gough, Trends in
`Biochem Sci, 6: 203 (1981). The resulting rearranged
`genome is capable of expression in the mature‘B lym-
`phocyte to produce the desired antibody. Even when
`only a single antigen is introduced into the sphere of the
`immune system for a particular mammal, however, a
`uniform population of antibodies does not result. The in
`situ immune response to any particular antigen is de-
`fined by the mosaic of responses to the various determi-
`nants which are present on the antigen. Each subset of
`homologous antibody is contributed by a single popula-
`tion of B cells—hence in situ generation of antibodies is
`“polyclonal”.
`'
`This limited but
`inherent heterogeneity has been
`overcome in numerous particular cases by use of hy-
`bridoma technology to create “monoclona ” antibodies
`(Kohler, et al., Eur. J. Immunol., 6: 511 (1976)). In this
`process, splenocytes or lymphocytes from a mammal
`which has been injected with antigen are fused with a
`tumor cell line, thus producing hybrid cells or “hy-
`bridomas” which are both immortal and capable of
`producing the genetically coded antibody of the B cell.
`
`4,816,567
`
`2
`The hybrids thus formed are segregated into single
`genetic strains by selection, dilution, and regrowth, and
`each strain thus represents a single genetic line. They
`therefore produce immunoreactive antibodies against a
`desired antigen which are assured to be homogenous,
`and which antibodies, referencing their pure genetic
`parentage, are called “monoclonal”. Hybridoma tech-
`nology has to this time been focused largely on the
`fusion of murine lines, but human-human hybridomas
`(Olsson, L. et al., Proc. Natl. Acad. Sci. (USA), 77: 5429
`(1980)); human-murine hybridomas (Schlom, J., et al.
`(ibid) 77: 6841 (1980)) and several other xenogenic hy-
`brid combinations have been prepared as well. Alterna-
`tively, primary, antibody producing, B cells have been
`immortalized in vitro by transformation with viral
`DNA.
`Polyclonal, or, much more preferably, monoclonal,
`antibodies have a variety of useful properties similar to
`those of the present invention. For example, they can be
`used as specific immunoprecipitating reagents to detect
`the presence of the antigen which elicited the initial
`processing of the B cell genome by coupling this anti-
`gen-antibody reaction with suitable detection tech-
`niques such as labeling with radioisotopes or with en-
`zymes capable of assay (RIA, EMIT, and ELISA).
`Antibodies are thus the foundation of immuno diagnos-
`tic tests for many antigenic substances. In another im-
`portant use, antibodies can be directly injected into
`subjects suffering from an attack by a substance or or-
`ganism containing the antigen in question to combat this
`attack. This process is currently in its experimental
`stages, but its potential is clearly seen. Third, whole
`body diagnosis and treatment is made possible because
`injected antibodies are directed to specific target disease
`tissues, and thus can be used either to determine the
`presence of the disease by carrying with them a suitable
`label, or to attack the diseased tissue by carrying a suit-
`able drug.
`Monoclonal antibodies produced by hybridomas,
`while theoretically effective as suggested above and
`clearly preferable to polyclonal antibodies because of
`their specificity, suffer
`from certain disadvantages.
`First, they tend to be contaminated with other proteins
`and cellular materials of hybridoma, (and, therefore,
`mammalian) origin. Second, hybridoma lines producing
`monoclonal antibodies tend to be unstable and may alter
`the structure of antibody produced or stop producing
`antibody altogether (Kohler, G., et al., Proc. Am]. Acad.
`Sci (USA) 77: 2197 (1980); Morrison, S. L., J. Immunol.
`123: 793 (1979)). The cell line genome appears to alter
`itself in response to stimuli whose nature is not cur-
`rently known, and this alteration may result in produc-
`tion of incorrect sequences. Third, both hybridoma and
`B cells inevitably produce certain antibodies in glycosy-
`lated form (Melchers, F., Biochemistry. 10: 653 (1971))
`which, under some circumstances, may be undesirable.
`Fourth, production of both monoclonal and polyclonal
`antibodies is relatively expensive. Fifth, and perhaps
`most important, production by current techniques (ei-
`ther by hybridoma or by B cell response) does not per-
`mit manipulation of the genome so as to produce anti-
`bodies with more effective design components than
`those normally elicited in response to antigens from the
`mature B cell in situ. The antibodies of the present in-
`vention do not suffer from the foregoing drawbacks,
`and, furthermore, offer the opportunity to provide mol-
`ecules of superior design.
`
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`

`
`4,816,567
`
`3
`Even those immunoglobulins which lack the specific-
`ity of atibodies are useful, although over a smaller spec-
`trum of potential uses than the antibodies themselves. In
`presently understood applications, such immunoglobu-
`lins are helpful in protein replacement therapy for glob-
`ulin related anemia. In this context, an inability to bind
`to antigen is in fact helpful, as the therapeutic value of
`these proteins would be impaired by such functionality.
`At present, such non-specific antibodies are derivable in
`quantity only from myeloma cell cultures suitably in-
`duced. The present invention offers an alternative, more
`economical source. It also offers the opportunity of
`cancelling out specificity by manipulating the four
`chains of the tetramer separately.
`A.2 General Structure Characteristics
`The basic immunoglobin structural unit in vertebrate
`systems is now well understood (Edelman, G. M., Ann.
`N. I’. Acad. Sci, 190: 5 (1971)). The units are composed
`to two identical light polypeptide chains of molecular
`weight approximately 23,000 daltons, and two identical
`heavy chains of molecular weight 53,000—70,000. The
`four chains are joined by disulfide bonds in a “Y” con-
`figuration wherein the light chains bracket the heavy
`chains starting at the mouth of the Y and continuing
`through the divergent region as shown in FIG. 1. The
`“branch” portion, as there indicated, is designated the
`Fab region. Heavy chains are classified as gamma, mu,
`alpha, delta, or epsilon, with some subclasses among
`them, and the nature of this chain, as it has a long con-
`stant region, determines the “class” of the antibody as
`IgG, IgM, IgA, IgD, or IgE. Light chains are classified
`as either kappa or lambda. Each heavy chain class can
`be prepared with either kappa or lambda light chain.
`The light and heavy chains are covalently bonded to
`each other, and the “tail” portions of the two heavy
`chains are bonded to each other by covalent disulfide
`linkages when the immunoglobulins are generated ei-
`ther by hybridomas or by B cells. However, if non-
`covalent association of the chains can be effected in the
`correct geometry, the aggregate will still be capable of
`reaction with antigen, or of utility as a protein supple-
`ment as a non-specific immunoglobulin.
`The amino acid sequence runs from the N-terminal
`end at the top of the Y to the C-terminal end at the
`bottom of each chain. At the N-terminal end is a vari-
`able region which is specific for the antigen which elic-
`ited it, and is approximately 100 amino acids in length,
`there being slight variations between light and heavy
`chain and from antibody to antibody. The variable re-
`gion is linked in each chain to a constant region which
`extends the remaining length of the chain. Linkage is
`seen, at the genomic level, as occuring through a linking
`sequence known currently as the “J” region in the light
`chain gene, which encodes about 12 amino acids, and as
`a combination of “D" region and “J" region in the
`heavy chain gene, which together encode approxi-
`mately 25 amino acids.
`The remaining portions of the chain are referred to as
`constant regions and within a particular class do not to
`vary with the specificity of the antibody (ie, the anti-
`gen eliciting it).
`As stated above, there are five known major classes
`of constant regions which determine the class of the
`immunoglobulin molecule (IgG, IgM, IgA, IgD, and
`IgE corresponding to 'y, p., CL, 6, and 6 heavy chain
`constant regions). The constant region or class deter-
`mines subsequent effector function of the antibody,
`including activation of complement (Kabat, E. A.,
`
`l0
`
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`4
`Structural Concepts in Immunology and Immunoclzemz's-
`try, 2nd Ed., p. 413-436, Holt, Rinehart, Winston
`(1976)), and other cellular responses (Andrews, D. W.,
`et al., Clinical lmmunobiology, pp 1-18, W. B. Sanders
`(1980); Kohl, S., et al., Immunology, 48: 137 (1983));
`while the variable region determines the antigen with
`which it will react.
`B. Recombinant DNA Technology
`Recombinant DNA technology has reach sufficient
`sophistication that it includes a repertoire of techniques
`for cloning and expression of gene sequences. Various
`DNA sequences can be recombined with some facility,
`creating new DNA entities capable of producing heter-
`ologous protein product in transformed microbes and
`cell cultures. The general means and methods for the in
`vitro ligation of various blunt ended or “sticky" ended
`fragments of DNA, for producing expression vectors,
`and for transforming organisms are now in hand.
`DNA recombination of the essential elements (i.e., an
`origin of replication, one or more phenotypic selection
`characteristics, expression control sequence, heterolo-
`gous gene insert and remainder vector) generally is
`performed outside the host cell. The resulting recombi-
`nant replicable expression vector, or plasmid, is intro-
`duced into cells by transformation and large quantities
`of the recombinant vehicle is obtained by growing the
`transformant. Where the gene is properly inserted with
`reference to portions which govern the transcription
`and translation of the encoded DNA message, the re-
`sulting expression vector is useful to produce the poly-
`peptide sequence for which the inserted gene codes, a
`process referred to as “expression.” The resulting prod-
`uct may be obtained by lysis, if necessary, of the host
`cell and recovery of the product by appropriate purifi-
`cations from other proteins.
`In practice, the use of recombinant DNA technology
`can express entirely heterologous polypeptides—so-
`called direct expression—or alternatively may express a
`heterologous polypeptide fused to a portion of the
`amino acid sequence of a homologous polypeptide. In
`the latter cases, the intended bioactive product is some-
`times rendered bioinactive within the fused, homolo-
`gous/heterologous polypeptide until it is cleaved in an
`extracellular environment.
`The art of maintaining cell or tissue cultures as well as
`microbial systems for studying genetics and cell physi-
`ology is well established. Means and methods are avail-
`able for maintaining permanent cell lines, prepared by
`successive serial transfers from isolated cells. For use in
`research, such cell lines are maintained on a solid sup-
`port in liquid medium, or by growth in suspension con-
`taining support nutriments. Scale-up for large prepara-
`tions seems to pose only mechanical problems.
`SUMMARY OF THE INVENTION
`The invention relates to antibodies and to non-
`specific immunoglobulins (NSIS) formed by recombi-
`nant techniques using suitable host cell cultures. These
`antibodies and NSIs can be readily prepared in pure
`“monoclonal" form. They can be manipulated at the
`genomic level to produce chimeras of variants which
`draw their homology from species which differ from
`each other. They can also be manipulated at the protein
`level, since all four chains do not need to be produced
`by the same cell. Thus, there are a number of “types” of
`immunoglobulins encompassed by the invention.
`First, immunoglobulins, particularly antibodies, are
`produced using recombinant techniques which mimic
`
`Sanofi/Regeneron Ex. 1007, pg 168
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`

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`4,816,567
`
`6
`FIG. 9 shows the results of western blots of extracts
`of cells transformed as those in FIGS. 8.
`FIG. 10 shows a standard curve for ELISA assay of
`anti CEA activity.
`FIGS. 11 and 12 show the construction of a plasmid
`for expression of the gene encoding a chimeric heavy
`chain.
`FIG. 13 shows the construction of a plasmid for ex-
`pression of the gene encoding the Fab region of heavy
`chain.
`
`5
`the amino acid sequence of naturally occuring antibod-
`ies produced by either mammalian B cells in situ, or by
`B cells fused with suitable immortalizing tumor lines,
`i.e., hybridomas. Second, the methods of this invention
`produced, and the invention is directed to, immuno-
`globulins which comprise polypeptides not hitherto
`found associated with each other in nature. Such reas-
`sembly is particularly useful
`in producing “hybrid”
`antibodies capable of binding more than one antigen;
`and
`in
`producing
`“composite”
`immunoglobuins
`wherein heavy and light chains of different origins es-
`sentially damp out specificity. Third, by genetic manip-
`ulation, “chimeric" antibodies can be formed wherein,
`for example, the variable regions, correspond to the
`amino acid sequence from one mammalian model sys-
`tem, whereas the constant region mimics the amino acid
`sequence of another. Again, the derivation of these two
`mimicked sequences may be from different species.
`Fourth, also by genetic manipulation, “altered” anti-
`bodies with improved specificity and other characteris-
`tics can be formed.
`Two other types of irnmunoglobulin-like moieties
`may be produced: “univalent” antibodies, which are
`useful as homing carriers to target tissues, and “Fab
`proteins” which include only the “Fab" region of an
`immunoglobulin molecule i.e, the branches of the “Y”.
`These univalent antibodies and Fab fragments may also
`be “mammalian” i.e., mimic mammalian amino acid
`sequences; novel assemblies of mammalian chains, or
`chimeric, where for example, the constant and variable
`sequence patterns may be of different origin. Finally,
`either the light chain or heavy chain alone, or portions
`thereof, produced by recombinant techniques are in-
`cluded in the invention and may be mammalian or chi-
`meric.
`In other aspects, the invention is directed to DNA
`which encodes the aforementioned NSIs, antibodies,
`and. portions thereof, as well as expression vectors or
`plasmids capable of effecting the production of such
`immunoglobulins in suitable host cells. It includes the
`host cells and cell cultures which result from transfor-
`mation with these vectors. Finally, the invention is
`directed to methods of producing these NSIs and anti-
`bodies, and the DNA sequences, plasmids, and trans-
`formed cells intermediate to them.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 is a representation of the general structure of
`immunoglobulins.
`FIG. 2A and 2B show the detailed sequence of the
`cDNA insert of pK17G4 which encodes kappa anti
`CEA chain.
`FIG. 3 shows the coding sequence of the fragment
`shown in FIG. 2, along with the corresponding amino
`acid sequence.
`FIGS. 4A, 4B and 4C show the combined detailed
`sequence of the cDNA inserts of p-y298 and p711 which
`encode gamma anti CEA chain.
`FIGS. 5A and 5B show the corresponding amino acid
`sequence encoded by the fragment in FIG. 4.
`FIGS. 6 and 7 outline the construction of expression
`vectors for kappa and gamma anti-CEA chains respec-
`tively.
`FIGS. 8A, 8B, and SC show the results of sizing gels
`run on extracts of E. coli expressing the genes for
`gamma chain, kappa chain, and both kappa and gamma
`chains respectively.
`
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`
`DETAILED DESCRIPTION
`A. Definitions
`As used herein, “antibodies” refers to tetramers or
`aggregates thereof which have specific immunoreactive
`activity, comprising light and heavy chains usually
`aggregated in the “Y” configuration of FIG. 1, with or
`without covalent linkage between them; “immunoglob-
`ulins” refers to such assemblies whether or not specific
`immunoreactive activity is a property. “Non-specific
`immunoglobulin” (“NSI”) means those immunoglobu-
`lins which do not possess specificity—i.e., those which
`are not antibodies.
`“Mammalian antibodies” refers to antibodies wherein
`the amino acid sequences of the chains are homologous
`with those sequences found in antibodies produced by
`mammalian systems, either in situ, or in hybridomas.
`These antibodies antibodies mimic antibodies which are
`otherwise capable of being generated, although in im-
`pure form, in these traditional systems.
`“Hybrid antibodies” refers to antibodies wherein
`chains are separately homologous with reference mam-
`malian antibody chains and represent novel assemblies
`of them, so that two different antigens are precipitable
`by the tetramer. In hybrid antibodies, one pair of heavy
`and light chain is homologous to antibodies raised
`against one antigen, while the other pair of heavy and
`light chain is homologous to those raised against an-
`other antigen. This results in the property of "diva-
`lence” i.e., ability to bind two antigens simultaneously.
`Such hybrids may, of course, also be formed using chi-
`meric chains, as set forth below.
`“Composite” immunoglobulins means those wherein
`the heavy and light chains mimic those of different
`species origins or specificities, and the resultant is thus
`likely to be a non-specific immunoglobulin (NSI), i.e.——-
`lacking in antibody character.
`“Chimeric antibodies" refers to those antibodies
`wherein one portion of each of the amino acid sequen-
`ces of heavy and light chains is homologous to corre-
`sponding sequences in antibodies derived from a partic-
`ular species or belonging to a particular class, while the
`remaining segment of the chains is homologous to cor-
`responding sequences in another. Typically,
`in these
`chimeric antibodies, the variable region of both light
`and heavy chains mimics the variable regions of anti-
`bodies derived from one species of mammals, while the
`constant portions are homologous to the sequences in
`antibodies derived from another. One clear advantage
`to such chimeric forms is that, for example, the variable
`regions can conveniently be derived from presently
`known sources using readily available hybridomas or B
`cells from non human host organisms in combination
`with constant
`regions derived from,
`for example,
`human cell preparations. While the variable region has
`the advantage of ease of preparation, and the specificity
`is not affected by its source, the constant region being
`human, is less likely to elicit an immune response from
`
`Sanofi/Regeneron Ex. 1007, pg 169
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`Mylan Ex. 1007, pg 169
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`4,816,567
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`7
`a human subject when the antibodies are injected than
`would the constant region from a non-human source.
`However, the definition is not limited to this particu-
`lar example. It includes any antibody in which either or
`both of the heavy or light chains are composed of com-
`binations of sequences mimicking the sequences in anti-
`bodies of different sources, whether these sources be
`differing classes, differing antigen respones, or differing
`species of origin and whether or not the fusion point is
`at the variable/constant boundary. Thus, it is possible to
`produce antibodies in which neither the constant nor
`the variable region mimic known antibody sequences. It
`then becomes possible, for example, to construct anti-
`bodies whose variable region has a higher specific affin-
`ity for a particular antigen, or whose constant region
`can elicit enhanced complement fixation or to make
`other improvements in properties possessed by a partic-
`ular constant region.
`“Altered antibodies” means antibodies wherein the
`amino acid sequence has been varied from that of a
`mammalian or other vertebrate antibody. Because of
`the relevance of recombinant DNA techniques to this
`invention, one need not be confined to the sequences of
`amino acids found in natural antibodies; antibodies can
`be redesigned to obtain desired characteristics. The
`possible variations are many and range from the chang-
`ing of just one or a few amino acids to the complete
`redesign of, for example, the constant region. Changes
`in the constant region will, in general, be made in order
`to improve the cellular process characteristics, such as
`complement fixation, interaction with membranes, and
`other effector functions. Changes in the variable region
`will be made in order to improve the antigen binding
`characteristics. The antibody can also be engineered so
`as to aid the specific delivery of a toxic agent according
`to the “magic bullet” concept. Alterations, can be made
`by standard recombinant
`techniques and also by
`oligonucleotide—directed mutagenesis techniques (Dal-
`badie-McFarland, et al Proc. Natl. Actzd. Sci.(USA),
`79:64-09 (1982)).
`“Univalent antibodies” refers to aggregations which
`comprise a heavy chain/light chain dimer bound to the
`Fc (or stem) region of a second heavy chain. Such anti-
`bodies are specific for antigen, but have the additional
`desirable property of targeting tissues with specific
`antigenic surfaces, without causing its antigenic effec-
`tiveness to be impaired—-i.e., there is no antigenic mod-
`ulation. This phenomenon and the property of univalent
`antibodies in this regard is set forth in Glennie, M. J., et
`al., Nature, 295: 712 (1982). Univalent antibodies have
`heretofore been formed by proteolysis.
`“Fab” region refers to those portions of the chains
`which are roughly equivalent, or analogous,
`to the
`sequences which comprise the Y branch portions of the
`heavy chain and to the light chain in its entirety, and
`which collectively (in aggregates) have been shown to
`exhibit antibody activity. “Fab protein”, which protein
`is one of the aspects of the invention, includes aggre-
`gates of one heavy and one light chain (commonly
`known as Fab’), as well as tetramers which correspond
`to the two branch segments of the antibody Y, (com-
`monly known as F(ab)z), whether any of the above are
`covalently or non-covalently aggregated, so long as the
`aggregation is capable of selectively reacting with a
`particular antigen or antigen family. Fab antibodies
`have, as have univalent ones, been formed heretofore by
`proteolysis, and share the property of not eliciting anti-
`gen modulation on target tissues. However, as they lack
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`8
`the “effector” Fc portion they cannot effect, for exam-
`ple, lysis of the target cell by macrophages.
`“Fab protein” has similar subsets according to the
`definition of the present invention as does the general
`term “antibodies” or “immunoglobulins”. Thus, “mam-
`malian” Fab protein, “hybrid” Fab protein “chimeric”
`Fab and “altered” Fab protein are defined analogously
`to the corresponding definitions set forth in the previ-
`ous paragraphs for the various types of antibodies.
`Individual heavy or light chains may of course be
`“mammalian”, “chimeric” or “altered“ in accordance
`with the above. As will become apparent from the de-
`tailed description of the invention, it is possible, using
`the techniques disclosed to prepare other combinations
`of the four-peptide chain aggregates, besides those spe-
`cifically defined, such as hybrid antibodies containing
`chimeric light and mammalian heavy chains, hybrid
`Fab proteins containing chimeric Fab proteins of heavy
`chains associated with mammalian light chains, and so
`forth.
`“Expression vector" includes vectors which are ca-
`pable of expressing DNA sequences contained therein,
`i.e., the coding sequences are operably linked to other
`sequences capable of effecting their expression. It
`is
`implied, although not always explicitly stated,
`that
`these expression vectors must be replicable in the host
`organisms either as episomes or as an integral part of the
`chromosomal DNA. Clearly a lack of replicability
`would render them effectively inoperable. A useful, but
`not a necessary, element of an effective expression vec-
`tor is a marker encoding sequence——i.e. a sequence en-
`coding a protein which results in a phenotypic property
`(e.g. tetracycline resistance) of the cells containing the
`protein which permits those cells to be readily identi-
`fied. In sum, “expression vector” is given a functional
`definition, and any DNA sequence which is capable of
`effecting expression of a specified contained DNA code
`is included in this term, as it is applied to the specified
`sequence. As at present, such vectors are frequently in
`the form of plasmids, thus “plasmid” and “expression
`vector” are often used interchangeably. However, the
`invention is intended to include such other forms of
`expression vectors which serve equivalent functions
`and which may, from time to time become known in the
`art.
`“Recombinant host cells” refers to cells which have
`been transformed with vectors constructed using re-
`combinant DNA techniques. As defined herein,
`the
`antibody or modification thereof produced by a recom-
`binant host cell
`is by virtue of this transformation,
`rather than in such lesser amounts, or more commonly,
`in such less than detectable amounts, as would be pro-
`duced by the untransformed host.
`In descriptions of processes for isolation of antibodies
`from recombinant hosts, the terms “cell" and “cell cul-
`ture” are used interchangeably to denote the source of
`antibody unless it is clearly specified otherwise. In other
`words, recovery of antibody from the “cells" may mean
`either from spun down whole cells, or from the cell
`culture containing both the medium and the suspended
`cells.
`B. Host Cell Cultures and Vectors
`The vectors and methods disclosed herein are suit-
`able for use in host cells over a wide range of prokary-
`otic and eukaryotic organisms.
`In general, of course, prokaryotes are preferred for
`cloning of DNA sequences in constructing the vectors
`useful in the invention. For example, E. colt’ K12 strain
`
`Sanofi/Regeneron Ex. 1007, pg 170
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`Mylan Ex. 1007, pg 170
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`9
`294 (ATCC No. 31446) is particularly useful. Other
`microbial strains which may be used include E. colt‘
`strains such as E. coli B, and E. coli X1776 (ATTC No.
`31537). These examples