`
`[19]
`
`[111 Patent Number:
`
`4,816,567
`
`Cabilly et al.
`[45] Date of Patent: Mar. 28, 1989
`
`Nisonolf, A. er. a.l., Arch. Biockem. Biophyaz. vol. 93, pp.
`1160-462, 1960.
`Glennie. M. J. et al., Nature. vol. 295. pp. 712-‘.-' 14. 1982.
`Eisen, I-I. N.. Immunaicgy. Harper & Row. Publishers,
`pp. 415 and 428-436. 1974.
`I-Iozumi. N. et
`31. No.1 Acids Res. vol. 5(6) pp.
`1779-1799, Chem. Abst. B9:3792Bt, 1978.
`Wetzel. R. et ai. Gene, vol. 16. pp. 63-11, 1931.
`Williams et al., Science, vol. 215. pp. 687-639. 1982.
`Falkner. F. G. et al.. Nature. vol. 298. pp. 286-288,
`1982.
`
`B035 et 3.1. I-lamer et al., Ed. “Gene Expreasion5—Proc.
`Cetus-UCLA Symposium .
`.
`. Mar. 26-Apr. 1. 1983"
`pp. 513-522.
`Amster et al.. "Nucleic Acid Research" 13(9): 2055-2065
`(1930).
`DeBoer et a1., Rodriguez et
`462431 (1982).
`Gough. “Tibs" 6(3): 203-205 (Aug. 1981).
`Morrison, "J. of Immunology" 123(2): 793-300 (Aug.
`1979).
`
`111.. Ed. "Promoters"
`
`Kohler “P.N.A.S. USA" 77(4): 2197-2199 (Apr. 1930).
`Thomas M. Roberts. Promoters, Rodriguez et al., Eds.
`(1982), pp. 452461.
`Kemp et al.. “Proc. Natl. Acad. Sci. USA" 73(7):
`45204524 (Jul. 1981).
`Valle et al.. "Nature" 300:"t'l—74 (4 Nov. 1982).
`Microbiology 3rd ed.. Harper Int. Ed. 333-379 (1980).
`I-litzeman el: al. Science 219: 620-625 (1933).
`Mercereau—Puija1on et 31..
`in “Expression of Eukary-
`otic Viral and Cellular Genes" Pettersson et al. (E.D.)
`295-303 (1981) Academic Pr.
`Primary Examt‘ner—Jayme Huleatl
`
`[57]
`
`(List continued on next page.)
`ABSTRAC1‘
`
`including con-
`Altered and native immunoglobulins.
`stant-variable region chimeras. are prepared in recombi-
`nant cell culture. 'T'he irnmunoglobulins contain variable
`regions which are immunologically capable of binding
`predetermined antigens. Methods are provided for re-
`folding directiy expressed irnmunoglohulins into immu-
`nologically active form.
`
`7 Claims. 15 Drawing Sheets
`
`SANOFI V. GENENTECH
`SANOFI v. GENENTECH
`IPR2015-O 1624
`IPR2015-01624
`EXHIBIT 21 1 1
`EXHIBIT 2111
`
`[54] RECOMBINANI‘ [MMUNDGLOBIN
`PREPARATIONS
`
`[75]
`
`Inventors: Slunuel Cahilly, Monrovia; Herbert
`L. Heynelrer. Burlingame; William E.
`Holmes. Pacifica; Arthur D. Riggs.
`La Verne; Ronald B. Wetzel. San
`Francisco, all of Calif.
`
`[73] Assignee: Genentech. Inc.. South San
`Francisco, Calif.
`
`[21] Appl. No.: 483.457
`
`[22] Filed:
`
`Apr. 8. 1933
`
`[51]
`
`Int. Cl.‘
`
`[56]
`
`CDTK 15/14; CUTK l5/06;
`C12? 21/00; Cl2N 1'5/00; CIZN 1/20
`530/387; 435/68;
`[52] U.S. Cl.
`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
`........... .. 435/68, 172.3, 240.
`[58] Field of Search
`435/253. 172.2, 317. 320; 260/112 B; 536/27;
`935/11, 15, 27. 29. 73; 530/387. 383
`References Cited
`U.S. PATENT DOCUMENTS
`4.444.818
`4/1934 Paulus
`4.512.922 4/1985 Jones et al.
`4.518.584 5/1985 Mark et all.
`4.104.362 ll/‘I987 Ilakum et al.
`
`.
`
`
`
`FOREIGN PATENT DOCUMENTS
`
`005'Ir'10'l' 8/I982 European Pat. Off.
`00’l3656 8/1982 European Pat. Off.
`0068163
`[/1983 European Pat. Off.
`0110694 10/1984 European Pet. Off.
`
`.
`
`_
`.
`
`OTHER PUBLICATIONS
`
`435/i72.3
`
`Dolby. T. W. ct al. Prcc. Nari. Amid. Sct'.. vol. 7'.-', (10)
`pp. 6027-6031. 1980.
`Rice, D. et al. Prue. Natl. Acad. Sci. vol. 79 pp.
`7862-7865, 1982.
`
`Accolla. R. S. ct al., Pr-ac. Nari. Accra‘. Sci, vol. 7'.-'. (1)
`pp. 563-566. 1930.
`Reso, V. et al.. Cancer Res. vol. 41, PP‘
`I981.
`
`i073-2078.
`
`
`
`4,816,567
`
`Page 2
`
`OTHER PUBLICATIONS
`
`Keshet et al. Nucleic Acids Res. 9(1): 194.0 (19313.
`Taniguchi et al.. Proc. Natl. Acad. Sci. USA, 77(9):
`5230-5233 (1980).
`Dhsuye et a1.. Nucleic Acids Res. 11(5): 1233-1295
`(I 933).
`
`Kadonags. at al., J. Biol. Chem. 259(4): 2149-2154
`(I984).
`Maniatis. T. "Molecular Cloning“ p. 433 (Sep. 1935).
`Fujisawa Y. et ai.
`.‘‘Nucleic Acids Res.”
`11(11):
`3581-3591 (1983).
`Roberts, T. M. in “Promoters Structures and Function"
`Rodriguez, R. L. (ED.) 452-461 (1982).
`
`
`
`
`
`U.S. PatentU.S. Patent
`
`
`
`Mar. 23, 1939Mar. 23, 1939
`
`
`
`Sheet 1 of 4Sheet 1 of 4
`
`
`
`4,816,5674,816,567
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`
`
`
`
`
`
`
`
`U.S. Patent Mar. 23, 1939U.S. Patent Mar. 23, 1939
`
`
`
`Sheet 2 of 4Sheet 2 of 4
`
`
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`4,816,5674,816,567
`
`
`
`99
`
`
`
`Fig.88.Fig.88.
`
`
`
`
`
`U.S. Patent Mar. 23, 1939U.S. Patent Mar. 23, 1939
`
`
`
`Sheet 3 of 4Sheet 3 of 4
`
`
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`4,816,5674,816,567
`
`
`
`Fr'g.8C.Fr'g.8C.
`
`
`
`U.S. Patent Mar. 23, 1939
`
`Sheet 4 of 4
`
`4,816,567
`
`?Ong
`
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`
`RECOMBINANT IMNIUNOGLDBIN
`PREPARATIONS
`
`4,816,567
`
`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 myo-
`lomas.
`A.l Source and Utility
`Two major sources of vertebrate B.IltllJ0dlt.'.S are pres-
`ently utilized—generat.ion 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 asingle 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 oella—hence in situ generation of antibodies is
`“polycl.onal".
`'
`This limited but
`inherent heterogeneity has been
`overcome in numerous particular cases by use of hy-
`bridoma technology to create ‘‘monoclonal'‘ antibodies
`(Kohler, et al.. Eur. J’. Immunol. 6: 511 (1916)). In this
`process, splenocytes or lymphocytes from a rnarnmai
`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-.
`
`5
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`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 hcmogenous.
`and which antibodies, referencing their pure genetic
`parentage, are called "rnonoclonal". Hybridorna tech-
`nology has to this time been focused largely on the
`fusion of murine lines, but human-human hybridornas
`(Olsson. L. et al., Proc Natl. Acad. Sci. (USA). Tr‘: 5429
`(1980)); human-marine hybridornas {Schlorn. 1., et al.
`(ibid) 77: 6841 (1980)) and several other xenogenic hy-
`brid combinations have been prepared as well. Alterna-
`tively, primary, antibody producing. 13 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 imrnuno 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}. Acori
`Sci (USA) Tr‘: 2197 (1980); Morrison, 8. I... 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, 17., Bt‘ocbem:'srry, 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-
`mil 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.
`
`
`
`3
`Even those immunoglobulins which lack the specific-
`ity of atibodies are useful, although over a smaller spec-
`trum ofpotential uses than the antibodies themselves. In
`presently understood applications, such immunog1obu-
`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 tetrnrner separately.
`A.2 General Structure Characteristics
`The basic imrnunoglobin structural unit in vertebrate
`systems is now well understood (Edelman, G. M.. Arm.
`N. I’. attend. Ser'.. 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 disuifide 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. Iglvl. 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 irnrnunoglobulin.
`The amino acid sequence runs from the N-terminal
`and at the top of the Y to the C-terrninal 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 ‘T’ 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 (i.e., 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. Iglvl. IgA. Igl), and
`IgE corresponding to 7. u, a, 3. and 5 heavy chain
`constant regions). The constant region or class deter-
`mines subsequent effector Function of the antibody,
`including activation of complement (Kabat. E. A...
`
`10
`
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`4,816,567
`
`4
`Structural Concepts in Immunology and Immnrrccliernm
`try. 2nd Ed.. p. 413-436. Holt. Rinehart, Winston
`(1976)). and other cellular responses (Andrews. D. W.,
`et al., C'Ir'm'cal Imrnunobfclcgv. pp 1-13. W. B. Sanders
`(1930); Kohl. S.. et al.. Immunology, 43: 13? (1933)):
`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 :1
`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 irnnmnoglobulins (N515) 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
`
`
`
`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. 1! and 12 show the construction of a plasmid
`for expression of the gene encoding a chimeric heavy
`chain.
`
`5
`
`the amino acid sequence of naturally oocuring 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 immunoglobulin-like moieties
`may be produced: “tInivalent" 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 pKl7Ci4 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, 413 and 4C show the combined detailed
`sequence of the CDNA inserts of p-y293 and pyll 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 ‘T outline the construction of expression
`vectors for kappa and gamma anti-CEA chains respec-
`tively.
`FIGS. EA, SB. and 8C 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.
`
`FIG. 13 shows the construction of a plasmid for ex-
`pression of the gene encoding the Fab region of heavy
`chain.
`
`ll]
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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 imrnunoglobu-
`lins which do not possess speciiicity—-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 ‘nybridornas.
`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 tetrarner. 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" irnmunoglobulins means those wherein
`the heavy and light chains mimic those of different
`species origins or specificitles. 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
`
`
`
`4,316.56?
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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 ailin-
`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 antibodia 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 ruutagenesis techniques (Dal-
`badie-McFarland, et al Prat: Natl. Acad. Sc:‘.{USA).
`7916409 (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 impai.red——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. 1.. et
`al.. Nature. 295: ‘M2 (1932). 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)2). 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
`
`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 “imn1unoglobulins". Thus. “mam-
`u:w.lian" Fab protein. "hybrid" Fab protein “cl1imeric"
`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
`"rnam.malian", “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
`irnplied. 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, prolraryotes are preferred for
`cloning of DNA sequences in constructing the vectors
`useful in the invention. For egtample. E. coli K12 strain
`
`IO
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`15
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`20
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`25
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`3|]
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`35
`
`40
`
`45
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`55
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`60
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`55
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`9
`294 (ATCC No. 314-46) is particularly useful. Other
`microbial strains which may be used include E. colt’
`strains such as E. colt‘ B, and E. coir‘ X1776 (ATTC No.
`3153?). These examples are, of course, intended to be
`illustrative rather than limiting.
`Prokaryotes may also be used for expression. The
`aforementioned strains, as well