5/12/2016
`
`Patent US6331415 - Methods of producing immunoglobulins, vectors and transformed host cells for
`
`- Google Patents
`
`6331415
`
`Patents
`
`Methods of producing immunoglobulins,
`vectors and transformed host cells for use
`therein
`US 6331415 B1
`
`ABSTRACT
`
`The invention relates to processes for producing an immunoglobulin or an
`immunologically functional immunoglobulin fragment containing at least the
`van'ab|e domains of the immunoglobulin heavy and light chains. The processes
`can use one or more vectors which produce both the heavy and light chains or
`fragments thereof in a single cell. The invention also relates to the vectors used
`to produce the immunoglobulin or fragment, and to cells transfonned with the
`vectors.
`
`IMAGES (19)
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`US6331415 B1
`Grant
`US 07/205,419
`Dec 18, 2001
`Jun 10, 1988
`Apr 8, 1983
`Paid
`
`CA1218613A, 7 More »
`
`Publication number
`Publication type
`Application number
`Publication date
`Filing date
`Priority date
`Fee status
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`Also published as
`Inventors
`
`Shmuel Cabilly, 4 More »
`Genentech, Inc.
`BiBTeX, EndNote, RefMan
`
`Original Assignee
`Export Citation
`Patent Citations (42), Non—Patent Citations (298), Referenced by (670),
`Classifications (55), Legal Events (11)
`
`External Links: USPTO, USPTO Assignment, Espacenet
`
`DESCRIPTION
`
`CROSS—REFERENCE TO RELATED APPLICATIONS
`
`CLAIMS (36)
`
`What is claimed is:
`
`This application is a continuation of U.S. patent application Ser. No. 06/483,457,
`filed Apr. 8, 1983, now U.S. Pat. No. 4,816,567, issued Mar. 28, 1989.
`
`BACKGROUND OF THE INVENTION
`
`1. A process for producing an immunoglobulin molecule or an immunologically
`functional immunoglobulin fragment comprising at least the variable domains of
`the immunoglobulin heavy and light chains, in a single host cell, comprising the
`steps of:
`
`This invention relates to the field of immunoglobulin production and to
`modification of naturally occurring 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 fonns.
`
`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 invasion 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 substance. The binding specificity of such polypeptides to a particular
`antigen is highly refined, and the multitude of specificities capable of being
`generated by the individual vertebrate is remarkable in its complexity and
`van'abiIity. Thousands of antigens are capable of eliciting responses, each almost
`exclusively directed to the particular antigen which elicited it.
`
`(i) transforming said single host cell with a first DNA sequence encoding at
`least the variable domain of the immunoglobulin heavy chain and a second
`DNA sequence encoding at least the variable domain of the immunoglobulin
`light chain, and
`
`(ii) independently expressing said first DNA sequence and said second DNA
`sequence so that said immunoglobulin heavy and light chains are produced
`as separate molecules in said transformed single host cell.
`
`2. The process according to claim 1 wherein said first and second DNA
`sequences are present in different vectors.
`
`3. The process according to claim 1 wherein said first and second DNA
`sequences are present in a single vector.
`
`4. A process according to claim 3 wherein the vector is a plasmid.
`
`5. The process according to claim 4 wherein the plasmid is pBR322.
`
`6. The process according to claim 1 wherein the host cell is a bacterium
`or yeast.
`
`Immunoglobulins include both antibodies, as above described, and analogous
`
`7. The process according to claim 6 wherein the host cell is E. coli or S.
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`https://www.google.com/patents/U $6331415?dq=6331415&hl=en&sa=X&ved=OahU KEwjX3buRvN TMAhVB6CYKH b9M D FEQGAEIH DAA
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`5/12/2016
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`Patent US6331415 - Methods of producing immunoglobulins, vectors and transformed host cells for
`cerevisiae.
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`- Google Patents
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`protein substances which lack antigen specificity. The latter are produced at low
`levels by the lymph system and in increased levels by myelomas.
`
`A.1 Source and Utility
`
`Two major sources of vertebrate antibodies are presently uti|ized—generation in
`situ by the mammalian B lymphocytes and in cell culture by B-cell hybrids.
`Antibodies 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
`review 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
`lymphocyte 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 defined by the mosaic of responses to the
`various determinants which are present on the antigen. Each subset of
`homologous antibody is contributed by a single population of B-ce||s—hence in
`situ generation of antibodies is “po|yc|ona|”.
`
`This limited but inherent heterogeneity has been overcome in numerous particular
`cases by use of hybridoma technology to create “monoclonal” 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 “hybridomas” which are both
`immortal and capable of producing the genetically coded antibody of the B cell.
`The hybrids thus formed we 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 technology has to this time been
`focused largely on the fusion of murine lines, but human-human hybridomas
`(O|sson, L. et al., Proc. Natl. Acad. Sci. (USA), 77: 5429 (1980)); human-murine
`hybridomas (Sch|om, J. , et al. (ibid) 77: 6841 (1980)) and several other
`xenogenic hybrid combinations have been prepared as well. Alternatively,
`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
`antigen-antibody reaction with suitable detection techniques such as labeling with
`radioisotopes or with enzymes capable of assay (RIA, EMIT, and ELISA).
`Antibodies are thus the foundation of immuno diagnostic tests for many antigenic
`substances. In another important use, antibodies can be directly injected into
`subjects suffering from an attack by a substance or organism 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 suitable 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. These cells contain additional materials, notably nucleic acid
`fragments, but protein fragments as well, which are capable of enhancing,
`causing, or mediating carcinogic responses. Second, hybridoma lines producing
`
`8. A process according to claim 7 wherein the host cell is E. coli strain
`X1776 (ATCC No. 31537).
`
`9. A process according to claim 1 wherein the immunoglobulin heavy
`and light chains are expressed in the host cell and secreted therefrom
`as an immunologically functional immunoglobulin molecule or
`immunoglobulin fragment.
`
`10. A process according to claim 1 wherein the immunoglobulin heavy
`and light chains are produced in insoluble form and are solubilized and
`allowed to refold in solution to form an immunologically functional
`immunoglobulin molecule or immunoglobulin fragment.
`
`11. A process according to claim 1 wherein the DNA sequences code
`for the complete immunoglobulin heavy and light chains.
`
`12. The process according to claim 1 wherein said first or said second
`DNA sequence further encodes at least one constant domain, wherein
`the constant domain is derived from the same source as the variable
`domain to which it is attached.
`
`13. The process according to claim 1 wherein said first or said second
`DNA sequence further encodes at least one constant domain, wherein
`the constant domain is derived from a species or class different from
`that from which the variable domain to which it is attached is derived.
`
`14. The process according to claim 1 wherein said first and second
`DNA sequences are derived from one or more monoclonal antibody
`producing hybridomas.
`
`15. A vector comprising a first DNA sequence encoding at least a variable
`domain of an immunoglobulin heavy chain and a second DNA sequence
`encoding at least a variable domain of an immunoglobulin light chain wherein said
`first DNA sequence and said second DNA sequence are located in said vector at
`different insertion sites.
`
`16. A vector according to claim 15 which is a plasmid.
`
`17. A host cell transfonned with a vector according to claim 15.
`
`18. A transformed host cell comprising at least two vectors, at least one of said
`vectors comprising a DNA sequence encoding at least a variable domain of an
`immunoglobulin heavy chain and at least another one of said vectors comprising
`a DNA sequence encoding at least the variable domain of an immunoglobulin
`light chain.
`
`19. The process of claim 1 wherein the host cell is a mammalian cell.
`
`20. The transformed host cell of claim 18 wherein the host cell is a
`mammalian cell.
`
`21. A method comprising
`
`a) preparing a DNA sequence consisting essentially of DNA encoding an
`immunoglobulin consisting of an immunoglobulin heavy chain and light chain
`or Fab region, said immunoglobulin having specificity for a particular known
`antigen;
`
`b) inserting the DNA sequence of step a) into a replicable expression vector
`operably linked to a suitable promoter;
`
`c) transforming a prokaryotic or eukaryotic microbial host cell culture with
`the vector of step b);
`
`d) culturing the host cell; and
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`monoclonal antibodies tend to be unstable and may alter the structure of antibody
`produced or stop producing antibody altogether (Kohler, G., et al. Proc. Natl.
`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 currently known, and this alteration may result in production of incorrect
`sequences. Third, both hybridoma and B cells inevitably produce certain
`antibodies in glycosylated 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 (either by hybridoma or
`by B cell response) does not pennit manipulation of the genome so as to produce
`antibodies 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
`invention do not suffer from the foregoing drawbacks, and, furthermore, offer the
`opportunity to provide molecules of superior design.
`
`Even those immunoglobulins which lack the specificity of antibodies are useful,
`although over a smaller spectrum of potential uses than the antibodies
`themselves. In presently understood applications, such immunoglobulins are
`helpful in proteins replacement therapy for globulin 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 induced. The present invention offers an alternative, more economical
`source. It also offers the opportunity of canceling 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.Y Acad. Sci., 190: 5 (1971)). The units are
`composed of 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”
`configuration 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 constant
`region, determines the “c|ass"of the antibody as |gG,
`|gM,
`|gA,
`|gD, or |gE. 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 either 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 supplement 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 variable
`
`e) recovering the immunoglobulin from the host cell culture, said
`immunoglobulin being capable of binding to a known antigen.
`
`22. The method of claim 21 wherein the heavy and light chain are the
`heavy and light chains of anti-CEA antibody.
`
`23. The method of claim 21 wherein the heavy chain is of the gamma
`family.
`
`24. The method of claim 21 wherein the light chain is of the kappa
`family.
`
`25. The method of claim 21 wherein the vector contains DNA encoding
`both a heavy chain and a light chain.
`
`26. The method of claim 21 wherein the host cell is E. coli or yeast.
`
`27. The method of claim 26 wherein the heavy chain and light chains or
`Fab region are deposited within the cells as insoluble particles.
`
`28. The method of claim 27 wherein the heavy and light chains are
`recovered from the particles by cell lysis followed by solubilization in
`denaturant.
`
`29. The method of claim 21 wherein the heavy and light chains are
`secreted into the medium.
`
`30. The method of claim 21 wherein the host cell is a gram negative
`bacterium and the heavy and light chains are secreted into the
`periplasmic space of the host cell bacterium.
`
`31. The method of claim 21 further comprising recovering both heavy
`and light chain and reconstituting light chain and heavy chain to form an
`immunoglobulin having specific affinity for a particular known antigen.
`
`32. The insoluble particles of heavy chain and light chains or Fab region
`produced by the method of claim 27.
`
`33. A process for producing an immunoglobulin molecule or an immunologically
`functional immunoglobulin fragment comprising at least the variable domains of
`the immunoglobulin heavy and light chains, in a single host cell, comprising:
`
`independently expressing a first DNA sequence encoding at least the
`variable domain of the immunoglobulin heavy chain and a second DNA
`sequence encoding at least the variable domain of the immunoglobulin light
`chain so that said immunoglobulin heavy and light chains are produced as
`separate molecules in said single host cell transformed with said first and
`second DNA sequences.
`
`34. The process of claim 9, further comprising the step of attaching the
`immunoglobulin molecule or immunoglobulin fragment to a label or drug.
`
`35. The process of claim 10, further comprising the step of attaching the
`immunoglobulin molecule or immunoglobulin fragment to a label or drug.
`
`region which is specific for the antigen which elicited 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 region 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 occurring 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 approximately 25 amino acids.
`
`36. The process of claim 33, further comprising the step of attaching the
`immunoglobulin molecule or immunoglobulin fragment to a label or drug.
`
`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 antigen eliciting it),
`
`As stated above, there are five known major classes of constant regions which determine the class of the immunoglobulin
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`|gD, and |gE corresponding to v, u, a, 6, and a heavy chain constant regions). The constant region
`|gA,
`|gM,
`molecule (|gG,
`or class determines subsequent effector function of the antibody, including activation of complement (Kabat, E. A.,
`Structural Concepts in Immunology and Immunochemistry, 2nd Ed., p. 413-436, Holt, Rinehart Winston (1976)), and other
`cellular responses (Andrews, D. W., et al., Clinical Immunobiology pp 1-18, W. B. Sanders (1980); Kohl, 3., et al.,
`Immunology, 48: 187 (1983)); while the variable region determines the antigen with which it will react.
`
`B. Recombinant DNA Technology
`
`Recombinant DNA technology has reached 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 heterologous 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, heterologous gene insert and remainder vector) generally is performed outside the host cell.
`The resulting recombinant replicable expression vector, or plasmid, is introduced into cells by transformation and large
`quantities of the recombinant vehicle is obtained by growing the transformants. Where the gene is properly inserted with
`reference to portions which govern the transcription and translation of the encoded DNA message, the resulting expression
`vector is useful to produce the polypeptide sequence for which the inserted gene codes, a process referred to as
`“expression." The resulting product may be obtained by lysis, if necessary, of the host cell and recovery of the product by
`appropriate purifications from other proteins.
`
`In practice, the use of recombinant DNA technology can express entirely heterologous po|ypeptides—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 sometimes rendered bioinactive within the
`fused, homologous/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 physiology is well
`established. Means and methods are available 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 support in liquid medium, or by growth in
`suspension containing support nutriments. Scale-up for large preparations seems to pose only mechanical problems.
`
`SUMMARY OF THE INVENTION
`
`The invention relates to antibodies and to non-specific immunoglobulins (NS|s) formed by recombinant techniques using
`suitable host cell cultures. These antibodies and NS|s can be readily prepared in pure “monoc|ona|" 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 the amino acid
`sequence of naturally occurring antibodies 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 produce, and the invention is directed to,
`immunoglobulins which comprise polypeptides not hitherto found associated with each other in nature. Such reassembly is
`particularly useful in producing “hybrid" antibodies capable of binding more than one antigen; and in producing “composite”
`immunoglobulins wherein heavy and light chains of different origins essentially damp out specificity. Third, by genetic
`manipulation, “chimeric” antibodies can be formed wherein, for example, the variable regions correspond to the amino acid
`sequence from one mammalian model system, 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” antibodies with improved specificity and other characteristics can be formed.
`
`Two other types of immunoglobulin-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
`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 included in the invention and may be mammalian or chimeric.
`
`In other aspects, the invention is directed to DNA which encodes the aforementioned NS|s, 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 transformation with these vectors. Finally, the invention is
`directed to methods of producing these NS|s and antibodies, and the DNA sequences, plasmids, and transformed cells
`intermediate to them.
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`BRIEF DESCRIPTION OF THE DRAWINGS
`
`- Google Patents
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`FIG. 1 is a representation of the general structure of immunoglobulins.
`
`FIGS. 2A-B shows 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-C shows the combined detailed sequence of the cDNA inserts of pv298 and pv11 which encode gamma anti CEA
`chain.
`
`FIGS. 5A-B shows 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 respectively.
`
`FIGS. 8A, 8B, 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. 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 constmction of a plasmid for expression of the gene encoding the Fab region of heavy chain.
`
`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; “immunog|obu|ins" refers to such assemblies whether or not specific immunoreactive activity is a property.
`“Non-specific immunog|obuIin” (“NSI”) means those immunoglobulins 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 mimic
`antibodies which are othenrvise capable of being generated, although in impure form, in these traditional systems.
`
`“Hybrid antibodies” refers to antibodies wherein chains are separately homologous with referenced mammalian 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 another antigen. This results in the property of “diva|ence" i.e.,
`ability to bind two antigens simultaneously. Such hybrids may, of course, also be formed using chimeric 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.—Iacking in antibody character.
`
`“Chimeric antibodies” refers to those antibodies wherein one portion of each of the amino acid sequences of heavy and light
`chains is homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular
`class, while the remaining segment of the chains is homologous to corresponding sequences in another. Typically, in these
`chimeric antibodies, the variable region of both light and heavy chains mimics the variable regions of antibodies 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 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 particular example. It includes any antibody in which either or both of the heavy
`or light chains are composed of combinations of sequences mimicking the sequences in antibodies of different sources,
`whether these sources be differing classes, differing antigen responses, 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 antibodies whose
`variable region has a higher specific affinity for a particular antigen, or whose constant region can elicit enhanced
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`complement fixation or to make other improvements in properties possessed by a particular 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 changing 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 (Dalbadie-McFarland, et al
`Proc. Natl. Acad. Sci. (USA), 7926.409 (1982)).
`
`“U nivalent 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 antibodies are specific for antigen but have the additional desirable property of
`targeting tissues with specific antigenic surfaces, without causing its antigenic effectiveness to be impaired—i.e., there is
`no antigenic modulation. 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, in