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`Fig. 30 depicts the percentage of monomer recovered after thermal acceleration tests at 60°C-1W on BiAb
`[0043]
`purified from humanized bispecific antibodies (IgG4-type) whose CH3 interface has been modified.
`[0044]
`Fig. 31 depicts the results of an assay evaluating coagulation activity of humanized bispecific antibodies (IgG4-
`type) whose CH3 interface has been modified. The results demonstrate that the coagulation activities are equivalent to
`that of the unmodified bispecific antibody.
`[0045]
`Fig. 32 depicts the formation ratio obtained through IEX analysis for A-Homo, BiAb, and B-Homo, which are
`humanized bispecific antibodies (lgG1-type) whose CH3 interface has been modified.
`
`Best Mode for Carrying Out the Invention
`
`[0046] The present invention relates to methods for regulating the association of polypeptides or association of heter-
`omultimers composed of polypeptides.
`[0047]
`First, the present invention provides methods for regulating polypeptide association, such methods including
`the step of modifying amino acid residues in an original peptide forming an interface so as to inhibit the association
`within the polypeptide.
`[0048]
`In the present invention, the term "polypeptides" ordinarily refers to peptides and proteins whose length is about
`ten amino acids or longer. Polypeptides are ordinarily derived from organisms but are not particularly limited thereto,
`and for example, they may be composed of an artificially designed sequence. They may also be any of naturally derived
`polypeptides, synthetic polypeptides, recombinant polypeptides, orsuch. Additionally, fragments ofthe above—mentioned
`polypeptides are also included in the polypeptides of the present invention.
`[0049]
`In the present invention, the phrase "polypeptide association" refers to, for example, a condition in which two
`or more polypeptide regions interact.
`[0050]
`In the present invention, the phrase "regulating association" refers to regulating to achieve a desired association
`condition, and more specifically refers to regulating so that undesirable associations are not formed in the polypeptides.
`[0051]
`In the present invention, the term "interface" ordinarily refers to the association surface that results from asso-
`ciation (interaction), and amino acid residues that form the interface are ordinarily one or more amino acid residues
`included in the polypeptide regions which participate in the association, and are more preferably amino acid residues
`that approach each other during association and are involved in the interaction. More specifically, this interaction includes,
`for example, instances where the amino acid residues come close during the association to form hydrogen bonds,
`electrostatic interactions, or salt bridges with each other.
`[0052]
`In the present invention, the phrase, "amino acid residues forming an interface" more specifically refers to
`amino acid residues included in the polypeptide region that constitutes the interface. For example, polypeptide regions
`constituting the interface referto polypeptide regions responsible for selective binding within or between molecules such
`as in antibodies, ligands, receptors, or substrates. More specifically, in antibodies, such examples include heavy chain
`variable regions and light chain variable regions.
`[0053]
`"Modification" of amino acid residues in the methods of the present invention specifically refers to substituting
`original amino acid residue(s) for other amino acid residue(s), deleting original amino acid residue(s), adding new amino
`acid residue(s), and such, but preferably refers to substituting one or more original amino acid residues for other amino
`acid residues.
`
`In the present invention, the term "polypeptides" preferably refers to polypeptides that form two or more types
`[0054]
`of conformational isomers. Conformational isomers are proteins whose amino acid sequences are identical but their
`three—dimensional (tertiary) structures are different. Ordinarily, among conformational isomers, at least either one of
`chemical or physical properties is also different.
`[0055]
`A preferred embodiment of the present invention relates to methods for preferentially (efficiently) obtaining
`desirable conformational isomers from among two or more types of potential conformational isomers. More specifically,
`an embodiment relates to methods for modifying the one or more amino acid residues that form an interface between
`the polypeptides so as to inhibit an association between polypeptides forming one or more types of conformational
`isomers from among those polypeptides that may form two or more types of conformational isomers.
`[0056]
`For example, when the first to fourth peptide regions exist in a polypeptide, and any two of these regions can
`associate, the following cases are conceivable where mainly three types of conformational isomers can exist: (1) the
`first and second polypeptide regions associate and the third and fourth polypeptide regions associate, (2) the first and
`third polypeptide regions associate, and the second and fourth polypeptide regions associate, and (3) the first and fourth
`polypeptide regions associate, and the second and third polypeptide regions associate.
`[0057] Under the above—mentioned circumstance, when one wishes to preferentially obtain a polypeptide (conforma—
`tional isomer) associated with the interaction of (1), for example, amino acid residues forming the interfaces present in
`the first, third, orfourth polypeptide regions are modified so that association of the first polypeptide region with the third
`and fourth polypeptide regions is inhibited.
`[0058] The methods of the present invention also relates to methods for regulating heteromultimer association, such
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`methods including the step of modifying amino acid residues that form the interface between the original polypeptides,
`such that the association between the polypeptides is inhibited.
`[0059]
`In the present invention, the term "heteromultimer" refers to a protein multimer composed of more than one
`type of polypeptide, in which the polypeptides can associate with each other. More specifically, a "heteromultimer"
`includes at least a first polypeptide and a second polypeptide; in this context, the second polypeptide is a molecule which
`differs from the first polypeptide by at least one amino acid residue. Furthermore, without particular limitation, the heter-
`omultimers preferably have binding specificity toward at least two different types of ligands, antigens, receptors, sub-
`strates, or such.
`In addition to a "heterodimer" formed by a first and second polypeptide, another different type of
`polypeptide may exist in the heteromultimer. More specifically, "heteromultimers" ofthe present invention are not limited
`to heterodimers and include for example heterotrimers and heterotetramers.
`[0060]
`Preferred embodiments of the above-mentioned methods are methods of modifying amino acid residues that
`form the interface between polypeptides in heteromultimers that may form two or more types of multimers, such that
`association between polypeptides forming one or more types of multimers is inhibited.
`[0061]
`For example, when any two of the polypeptides can associate in the protein multimers composed of the first
`to fourth polypeptides, the following multimers can mainly exist: (1) multimers in which the first and second polypeptides
`are associated and the third and fourth polypeptides are associated, (2) multimers in which the first and third polypeptides
`are associated and the second and fourth polypeptides are associated, or (3) multimers in which the first and fourth
`polypeptides are associated and the second and third polypeptides are associated.
`[0062] Underthe above—mentioned circumstance, when one wishes to preferentially obtain multimers associated with
`the interaction of (1), for example, amino acid residues included in the first, third, or fourth polypeptide can be modified
`so that association of the first polypeptide with the third and fourth polypeptides is inhibited.
`[0063]
`Preferred embodiments of the methods of the present invention for regulating polypeptide association include,
`for example, methods in which modification of amino acid residues forming the interface of polypeptides include intro—
`ducing amino acid residue mutations to the interface so that two or more amino acid residues forming an interface will
`have the same type of charge.
`[0064]
`In the methods mentioned above, by modifying two or more amino acid residues involved in an association at
`the interface such that they carry the same kind of charge, repulsive forces among those charges will inhibit association
`among these amino acid residues.
`[0065] Therefore, in the method mentioned above, the amino acid residues that are to be modified are preferably two
`or more amino acid residues that come close to each other during association in the region between the polypeptide
`regions that form the interface.
`[0066] Amino acid residues that come close to each otherduring association can be identified, forexample, by analyzing
`the three dimensional structures of the polypeptides, and investigating the amino acid sequences of the polypeptide
`regions forming the interface when these polypeptides associate. Amino acid residues that come close to each other at
`the interface will be preferred targets for "modifications" in the methods ofthe present invention.
`[0067]
`Some amino acids are known to be charged amino acids. Generally, lysine (K), arginine (R), and histidine (H)
`are known as positively charged amino acids (cationic amino acids) whereas aspartate (D), glutamate (E), and such are
`known as negatively charged amino acids (anionic amino acids). Therefore, in the context of the present invention,
`amino acids carrying the same type of charge preferably refer to amino acids that are either positively charged or
`negatively charged.
`[0068]
`In the methods of the present invention, all of the mutated amino acid residues are preferably modified to have
`the same type of charges, but the methods are not necessarily limited to such cases. For example, when a number of
`amino acid residues are introduced by the modification, there may be a few uncharged amino acid residues among
`these amino acid residues.
`
`[0069] The number of amino acid residues that undergo modification in the methods of the present invention is not
`particularly limited. However, when modifying the variable region(s) of an antibody, it is preferable that only a few amino
`acid residues are modified so as not to decrease the antigen binding activity or increase the antigenicity of the resulting
`antibody. The methods of the present invention can regulate association by modifying one or both of the two amino acid
`residues that come close to each other at the interface, as indicated in the Examples described below. The term "few"
`as used in the above-mentioned context refers to about one to ten for example, preferably about one to five, more
`preferably about one to three, and even more preferably about one to two.
`[0070]
`In a preferred embodiment, the amino acid residues that are introduced by modification (i.e., subjected to
`modification) are preferably all selected from among the above-mentioned positively charged amino acids, or, alterna-
`tively, are all selected from among the above—mentioned negatively charged amino acids.
`[0071]
`Furthermore, in the present invention, preferred amino acid residues to be introduced include glutamic acid
`(E), asparagine (D), lysine (K), arginine (R), or histidine (H).
`[0072]
`In another preferred embodiment of the present invention, when an interface-forming amino acid residue (X)
`in an original polypeptide (before modification) is already charged, it is preferable thatthe amino acid residue that comes
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`close to and faces this amino acid residue (X) during association is modified to be the same amino acid residue (or an
`amino acid residue with the same type of charge) as the amino acid residue (X). In this embodiment, it is only necessary
`to modify one of the amino acid residues that form the interface.
`[0073]
`Preferred embodiments of the methods of the present invention for regulating association include methods in
`which modification of amino acid residues forming the interface of the polypeptides that feature the introduction of amino
`acid residue mutations to the interface such that the amino acid residues forming a hydrophobic core present at the
`interface are transformed into charged amino acid residues.
`[0074]
`In general, the term "hydrophobic core" refers to a part of a polypeptide that is formed by an assembly of
`hydrophobic amino acid side chains at the interior of the associated polypeptides. Examples of hydrophobic amino acids
`include alanine, isoleucine, Ieucine, methionine, phenylalanine, proline, tryptophan, and valine. Furthermore, amino acid
`residues otherthan hydrophobic amino acids (for example tyrosine) may be involved in the formation of a hydrophobic
`core. This hydrophobic core together with a hydrophilic surface, in which hydrophilic amino acid side chains are exposed
`to the exterior, becomes a driving force for promoting association of water-soluble polypeptides. When hydrophobic
`amino acids of two different domains are present on a molecular surface and are exposed to water molecules, the
`entropy will increase and the free energy will increase. Accordingly, the two domains will associate with each other to
`decrease the free energy and become stable, and hydrophobic amino acids at the interface will be buried into the interior
`of the molecule to form a hydrophobic core.
`[0075] When polypeptide associations take place, modification of hydrophobic amino acids forming the hydrophobic
`core to charged polar amino acids inhibits the formation ofthe hydrophobic core, and as a result, inhibits the polypeptide
`association.
`
`[0076] Those skilled in the art can identify the organized sites (regions) and such, as well as the presence of the
`hydrophobic core, by analyzing the amino acid sequence of the desired polypeptides. Thus, the present invention relates
`to methods for regulating association that feature the step of modifying amino acid residues involved with the formation
`of the hydrophobic core at the interface into charged amino acid residues.
`[0077] Examples of charged amino acid residues suitable for use in the methods described above preferably include
`glutamic acid (E), aspartic acid (D), lysine (K), arginine (R), and histidine (H).
`[0078] The methods of the present invention for regulating association can be used as methods for preferentially
`obtaining (producing) antibodies (polypeptides) of interest and in the production of antibodies, antibody fragments,
`polypeptides having antibody-like activity, and the like.
`[0079] Herein, the term "antibody" is used in the broadest sense, and includes monoclonal antibodies, polyclonal
`antibodies, and mutant antibodies (chimeric antibodies, humanized antibodies, minibodies (including antibody frag—
`ments), and multispecific antibodies), so long as they exhibit a desired biological activity. Furthermore, in the context of
`the present invention, the "antibody" can be a polypeptide or heteromultimer. Preferred antibodies include monoclonal
`antibodies, chimeric antibodies, humanized antibodies, and minibodies, such as antibody fragments.
`[0080]
`In the context of the present invention, the term "multispecific antibody" (used in the present description to have
`the same meaning as "polyspecific antibody") refers to an antibody that may bind specifically to differenttypes of epitopes.
`More specifically, multispecific antibodies are antibodies having specificity to at least two different types of epitopes,
`and,
`in addition to antibodies recognizing different antigens, antibodies recognizing different epitopes on the same
`antigen are also included. For example, when the antigens are heterologous receptors, multispecific antibodies can
`recognize different domains constituting the heterologous receptors; alternatively, when the antigens are monomers,
`multispecific antibodies recognize multiple sites on the monomerantigens. Ordinarily, such molecules bindto two antigens
`(bispecific antibodies; used in the present description to have the same meaning as "dual—specific antibodies"), but they
`may even have specificity toward more antigens (for example three types).
`[0081]
`In addition to the antibodies described above, the antibodies of the present invention include antibodies whose
`amino acid sequences have been modified by amino acid substitutions, deletions, additions, and/or insertions, or chi-
`merization, humanization, and such. Such amino acid sequence modifications, such as amino acid substitutions, dele-
`tions, additions, and/or insertions, and humanization and chimerization, can be achieved by methods known to those
`skilled in the art. When the antibodies of the present invention are prepared as recombinant antibodies, likewise, the
`amino acid sequences of the antibody variable and constant regions may also be modified by amino acid substitutions,
`deletions, additions, and/or insertions, or chimerization, humanization and the like.
`[0082] The antibodies of the present invention may be derived from any animal, such as a mouse, human, rat, rabbit,
`goat, orcamel. Furthermore, the antibodies may be modified, for example, chimeric antibodies, and in particular, modified
`antibodies that include amino acid substitutions in their sequence, such as humanized antibodies. The antibodies may
`be any type of antibody, such as antibody modification products linked with various molecules, antibodyfragments, and
`minibodies.
`
`"Chimeric antibodies" are antibodies prepared by combining sequences derived from different animals. An
`[0083]
`example is an antibody having heavy and light chain variable (V) regions from a mouse antibody and heavy and light
`chain constant (C) regions from a human antibody. Chimeric antibodies can be prepared by known methods. To obtain
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`such chimeric antibodies, for example, a DNA encoding an antibody V region may be ligated with a DNA encoding a
`human antibody C region; the resulting ligation product can be inserted into an expression vector; and the construct can
`be introduced into a host to produce the chimeric antibody.
`[0084]
`"Humanized antibodies" are also referred to as reshaped human antibodies, and can be obtained by substituting
`the complementarity determining region (CDR) of a human antibodyforthe CDR of an antibody derived from a nonhuman
`mammal, for example, a mouse. Methods for identifying CDRs are known in the art (Kabat et al., Sequence of Proteins
`of Immunological Interest (1987), National Institute of Health, Bethesda, Md.; Chothia et al., Nature (1989) 342:877).
`General genetic recombination techniques suitable for this purpose are also known (see European Patent Application
`EP 125023; and WO 96/02576). For example, the CDR of a mouse antibody can be determined by known methods,
`and a DNA can be prepared such that it encodes an antibody in which the CDR is ligated with the framework region
`(FR) of a human antibody. A humanized antibody can then be produced using a system that uses conventional expression
`vectors. Such DNAs can be synthesized by PCR, using as primers several oligonucleotides designed to include portions
`that overlap the ends of both the CDR and FR regions (see the method described in WO 98/13388). Human antibody
`FRs linked via CDRs are selected such that the CDRs form a suitable antigen binding site. If required, amino acids in
`the PBS of an antibody variable region may be substituted so that the CDRs ofthe reshaped human antibody can form
`a suitable antigen binding site (Sato, K. et al., Cancer Res. (1993) 53:851-856). Modifiable amino acid residues in the
`FRs include portions that directly bind to an antigen via non-covalent bonds (Amit et al., Science (1986) 233: 747-53),
`portions that have some impact or effect on the CDR structure (Chothia et al., J. Mol. Biol. (1987) 196: 901-17), and
`portions involved in the interaction between VH and VL (EP 239400).
`[0085] When the antibodies of the present invention are chimeric antibodies or humanized antibodies, the C regions
`of these antibodies are preferably derived from human antibodies. For example, 071, 072, Cy3, and 074 can be used
`forthe H chain, while CK and CA can be used forthe L chain. Meanwhile, the human antibody C region may be modified
`as required to improve antibody or production stability. A chimeric antibody of the present invention preferably includes
`a variable region of an antibody derived from a nonhuman mammal and a constant region of a human antibody. A
`humanized antibody preferably includes CDFis of an antibody derived from a nonhuman mammal and FRs and C regions
`of a human antibody. The variable regions are described in detail in (3)—3. The constant regions of the human antibodies
`include specific amino acid sequences, which vary depending on the isotype of the antibody, for example, lgG (lgG1,
`lgG2, lgG3, and lgG4), lgM,
`lgA, lgD, and lgE. The constant regions used to prepare the humanized antibodies of the
`present invention may be the constant regions of antibodies of any isotype. A constant region of human lgG is preferably
`used, though the invention is not limited thereto. The FRs derived from a human antibody, which are used to prepare
`the humanized antibodies, are not particularly limited, and thus may be derived from an antibody of any isotype.
`[0086] The variable and constant regions of chimeric or humanized antibodies ofthe present invention may be modified
`by deletion, substitution, insertion, and/or addition, so long as the antibodies exhibit the same binding specificity as that
`of the original antibodies.
`[0087]
`Since their antigenicity in the human body has been attenuated, chimeric and humanized antibodies using
`human-derived sequences are expected to find utility when administered to humans for therapeutic purposes or such.
`[0088]
`In addition, minibodies are useful as the antibodies because of their in vivo kinetic characteristics and low-cost
`production using E. coli, plant cells, or such.
`[0089] Antibodyfragments are one type of minibody. Theterm "minibodies" includes antibodies thatinclude an antibody
`fragment as a partial structural unit. The minibodies of the present invention are not particularly limited by their structure
`northeir method of production, so long as they have antigen binding activity. Some minibodies have an activity greater
`than that of awhole antibody (Orita et al., Blood (2005) 105:562—566). Herein,the "antibodyfragments" are not particularly
`limited, so long as they are a portion of a whole antibody (for example, whole lgG). However, the antibody fragments
`preferably include a heavy chain variable region (VH) or a light chain variable region (VL). Examples of preferred antibody
`fragments are: Fab, F(ab’)2, Fab’, and Fv. The amino acid sequence of a VH or VL in an antibody fragment may be
`modified by substitution, deletion, addition, and/or insertion. Furthermore, some portions of aVH and VL may be deleted,
`so long as the resulting fragments retain their antigen binding ability. For example, of the antibody fragments described
`above, "Fv" is a minimal antibody fragment composed of the complete antigen recognition and binding sites. "Fv" is a
`dimer (VH—VL dimer) composed of one unit of VH and one unit of VL bound very strongly by non—covalent bonding. An
`antigen binding site is formed on the surface of the VH-VL dimer by the three complementarity determining regions
`(CDRs) of each variable region. Six CDRs confer an antigen binding site to the antibody. However, even one variable
`region (or half of an Fv composed of onlythree antigen-specific CD Rs) has the ability to recognize and bind to an antigen,
`although its affinity is lowerthan that of the complete binding site. Thus, molecules smaller than Fv are also included in
`the context of antibody fragments of the present invention. The variable regions of an antibody fragment may also be
`chimerized or humanized.
`
`[0090] The minibodies preferably include both VH and VL. Examples of suitable minibodies include antibodyfragments
`such as Fab, Fab’, F(ab’)2, and Fv, and scFv (single-chain Fv), which can be prepared using antibodyfragments, (Huston
`et al., Proc. Natl. Acad. Sci. USA (1988) 85: 5879-83; Plickthun "The Pharmacology of Monoclonal Antibodies" Vol. 113,
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`Resenburg and Moore (eds), Springer Verlag, New York, pp. 269-315, (1994)); diabodies (Holliger et al., Proc. Natl.
`Acad. Sci. USA (1993) 90:6444-8; EP 404097; W093/11 161; Johnson et al., Method in Enzymology (1991) 203: 88-98;
`Holliger et al., Protein Engineering (1996) 9:299-305; Perisic et al., Structure (1994) 2:1217-26; John et al., Protein
`Engineering (1999) 12(7):597-604; Atwell et al., Mol.lmmunol. (1996) 33:1301-12); sc(Fv)2 (Hudson et al, J Immunol.
`Methods (1999) 231:177—89; Orita et al., Blood (2005) 105:562—566); triabodies (Journal of Immunological Methods
`(1999) 231: 177-89); and tandem diabodies (Cancer Research (2000) 60:4336-41).
`[0091] An antibody fragment can be prepared by treating an antibody with an enzyme, for example, a protease such
`as papain or pepsin (see Morimoto et al., J. Biochem. Biophys. Methods (1992) 24: 107-17; Brennan et al., Science
`(1985) 229:81). Alternatively, antibody fragments can also be produced by genetic recombination based on its amino
`acid sequence.
`[0092]
`A minibody having a structure that results from modification of an antibody fragment can be prepared using
`antibody fragments obtained by enzyme treatment or genetic recombination. Alternatively, after constructing a gene
`which encodes awhole minibody, and introducing the constructinto an expression vector, the minibody maybe expressed
`in appropriate host cells (see, for example, Co et al., J. Immunol. (1994) 152: 2968-76; Better and Horwitz, Methods
`Enzymol. (1989) 178:476-96; Pluckthun and Skerra, Methods Enzymol. (1989) 178:497-515; Lamoyi, Methods Enzymol.
`(1986) 121: 652-63; Rousseaux et al., Methods Enzymol. (1986) 121:663-9; Bird and Walker, Trends Biotechnol. (1991)
`9: 132-7).
`[0093] The above described scFVs are single-chain polypeptides that include two variable regions linked together via
`a linker or such, as required. The two variable regions in an scFv are typically one VH and one VL, but an scFv may
`include two VH or two VL.
`In general, scFv polypeptides include a linker between the VH and VL domains, thereby
`forming a paired portion of VH and VL required for antigen binding. A peptide linker composed of ten or more amino
`acids is typically used as the linker between VH and VL when forming an intramolecular paired portion between VH and
`VL. However, the linkers of the scFv of the present invention are not limited to such peptide linkers, so long as they do
`not inhibit the formation of an scFv. To review scFv, see Pluckthun "The Pharmacology of Monoclonal Antibody", Vol.
`113 (Rosenburg and Moore ed., SpringerVerlag, NY, pp.269-315 (1994)).
`[0094] The term, "diabodies (Db)" refers to bivalent antibody fragments constructed by gene fusion (P. Holliger et al.,
`Proc. Natl. Acad. Sci. USA 90: 6444—6448 (1993); EP 404,097; W093/1 1 161 and such). Diabodies are dimers composed
`of two polypeptide chains, wherein each polypeptide chain includes within the same chain a light chain variable region
`(VL) and a heavy chain variable region (VH) connected with a linker short enough to disable interaction of these two
`regions, for example a linker of about five amino acid residues. VL and VH encoded on the same polypeptide chain will
`form a dimer because the linker between VL and VH is too short to form a single chain V region fragment. Therefore,
`the resulting diabody has two antigen-binding sites. Herein, when VL and VH directed against two different epitopes (a
`and b) are expressed simultaneously as combinations of VLa-VHb and VLb-VHa connected with a linker of about five
`residues, they are secreted as bispecific Db. In this case, the two different epitopes may be epitopes at two different
`sites on the same antigen, or epitopes at two different sites, each on two different antigens.
`[0095]
`Since diabodies include two molecules of scFvs, they thus composed of four variable regions, and as a result
`have two antigen binding sites. When the objective is to form a diabody, unlike as in the case with scFvs that do not
`form dimers, ordinarily, linkers forming a connection between VH and VL in each scFv molecules are linkers of about
`five amino acids when used as peptide linkers. However, scFv linkers for diabody formation are not limited to such
`peptide linkers so long as they do not interfere with scFv expression and diabody formation.
`[0096] Examples of preferred polypeptides or heteromultimers subjected to the methods of the present invention
`include polypeptides or heteromultimers composed of antibody heavy chain variable regions and light chain variable
`regions. More preferably, preferred embodiments of the present invention are methods for regulating association when
`polypeptides or heteromultimers of the present invention include two or more types of heavy chain variable regions and
`two or more types of Iightchain variable regions. Such polypeptides or heteromultimers are preferablythose that recognize
`two or more types of epitopes, and examples include multispecific antibodies.
`[0097] More preferably, examples of multispecific antibodies in the present invention include bispecific antibodies.
`[0098] More specifically, preferred embodiments ofthe present invention relate to, for example, methods for regulating
`association of bispecific antibodies composed of two types of heavy chain variable regions (first heavy chain and second
`heavy chain) and two types of light chain variable regions (first light chain and second light chain).
`[0099] Describing the "bispecific antibodies" of the preferred embodiments of the present invention more precisely,
`the above-mentioned "first heavy chain" refers to one of the two H chains forming the antibody, and the second H chain
`refers to the other H chain that is different from the first H chain. That is, of the two H chains, one of them can be arbitrarily
`defined as the first H chain and the other can be defined as the second H chain. Similarly, the "first light chain" refers
`to one of the two L chains forming the bispecific antibody, and the "second L chain" refers to the other L chain that is
`different from the first L chain. Of the two L chains, one of them can be arbitrarily defined as the first L chain and the
`other can be defined as the second L chain. Ordinarily, the first L chain and the first H chain are derived from the same
`antibody that recognizes a certain antigen (or epitope), and the second L chain and the second H chain are also derived
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`from the same antibody that recognizes a certain antigen (or epitope). Herein, the L chain-H chain pair formed by the
`first H chain and L chain is called as the first pair, and the L chain-H chain pair formed by the second H chain and L
`chain is called as the second pair. An antigen (or epitope) used to produce the antibody from which the second pair
`derives is preferably different from the antigen used to produce the antibody from which the first pair is derives. More
`specifically, antigens recognized by the first pair and the second pair may be the same but different antigens (or epitopes)
`are preferred to be recognized. Herein, the H chains and L chains of the first pair and second pair preferably have amino
`acid sequences that differ from each other. When the first pair and the second pair recognize different epitopes, the first
`and the second pairmay recognize a completely different antigen, orthey may recognize different sites (different epitopes)
`on the same antigen. Furthermore, one of them may recognize an antigen such as a protein, peptide, gene, or sugar,
`and the other may recognize cytotoxic substances such as radioactive substances, chemotherapeutic agents, or cell-
`derived toxins. However, when one wishes to produce an antibody having pairs formed by specific combinations of H
`chains and L chains, those specific H chains and L chains may be arbitrary determined to be the first pair and second pair.
`[0100] The above-mentioned "bispecific antibodies" are not necessarily limited to antibodies composed of two types
`of heavy chains and two typ