PCT
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PC1)
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`(51) International Patent Classification 5 :
`C12N 15/13, C12P 21/08
`C07K 13/00, Cl2N 5/10
`G06Fl5/00
`
`(11) International Publication Number:
`
`WO 92/22653
`
`Al
`
`(43) International Publicatiolil Date:
`
`23 December 1992 (23.12.92)
`
`(21) International AppJication Number:
`
`PCT/US92/05126
`
`(22) International Filing Date:
`
`15 June 1992 (15.06.92)
`
`(30) Priority data:
`715,272
`
`14 June 1991 (14.06.91)
`
`US
`
`(60) Parent Application or Grant
`(63) Related by Continuation
`us
`Filed on
`
`715,272 (CIP)
`14 June 1991 (14.06.91)
`
`(71) Applicant (for all designated States except US): GENEN(cid:173)
`TECH, INC. [US/US]; 460 Point San Bruno Boulevard,
`South San Francisco, CA 94080 (US).
`
`(72) Inventors; and
`(75) Inventors/ Applicants (for US only) : CARTER, Paul, J.
`[GB/US]; 2074 18th Avenue, San Francisco, CA 94116
`(US). PRESTA, Leonard, G. [US/US]; 1900 Gough
`Street, #206, San Francisco, CA 94109 (US).
`
`(74)Agents: ADLER, Carolyn, R. et al.; Genentech, Inc., 460
`Point San Bruno Boulevard, South San Francisco, CA
`94080 (US).
`
`(81) Designated States: AT (European patent), AU, BE (Euro(cid:173)
`pean patent), CA, CH (European patent), DE (Euro(cid:173)
`pean patent), DK (European patent), ES (European pa(cid:173)
`tent), FR (European patent), GB (European patent), GR
`(European patent), IT (European patent), JP, LU (Euro(cid:173)
`pean patent), MC (European patent), NL (European pa(cid:173)
`tent), SE (European patent), US.
`
`Published
`With international search report.
`Before the expiration of the time limit for amending the
`claims and to be republished in the event of the receipt of
`amendments.
`
`(54)Title: METHOD FOR MAKING HUMANIZED ANTIBODIES
`
`(57) Abstract
`
`Variant immunoglobulins, particularly humanized
`antibody polypeptides are provided, along with methods
`for their preparation and use. Consensus immunoglobulin
`sequences and structural models are also provided.
`
`Anneal huVL or huVH oligomers to pAKl template
`- · -A--> -· --A...1! 5'
`
`4
`3'-
`
`1. Ligate
`2. Isolate usembled oligomers
`3. Anneal topAKl template (Xhol~Stuf+)
`4. Extend and ligate
`
`_fl
`
`1. Transform E. coli
`2. Isolate phagemid pool
`3. Enrich for hu\i_ and buVaCXho I"; Stu!-)
`4. Sequence verify
`
`Xhol
`
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`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify S1att:s party 10 the PCr on lhe front pages of pamphlets publishing international
`applications under the PCL
`
`AT
`AU
`BB
`BE
`BF
`BG
`BJ
`BR
`CA
`CF
`CG
`CH
`Cl
`CM
`cs
`DE
`UK
`ES
`
`Aus1ria
`Auslralia
`Barhados
`Belgium
`Burkina 1-aso
`Bulgaria
`Benin
`Brwil
`Canada
`Ccnlral African Republic
`Congo
`Swil7crland
`Cote d'Ivoire
`Cameroon
`(J.t:eboslovaL.ia
`Germany
`Denmark
`Spain
`
`Fl
`FR
`GA
`GB
`GN
`GR
`HU
`IE
`IT
`JP
`KP
`
`KR
`LI
`LK
`LU
`MC
`MG
`
`Finland
`Fram;~
`Oabon
`United Kingduru
`Guinea
`Greece
`Hungary
`Ireland
`Italy
`Japan
`Democratic People'> Republic
`of Korea
`Republic of Korea
`L.iechtcn>lein
`Sri Lanka
`Lu~cmbourg
`Monaco
`Madagas.:ar
`
`ML
`MN
`MR
`MW
`NL
`NO
`PL
`RO
`RU
`SD
`SE
`SN
`SU
`TD
`TC
`us
`
`Mali
`Mongolia
`Mauritania
`Malawi
`Nciherlands
`Norway
`Poland
`Romania
`Russian Federation
`Sudan
`Sweden
`Senegal
`Soviet Union
`Chad
`Togo
`United States of America
`
`...
`. .,
`,-·
`...
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`W092/22653
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`PCT/US92/05126
`
`I
`
`METHOD FOR MAKING-~UMANIZED ANTIBODIES.
`
`5
`
`•
`
`Field of the Invention
`
`This invention relates to methods for the preparation and use of variant antibodies and
`finds application particularly in the fields of immunology and cancer diagnosis and therapy.
`
`IO
`
`Background of the Invention
`
`Naturally occurring antibodies (immunoglobulins) comprise two heavy chains linked
`
`15
`
`together by disulfide bonds and two light chains, one light chain being linked to each of the
`
`heavy chains by disulfide bonds. Each heavy chain has at one end a variable domain (VH)
`
`followed by a number of constant domains. Each light chain has a variable domain (VL) at one
`
`end and a constant domain at its other end; the constant domain of the light chain is aligned
`
`with the first constant domain of the heavy chain, and the light chain variable domain is
`
`io
`
`aligned with the variable domain of the heavy chain. Particular amino acid residues are
`
`believed to form an interface between the light and heavy chain variable domains, see e.g.
`
`Chothia eta/., J. Mo/. Biol. 186:651-663 (1985); Novotny and Haber, Proc. Natl. Acad. Sci.
`'
`USA 82:4592-4596 (1985).
`
`The constant domains are not involved directly in binding the antibody to an antigen,
`
`25
`
`but are involved in various effector functions, such as participation of the antibody in antibody(cid:173)
`
`dependent cellular cytotoxicity. The variable domains of each pair of light and heavy chains
`
`are involved directly in binding the antibody to the antigen. The domains of natural light and
`
`heavy chains have the same general structure, and each domain comprises four framework
`
`(FR) regions, whose sequences are somewhat conserved, connected by three hyper-variable
`
`30
`
`or complementarity determining regions (CDRs) (see Kabat, E. A. et al., Sequences of Proteins
`
`...
`
`of Immunological Interest, National Institutes of Health, Bethesda, MD, (1987)). The four
`
`framework regions largely adopt a P-sheet conformation and the CDRs form loops connecting,
`
`and in some cases forming part of, the P-sheet structure. The CDRs in each chain are held in
`
`close proximity by the framework regions and, with the CDRs from the other chain, contribute
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`to the formation of the antigen binding site.
`Widespread use has been made of monoclonal antibodies, particularly those derived
`
`from rodents including mice, however they are frequently antigenic in human clinical use. For
`
`example, a major limitation in the clinical use of rodent monoclonal antibodies is an
`
`5
`
`anti-globulin response during therapy {Miller, R. A. et al., Blood 62:988-995 (1983); Schroff,.
`
`R. W. et al, Cancer Res. 45:879-885 (1985)).
`
`The art has attempted to overcome this problem by constructing "chimeric" antibodies
`
`in which an animal antigen-binding variable domain is coupled to a human constant domain
`
`{Cabilly et al, U.S. patent No. 4,816,567; Morrison, S. L. eta/., Proc. Natl. Acad. Sci. USA
`
`~
`J
`
`10
`
`81 :6851-6855 (1984); Boulianne, G. L. et al., Nature 312:643-646 (1984); Neuberger, M. S.
`
`et al., Nature 314:268-270 (1985)). The term "chimeric" antibody is used herein to describe
`
`a polypeptide comprising at least the antigen binding portion of an antibody molecule linked
`
`to at least part of another protein (typically an immunoglobulin constant domain).
`
`The isotype of the human constant domain may be selected to tailor the chimeric
`
`15
`
`antibody
`
`for participation
`
`in antibody-dependent cellular cytotoxicity
`
`(ADCC) and
`
`complement-dependent cytotoxicity (see e.g. Bruggemann, M. et al., J. Exp. Med.
`
`166:1351-1361 (1987); Riechmann, L. et al., Nature 332:323-327 (1988); Love et al.,
`
`Methods in Enzymology 178:515-527 (1989); Bindon et al., J. Exp. Med. 168:127-142
`
`2o
`
`25
`
`(1988).
`
`In the typical embodiment, such chimeric antibodies contain about one third rodent (or
`
`other non-human species) sequence and thus are capable of eliciting a significant anti-globulin
`
`response in humans. For example, in the case of the murine anti-CD3 antibody, OKT3, much
`
`of the resulting anti-globulin response is directed against the variable region rather than the
`
`constant region (Jaffers, G. J. et al, Transplantation 41 :572-578 (1986)).
`
`In a further effort to resolve the antigen binding functions of antibodies and to minimize
`
`the use of heterologous sequences in human antibodies, Winter and colleagues (Jones, P. T.
`
`et al., Nature 321 :5·22-525 (1986); Riechmann, L. et al., Nature 332:323-327 (1988);
`
`Verhoeyen, M. et al., Science 239:1534-1536 (1988)) have substituted rodent CDRs or CDR
`
`sequences for the corresponding segments of a human antibody. As used herein, the term
`
`30
`
`"humanized" antibody is an embodiment of chimeric antibodies wherein substantially less than
`
`an intact human variable domain has been substituted by the corresponding sequence from a
`
`non-human species. In practice, humanized antibodies are typically human antibodies in which
`
`some CDR residues and possibly some FR residues are substituted by residues from analogous
`
`sites in rodent antibodies.
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`W092/22653
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`PCT /US92/05126
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`The therapeutic promise of this approach is supported by the clinical efficacy of a
`
`humanized antibody specific for the CAMPATH-1 antigen with two non-Hodgkin lymphoma
`
`patients, one of whom had previously developed an anti-globulin response to the parental rat
`
`antibody (Riechmann, L. et al., Nature 332:323-327 (1988); Hale, G. et al., Lancet
`
`5
`
`i:1394-1399 (1988)). A murine antibody to the interleukin 2 receptor has also recently been
`
`f
`
`;.,
`
`humanized (Queen, C. et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989)) as a
`
`potential immunosuppressive reagent. Additional references related to humanization of
`
`antibodies include Co et al., Proc. Natl. Acad. Sci. USA 88:2869-2873 (1991 ); Gorman et al.,
`
`Proc. Natl. Acad. Sci. USA 88:4181-4185 (1991 ); Daugherty et al., Nucleic Acids Research
`19(9):2471-2476 (1991 ); Brown et al., Proc. Natl. Acad. Sci. USA 88:2663-2667 (1991 );
`
`10
`
`Junghans et al., Cancer Research 50:1495-1502 (1990).
`
`In some cases, substituting CDRs from rodent antibodies for the human CD Rs in human
`frameworks is sufficient to transfer high antigen binding affinity (Jones, P. T. et al., Nature
`
`321 :522-525 (1986); Verhoeyen, M. eta/., Science 239:1534-1536 (1988)), whereas in other
`
`15
`
`cases it has been necessary to additionally replace one (Riechmann, L. et al., Nature
`
`(Queen, C. et al., Proc. Natl. Acad. Sci. USA
`332:323-327 (1988)) or several
`86:10029-10033 (1989)) framework region (FR) residues. See also Co et al., supra.
`
`For a given antibody a small number of FR residues are anticipated to be important for
`
`antigen binding. Firstly for example, certain antibodies have been shown to contain a few FR
`residues which directly contact antigen in crystal structures of antibody-antigen complexes
`
`2.o
`
`(e.g., reviewed in Davies, D. R. et al., Ann. Rev. Biochem. 59:439-473 (1990)). Secondly,
`
`a number of FR residues have been proposed by Chothia, Lesk and colleagues (Chothia, C. &
`Lesk, A. M., J. Mo/. Biol. 196:901-917 (1987); Chothia, C. et al., Nature 342:877-883
`(1989); Tramontano, A. et al., J. Mo/. Biol. 215:175-182 (1990)) as critically affecting the
`
`25
`
`conformation of particular CDRs and thus their contribution to antigen binding. See also
`
`Margolies eta/., Proc. Natl. Acad. Sci. USA 72:2180-2184 (1975).
`
`It is also known that, in a few instances, an antibody variable domain (either VH or VL)
`may contain glycosylation sites, and that this glycosylation may improve or abolish antigen
`
`binding, Pluckthun, Biotechnology 9:545-51 (1991 ); Spiegelberg eta/., Biochemistry 9:4217-
`
`30
`
`4223 (1970); Wallie et al., J. Exp. Med. 168: 1099-1109 (1988); Sox et al., Proc. Natl. A cad.
`
`Sci. USA 66:975-982 (1970); Margni et al., Ann. Rev. lmmunol. 6:535-554 (1988).
`
`Ordinarily, however, glycosylation has no if!tluence on the antigen-binding properties of an
`
`antibody, Pluckthun, supra, (1991 ).
`
`The three-dimensional structure of immunoglobulin chains has been studied, and crystal
`
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`structures for intact immunoglobulins, for a variety of immunoglobulin fragments, and for
`antibody-antigen complexes have been published (see e.g., Saul et al., Journal of Biological
`
`Chemistry 25:585-97 (1978); Sheriff et al., Proc. Natl. Acad. Sci. USA 84:8075-79 (1987);
`
`Segal et al., Proc. Natl. Acad. Sci. USA 71 :4298-4302 (1974); Epp et al., Biochemistry
`
`5
`
`14(22):4943-4952 (1975); Marquart eta/., J. Mo/. Biol. 141:369-391 (1980); Furey eta/.,
`J. Mo/. Biol. 167:661-692 (1983); Snow and Amzel, Protein: Structure, Function, and l
`Genetics 1 :267-279, Alan R. Liss, Inc. pubs. (1986); Chothia and Lesk, J. Mo/. Biol. 196:901-
`
`,.
`
`917 (1987); Chothia et al., Nature 342:877-883 (1989); Chothia et al., Science 233:755-58
`
`(1986); Huber et al., Nature 264:415-420 (1976); Bruccoleri et al., Nature 335:564-568
`
`10
`
`(1988) and Nature 336:266 { 1988); Sherman et al., Journal of Biological Chemistry 263:4064-
`
`4074 (1988); Amzel and Poljak, Ann. Rev. Biochem. 48:961-67 (1979); Silverton et al., Proc.
`
`Natl. Acad. Sci. USA 74:5140-5144 (1977); and Gregory et al., Molecular Immunology
`
`24:821-829 (1987). It is known that the function of an antibody is dependent on its three
`
`dimensional structure, and that amino acid substitutions can change the three-dimensional
`
`15
`
`structure of an antibody, Snow and Amzel, supra.
`
`It has previously been shown that the
`
`antigen binding affinity of a humanized antibody can be increased by mutagenesis based upon
`
`molecular modelling (Riechmann, L. et al., Nature 332:323-327 (1988); Queen, C. et al., Proc.
`
`Natl. Acad. Sci. USA 86:10029-10033 (1989)).
`
`Humanizing an antibody with retention of ~igh affinity for antigen and other desired
`
`20
`
`biological activities is at present difficult to achieve using currently available procedures.
`
`25
`
`30
`
`Methods are needed for rationalizing the selection of sites for substitution in preparing such
`
`antibodies and thereby increasing the efficiency of antibody humanization.
`
`(human epidermal growth factor receptor 2) encodes a
`The proto-oncogene HER2
`protein tyrosine kinase (p185HER2> that is related to and somewhat homologous to the human
`epidermal growth factor receptor (see Coussens, L. et al., Science 230:1132-1139 (1985);
`
`Yamamoto, T. et al., Nature 319:230-234 (1986); King, C. R. et al., Science 229:974-976
`
`(1985)). HER2 is also known in the field as c-erbB-2, and sometimes by the name of the rat
`
`homolog, neu. Amplification and/or overexpression of HER2 is associated with multiple human
`
`malignancies and appears to be integrally involved in progression of 25-30% of human breast
`and ovarian cancers CSlamon, D. J. et al., Science 235:177-182 (1987), Slamon, D. J. et al.,
`
`Science 244:707-712 (1989)). Furthermore, the extent of amplification is inversely correlated
`
`with the observed median patient survival time CSlamon, supra, Science 1989).
`
`The murine monoclonal antibody known as muMAb4D5 (Fendly, B. M. et al., Cancer
`
`Res. 50:1550-1558 (1990)), directed against the extracellular domain CECD) of p185HER2,
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`5
`specifically inhibits the growth of tumor cell lines overexpressing p185HER2 in monolayer
`
`culture or in soft agar (Hudziak, R. M. eta/., Malec. Cell. Biol. 9:1165-1172 (1989); Lupu, R.
`
`et al., Science 249:1552-1555 (1990)). MuMAb4D5 also has the potential of enhancing
`
`tumor cell sensitivity to tumor necrosis
`
`factor, an
`
`important effector molecule
`
`in
`
`5
`
`macrophage-mediated tumor cell cytotoxicity (Hudziak, supra, 1989; Shepard, H. M. and
`
`~,..
`
`Lewis, G.D. J. Clinical Immunology 8:333-395 (1988)). Thus muMAb4D5 has potential for
`clinical intervention in and imaging of carcinomas in which p1 s5HER2 is overexpressed. The
`muMAb4D5 and its uses are described in PCT application WO 89/06692 published 27 July
`
`1989. This murine antibody was deposited with the ATCC and designated A TCC CRL 10463.
`
`10
`
`However, this antibody may be immunogenic in humans.
`
`It is therefore an object of this invention to provide methods for the preparation of
`
`antibodies which are less antigenic in humans than non-human antibodies but have desired
`
`antigen binding and other characteristics and activities.
`
`It is a further object of this invention to provide methods for the efficient humanization
`
`15
`
`of antibodies, i.e. selecting non-human amino acid residues for importation into a human
`
`antibody background sequence in such a fashion as to retain or improve the affinity of the non(cid:173)
`
`human donor antibody for a given antigen.
`
`It is another object of this invention to provide humanized antibodies capable of binding
`p185HER2.
`Other objects, features, and characteristics of the present invention will become
`apparent upon consideration of the following description and the appended claims.
`
`20
`
`Summary of the Invention
`
`25
`
`The objects of this invention are accomplished by a method for making a humanized
`
`antibody comprising amino acid sequence of an import, non-human antibody and a human
`
`antibody, comprising the steps of:
`
`30
`
`a.
`
`b.
`
`obtaining the amino acid sequences of at least a portion of an import antibody
`
`variable domain and of a consensus variable domain;
`
`identifying Complementarity Determining Region (CDR) amino acid sequences
`
`in the import and the human variable domain sequences;
`
`c.
`
`substituting an import CDR amino acid sequence for the corresponding human
`
`CDR amino acid sequence;
`
`d.
`
`aligning the amino acid sequences of a Framework Region (FR) of the import
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`antibody and the cor_responding FR of the consensus antibody;
`
`e.
`
`identifying import antibody FR residues in the aligned FR sequences that are
`
`f.
`
`g.
`
`5
`
`10
`
`non-homologous to the corresponding consensus antibody residues;
`
`determining if the non-homologous import amino acid residue is reasonably
`
`expected to have at least one of the following effects:
`
`1 .
`
`2.
`
`3.
`
`non-covalently binds antigen directly,
`
`interacts with a CDR; or
`
`participates in the VL - VH interface; and
`
`for any non-homologous import antibody amino acid residue which is reasonably
`
`expected to have at least one of these effects, substituting that residue for the
`
`corresponding amino acid residue in the consensus antibody FR sequence.
`
`Optionally, the method of this invention comprises the additional steps of determining
`
`if any non-homologous residues identified in step (e) are exposed on the surface of the domain
`
`or buried within it, and if the residue is exposed but has none of the effects identified in step
`
`15
`
`(f), retaining the consensus residue.
`
`Additionally, in certain embodiments the method of this invention comprises the feature
`
`wherein the corresponding consensus antibody residues identified in step (e) above are
`
`selected from the group consisting of 4L, 35L, 36L, 38L, 43L, 44L, 46L, 58L, 62L, 63L, 64L,
`
`65L,66L,67L,68L,69L,70L,71L,73L,85L,87L,98L,2H,4H,24H,36H,37H,39H,43H,
`
`io
`
`45H,49H,58H,60H,67H,68H,69H,70H,73H,74H,75H,76H,78H,91H,92H,93H,and
`
`103H (utilizing the numbering system set forth in Kabat, E. A. et al., Sequences of Proteins
`
`of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987)).
`
`In certain embodiments, the method of this invention comprises the additional steps of
`
`searching either or both of the import, non-human and the consensus variable domain
`
`25
`
`sequences for glycosylation sites, determining if the glycosylation is reasonably expected to
`
`be important for the desired antigen binding and biological activity of the antibody (i.e.,
`
`determining if the glycosylation site binds to antigen or changes a side chain of an amino acid
`
`residue that binds to antigen, or if the glycosylation enhances or weakens antigen binding, or
`
`is important for maintaining antibody affinity). If the import sequence bears the glycosylation
`
`30
`
`site, it is preferred to substitute that site for the corresponding residues in the consensus
`
`human if the glycosylation site is reasonably expected to be important. If only the consensus
`
`sequence, and not the import, bears the glycosylation site, it is preferred to eliminate that
`
`glycosylation site or substitute therefor the corresponding amino acid residues from the import
`
`sequence.
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`W092/22653
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`7
`Another embodiment of this invention comprises aligning import antibody and the
`
`PCT /US92/05126
`
`consensus antibody FR sequences, identifying import antibody FR residues which are non(cid:173)
`
`homologous with the aligned consensus FR sequence, and for each such non-homologous
`
`import antibody FR residue, determining if the corresponding consensus antibody residue
`
`" s
`
`represents a residue which is highly conserved across all species at that site, and if it is so
`
`conserved, preparing a humanized antibody which comprises the consensus antibody amino
`
`acid residue at that site.
`
`Certain alternate embodiments of the methods of this invention comprise obtaining the
`
`amino acid sequence of at least a portion of an import, non-human antibody variable domain
`
`10
`
`having a CDR and a FR, obtaining the amino acid sequence of at least a portion of a consensus
`
`antibody variable domain having a CDR and a FR, substituting the non-human CDR for the
`
`human CDR in the consensus antibody variable domain, and then substituting an amino acid
`
`residue for the consensus amino acid residue at at least one of the following sites:
`
`a.
`
`(in the FR of the variable domain of the light chain) 4L, 35L, 36L, 38L, 43L,
`
`15
`
`44L,58L,46L,62L,63L,64L,65L, 66L,67L, 68L, 69L, 70L, 71L, 73L,85L,
`
`87L, 98L, or
`
`b.
`
`(in the FR of the variable domain of the heavy chain) 2H, 4H, 24H, 36H, 37H,
`
`39H,43H,45H,49H,58H,60H,67H,68H,69H,70H,73H,74H,75H,76H,
`
`78H, 91 H, 92H, 93H, and 103H.
`
`2.o
`
`In preferred embodiments, the non-CDR residue substituted at the consensus FR site is the
`
`residue found at the corresponding location of the non-human antibody.
`
`Optionally, this just-recited embodiment comprises the additional steps of following the
`
`method steps appearing at the beginning of this summary and determining wheth~r a particular
`
`amino acid residue can reasonably be expected to have undesirable effects.
`
`25
`
`This invention also relates to a humanized antibody comprising the CDR sequence of
`
`an import, non-human antibody and the FR sequence of a human antibody, wherein an amino
`
`acid residue within the human FR sequence located at any one of the sites 4L, 35L, 36L, 38L,
`
`43L,44L,46L,58L,62L,63L,64L,65L,66L,67L,68L,69L, 70L, 71L, 73L,85L,87L,98L,
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`2H,4H,24H,36H,37H,39H,43H,45H,49H,58H,60H,67H,68H,69H,70H,73H, 74H,
`
`30
`
`75H, 76H, 78H, 91 H, 92H, 93H, and 103H has been substituted by another residue.
`
`In
`
`preferred embodiments, the residue substituted at the human FR site is the residue found at
`
`the corresponding location of the non-human antibody from which the non-human CDR was
`
`obtained. In other embodiments, no human FR residue other than those set forth in this group
`
`has been substituted.
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`This invention also encompasses specific humanized antibody variable domains, and
`
`isolated polypeptides having homology with the following sequences.
`
`1. SEQ. ID NO. 1, which is the light chain variable domain of a humanized version of
`
`muMAb4D5:
`
`5
`
`DI QMTOSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLESGVP
`
`SRFSGSRSGTDFTL TISSLOPEDFATYYCQQHYTTPPTFGQGTKVEIKRT
`
`2. SEO. ID NO. 2, which is the heavy chain variable domain of a humanized version of
`
`muMAb4D5):
`
`10
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`EVQLVESGGGL VOPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTR
`
`Y ADSVKGRFTISADTSKNTA YLQMNSLRAEDTAVYYCSRWGGDGFY AMDVWGOGTLV
`
`TVSS
`
`In another aspect, this invention provides a consensus antibody variable domain amino
`
`15
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`acid sequence for use in the preparation of humanized antibodies, methods for obtaining,
`
`using, and storing a computer representation of such a consensus sequence, and computers
`
`comprising the sequence data of such a sequence.
`
`In one embodiment, the following
`
`consensus antibody variable domain amino acid sequences are provided:
`
`20
`
`SEO. ID NO. 3 (light chain):
`
`DI OMTQSPSSLSASVGDRVTITCRASQDVSSYLAWYQQKPGKAPKLLIY AASSLESGVP
`
`SRFSGSGSGTDFTL TISSLQPEDFATYYCOQYNSLPYTFGQGTKVEIKRT, and
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`SEQ. ID NO. 4 (heavy chain):
`
`25
`
`EVOLVESGGGLVQPGGSLRLSCAASGFTFSDY AMSWVROAPGKGLEWVAVISENGGYT
`
`RY ADSVKGRFTISADTSKNTA YLOMNSLRAEDTAVYYCSRWGGDGFY AMDVWGQGTL
`
`VTVSS
`
`30
`
`Brief Description of the Drawings
`
`FIGURE 1 A shows the comparison of the VL domain amino acid residues of
`
`muMAb4D5, huMAb4D5, and a consensus sequence (Fig. 1A, SEQ.ID NO. 5, SEQ. ID NO. 1
`
`and SEQ. ID NO. 3, respectively). FIGURE 1 B shows the comparison between the VH domain
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`amino acid residues of the muMAb4d5, huMAb4D5, and a consensus sequence (Fig. 1 B, SEO.
`
`ID NO. 6, SEO. ID NO. 2 and SEO. ID NO. 4, respectively). Both Figs 1 A and 1 B use the
`
`generally accepted numbering scheme from Kabat, E. A., et al., Sequences of Proteins of
`
`Immunological Interest (National Institutes of Health, Bethesda, MD (1987)). In both Fig. 1 A
`
`5
`
`and Fig. 1 B, the CDR residues determined according to a standard sequence definition (as in
`
`Kabat, E. A. et al., Sequences of Proteins of Immunological Interest (National Institutes of
`
`Health, Bethesda, MD, 1987)) are indicated by the first underlining beneath the sequences, and
`
`the CDR residues determined according to a structural definition (as in Chothia, C. & Lesk, A.
`
`M., J. Mo/. Biol. 196:901-917 (1987)) are indicated by the second, lower underlines. The
`
`10
`
`mismatches between genes are shown by the vertical lines.
`
`FIGURE 2 shows a scheme for humanization of muMAb4D5 VL and VH by gene
`
`conversion mutagenesis.
`
`FIGURE 3 shows the inhibition of SK-BR-3 proliferation by MAb4D5 variants. Relative
`
`cell proliferation was determined as described (Hudziak, R. M. et al., Molec. Cell. Biol.
`
`15
`
`9:1165-1172 (1989)) and data (average of triplicate determinations) are presented as a
`
`percentage of results with untreated cultures for muMAb4D5 (I), huMAb4D5-8 (n) and
`
`huMAb4D5-1 (I).
`
`FIGURE 4 shows a stereo view of a-carbon tracing for a model of huMAb4D5-8 VL and
`
`V H . The CDR residues (Kabat, E. A. et al., Seque.nces of Proteins of Immunological Interest
`
`10
`
`(National Institutes of Health, Bethesda, MD, 1987)) are shown in bold and side chains of VH
`residues A71, T73, A78, S93, Y102 and VL residues Y55 plus R66 (see Table 3) are shown.
`
`FIGURE 5 shows an amino acid sequence comparison of VL (top panel) and VH (lower
`
`panel) domains of the murine anti-CD3 monoclonal Ab UCHT1 (muxCD3, Shalaby et al., J.
`
`Exp. Med. 175, 217-225 (1992) with a humanized variant of this antibody (huxCD3v9). Also
`
`25
`
`shown are consensus sequences (most commonly occurring residue or pair of residues) of the
`
`most abundant human subgroups, namely VL K 1 and VH Ill upon which the humanized
`
`sequences are based (Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th
`
`edition, National Institutes of Health, Bethesda, MD, USA (1991 )). The light chain sequences-(cid:173)
`
`muxCD3, huxCD3v9 and huKl--correspond to SEO.ID.NOS 16, 17, and 18, respectively. The
`
`30
`
`heavy chain sequences--muxCD3, huxCD3v9 and huKl--correspond to SEO.ID.Nos 19, 20, and
`
`21 , respectively. Residues which differ between muxCD3 and huxCD3v9 are identified by an
`
`asterisk (*), whereas those which differ between humanized and consensus sequences are
`
`identified by a sharp sign (#). A bullet (o) denotes that a residue at this position has been
`
`found to contact antigen in one or more crystallographic structures of antibody/antigen
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`complexes (Kabat eta/., 1991; Mian, I. S. eta/., J. Mo/. Biol. 217, 133-151 (1991)). The
`
`location of CDR residues according to a sequence definition {Kabat et al., 1991) and a
`structural definition {Chothia and Lesk, supra 1987) are shown by a line and carats("') beneath
`
`the sequences, respectively.
`
`FIGURE SA compares murine and humanized amino acid sequences for the heavy chain
`
`of an anti-CD18 antibody. H52H4-160 {SEQ. ID. NO. 22) is the murine sequence, and pH52-
`
`8.0 {SEQ. ID. NO. 23) is the humanized heavy chain sequence. pH52-8.0 residue 143S is the
`
`final amino acid in the variable heavy chain domain VH, and residue 144A is the first amino
`acid in the constant heavy chain domain CH 1 .
`FIGURE 68 compares murine and humanized amino acid sequences for the light chain
`
`of an anti-CD18 antibody. H52L6-158 (SEQ. ID. NO. 24) is the murine sequence, and pH52-
`
`9.0 (SEQ. ID. NO. 25) is the humanized light chain sequence. pH52-9.0 residue 128T is the
`
`final amino acid in the light chain variable domain VL, and residue 129V is the first amino acid
`
`in the light chain constant domain CL.
`
`Detailed Description of the Invention
`
`s
`
`10
`
`15
`
`In general, the following words or phrases have the indicated definitions when used in
`
`Definitions
`
`20
`
`the description, examples, and claims:
`
`The murine monoclonal antibody known as muMAb405 {Fendly, B. M. et al., Cancer
`Res. 50:1550-1558 (1990)) is directed against the extracellular domain CECO) of p1 s5HER2.
`The muMAb4D5 and its uses are described in PCT application WO 89/06692 publ!shed 27 July
`
`1989. This murine antibody was deposited with the ATCC and designated ATCC CRL 10463.
`
`25
`
`In this description and claims, the terms muMAb405, chMAb405 and huMAb405 represent
`
`murine, chimerized and humanized versions of the monoclonal antibody 405, respectively.
`
`A humanized antibody for the purposes herein is an immunoglobulin amino acid
`
`sequence variant or fragment thereof which is capable of binding to a predetermined antigen
`
`and which comprises a FR region having substantially the amino acid sequence of a human
`
`30
`
`immunoglobulin and a CDR having substantially the amino acid sequence of a non-human
`
`immunoglobulin.
`
`Generally, a humanized antibody has one or more amino acid residues introduced into
`
`it from a source which is non-human. These non-human amino acid residues are referred to
`
`herein as "import" residues, which are typically taken from an "import" antibody domain,
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`particularly a variable domain. An import residue, sequence, or antibody has a desired affinity
`
`and/or specificity, or other desirable antibody biological activity as discussed herein.
`
`In general, the humanized antibody will comprise substantially all of at least one, and
`typically two, variable domains (Fab, Fab', F(ab') 2, Fabe, Fv) in which all or substantially all
`of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially
`
`all of the FR regions are those of a human immunoglobulin consensus sequence. The
`
`humanized antibody optimally also will comprise at least a portion of an immunoglobulin
`
`constant region (Fe), typically that of a human immunoglobulin. Ordinarily, the antibody will
`
`contain both the light chain as well as at least the variable domain of a heavy chain. The
`antibody also may include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain.
`
`The humanized antibody will be selected from any class of immunoglobulins, including
`
`lgM, lgG, lgD, lgA and lgE, and any isotype, including lgG 1, lgG2, lgG3 and lgG4. Usually the
`
`constant domain is a complement fixing constant domain where it is desired that the
`humanized antibody exhibit cytotoxic activity, and the class is typically lgG 1• Where such
`cytotoxic activity is not desirable, the constant domain may be of the lgG 2 class. The
`humanized antibody may comprise sequences from more than one class or isotype, and
`
`selecting particular constant domains to optimize desired effector functions is within the
`
`ordinary skill in the art.
`
`5
`
`..
`
`10
`
`15
`
`The FR and CDR regions of the humanized antibody need not correspond precisely to
`
`io
`
`the parental sequences, e.g., the import CDR or the consensus FR may be mutagenized by
`
`25
`
`substitution, insertion or deletion of at least one residue so that the CDR or FR residue at that
`
`site does not correspond to either the consensus or the import antibody. Such mutations,
`
`I
`
`however, will not be extensive. Usually, at least 75% of the humanized antibody residues will
`correspond to those of the parental FR and CDR sequences, more often 90%, and most
`preferably greater than 95%.
`
`In general, humanized antibodies prepared by the method of this invention are produced
`
`by a process of analysis of the parental sequences and various conceptual humanized products
`
`using three dimensional models of the parental and hum