United States Patent [19]
`do Couto et al.
`
`(54] POLYNUCLEOTIDES ENCODJNG
`MODIFlED ANTIBODIES WITH HUMAN
`MlLK FAT GLOBULE SPECIFICITY
`
`(75]
`
`Inventors: Fernando J. R. do Couto. Pleasanton:
`Roberto L Ceriani; JerTY A.
`Peterson. both of Lafayette. all of
`Calif.: Eduardo A. Padlan. Kensington.
`Md.
`
`[73) Assignee: Cancer Research Fund of Contra
`Costa. Walnut Creek. Calif.
`
`[21] Appl. No.: m ,696
`
`Nov. 16, 1992
`
`(22] Filed:
`lnt.Cl.6
`
`(51]
`
`........................... C07H21/04;Cl2P21/08:
`A61K 39/695; A61K 39/40
`(52] U.S. Cl . .................. 536/23.53; 536123.5; 5301387.3:
`424/133. l ; 424/134.1; 4241135.1
`[58) Field of Search .............................. 5301387.3. 387.7.
`530/388.15. 388.8: 424/133.1. 134.1. 135.1.
`136.1. 138.1. 155.l; 536123.5. 23.53
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`ll/1987 Kortright ..................................... 435n
`4,708,930
`5.075,219 1211991 Ceciani et al .
`......................... 435n.23
`5,077,220 1211991 Ceciani et al .
`......................... 435n.23
`
`FOREIGN PATENT DOCUMENTS
`
`2 188 638 10/1988 United Kingdom .
`WO 92/04380
`311992 WlPO .
`311992 WIPO.
`W092/04380
`anIER PUBLICATIONS
`PadJan. E.A. "A possible procedure for reducing the immu(cid:173)
`nogenicity of antibody variable domains while preserving
`their ligand-binding properties" Molecular Immunology.
`vol. 28. No. 4/5. pp. 48~98. 1991.
`Couto. J.R.. et al. "Humanization of KC4G3. an Anti-Hu(cid:173)
`man Carcinoma Antibody". Hybridoma. vol 13. No. 3. pp.
`215-219 (1994).
`Bowie. J.U .. et al. "Deciphering the Message in Protein
`Sequences: Tolerance to Amino Acid Substitutions". Sci(cid:173)
`ence. 247: 1306--1310 (1990).
`Baggiiolini. M .• et al. "Tumi.ng on the Respiratory Burst".
`Elsevier Science Publishers Ltd. (UK). pp. 69-72. (1990).
`Friend. R.. "polymers from the Soviet Union". Nature. 326:
`335 (1987).
`Steiner. Lisa A.. "ImmunogJobuli.n Disulfide Bridges:
`Theme and Variations". Bioscience Reports 5: 973-989
`(1985).
`Burton. D. R.. "Antibody: the Flexible Adaptor Molecule".
`Elsevier Science Publishers Lul. (UK). pp. 65-69 (1990).
`Bhat. T.N .. "Bound Water Molecules and Conformational
`Stabilization Help Mediate an Antigen-Antibody Associa(cid:173)
`tion". Proc. Natl. Acd. Sci (USA) 91:1089-1093.
`Kettleborough et al.. (Protein Eng. 4:773-783 ( 1991)
`Humanization of a mouse monoclonal .. .
`
`I lllll llllllll Ill lllll lllll lllll lllll lllll lllll lllll lflll llllll Ill lllll llll
`US005792852A
`S,792,852
`Patent Number:
`Date of Patent:
`Aug. 11, 1998
`
`[ 11]
`
`[45)
`
`Eigenbrot et al .. (Proteins: Structures. Function and Genetics
`18:49-{)2 (1994» X-ray Structure of ...
`Co and Queen. Nature 351:501-502 (199J ) Humanized
`antibodies for therapy.
`Presta et al.. Jour. of Immuno. 151 :2623-2632 (1993)
`Humanization of an antibody directed against ...
`Huber & Bennett. (Nature 326:334 (1987) Antibody-anti(cid:173)
`gen flexibility.
`Fischm.ann et al.. (J. Bio. Chem .. 266:1291512920 (1991)
`Crystallographic Refinment of the Three- . ..
`Brady et al .. (J. Mo!. Biol. 227:253-264 ( 1992) Crystal
`Structure of a Chimeric Fab'Fragment of an ...
`Bhat et al.. Nature 347:483-485 (1990) Small rearrange(cid:173)
`ments in structure of Fv and Fab fragments ...
`Peter J. Delves. Encyclopedia of Immunology. Academic
`Press. p. 207 (Avidity).
`Davies & Padlan. (Current Biology. 2:254-256 (1992)
`Twisting into Shape.
`Varhoeyen. et al .. "Reshaping Human Antibodies: Grafting
`an AntiJysozyme Activity". Science 239: 1534-1536 (Mar.
`23. 1988).
`Tempest. et al.. "reshaping a Human Monoclonal Antibody
`to Inhibit Human Respiratory SynctiaJ Virus Infection In
`Vivo". Biotechnology 9: 266-271 (Mar. 1991).
`Peterson. J.A.. et al .. "Biochemical and Histogical Charac(cid:173)
`terization of Antigens Preferentially Expressed on the Sur(cid:173)
`face and Cytoplasm of Breast Carcinoma Cells lndentified
`by Monoclonal Antibodies Against the Human Milk Fat
`Globule". Hybridoma 9:221-235 ( 1990).
`Davies, D.R. and Padlan. E.A.. "Antibody-Antigen Com(cid:173)
`plexes". Annu. Rev. Biochem. 59:439-73 (1990).
`Riechmann. L .. et aJ .. "Reshaping Human Antibodies for
`Therapy". Nature 332:323-327 ( 1988).
`
`Primary Examiner-Y. Eyler
`Attome.>; Agent; or Firm-Viviana Arozel; Pretty. Schroeder
`& Poplawski
`
`[57]
`
`ABSTRACT
`
`A polynucleotide encodes a modified anul>ody. or single
`chains thereof. The modified antibody has a non-antigen(cid:173)
`binding peptide such as the constant regions of an antibody
`of a first species. peptide hormones. enzymes. and peptide
`transmitters; and a binding peptide such as the unsubstituted
`light and heavy chains of the variable region of an antibody
`of a second species which binds the human milk fat globule
`(HMF'G) antigen. The non-antigen-binding peptide is linked
`to at least one chain of the binding peptide. the chains may
`be linked to one another at a site other than the antigenic
`binding site. and at least one chain of the binding peptide has
`1 to 46 amino acids substituted with amino acids selected
`from specific ones assigned to each site. The polynucleotide
`and other products are also provided in the form of
`compositions. with a carrier. The polynucleotides may be
`RNAs and DNAs. and are also provided as hybrid vectors
`carrying them. and as transfected cells exp!'essing the modi(cid:173)
`fied antibodies or their single chains.
`
`63 Claims, No Drawings
`
`1 of 65
`
`BI Exhibit 1122
`
`

`

`5.792.852
`
`1
`POLYNUCLEOTIDES ENCODING
`MODlFIED ANTIBODIES WITH HUMAN
`MJLK FAT GLOBULE SPECIFICITY
`
`BACKGROUND OF THE INVENTION
`1. Field of the Invention
`This invention relates to the diagnosis. immunization. and
`therapy of neoplastic tumors. particularly carcinomas by
`means of specifically targeted analogue peptides comprising
`amino acid sequences encompassing. for instance. the
`complementarity determining regions (CDRs). and ana(cid:173)
`logues of the variable (Fv) region of anti-carcinoma
`antibodies. among others. The carcinoma specific peptides
`are provided as a single amino acid chain having the
`specificity of the non-human antibody F .. regions of the light
`or heavy chains. or as paired chains. either by themselves oc
`bound to other polymers such as synthetic polymers or
`oligopeptides resulting in chimeric antibodies. and more
`particularly analogues of human/non-human chimeric anti(cid:173)
`bodies or other polymeric constructs. The analogue peptides
`comprise sequences derived from variable regions of het(cid:173)
`erogeneous antibodies specific for human carcinoma anti(cid:173)
`gens that elicit a lesser immunological response in humans
`than whole heterologous non-human antibodies. The pep(cid:173)
`tides of the invention are useful for in vivo and in vitro
`diagnosis and therapy of carcinoma. Anti-idiotype polYJ>l!p(cid:173)
`tide and analogues thereof are suitable for inununiting
`humans or other animals against carcinoma. Polynucleotide
`sequences. hybrid vectors and host cells encoding the ana(cid:173)
`logue peptide and anti-idiotype polypeptides. hybrid vectors
`and transfected hosts are useful for preparing the peptides
`disclosed herein.
`2. Description of the Background
`Carcinomas result from the carcinogenic transformation
`of cells of different epithelia. Two of the roost damaging
`characteristics of carcinomas are their uncontrolled growth
`and their ability to create metastases in distant sites of the
`host. particularly a human host It is usually these distant
`metastases that cause serious consequences to the host. since
`frequently the primary carcinoma may be. in most cases.
`removed by surger y. The treatment of metastatic
`carcinomas. that are seldom removable. depends on irradia(cid:173)
`tion therapy and systemic therapies of different natures. The
`systemic therapies cWTently include. but not fully comprise.
`chemotherapy, different immunity-boosting medicines and
`procedures. hyperthennia and systemic monoclonal anti(cid:173)
`body treatment. The latter can be labeled with radioactive
`elements, immunotoxi.ns and chemotherapeutic drugs.
`Radioactively labeled monoclonal antibodies were ini(cid:173)
`tially used with success in lymphomas and leukemia. and
`recently in some carcinomas. The conoept underlying the
`use of labeled antibodies is that the labeled antibody will
`specifically seek and bi.nd to the carcinoma and. the radio(cid:173)
`active element. through its decay. will irradiate the rumor in
`siru. Since radioactive rays travel some distance in tumors it
`is not necessary that every carcinoma cell bind the labeled
`antibody. The specificity of the monoclonal antibodies will
`permit a selective treatment of the tumor while avoiding the
`irradiation of innocent by-stander norm.al tissues. that could
`be dose limiting. Chemotherapy produces serious toxic
`effects on normal tissues, making the chemotherapy of
`carcinomas less than desirable. and the use of radiolabeled
`monoclonal antibodies a valid alternative.
`Non-human antibodies raised against human epitopes 65
`have been used for the diagnosis and therapy of carcinomas
`as is known in the art. Also known are the methods for
`
`2
`preparing both polyclonal and monoclonal antibodies.
`Examples of the latter are BrE-2. BrE-3 ancil KC--4 (e.g .. U.S.
`Pat. Nos. 5.077.220: 5.075.219 and 4.708.930.
`The KC-4 murine monoclonal antibody is specific to a
`s unique antigenic determinant. the .. antigen'". and selectively
`binds strongly to neoplastic carcinoma cells and not to
`normal human tissue (U.S. Pat. No. 4.708.930 to Coulter).
`The antigen appears in two forms in carcinoma cells. only
`the smaller of these forms being expressed in the cell
`10 membrane. The larger form appears only in the cytoplasm
`and bas an approximate 490 Kdalton molecular weight
`(range of 480.000-510.000). The second form occurs at a
`higher density of expression. is found both in the cytoplasm
`and the membrane of carcinoma cells and has an approxi-
`15 mate 438 Kdalton molecular weight (range of 390.000-450.
`000) as determined by gel electrophoresis with marker
`proteins of known molecular weights. Labeled KC-4 was
`applied to the diagnosis and medical treatment of various
`carcinomas. particularly adenocarcinoma and squamous cell
`20 carcinoma regardless of the human organ site of origin.
`The BrE-3 antibody (Peterson et al .. Hybridoma 9:221
`(1990); U.S. Pat No. 5.075.219) was shown to bind to the
`tandem repeat of the polypeptide core of hwuan breast
`epithelial mucin. When the mucin is deglycosylated. the
`2s presence of moce tandem repeat epitopes is exposed and the
`binding of the antibody increases. Thus. arntibodies such as
`BrE-3 bind preferent:ially to neoplastic carcinoma tumors
`because these express an unglycosylated form of the breast
`epithelial mucin that is not expressed in normal epithelial
`30 tissue. This preferential binding combined with an observed
`low concenttation of epitope for these antibodies in the
`circulation of carcinoma patients. such as breast cancer
`patients. makes antibodies having specifidty for a mucin
`epitope highly effective for carcinoma radioimmunotherapy.
`3S A 'X>y-BrE-3 radioimmunoconjugate proved highly effective
`against hwnan breast carcinomas transplanted into nude
`.mice. Human clinical studies showed the 90y-BrE-3 radio(cid:173)
`immunoconjugate to considerably reduce tthe size of breast
`tumor metastases without any immediate toxic side effects.
`40 Moreover. an u 11n-BrE-3 radioimmunoconjugate was suc(cid:173)
`cessfully used for imaging 15 breast cancer patients. pro(cid:173)
`viding excellent twnor targeting in 13 out of 15 of the
`patients. Out of all the breast tumor metastases occurring in
`a nother study. 86% were detected by 111In-BrE-3.
`45 Unfortunately. 2 to 3 weeks after treatment. the patients
`developed a strong human anti-mouse antibody (HAMA)
`response that prevented further administration of the radio(cid:173)
`immuooconjugate. The HAMAresponse. which is observed
`for numerous murine monoclonal antibodies. precludes any
`so long-term administration of murine antibodies to hwnan
`patients. Similarly. other heterologous antibodies. when
`administered to humans. elicited similar antibody responses.
`The anti-hetc:rologous human response is. thus. a substantial
`limiting factor hindering the successful use of heterologous
`ss monoclonal antibodies as therapeutic agents. which could.
`otherwise. specifically annihilate breast carcinomas. causing
`little or no damage to normal tissue and having no other
`toxic effects.
`Chimeric antibodies are direct fusions between variable
`60 domains of one species and constant domains of another.
`Mouse/human chimeric antibodies prepared from other
`types of B cells binding to other types of antigenic deter(cid:173)
`minants have been shown to be less immuoogenic in humans
`than whole mouse antibodies. These proved to be less
`immunogenic but still in some cases there is a mounted
`immune response to the rodent variable region framework
`region (FR). A further reduction of the .. foreign'" nature of
`
`2 of 65
`
`BI Exhibit 1122
`
`

`

`5.792.852
`
`10
`
`3
`the chimeric antibodies was achieved by grafting only the
`CDRs from a rodent monoclonal into a human supporting
`framework prior to its subsequent fusion with an appropriate
`constant domain. (European Patent Application. Publication
`No. 239.400 to Winter: Riechmann. et al.. Nature
`332:323-327 (1988). However. the procedures employed to
`accomplish CDR-grafting often result in imperfectly
`"humanized" antibodies. That is to say. the resultant anti(cid:173)
`body loses avidity (usually 2-3 fold. at best).
`The ligand b.inding characteristics of an antibody com(cid:173)
`bining site are determined primarily by the structure and
`relative disposition of the CDRs. although some neighboring
`residues also have been found to be involved in antigen
`binding (Davies. et al .. Ann. Rev. Biochem. 59:439-473
`(1990)).
`The technologies of molecular biology have further
`expanded the utility of many antibodies by allowing for the
`aeation of class switched molecules whose functionality has
`been improved by the acquisition or loss of complement
`fixation. The size of the bioactive molecule may also be 20
`reduced so as to increase the tissue target availability of the
`antibody by either changing the class from an lgM to an IgG.
`or by removing most of the heavy and light chain constant
`regions to form an Fv antibody. Common to all of these
`potentially therapeutic forms of antibody are the required
`complementary determining regions (CDRs). which guide
`the molecule to its ligand. and the framework residues (FRs)
`which support the CDRs and dictate their disposition rela(cid:173)
`tive to one another. The crystallographic analysis of numer(cid:173)
`ous antibody structures revealed that the antigen combining
`site is composed almost entirely of the CDR residues
`arranged in a limited number of loop motifs. The necessity
`of the CDRs to form these structures. combined with the
`appreciated hypervariability of their primary sequence. leads
`to a great diversity in the antigen combining site. but one
`which has a finite number of possibilities. Thus. its hyper(cid:173)
`mutability and the limited primary sequence repertoire for
`each CDR would suggest that the CDRs derived for a given
`antigen from one species of animal would be the same
`derived from another species. Hence. they should be poorly
`immunogenic. if at all. when presented to a recipient organ(cid:173)
`ism.
`Accordfagly. there is still need for a product of high
`affinity and/or specificity for carcinoma antigens suitable for
`the detection and therapy of carcinomas which elicits a
`lesser antibody response than whole non-human antibodies
`orchimeric antibodies containing. for instance the entire
`non-huma.o variable region.
`
`4
`with variable regions or analogues thereof. wherein each
`analogue peptide is operatively linked to at least one other
`peptide or analogue thereof. or mixtures thereof. T he ana(cid:173)
`logue is also provided as a fusion protein. their correspond-
`s ing DNAs hybrid vectors. transfected hosts and RNAS. This
`invention also encompasses a method of producing an
`analogue peptide or hybrid analogue peptide by recombinant
`technology. Also provided herein are in vivo an in vitro
`methods of diagnosing and for the therapy of a carcinoma.
`Also disclosed herein is an anti-idiotype polypeptide.
`comprising polyclonal antibodies raised against the ana(cid:173)
`logue peptide of this invention. monoclonal antibodies
`thereof. fragments thereof selected from the group consist(cid:173)
`ing of Fab. Fab'. (Fab')~. CDRs. variable regions and ana(cid:173)
`logues thereof described above. an anti-carcinoma vaccine.
`15 a vaccination kit, a method of vaccinating against
`carcinoma. and a method of lowering the serum concentra(cid:173)
`tion of a circulating antibody with the anti-idiotype polypep(cid:173)
`tide of this invention.
`DESCR1PTION OF THE PREFERRED
`EMBODIMENTS
`This invention arose from a desire by the inventors to
`improve on antibody technology suitable for use in
`diagnostic. vaccine and therapeutic applications. The mono-
`25 clonal antibodies obtained up to the present time have been
`prepared by fusing immortalized cell Jines with B-cells of
`non-human origin such as murine. rat. rabbit. goat. and the
`like. Many of these hybridomas can produce monoclonal
`antibodies that have desirable binding properties such as
`30 high affinity and/or specificity for human carcinoma
`antigens. and are also produced in large amounts. However.
`in general. non-human antibodies may only be administered
`once to humans due to the detrimental effects they produce.
`This is true foe most xenogeneic antibodies being adminis-
`35 tered to a mammalian animal. For example. the repeated
`administration of mouse antibodies to a human subject
`elicits a strong human anti-mouse antibody (HAMA)
`response. which precludes their further utilization as thera(cid:173)
`peutic agents in humans. These non-human antibodies i.ni-
`40 tiate an immediate adverse reaction in many patients and are.
`thus. rendered ineffective as therapeutic agents. Non(cid:173)
`human-human chimeric antibodies and non-human CDR
`"grafted"-human antibodies may have low affinity and/or
`specificity for their antigens. On the other hand. human
`45 monoclonal hybridoma cell lines have not been very stable
`and have. therefore. not been suitable for the large scale.
`repeated production of monoclonal antibodies.
`The present inventors. thus. have undertaken the prepa(cid:173)
`ration of anti-carcinoma human and non-human CDRs and
`so non-human variable regions of antibodies. having affinity
`and specificity for an antigen found on the surface or the
`cytoplasm of a human carcinoma cell or released by the
`cells. wherein about l to 46 amino acids in the FR are
`substituted per chain with amino acids selected from the
`ss group consisting of amino acids present in equivalent posi(cid:173)
`tions in hum.an antibodies. or fragments thereof comprising
`l to 3 CDRs per chain and flanking regions thereof. each of
`about J to at least up to JO amino acids. alone or with an
`N-termioal fragment of about 1 to at least up to 10 amino
`acids. to lower or even circumvent the anti-xenogeneic
`response. To preserve substantial binding specificity the
`present invention utilizes CDRs and/or analogues of varying
`lengths of the variable regions of light and/or heavy chains
`of mouse. rat. rabbit. goat. horse. primate such as human and
`65 simian. bovine. and guinea pig antibodies. among others.
`The present inventors have found. surprisingly. that these
`analogue anttbody fragments substantially preserve the
`
`SUMMARY OF THE INVENTION
`
`This invention relates to a.n analogue peptide or a glyco(cid:173)
`sylated derivative which specifically binds to an antigen
`found on the surface or in the cytoplasm of carcinoma cells
`or released by the cells. the analogue peptide consisting
`essentially of at least one CDR or variable region of the light
`or heavy chains of an antibody of a first species having
`affinity and specificity for an antigen found on the surface or
`the cytoplasm of a carcinoma cell or released by the cells.
`wherein preferably about l to at least 46 amino acids in the 60
`FR are substituted per chain with amino acids selected from
`the group consisting of amino acids present in equivalent
`positions in antibodies of a second species. or fragments
`thereof comprising 1 to 3 CDRs per chain and flan.king
`regions thereof. each of about 1 to at least 10 amino acids.
`alone or with an N-terminal fragment of to about l to at least
`10 amino acids. combinations thereof. combinations thereof
`
`3 of 65
`
`BI Exhibit 1122
`
`

`

`5.792.852
`
`6
`5
`are not available. it is. fortunately. stiU possible to predict the
`binding and specificity characteristics of the whole antibody
`location of these important amino acids from the knowledge
`while eliciting a lesser detrimental inununological.
`However. the simple preservation of the binding region of an
`of other antibody slructures. especially those whose variable
`light and heavy regions belong to the same class. The classes
`antibody does not ensure that the binding characteristics of
`of variable regions can be determined from their amino acid
`the antibody will be maintained. Antibodies are glyc<r
`sequence.
`polypeptides that are folded into specific conformations.
`A method by which these important .amino acids are
`When the glycoside portion of the molecule or portions of
`identified has been described for the case of the amino acids
`the amino acid sequence are perturbed or excised. the
`with buried side chains by Padlan. E. A. (Padlan. E. A .... A
`folding pattern of the molecule is generaUy perturbed. Thus.
`any deletion or modification of sequences of an antibody 10 Possible Procedure for Reducing the lrnmunogenicity of
`must be made taking into consideration that its folding-
`Antibody Variable Domains While Preserving Their Ligand-
`dependent properties may be diminished or even obliterated
`Binding Binding Properties". Molecular lmmmunology.
`if die folding is affected. even though the amino acid
`28:48~948 (1991)). Various antibody variable region
`sequences involved in the binding of the antigen are pre-
`structures were compared using a computer program that
`15 determines the solvent accessibility of the framework resi-
`served.
`The present inventors have selected the following strategy
`dues as well as their contacts with the .opposite domain
`for the preparation and manufacture of the analogue peptides
`(Padlan. E. A. ( 1991). supra). Swprisingly. a close exami-
`and hybrid peptides of this invention. The cDNAs that
`nation of the fractional solvent accessibility reveals a very
`encode the variable chains of an antibody may be obtained
`close similarity in the exposure patterns of the V Hand the V L
`by isolation of mRNA from a hybridoma cell and reverse 20 domains. Put in simple terms. this means that regardless of
`the particular antibody in question. and of its amino acid
`transcription of the mRNA. amplification of the cDNA by
`PCR and insertion of the DNA into a vector for sequencing
`sequence. the buried residues occupy the same relative
`and restriction enzyme cutting. The cDNAs encoding the
`positions in most antibodies.
`CDR or F, region fragments of the light (VJ and heavy
`A similar analysis can be done by computer modeling. to
`('I H) chains of an antibody having affinity and specificity for 25 detennine which amino acids contact the CDRs and which
`a carcinoma cell antigen may be reverse transcribed from
`contact the opposite domain. At this point. the Fab slructures
`that are currently in the Protein Data bank (Bernstein. F. C..
`isolated mRNA. The variable region cDNAs may then be
`modified with predesigncd primers used to PCR amplify
`et al .. J. Mo. Biol. 112:535-542 ( 1977)) ma:y be examined to
`them. cloned. into a vector optionally carrying DNA
`detennine which FRs are probably imponant in maintaining
`sequences encoding. e.g .. a constant region(s). optionally 30 the structure of the combining site. Thus. after a close
`sequenced. and then transfected into a host cell for expres-
`inspection of many high resolution three-dimensional struc-
`sion of the analogue gene products. The binding specificity
`lures of variable regions. the positions of all important
`framework amino acids. that is. those that oontact the CDRS.
`characteristics of the analogue peptides may be then deter-
`mined and compared to those of the whole antibodies.
`the opposite domain. and those whose side chains are
`X-ray crystalographic srudies have repeatedly demon- 35 inwardly pointed. may be tabulated. Keeping these amino
`strated that the framework structures of the F~ of different
`acids. as well as those from the CDRs. and finally those FR
`antibodies assume a canonical structure regardless of the
`amino acids that may be involved in ligand binding. should
`species of origin. amino acid sequence. or ligand specificity.
`insure to a great extent the preservation of affinity. The
`This is generally taken as evidence that the ligand-binding
`precise identification of FR amino acids that are involved in
`characteristics of an antibody combining site are determined 40 ligand-binding cannot be generalized since it varies for
`different antibodies. Nevertheless. conservative decisions
`primarily by the structure and relative disposition of the
`can be made to preserve the amino acids located in FR that
`CDRs. although some neighboring framework residues also
`have been found to be involved in antigen-binding. Thus. if
`have a high probability of cont.acting the antigen. These
`the fine specificity of an antibody is to be preserved. its CDR
`regions are located immediately adjacent to the CDRs and at
`structures. and probably some of the neighboring residues. 45 the N-terminus of both chains. because the surfaces of these
`their interaction with each other and with the rest of the
`regions are contiguous with the CDR surfaces.
`variable domains. must also be maintained. These crystal-
`S111prisingly. it is possible to keep all of these important
`lographic studies point to the possible need for retaining
`amino acids in a heterologous humanized antibody and still
`most. if not all. of the many interior and inter-domain
`inaease dramatically the similarity with a buman consensus
`contact residues since the structural effects of replacing only so sequence. That is. the final number of amino acids with
`mouse identities differing from human identities that are
`a few of them cannot be predicted.
`While at first the necessity of keeping these amino acids
`kept is typically sm.all. This is usually possible because
`might seem to defeat the hwnanization goal of decreasing
`human frameworks that are similar to the mouse
`immunogenicity. the actual number of amino acids that must
`frameworks. especially at the positions of the important
`be retained is usually srnal.l because of the striking similarity 55 amino acids. can be found. This is because many of the
`between human and murine variable regions. Moreover.
`import.ant amino acids have the same identities in both
`many. if not most. of the retained amino acids possess side
`mouse and hwnan antibodies.
`chains that are not exposed on the surface of the molecule
`All the amino acids that are determined to be not impor-
`and. therefore. may not contribute to the antigenicity.
`tant by the method described above may be completely
`The challenge in humanizing the variable regions of a 60 replaced by their corresponding human counterparts. The
`surface of the finally humanized anti.body should look very
`non-human antibody. e.g .. a murine antibody. thus begins
`with the identification of the "important" xenogenenic
`much like that of a human antibody except for the antigen
`amino acids. ''Important" amino acids are those that are
`binding surfaces. The orJginal shape of those binding
`involved in antigen binding. contact the CDRs and the
`surfaces. however. is maintained by leaving the internal
`opposite chains. and have buried side-chains. Ideally. lhese 65 composition of the antibody intacl preserving inter-domain
`contacts and by keeping very few key amino acids that
`residues would be readily identified from a weU character-
`ized three-dimensional structure. When direct structural data
`contact the CDRs.
`
`4 of 65
`
`BI Exhibit 1122
`
`

`

`5.792.852
`
`7
`a) Choosing the Best Human Framework to Use in lhe
`"Humanization" of an Antibody When lls Structure Is
`Known
`At the present time. there a.re I 1 Fab structures for which
`the alomic coordinates are known and have been placed in
`the Protein Data Banlc as shown in Table l below. 2 from
`human and 9 from murine antibodies.
`
`TABLE 1
`
`Fab Strucrures for Which Coordinates
`are in the Protein Dai.a llank
`
`ANIIBODY RESOLUTION (A) R-VALUE PDB CODE
`
`HUMAN: NEWM
`KOL
`MU1UNE: McPC603
`1539
`HyHEL-5
`HyHEL-10
`
`2.0
`1.9
`2.7
`1.95
`2.54
`3.0
`
`0.46
`3FAB
`0.189 2FB4
`0.225
`lMCP
`0.194 2FB1
`0.245 2HFL
`0.24
`3HFM
`
`5
`
`10
`
`15
`
`8
`
`TABLE I-continued
`
`Fab Structures for Which Coordina1es
`are in the Prolein Data Bank
`
`ANTIBODY RESOLlTllON (A) R-VALUE PDB CODE
`
`RI9.9
`44-20
`36-71
`81312
`Dl.3
`
`2.8
`2.7
`l.85
`2.8
`2.5
`
`0.30
`0.215
`0.248
`0.197
`0.184
`
`lFJ9
`4FAB
`6FAB
`llGF
`!FOL
`
`The contacts between side chains in lhe variable domains
`of the 11 Fabs have been collected and are presented in
`Tables 2 to 4 below. The FR in the V L domains that contact
`CDRs are listed in Table 2 below.
`
`TABLE 2
`
`V L Frame-..·orl.: Residues Thar Contact CDR Residues
`in Fabs of Known Thtte-Dimensiooal S!tuc:turc
`
`ANTIBODY
`
`POSmON J539
`
`McPCtS03 HyHEL-10 HyHEl.r5 Rl9.9
`
`4-4-20
`
`36-71
`
`81312
`
`DJ.3
`
`NEWM
`
`KOL
`
`GLU(2) ASP(S)
`ll.E(ll)
`II..F.(lS)
`VAL(3)
`MET(6)
`TIIR(l)
`
`LEU(7)
`
`ASP(lO)
`ll.E(17)
`VAL(2)
`LEU(6)
`
`ASP(4)
`ASP(8)
`ASP(3)
`VAL(9)
`VAL(9)
`ll.E(S)
`II..F.(13)
`ll..E{20)
`LEU(6)
`GLN(2)
`VAL(2)
`Gl.N(2)
`VAL(3)
`LEU(lO) MEI'(9) MET(l3) MET(7) MET(6) MET(?)
`TIIR(l)
`TIIR(2)
`
`ASP(ll)
`Il..F.(10)
`
`SER(3)
`VAL(2)
`LEU(4)
`THR(l)
`
`LEU(6)
`
`TIIR(4)
`SER(6)
`
`I
`2
`3
`4
`s
`7
`22
`23
`35
`36
`45
`46
`4$
`49
`58
`(,()
`62
`66
`67
`@
`70
`71
`88
`98
`
`CYS(2)
`CYS(l)
`CYS(l)
`TRP(3)
`TRP(2)
`TRP(4)
`TYR(12) TYR(16) TYR(8)
`
`PR0(3)
`LEU(4)
`LEU(6)
`Il.E(l)
`II..F.(J)
`II..F.(J)
`TYR(28) TYR(29) LYS(13)
`VAL(3)
`VAL(3)
`ll.E(J)
`ASP(!)
`
`SER(3)
`l11R(3)
`ASP(2)
`1YR(l4) PHE(23)
`CYS(l)
`PHE(8)
`
`PHE(8)
`
`1liR(3)
`
`PHE(17)
`CYS(2)
`PHE(IO)
`
`CYS(2)
`
`CYS(l)
`
`CYS(l)
`TRP(2)
`TRP(4)
`TRP(l)
`TYR(l4) TYR(l3) TYR(ll)
`
`CYS(l)
`CYS(l)
`TRP(4)
`TRP(6)
`TRP(2)
`TYR(lO) TYR(22) 1YR(l3) TYR(IS) TYRC8)
`LYS(J2) LYS(S)
`LEU( JO) LEU(6)
`ARG(JS) LEU(5)
`VAL(l4) LEU(5)
`Il.E(2)
`VAL(!)
`ll..E{3)
`TYR(12) TYR(40) TYR(22) TYR(22) TYR(l6)
`1YR(25)
`VAL(6)
`VAL(6)
`VAL(S)
`VAL(4)
`VAL(S)
`VAL(!)
`ASP(2)
`ASP(4)
`PHE( l)
`
`PHE(l)
`
`PHE(l )
`
`TIIR(5)
`
`1HR(4)
`
`SER(l)
`TIIR(l)
`
`PHE(S)
`
`!l!R(I)
`SER(l)
`ASP(l)
`ASP(6)
`SER(2)
`TYR(l7) TYR(24) PHE(17) TYR(l7) PHE(l9) TYR(l6) ALA(3)
`CYS(l)
`CYS(l)
`CYS(J)
`CYS(l)
`CYS(2)
`PHE(8)
`PHE(4)
`PHE(8)
`PHE(l4)
`PHE(l4)
`
`LEU(2)
`
`LEU(6)
`Il.E(l)
`TYR(2S)
`VAL(6)
`ASP(2)
`
`LYS(2)
`
`LYS(ll)
`
`ALA(4)
`CYS(l)
`PHE('7)
`
`PHE(3)
`
`Those f'R in the V H domains that contact CDRs are listed
`in Table 3 below.
`
`TABLE3
`
`VH Frameworl.: Residues That Contact CDR Residues
`in Fabs of Koowo Thtee-Dimeosiooal Structure
`
`ANTIBODY
`
`POSmON 1539
`
`McPC603 HyHEL-10 HyHEL-5 Rl9.9
`
`4-4-20
`
`36-7l
`
`Bi312
`
`Dl.3
`
`NEWM
`
`KOL
`
`I
`2
`4
`24
`27
`
`VAL(ll) VAL(3)
`l.EU(2)
`LEU(S)
`THR(2)
`PHE(2)
`
`PHE{3)
`
`VAL(8)
`LEU(S)
`VAL(6)
`
`VAL(!)
`LEU(2)
`
`LEU(!)
`ALA(!)
`TYR(J4) TYR(ll) PHE(26)
`
`Gl.U(3)
`VAL(?)
`LEU(!)
`
`VAL(3)
`LEU(J)
`
`VAL(J2)
`LEU(! )
`
`VAL(9)
`LEU(l)
`
`TYR(4)
`
`PHE(4)
`
`PHE(4)
`
`THR(l)
`
`PHE(3)
`
`5 of 65
`
`BI Exhibit 1122
`
`

`

`9
`
`5.792.852
`
`10
`
`TABLE :l-conrinued
`
`V" Framewo rk Residues 1liat Contact COR Residues
`in F abs of Known Tivu-Oimensional Structure
`
`ANTIBODY
`
`POSffiON 1539
`
`McPC603 HyHEJ., 10
`
`HyHEL-5 Rl9.9
`
`4-4-20
`
`36-7 1
`
`BJ31 2
`
`Dt.3
`
`NEWM
`
`KOL
`
`ASP{9)
`PHE(4)
`
`1lfR(S1
`PHE(41
`
`1lfR(3)
`PHE(IO)
`
`rnR(2)
`
`1HR(6)
`PHE(7)
`1HRl6)
`
`VAL(!)
`LYS(I)
`
`lHR(4)
`PHE( 13)
`SER(7)
`
`lliR(2)
`PHE(6)
`
`1lfR(3)
`PHE(3)
`
`l.EU(I)
`
`SER(l1
`
`ASP(6)
`
`TRP(2)
`
`VAL(I)
`
`VAL(2)
`
`ARG(4)
`
`LYS(2)
`
`ARG(I)
`
`ARG(l )
`
`GLU(3)
`TRP(2 1)
`ll.E(I)
`
`PHE(4)
`
`Il..E(8)
`ARG(7)
`ASN(I )
`LEU(4)
`
`VAL(I)
`ARG(2)
`
`ARG(4)
`
`GLU(4) GLU( I )
`11lP(29) TYRl20
`MET(6)
`n.£(1)
`ALA(2}
`
`ARG(ll)
`ll.E(9)
`
`PHE(IO)
`ll.E( I)
`VAL(6)
`ll.E(8)
`ARG(l6) ARG(2)
`lliR(J)
`LEU(1)
`
`TYR(9)
`
`GLU(l)
`TRP(l9) TRP(22)
`Ill!(2)
`LEU(!)
`
`GLU(