PCT
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`Intematlonal Bureau
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(51) International Patent Classification 6 1
`
`(11) International Publication Number:
`
`WO 99/57134
`
`C07K 1/18, 16/32
`
`(43) International Publication Date:
`
`11 November 1999 (11.11.99)
`
`(21) International Application Number:
`
`PCT/US99/09637
`
`(22) International Filing Date:
`
`3 May 1999 (03.05.99)
`
`(30) Priority Data:
`60/084,459
`
`6 May 1998 (06.05.98)
`
`US
`
`(71) Applicant: GENENTECH, INC. [US/US]; 1 DNA Way, South
`San Francisco, CA 94080-4990 (US).
`
`(72) Inventors: BASEY, Carol, D.; 319 Hillview Lane, Winters,
`CA 95694 (US). BLANK, Greg, 8.; 1320 Hobart Street,
`Menlo Park, CA 94025 (US).
`
`(74) Agents: LEE, Wendy, M. et al.; Genentech, Inc., 1 DNA Way,
`South San Francisco, CA 94080—4990 (US).
`
`(81) Designated States: AE, AL, AM, AT, AU, AZ, BA, BB, BG,
`BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB,
`GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG,
`KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK,
`MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI,
`SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZA, ZW,
`ARIPO patent (GH, GM, KE, LS, MW, SD, SL, SZ, UG,
`ZW), Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ,
`TM), European patent (AT, BE, CH, CY, DE, DK, ES, FI,
`FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OAPI patent
`(BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE,
`SN, TD, TG).
`
`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: PROTEIN PURIFICATION BY ION EXCHANGE CHROMATOGRAPHY
`
`1.0 M NaCI
`
`(57) Abstract
`
`A method for purifying a polypeptide by ion exchange chro-
`matography is described which involves changing the conductivity
`and/or pH of buffers in order to resolve a polypeptide of interest
`from one or more contaminants.
`
`‘
`
`‘
`
`LOADING
`BUFFER
`50 mM NaCI
`
`INTERMEDIATE
`BUFFER
`70 mM NaCl
`
`I
`
`ELUTION
`BUFFER
`95 mM NaCI
`
`‘
`
`WASH
`BUFFER
`50 mM NaCI
`
`REGENERATION
`BUFFER
`
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`

`

`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`Slovenia
`SI
`LS
`Lesotho
`SK
`Slovakia
`LT
`Lithuania
`SN
`LU
`Senegal
`Luxembourg
`SZ
`Swaziland
`LV
`Latvia
`TD
`Chad
`MC
`Monaco
`TG
`MD
`Togo
`Republic of Moldova
`MG
`TJ
`Tajikistan
`Madagascar
`TM
`Turkmenistan
`MK
`The former Yugoslav
`TR
`Turkey
`Republic of Macedonia
`TT
`Mali
`Trinidad and Tobago
`UA
`Ukraine
`Mongolia
`UG
`Mauritania
`Uganda
`US
`United States of America
`Malawi
`UZ
`Uzbekistan
`Mexico
`VN
`Viet Nam
`Niger
`YU
`Netherlands
`Yugoslavia
`ZW
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`
`FOR THE PURPOSES OF INFORMATION ONLY
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`Ireland
`Israel
`Iceland
`Italy
`Japan
`Kenya
`Kyrgyzstan
`Democratic People’s
`Republic of Korea
`Republic of Korea
`Kazakstan
`Saint Lucia
`Liechtenstein
`Sri Lanka
`Liberia
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SG
`
`Zimbabwe
`
`Albania
`Armenia
`Austria
`Australia
`Azerbaijan
`Bosnia and Herzegovina
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`cote d’Ivoire
`Cameroon
`China
`Cuba
`Czech Republic
`Germany
`Denmark
`Estonia
`
`ES
`FI
`FR
`GA
`GB
`GE
`GH
`GN
`GR
`HU
`IE
`IL
`IS
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LC
`LI
`LK
`LR
`
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`WO 99/57134
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`PCT/US99/09637
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`PROTEIN PURIFICATION BY ION EXCHANGE CHROMATOGRAPHY
`
`BACKGROUND OF THE INVENTION
`
`Field ofthe Invention
`
`This invention relates generally to protein purification. In particular. the invention relates to a method
`
`for purifying a polypeptide (e. g. an antibody) from a composition comprising the polypeptide and at least one
`
`contaminant using the method of ion exchange chromatography.
`
`Description of Related Art
`
`The large-scale. economic purification of proteins is increasingly an important problem for the
`
`biotechnology industry. Generally, proteins are produced by cell culture, using either mammalian or bacterial cell
`
`lines engineered to produce the protein of interest by insertion of a recombinant plasmid containing the gene for
`
`that protein. Since the cell lines used are living organisms, they must be fed with a complex growth medium,
`
`containing sugars. amino acids, and growth factors, usually supplied from preparations of animal serum.
`
`Separation of the desired protein from the mixture of compounds fed to the cells and from the by-products of the
`
`cells themselves to a purity sufficient for use as a human therapeutic poses a formidable challenge.
`
`Procedures for purification of proteins from cell debris initially depend on the site of expression of the
`
`protein. Some proteins can be caused to be secreted directly from the cell into the surrounding growth media;
`
`others are made intracellularly. For the latter proteins. the first step of a purification process involves lysis of the
`
`cell. which can be done by a variety of methods. including mechanical shear, osmotic shock, or enzymatic
`
`treatments. Such disruption releases the entire contents of the cell into the homogenate. and in addition produces
`
`subcellular fragments that are difficult to remove due to their small size. These are generally removed by
`
`differential centrifugation or by filtration. The same problem arises. although on a smaller scale, with directly
`
`secreted proteins due to the natural death of cells and release of intracellular host cell proteins in the course of
`
`the protein production run.
`
`Once a clarified solution containing the protein of interest has been obtained. its separation from the
`
`other proteins produced by the cell
`
`is usually attempted using a combination of different chromatography
`
`techniques. These techniques separate mixtures of proteins on the basis of their charge, degree of hydrophobicity.
`
`or size. Several different chromatography resins are available for each of these techniques, allowing accurate
`
`tailoring of the purification scheme to the particular protein involved. The essence of each of these separation
`
`methods is that proteins can be caused either to move at different rates down a long column, achieving a physical
`
`separation that increases as they pass further down the column. or to adhere selectivelyto the separation medium,
`
`being then differentially eluted by different solvents. In some cases, the desired protein is separated from
`
`impurities when the impurities specifically adhere to the column. and the protein of interest does not, that is, the
`
`protein of interest is present in the "flow-through".
`
`Ion exchange chromatography is a chromatographictechnique that is commonly used for the purification
`
`of proteins. In ion exchange chromatography, charged patches on the surface of the solute are attracted by
`
`opposite charges attached to a chromatography matrix, provided the ionic strength of the surrounding buffer is
`
`low, Elution is generally achieved by increasingthe ionic strength (tie. conductivity) of the buffer to compete with
`
`the solute for the charged sites ofthe ion exchange matrix. Changing the pH and thereby altering the charge of
`
`l
`
`10
`
`15
`
`3O
`
`35
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`WO 99/57134
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`IO
`
`15
`
`20
`
`the solute is another way to achieve elution of the solute. The change in conductivity or pH may be gradual
`
`(gradient elution) or stepwise (step elution). In the past, these changes have been progressive; z'.e., the pH or
`
`conductivity is increased or decreased in a single direction.
`
`SUMMARY OF THE INVENTION
`
`The present invention provides an ion exchange chromatographic method wherein a polypeptide of
`
`interest is bound to the ion exchange material at an initial conductivity or pH and then the ion exchange material
`
`is washed with an intermediate buffer at a different conductivity or pH, or both. At a specific point followingthis
`
`intermediate wash, and contrary to ion exchange chromatography standard practice. the ion exchange material
`
`is washed with a wash buffer where the change in conductivity or pH, or both, from the intermediate buffer to
`
`the wash buffer is in an opposite direction to the change in conductivity or pH, or both. achieved in the previous
`
`steps. Only after washing with the wash buffer,
`
`is the ion exchange material prepared for the polypeptide
`
`molecule of interest to be eluted by the application of the elution buffer having a conductivity or pH, or both,
`
`which differ from the conductivity or pH, or both, ofthe buffers used in previous steps.
`
`This novel approach to ion exchange chromatography is particularly useful in situations where a product
`
`molecule must be separated from a very closely related contaminant molecule at full manufacturing scale, where
`
`both purity and high recovery of polypeptide product are desired.
`
`Accordingly,
`
`the invention provides a method for purifying a polypeptide from a composition
`
`comprising the polypeptide and a contaminant, which method comprises the following steps performed
`
`sequentially:
`
`(a) binding the polypeptide to an ion exchange material using a loading buffer, wherein the loading
`
`buffer is at a first conductivity and pH;
`
`(b) washing the ion exchange material with an intermediate buffer at a second conductivity and/or pH
`
`so as to elute the contaminant from the ion exchange material;
`
`(c) washing the ion exchange material with a wash buffer which is at a third conductivity and/or pH,
`
`wherein the change in conductivity and/or pH from the intermediate buffer to the wash buffer is in an opposite
`
`direction to the change in conductivity and/or pH from the loading buffer to the intermediate buffer; and
`
`(d) washing the ion exchange material with an elution buffer at a fourth conductivity and/or pH so as
`
`to elute the polypeptide from the ion exchange material. The first conductivity and/or pH may be the same as
`
`the third conductivity and/or pH.
`
`Where the ion exchange material comprises a cation exchange resin, the conductivity and/or pH ofthe
`
`intermediate buffer is/are preferably greater than the conductivity and/or pH of the loading buffer;
`
`the
`
`conductivity and/or pH of the wash buffer is/are preferably less than the conductivity and/or pH of the
`
`intermediate buffer: and the conductivity and/or pH of the elution buffer is/are preferably greater than the
`
`conductivity and/or pH of the intermediate buffer. Preferably, the conductivity and/or pH of the wash buffer is/are
`
`35
`
`about the same as the conductivity and/0r pH of the loading buffer.
`
`Preferably elution of the contaminant and of the polypeptide is achieved by modifying the conductivity
`
`of the intermediate buffer and of the elution buffer, respectively, while keeping the pH of these buffers
`
`approximately the same.
`
`The invention also provides a method for purifying a polypeptide from a composition comprising the
`2
`
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`WO 99/57134
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`PCT/US99/09637
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`polypeptide and a contaminant. which method comprises the following steps performed sequentially:
`
`(a) binding the polypeptide to a cation exchange material using a loading buffer. wherein the loading
`
`buffer is at a first conductivity and pH;
`
`(b) washing the cation exchange material with an intermediate buffer at a second conductivity and/or
`
`pH which is greaterthan that of the loading buffer so as to elute the contaminant from the ion exchange material;
`
`(c) washing the cation exchange material with a wash buffer which is at a third conductivity and/or pH
`
`which is less than that of the intermediate buffer; and
`
`(d) washing the cation exchange material with an elution buffer at a fourth conductivity and/or pH which
`
`is greater than that of the intermediate buffer so as to elute the polypeptide from the ion exchange material.
`
`In addition, the invention provides a method for purifying an antibody from a composition comprising
`
`the antibody and a contaminant. which method comprises loading the composition onto a cation exchange resin,
`
`wherein the amount of antibody loaded onto the cation exchange resin is from about 20mg to about 35mg ofthe
`
`antibody per mL of cation exchange resin and, optionally, further comprising eluting the antibody from the cation
`
`exchange resin. The method preferably further comprises an intermediate wash step for eluting one or more
`
`contaminants from the ion exchange resin. This intermediate wash step usually precedes the step of eluting the
`
`antibody.
`
`The invention further provides a composition comprising a mixture of anti-HER2 antibody and one or
`
`more acidic variants thereof, wherein the amount of the acidic variant(s) in the composition is less than about 25%
`
`and preferably less than about 20%. eg. in the range from about 1% to about 18%. Optionally, the composition
`
`further comprises a pharmaceutically acceptable carrier.
`
`Brief Description OfThe Drawings
`
`Figure l
`
`is a flow diagram showing how one could perform cation exchange chromatography by altering
`
`conductivity (e.g. to the NaCl concentrations of Example 1 below) or by altering pH (e.g. to the pH values as
`
`shown in the flow diagram).
`
`Figure 2 is a flow diagram showing how one could perform anion exchange chromatography by altering
`
`conductivity (e.g. to the NaCl concentrations as depicted in the figure) or by altering pH (e.g. to the pH values
`
`as shown).
`
`Figure 3 is an absorbance trace from a cation exchange chromatography run of Example 1 at full
`
`manufacturing scale. Points at which the column is washed with the different buffers described herein are marked
`
`10
`
`15
`
`20
`
`25
`
`30
`
`with arrows.
`
`Figure 4 depicts recombinant humanized anti-HERZ monoclonal antibody (rhuMAb HERZ) recovered
`
`in each chromatography fraction (calculated as the percentage of the sum total of all fractions of the relevant
`
`chromatography). Flow through, wash steps, and prepool fractions are all effluent samples collected from the
`
`onset of load to the initiation of pooling. The pool fraction is the five column volume effluent sample of elution
`
`35
`
`starting at the leading shoulder’s inflection point. The regeneration fraction contains effluent captured from the
`
`end of pooling to the end of regeneration.
`
`Figure 5 shows the quality of rhuMAb HERZ in each cation exchange chromatography pool sample as
`
`evaluated by carboxy sulfon cation exchange high pressure liquid chromatography (CSx HPIEX). Peaks a, b, and
`
`l are deamidated forms of rhuMAb HERZ. Peak 3 is nondeamidated rhuMAb HERZ. Peak 4 is a combination
`
`3
`
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`WO 99/57134
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`of C-terminal Lysine containing and iso-aspartate variants of rhuMAb HERZ.
`
`Figure 6 shows the absorbance (280 nm) profiles ofthe 0.025 M MES / 0.070 M NaCl, pH 5.6 wash for
`
`each chromatography. The mass of rhuMAb HER2 applied to the cation exchange resin effects the peak’s
`
`absorbance level at the apex as well as the amount of buffer required to reach the apex. Due to minor peaks which
`
`occur (as best seen in the 30 mg/mL load) in this wash, the apex is defined as absorbance levels of at least 0.5
`
`absorbance units (AU),
`
`Figures 7A and 7B show the amino acid sequences of humMAb4D5-8 light chain (SEQ ID NO: 1) and
`
`humMAb4D5-8 heavy chain (SEQ ID N022), respectively.
`
`Detailed Description Of The Preferred Embodiments
`
`10
`
`Definitions:
`
`15
`
`25
`
`30
`
`The “composition” to be purified herein comprises the polypeptide of interest and one or more
`
`contaminants. The composition may be “partially purified” (Le. having been subjected to one or more purification
`
`steps, such as Protein A Chromatography as in Example 1 below) or may be obtained directly from a host cell
`
`or organism producing the polypeptide (e. g. the composition may comprise harvested cell culture fluid).
`
`As used herein, "polypeptide" refers generally to peptides and proteins having more than about ten
`
`amino acids. Preferably, the polypeptide is a mammalian protein, examples of which include renin; a growth
`
`hormone. including human growth hormone and bovine growth hormone; growth hormone releasing factor;
`
`parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha- 1 -antitrypsin; insulin A-chain; insulin B—
`
`chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors such
`
`as factor VlllC; factor IX, tissue factor, and von Willebrands factor; anti-clotting factors such as Protein C; atrial
`
`natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or human urine or tissue-type
`
`plasminogen activator (t-PA); bombesin; thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and
`
`-beta; enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); human
`
`macrophage inflammatory protein (MlP-l-alpha); a serum albumin such as human serum albumin; Muellerian-
`
`inhibiting substance; relaxin A—chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; a
`
`microbial protein, such as beta-lactamase; DNase; lgE; a cytotoxic T-lymphocyte associated antigen (CTLA),
`
`such as CTLA-4; inhibin; activin: vascular endothelial growth factor (VEGF); receptors for hormones or growth
`
`factors; Protein A or D; rheumatoid factors; a neurotrophic factor such as bone-derived neurotrophic factor
`
`(BDNF), neurotrophin-S, -4, -5, or —6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF-B;
`
`platelet-derived growth factor (PDG F); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor
`
`(EGF); transforming growth factor (TG F) such as TG F-alpha and TGF-beta, including TGF-B l, TGF-BZ, TG F-
`
`133, TOP-[34, or TGF-BS; insulin-like growth factor-1 and -II (IGF-I and lGF-lI); des(l-3)—IGF-1 (brain IGF-I),
`
`insulin-like growth factor binding proteins (IGFBPs); CD proteins such as CD3, CD4, CD8, CD19 and CD20;
`
`erythropoietin; osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); an interferon such
`
`as interferon~alpha, -beta, and -gamma; colony stimulating factors (CSFs), e. g., M~CSF, GM-CSF, and G-CSF;
`
`interleukins (ILs), e. g., lL-l to lL—10;superoxide dismutase; T-cell receptors; surface membrane proteins; decay
`
`accelerating factor; viral antigen such as, for example, a portion of the AIDS envelope; transport proteins: homing
`
`receptors; addressins: regulatory proteins; integrins such as CD1 la, CD1 lb, CD1 1c, CD18, an ICAM, VLA-4
`
`and VCAM; a tumor associated antigen such as HER2, HER3 or HER4 receptor; and fragments and/or variants
`4
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`of any ofthe above~listed polypeptides. Most preferred is a full length antibody that binds human HER2.
`
`A “contaminant” is a material that is different from the desired polypeptide product. The contaminant
`
`may be a variant of the desired polypeptide (e. g. a deamidated variant or an amino-aspartate variant of the desired
`
`polypeptide) or another polypeptide. nucleic acid. endotoxin etc.
`
`A "variant" or "amino acid sequence variant" of a starting polypeptide is a polypeptide that comprises
`
`an amino acid sequence different from that of the starting polypeptide. Generally, a variant will possess at least
`
`80% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity,
`
`and most preferably at least 98% sequence identity with the native polypeptide. Percentage sequence identity is
`
`determined, for example, by the Fitch et al., Proc. Natl. Acad. Sci. USA 80: 1382-1386 (1983), version of the
`
`l0
`
`15
`
`algorithm described by Needleman et al., J. Mol. Biol. 48:443-453 (1970), after aligning the sequences to provide
`
`for maximum homology. Amino acid sequence variants of a polypeptide may be prepared by introducing
`
`appropriate nucleotide changes into DNA encoding the polypeptide, or by peptide synthesis. Such variants
`
`include. for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid
`
`sequence of the polypeptide of interest. Any combination of deletion, insertion. and substitution is made to arrive
`
`at the final construct. provided that the final construct possesses the desired characteristics. The amino acid
`
`changes also may alter post-translational processes ofthe polypeptide. such as changing the number or position
`
`of glycosylation sites. Methods for generating amino acid sequence variants of polypeptides are described in US
`
`Pat 5,534,615, expressly incorporated herein by reference, for example.
`
`An “acidic variant” is a variant ofa polypeptide ofinterest which is more acidic (e.g. as determined by
`
`cation exchange chromatography) than the polypeptide of interest. An example of an acidic variant is a
`deamidated variant.
`
`A “deamidated” variant of a polypeptide molecule is a polypeptide wherein one or more asparagine
`
`residue(s) ofthe original polypeptide have been converted to aspartate, Le. the neutral amide side chain has been
`
`converted to a residue with an overall acidic character. Deamidated humMAb4D5 antibody from the Example
`
`below has Asn30 in CDRl of either or both of the VL regions thereof converted to aspartate. The term
`
`"deamidated human DNase" as used herein means human DNase that is deamidated at the asparagine residue that
`
`occurs at position 74 in the amino acid sequence of native mature human DNase (US Patent 5,279,823; expressly
`
`incorporated herein by reference).
`
`The term "mixture" as used herein in reference to a composition comprising an anti-HERZ antibody,
`
`means the presence of both the desired anti-HER2 antibody and one or more acidic variants thereof. The acidic
`
`variants may comprise predominantly deamidated anti-HER2 antibody, with minor amounts of other acidic
`
`variant(s). It has been found, for example, that in preparations of anti-HER2 antibody obtained from recombinant
`
`expression, as much as about 25% of the anti-HER2 antibody is deamidated.
`
`In preferred embodiments of the invention,
`
`the polypeptide is a recombinant polypeptide. A
`
`35
`
`“recombinant polypeptide” is one which has been produced in a host cell which has been transformed or
`
`transfected with nucleic acid encoding the polypeptide, or produces the polypeptide as a result of homologous
`
`recombination. "Transformation" and "transfection" are used interchangeably to refer to the process of
`
`introducing nucleic acid into a cell. Following transformation or transfection, the nucleic acid may integrate into
`
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`the host cell genome, or may exist as an extrachromosomalelement. The “host cell” includes a cell in in vitro cell
`
`culture as well a cell within a host animal. Methods for recombinant production of polypeptides are described
`
`in US Pat 5,534,615, expressly incorporated herein by reference, for example.
`
`The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies
`
`(including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e. g., bispecific
`
`antibodies), and antibody fragments so long as they exhibit the desired biological activity.
`
`The antibody herein is directed against an “antigen” of interest. Preferably, the antigen is a biologically
`
`important polypeptide and administration of the antibody to a mammal suffering from a disease or disorder can
`
`result in a therapeutic benefit in that mammal. However, antibodies directed against nonpolypeptide antigens
`
`(such as tumor-associatedglycolipid antigens; see US Patent 5,091 , l 78) are also contemplated. Where the antigen
`
`is a polypeptide, it may be a transmembrane molecule (e. g. receptor) or ligand such as a growth factor. Exemplary
`
`antigens include those polypeptides discussed above. Preferred molecular targets for antibodies encompassed by
`
`the present invention include CD polypeptides such as CD3, CD4, CD8, CD19, CD20 and CD34; members of
`
`the HER receptor family such as the EGF receptor, HER2, HER3 or HER4 receptor; cell adhesion molecules such
`
`as LFA-l, Macl, p150.95, VLA—4, ICAM-l, VCAM and av/b3 integrin including either a or b subunits thereof
`
`(e.g. anti-CD1 la, anti—CD1 8 or anti-CD1 lb antibodies); growth factors such as VEGF; IgE; blood group antigens;
`
`flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; polypeptide C etc. Soluble antigens or fragments
`
`thereof, optionally conjugated to other molecules, can be used as immunogens for generating antibodies. For
`
`transmembrane molecules, such as receptors, fragments of these (e. g. the extracellular domain of a receptor) can
`
`be used as the immunogen. Alternatively, cells expressing the transmembrane molecule can be used as the
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`immunogen. Such cells can be derived from a natural source (e.g. cancer cell lines) or may be cells which have
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`been transformed by recombinant techniques to express the transmembrane molecule.
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`The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of
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`substantially homogeneous antibodies. i.e., the individual antibodies comprising the population are identical
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`except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies
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`are highly specific. being directed against a single antigenic site. Furthermore,
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`in contrast to conventional
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`(polyclonal) antibody preparations which typically include different antibodies directed against different
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`determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The
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`modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially
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`homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any
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`particular method. For example, the monoclonal antibodies to be used in accordance with the present invention
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`may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made
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`by recombinant DNA methods (see, e.g., US. Patent No. 4,816,567). In a further embodiment, “monoclonal
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`antibodies” can be isolated from antibody phage libraries generated using the techniques described in McCafferty
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`et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol,
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`222158 1-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries.
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`Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling
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`(Marks et al., Bio/Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination
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`as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266
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`(1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques
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`for isolation of monoclonal antibodies. Alternatively, it is now possible to produce transgenic animals (e. g., mice)
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`that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of
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`endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of
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`the antibody heavy-chain joining region (1“) gene in chimeric and germ-line mutant mice results in complete
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`inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in
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`such germ—line mutant mice will result in the production of human antibodies upon antigen challenge. See, e. g.,
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`Jakobovits et al., Proc. Natl. Acad. Sci. USA, 902255] (1993); Jakobovits et al., Nature, 362:255-258 (1993);
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`Bruggerrnann et a1., Year in lmmun0., 7:33 (1993); and Duchosal et al. Nature 3552258 (1992).
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`The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in
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`which a portion ofthe heavy and/or light chain is identical with or homologous to corresponding sequences in
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`antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the
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`remainder ofthe chain(s) is identical with or homologous to corresponding sequences in antibodies derived from
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`another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so
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`long as they exhibit the desired biological activity (US. Patent No. 4,816,567; and Morrison et al., Proc. Natl.
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`Acaa’. Sci. USA 81:6851-6855 (1984)).
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`The term “hypervariable region” when used herein refers to the amino acid residues of an antibody
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`which are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a
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`“complementaritydetermining region” or “CDR” (LC. residues 24-34 (L1 ), 50-56 (1.2) and 89-97 (L3) in the light
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`chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat
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`et a1., Sequences of Polypeptides of Immunological Interest, 5th Ed. Public Health Service, National Institutes
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`of Health. Bethesda. MD. (1991)) and/or those residues from a “hypervariable loop” (Le. residues 26-32 (L1),
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`50-52 (L2) and 9 1 -96 (L3) in the light chain variable domain and 26—32 (H1), 53-55 (H2) and 96-101 (H3) in the
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`heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-9 17 (1987)). "Framework" or "FR" residues
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`are those variable domain residues other than the hypervariable region residues as herein defined. The CDR and
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`FR residues ofthe rhuMAb HER2 antibody ofthe example below (humAb4D5-8) are identified in Caner et al.,
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`Proc. Natl. Acaa’. Sci. USA, 8914285 (1992).
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`"Humanized" forms of non-human (e. g., murine) antibodies are chimeric antibodies that contain minimal
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`sequence derived from non—human immunoglobulin. For the most part, humanized antibodies are human
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`immunoglobulins(recipient antibody) in which residues from a hypervariable region ofthe recipient are replaced
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`by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or
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`nonhuman primate having the desired specificity, affinity, and capacity. In some instances, Fv framework region
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`(FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore,
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`humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor
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`antibody. These modifications are made to further refine antibody performance. In general, the humanized
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`antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or
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`substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or
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`substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody
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`optionally also will comprise at least a portion of an immunoglobulin constant region (Fc). typically that ofa
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`human immunoglobulin.
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`The choice of human variable domains, both light and heavy, to be used in making the humanized
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`antibodies is very important to reduce antigenicity. According to the so-called "best—fit" method, the sequence
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`of the variable domain of a rodent antibody is screened against the entire library of known human variable-
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`domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human
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`framework (FR) for the humanized antibody (Sims et al., J. Immunol, 151 :2296 (1993); Chothia et al., J. Mol.
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`Biol., 1962901 (1987)).
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`Another method uses a particular framework derived from the consensus sequence of all human
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`antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several
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`different humanized antibodies (Carter 2! (1]., Proc. Natl. Acaa’. Sci. USA, 89:4285 (1992); Presta et al., J.
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`lmmnol.. 15122623 (1993)).
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`It is further important that antibodies be humanized with retention of high affinity for the antigen and
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`other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies
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`are prepared by a process of analysis of the parental sequences and various conceptual humanized products using
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`three-dimensionalmodels ofthe parental and humanized sequences. Three-dimensional immunoglobulin models
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`are commonly available and are familiar to those skilled in the art. Computer programs are available which
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`illustrate and di