(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`15 March 2012 (15.03.2012) (10) International Publication Number
`
`(43) International Publication Date
`
`WO 2012/033953 A1
`
`
`International Patent Classification:
`C07K 16/30 (2006.01)
`GolN 33/574 (2006.01)
`G01N 33/53 (2006.01)
`G01N 33/68 (2006.01)
`
`(81)
`
`International Application Number:
`PCT/US201 1/050891
`
`International Filing Date:
`8 September 2011 (08.09.2011)
`
`Filing Language:
`
`Publication Language:
`
`English
`
`English
`
`Priority Data:
`61/402,979
`
`8 September 2010 (08.09.2010)
`
`US
`
`except US):
`all designated States
`Applicant Uor
`HALOZYME, INC.
`[US/US]; 11388 Sorrento Valley
`Road, San Diego, CA 92121 (US).
`
`Inventors; and
`Inventors/Applicants (for US only): KODANDAPANI,
`Lalitha [US/US]; 6578 Dandelion Way, San Diego, CA
`92130 (US). BOOKBINDER, Louis, Howard [US/US];
`14823 Gable Ridge Rd., San Diego, CA 92128 (US).
`FROST, Gregory, Ian [US/US]; 13662 Mercado Drive,
`Del Mar, CA 92014 (US). SHERIDAN, Philip, Lee [US/
`US]; 13289 Deer Canyon Place, San Diego, CA 92129
`(US). SHEPARD, Harold, Michael
`[US/US]; 8124
`Montrose Way, San Diego, CA 92122 (US). WEI, Ge
`[US/US]; 11530 Miro Circle, San Diego, CA 92131 (US).
`HUANG, Lei [CN/US]; 3683 Caminito Carmel Landing,
`San Diego, CA 92130 (US).
`
`(74)
`
`Agents: SEIDMAN, Stephanie L. et a1.; MCKENNA
`LONG & ALDRIDGE LLP, 4435 Eastgate Mall, Suite
`400, San Diego, CA 92121 (US).
`
`Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ,
`CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO,
`DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP,
`KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD,
`ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI,
`NO, NZ, OM, PE, PG, PH, PL, PT, QA, RO, RS, RU,
`RW, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ,
`TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA,
`ZM, ZW.
`
`(84)
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GII,
`GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG,
`ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ,
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`EE, ES, FI, FR, GB, GR, IIR, IIU, IE, IS, IT, LT, LU,
`LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK,
`SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
`GW, ML, MR, NE, SN, TD, TG).
`Declarations under Rule 4.17 :
`
`as to the applicant’s entitlement to claim the priority of
`the earlier application (Rule 4.1 7(iii))
`Published:
`
`with international search report (Art. 21(3))
`
`before the expiration of the time limit for amending the
`claims and to be republished in the event of receipt of
`amendments (Rule 48.2(h))
`
`with sequence listingpart ofdescription (Rule 5.2(a))
`
`(51)
`
`(21)
`
`(22)
`
`(25)
`
`(26)
`
`(30)
`
`(71)
`
`(72)
`(75)
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`
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`W02012/033953A1|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
`
`(54) Title: METHODS FOR ASSESSING AND IDENTIFYING OR EVOLVING CONDITIONALLY ACTIVE THERAPEU—
`TIC PROTEINS
`
`(57) Abstract: Methods for evolving or selecting therapeutic proteins with reduced adverse side-effects and the resulting proteins
`are provided. For example, provided herein is an in vitro assay to identify conditionally active therapeutic proteins that exhibit
`better activity within one in viva environment compared to another in vivo environment. The methods include the steps of a) test-
`ing the activity of a protein under conditions in which normal or increased activity is desired; b) testing the activity of the protein
`under conditions in which reduced activity compared to normal is desired; and c) comparing the activity in a) with b) and select-
`ing/identifying a protein that has greater activity in a) compared to b). The selected/identified protein is a conditionally active pro—
`tein.
`
`

`

`WO 2012/033953
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`PCT/US2011/050891
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`METHODS FOR ASSESSING AND IDENTIFYING OR EVOLVING
`
`CONDITIONALLY ACTIVE THERAPEUTIC PROTEINS
`
`RELATED APPLICATIONS
`
`Benefit of priority is claimed to US. Provisional Application Serial No.
`
`61/402,979, entitled “METHODS FOR ASSESSING AND IDENTIFYING 0R
`
`EVOLVING CONDITIONALLY ACTIVE THERAPEUTIC PROTEINS AND
`
`CONDITIONALLY ACTIVE THERAPEUTIC PROTEINS,” filed on September 8,
`
`2010 to Laiitha Kodandapani, Philip Lee Sheridan, Harold Michael Shepard, Louis H.
`
`Bookbinder and Gregory 1. Frost.
`
`10
`
`FIELD OF THE INVENTION
`
`Methods for evolving or selecting therapeutic proteins with reduced adverse
`
`side-effects and the resulting proteins are provided.
`
`BACKGROUND
`
`Proteins have a role as pharmaceutical or therapeutic agents for the treatment
`
`15
`
`of a wide range of human diseases, such as cancer, hemophilia, anemia and diabetes,
`
`and for a number of diseases are the only effective treatment. As such, there is a need
`
`to identify protein therapeutics with altered or improved activities or properties. It is
`
`an object herein to provide a method to identify or generate such proteins.
`
`SUMMARY
`
`20
`
`Provided are methods for identifying/selecting conditionally active proteins.
`
`In the method, the activity of the protein is tested under conditions in which normal or
`
`increased activity is desired, and the activity of the protein is tested under conditions
`
`in which reduced activity compared to normal is desired. The activity of the protein
`
`under conditions in which normal or increased activity is desired can be compared to
`
`25
`
`the activity of the protein under conditions in which reduced activity compared to
`
`normal is desired. Proteins can be selected and/or identified that have greater activity
`
`under conditions in which normal or increased activity is desired compared to
`
`conditions in which reduced activity compared to normal is desired.
`
`In the methods, the activity of the protein under conditions in which reduced
`
`30
`
`activity compared to normal is desired can be reduced compared to normal. In the
`
`methods, the conditions in which normal or increased activity is desired and the
`
`conditions in which reduced activity compared to normal is desired can be identical,
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`except for a condition or conditions that render the protein conditionally active. In the
`
`methods herein, the activity that is tested can be binding to a target of the protein.
`
`The target can be immobilized on a solid support. In the methods herein, binding can
`
`be assessed by an immunoassay. Immunoassays include ELISA immunoassays,
`
`heterogeneous immunoassays and homogeneous immunoassays.
`
`In the methods herein, the conditions in which normal or increased activity of
`
`the protein is desired can simulate a disease microenvironment, and the conditions in
`
`which reduced activity compared to normal is desired can simulate a healthy tissue
`
`environment. Exemplary of a healthy tissue environment is a non-tumor tissue
`
`10
`
`environment, such as a systemic environment or a healthy tissue. Exemplary of
`
`healthy tissues are the GI tract, the skin, the vasculature, the blood, and the
`
`extracellular matrix. Exemplary of diseased microenvironments is a tumor
`
`microenvironment. A tumor or disease microenvironment can have lower than
`
`neutral pH or lower pH than a healthy tissue microenvironment. A tumor or disease
`
`15
`
`microenvironment can include one or more of increased vascularization, hypoxia,
`
`lowered pH, increased interstitial fluid pressure, altered metabolites or metabolism
`
`indicative of a tumor or other disease. For example, a tumor or other disease
`
`microenvironment can have elevated lactate concentration and/or increased pyruvate
`
`compared to a healthy microenvironment.
`
`20
`
`Also provided herein are methods in which conditions in which normal or
`
`'
`
`increased activity of the protein is desired can include lower than neutral pH and
`
`elevated lactic acid compared to the conditions in which reduced activity compared to
`
`normal is desired.
`
`In the methods herein, the protein tested can be a therapeutic protein and/or a
`
`25
`
`protein with undesirable side effects manifested in healthy tissue. In the methods
`
`herein, reducing the activity of the protein under conditions in which reduced activity
`compared to normal is desired can ameliorate or prevent the undesirable side-effects.
`
`In the methods provided herein, the activity of the protein can be tested in the
`
`presence of human serum. Human serum can be present in an amount that simulates
`
`30
`
`physiological conditions, and the amount of serum present under conditions in which
`
`normal or increased activity is desired can be the same as the amount of serum present
`
`under conditions in which reduced activity compared to normal is desired. For
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`For example, the methods provided herein can be performed in the presence of at
`
`least about or at any of 3 %—30 %, inclusive, or S%-30%, inclusive, or S%-25%,
`
`inclusive, 10%-30 %, inclusive, or 15%-30%, inclusive, or 15%—25%, inclusive, of
`
`human serum by volume, including at or about 25% (plus or minus 10%) of human
`
`serum by volume.
`
`In the methods provided herein, a plurality of proteins can be tested under
`
`conditions in which normal or increased activity is desired and under conditions in
`
`which reduced activity compared to normal is desired. In this particular method, each
`
`protein is tested under both conditions, and any proteins that have greater activity
`
`10
`
`under conditions in which normal or increased activity is desired than under
`
`conditions in which reduced activity compared to normal is desired can be selected.
`
`In some examples, the activity is greater by a predetermined amount or ratio. For
`
`example, the activity is increased by at least 5%, 10%, 15% , 20%, 25%, 35%, 50%,
`
`100%, 2-fold, 5-fold, 10-fold, 20—fold or more.
`
`15
`
`In the methods provided herein, the target protein can be a receptor or a
`
`portion thereof that binds to a ligand. Exemplary of a target protein is a receptor that
`
`is a tumor antigen. For example, the target protein is a member of the Her family of
`
`receptors or the target protein is the EGFR receptor or the extracellular domain
`
`thereof. In the methods provided herein, the protein whose activity is tested (the
`
`20
`
`tested protein) can be a therapeutic protein that treats a tumor or other disease. In
`
`some examples, the therapeutic protein is an antibody, an enzyme, a hormone, a
`
`cytokine or active portion thereof, and reference to an antibody herein refers to an
`
`antibody or antigen-binding fragment thereof. In other examples of the method
`
`provided herein, the proteins can be modified variants of a therapeutic protein.
`
`25
`
`Exemplary of a therapeutic protein is a ligand for a target receptor. In some examples
`
`of the method provided herein, the protein contains a multimerization domain, such
`
`as, for example, a multimerization domain that contains an Fc domain or modified Fc
`
`domain. In exemplary methods, the therapeutic protein is an antibody, an enzyme, a
`
`hormone or a cytokine. For example, the therapeutic protein is an antibody.
`
`30
`
`In the methods provided herein, the protein tested in the method can be an
`
`anti-tumor antibody, selected from among those listed in Table 5 (the table(s) in the
`
`specification that list exemplary anti-tumor antibodies). In some examples, the anti-
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`tumor antibody exhibits undesirable side effects in healthy tissues. For example, the
`
`antibody is an anti-EGFR antibody or an anti-CTLA4 antibody that exhibits
`
`undesirable side effects in healthy tissues.
`
`In other examples of the method provided herein, the proteins can be modified
`
`variants of a therapeutic protein. The modified variants can contain amino acid
`
`replacements, insertions and/or deletions. In some examples, a collection of variants
`
`are tested. In some examples, each variant differs from the wildtype or unmodified
`
`protein and all other variants by a single amino acid.
`
`In other examples, each variant
`
`contains two, three, four, five, six, seven, eight, nine or more different amino acids
`
`10
`
`from the unmodified or wildtype protein. In the methods provided herein, in the
`
`collection of variants, the amino acid at each changed position is replaced by up to l-
`
`19 other amino acids than the original amino acid. In other examples, histidine is a
`
`replacing amino acid or the histidines in the protein are replaced by a non-basic or
`
`uncharged amino acid. Each variant protein can be tested individually. For example,
`
`15
`
`each variant protein can be tested in a high throughput format or an automated
`
`method.
`
`In the methods provided herein, the selected protein can be conditionally
`
`active such that it has greater activity in the tumor or other disease microenvironment
`
`compared to the non-tumor environment. The methods provided herein can be
`
`20
`
`repeated a plurality of times, wherein in each repetition, further variants of a selected
`
`protein or proteins are tested, whereby the therapeutic protein is evolved to exhibit
`
`reduced toxicity or adverse side-effects. In the methods provided herein, variant
`
`proteins can be produced by expression from a vector that contains a nucleic acid
`
`molecule encoding a variant protein.
`
`25
`
`In some examples of the methods provided herein, the protein that is tested is
`
`a variant antibody that contains one or more amino acid replacements in a CDR. In
`
`specific examples, every amino acid along the length of the protein or a selected
`
`portion thereof is replaced, one-by-one with up to 19 other amino acids. In other
`
`examples, the protein is an antibody and the selected portion is a CDR.
`
`30
`
`In one example, the therapeutic protein is an anti-EGFR antibody and the
`
`reduced adverse side effects are reduced dermal toxicities associated with systemic
`
`exposure to the antibody. In some examples of the methods provided herein, the pH of
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`the tumor or other disease microenvironment is about or is 5.8-6.8, inclusive. In other
`
`examples, the selected protein is anti-EGFR antibody that preferentially binds to EGFR
`
`within the tumor microenvironment of reduced pH of 5.8-6.8 and lactate
`
`concentrations of about 12-20 mM compared to a normal physiologic pH of 7.3-7.4
`
`and normal lactate concentrations below 12 mM.
`
`Provided herein is a method for identifying a conditionally active protein in
`
`which the method is performed by contacting a solid support coated with EGFR or the
`
`EGFR ECD with buffer at about pH 7.3-7.4 containing 1 mM lactic acid and about
`
`25% human serum; contacting a second duplicate support with buffer at about pH 6
`
`10
`
`containing 12-20 mM, such as 16.6 mM, lactic acid and about 25% human serum;
`
`washing the supports with the corresponding buffer (pH 6.0 or pH 7.4); binding anti-
`
`EGFR-tagged, such as FLAG-tagged, standard in either the pH 7.4 buffer with lactic
`
`acid and human serum, or the pH 6.0 buffer with lactic acid and human serum to the
`
`corresponding support; and detecting binding of the anti-EGFR to the EGFR by
`
`15
`
`adding goat-anti—Tag-enzyme, such as horse radish peroxidase (HRP), in the
`corresponding buffer and enzyme substrate to detect or quantitate binding of the anti-
`
`EGFR to each support.
`
`Also provided herein are therapeutic proteins that are selected/identified or
`
`evolved by any of the methods provided herein. Also provided herein is a variant
`
`20
`
`anti-tumor antibody that exhibits reduced dermal toxicity compared to an unmodified
`
`antibody. Also provided herein is an anti-EGFR antibody that exhibits reduced
`
`dermal toxicity compared to Erbitux.
`
`Provided are methods for identifying/selecting a therapeutic protein that treats
`
`tumors and that is more active in low pH than at neutral pH. In the method, the
`
`25
`
`activity of the protein is tested under conditions the comprise a low pH, and the
`activity ofthe protein is tested under conditions that comprise a neutral pH. The
`activity of the protein under conditions that comprise low pH can be compared to the
`
`activity of the protein under conditions that comprise neutral pH. Proteins can be
`
`selected and/or identified that are more active at low pH than at high pH. Low pH can
`be any pH that is less.than 7.4, such as between or about between 5.8 to 6.8. Neutral
`
`30
`
`pH can also be any pH is or is about between 7.2 to 7.6, such as 7.4.
`
`5 .
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`RECTIFIED SHEET (RULE 91) ISA/EP
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`In the methods, conditions that comprise low pH can include one or more
`
`conditions selected from among increased lactate concentration, increased pyruvate
`
`concentration and hypoxia. For example, the conditions that comprise low pH can
`
`include increased lactate concentration selected from among 10 mM to 20 mMlactic
`
`acid or 15 mM to 18 mM lactic acid; or at least about or 16 mM, 16.5 mM or 17 mM
`
`lactic acid. In the methods, conditions that comprise neutral pH can include a lactate
`
`concentration selected from among 0.5 to 5 mM or 0.2 mM to 4 mM lactic acid; or at
`
`or about 0.5, l, 2, 3, 4, or 5 mM lactic acid.
`
`Also provided herein are methods for identifying/selecting a therapeutic
`
`10
`
`protein that is more active in a tumor microenvironment than in a non-tumor
`
`microenvironment. In the method, the activity of the protein is tested under a
`
`condition that exists in a tumor microenviromnent but not a non-tumor environment in
`
`which activity is desired, and the activity of the protein is tested under a condition that
`
`exists in a non-tumor microenvironment. The activity of the protein under a condition
`
`15
`
`that exists in a tumor microenvironment can be compared to the activity of the protein
`
`under a condition that exists in a non-tumor microenvironment. Proteins can be
`
`selected and/or identified that have greater activity under a condition that exists in a
`
`tumor microenvironment compared to under a condition that exists in a non-tumor
`
`microenvironment, thereby identifying a protein that is more active in a tumor
`
`20
`
`microenvironment than in a non-tumor microenvironment. The non-tumor
`
`microenvironment can be a systemic microenvironment and/or a healthy tissue, such
`
`as the gastrointestinal (GI) tract, the skin, the vasculature, the blood or the
`
`extracellular matrix.
`
`The testing of the activity of the protein under conditions that comprise low
`
`25
`
`pH and under conditions that comprise neutral pH can be performed under identical
`
`conditions, except for a condition or conditions that exists in a tumor
`
`microenvironment but not in a non-tumor microenvironment. Exemplary of
`
`conditions that exist in a tumor microenvironment include one or more properties
`
`such as increased vaseularization, hypoxia, lowered pH, increased lactate
`concentration, increased pyruvate concentration, increased interstitial fluid pressure
`
`30
`
`and altered metabolites or metabolism indicative of a tumor.
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`The conditions that exist in a tumor microenvironment can include lower than
`
`neutral pH or lower pH than the non-tumor microenvironment. For example, the
`
`condition that exists in a tumor microenvironment can be a pH below 7.4. In some
`
`examples, the pH of the tumor is‘about or is 5.8—6.8, inclusive, and the condition that
`
`exists in a tumor microenvironment is a pH between or about between 5.8 to 6.8. The
`
`conditions that exist in a tumor microenvironment can include elevated lactate
`
`concentration and/or increased pyruvate compared to the conditions that exist in a
`
`non-tumor microenvironment.
`
`The condition in which the protein is tested that exists in a tumor
`
`10
`
`microenvironment but not a non-tumor environment in which activity is desired, can
`
`include lower than neutral pH and elevated lactic acid concentration compared to the
`
`conditions in which the protein is tested that includes a condition that exists in a non-
`
`tumor microenvironment. For example, the lower than neutral pH can be between 5.8
`
`and 6.8, inclusive, or 5.8 and 6.5, inclusive. The condition in which the protein is
`
`15
`
`tested that exists in a tumor microenvironment but not a non-tumor environment in
`
`which activity is desired can include increased lactate concentration selected from
`
`among 10 mM to 20 mM lactic acid or 15 mM to 18 mM lactic acid; or at least about
`
`or 16 mM, 16.5 mM or 17 mM lactic acid. The condition in which the protein is
`
`tested that exists in a non-tumor microenvironment can include a lactate concentration
`
`20
`
`selected from among 0.5 to 5 mM or 0.2 mM to 4 mM lactic acid; or at or about 0.5,
`
`l, 2, 3, 4, or 5 mM lactic acid. In some examples of the methods, the protein is a
`
`therapeutic protein that treats a tumor. In some examples, a therapeutic protein is an
`
`antibody, an enzyme, a hormone, a cytokine or active portion thereof. Reference
`
`herein to an antibody herein includes an antibody or antigen-binding fragment thereof.
`
`25
`
`In some examples, the therapeutic protein is a ligand for a target receptor, and/or an
`
`anti-tumor antibody.
`
`Anti-tumor antibodies for use in the methods provided herein include
`
`Cetuxirnab (Erbitux®), Trastuzurnab (Herceptin®), Rituximab (Rituxan®,
`
`MabThera®), Bevacizumab (Avastin®), Alemtuzumab (Campath®), Panitumumab
`
`30
`
`(Vectibix®), Ranibizumab (Lucentis®), Ibritumomab, lbritumomab tiuxetan
`
`(Zevalin®), Tositumomab, Iodine 1131 Tositumomab (BEXXAR®), Catumaxomab
`
`(Removab®), Gemtuzumab, Gemtuzumab ozogamicine (Mylotarg®), Abatacept
`
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`(CTLA4-Ig, 0rencia®), Belatacept (L104EA29YIg; LEA29Y; LEA), Ipilimumab
`
`(MDX-OIO, MDX-lOl), Tremelimumab (ticilimumab, CP—675,206), PRS-OlO, PRS-
`
`050, Aflibercept (VEGF Trap, AVEOOS), Volociximab (M200), F200, MORAb-009,
`
`SSlP (CAT-5001), Cixutumumab (IMC-A12), Matuzumab (EMD72000),
`
`Nimotuzumab (h-R3), Zalutumumab (HuMax—EGFR), Necitumumab IMC-l IFS,
`
`mAb806/ch806, Sym004 and mAb-425. In some examples, the anti-tumor antibody
`
`is Cetuximab (Erbitux®).
`
`The methods provided herein can be performed in vitro or in vivo.
`
`In the methods provided, a plurality of proteins can be tested, and proteins that
`
`10
`
`have greater activity in low pH conditions compared to neutral pH can be selected. In
`
`the methods provided, a plurality of proteins can be tested and proteins that have
`
`greater activity under a condition that exists in a tumor microenvironment than a non-
`
`tumor microenvironment can be selected. The plurality of proteins can include
`
`modified variants of a therapeutic protein, and a collection of variants can be tested.
`
`15
`
`The therapeutic proteins can include a multimerization domain, and the
`
`multimerization domain can include an Fc domain or modified Fc domain. A
`
`therapeutic protein can be an antibody (including an anti-tumor antibody), an enzyme,
`
`a hormone or a cytokine. In the methods provided, an anti-tumor antibody can be
`
`selected from among Cetuximab (Erbitux®), Trastuzumab (Herceptin®), Rituximab
`
`20
`
`(Rituxan®, MabThera®), Bevacizumab (Avastin®), Alemtuzumab (Campath®),
`
`Panitumumab (Vectibix®), Ranibiiumab (Lucentis®), Ibritumomab, Ibritumomab
`
`tiuxetan (Zevalin®), Tositumomab, Iodine 1131 Tositumomab (BEXXAR®),
`
`Catumaxomab (Removab®), Gemtuzumab, Gemtuzumab ozogamicine (Mylotarg®),
`
`Abatacept (CTLA4-Ig, Orencia®), Belatacept (L104EA29YIg; LEA29Y; LEA),
`
`25
`
`Ipilimumab (MDX-OIO, MDX-101), Tremelimumab (ticilimumab, CP-675,206),
`
`PRS-OIO, PRS-050, Aflibercept (VEGF Trap, AVEOOS), Volociximab (M200), F200,
`
`MORAb-009, SSlP (CAT-5001), Cixutumumab (IMC—A12), Matuzumab
`
`(EMD72000), Nimotuzumab (h-R3), Zalutumumab (HuMax-EGFR), Necitumumab
`
`IMC-I 1F8, mAb806/ch806, Sym004 and mAb-425.
`
`30
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`Modified variants of a therapeutic protein or plurality of therapeutic proteins
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`can include amino acid replacements, insertions and/or deletions of an amino acid
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`U]
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`residue or residues compared to an unmodified form of the therapeutic protein. For
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`example, each variant protein can contain a single amino acid replacement compared
`
`to an unmodified form of the therapeutic protein. Each variant protein can contain
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`two, three, four, five, six, seven, eight, nine or more amino acid replacements
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`compared to an unmodified form of a variant protein, such as a therapeutic antibody.
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`In some examples, the protein that is tested is a variant antibody that
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`comprises one or more amino acid replacements in a complementarity determining
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`region (CDR) compared to an unmodified form of the antibody.
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`In the methods provided, variants of a therapeutic protein can be tested that
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`include replacement of the amino acid at each changed position by up to 1-19 other
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`amino acids than the original amino acid at the position, and every amino acid can be
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`replaced along the length of the therapeutic protein, or a selected portion thereof.
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`Provided are methods in which a modified protein is an antibody and the selected
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`portion that is modified is a CDR.
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`Replacement amino acids can be selected from among Ala, Arg, Asn, Asp,
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`Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val, with
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`the proviso that the replacement amino acid differs from the amino acid at the
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`corresponding position in the therapeutic protein. An example of a replacement
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`amino acid is histidine.
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`In some examples, the histidines in a protein are replaced by
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`a non-basic or uncharged amino acid.
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`In some methods, modifications comprise
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`amino acid replacement with an amino acid selected from among Arg, Asp, Glu, His
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`and Lys, in some examples, replacement with His.
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`In the methods provided, each variant protein, such as a variant protein in a
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`collection can be tested individually, such as, for example, in an array, including an
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`addrcsiblc array.
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`In the methods provided, the activity that is tested can be binding to a target
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`protein of the therapeutic protein. Binding can be assessed by an immunoassay, such
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`as, for example, an ELISA. Examples of an immunoassay is a heterogeneous
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`immunoassay that can include immobilizing the target protein on a solid support;
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`contacting the therapeutic protein with the target protein, wherein the therapeutic
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`protein is detectably labeled; removing unbound therapeutic protein; and detecting or
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`measuring the binding of the labeled therapeutic protein to the target protein. The
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`immunoassay can be homogenous, comprising contacting the therapeutic protein with
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`a target protein, wherein the therapeutic protein is detectably labeled; and detecting or
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`measuring the binding of the labeled therapeutic protein to the target protein.
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`Provided are methods in which binding activity is assessed using a cell surface
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`expression system comprising a cell or cells expressing therapeutic protein on the
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`surface. The therapeutic protein can be expressed on the surface of cells, a target
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`protein can be contacted with a population of the cells; and a cell or cells can be
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`identified that binds to the target protein, thereby identifying a therapeutic protein that
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`exhibits binding activity. The target protein can be detectably labeled or can be
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`detected. The target protein can be fluorescently labeled or detected by a secondary
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`reagent that is fluorescently labeled.
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`Binding can be detected or measured by fluorescence activated cell sorting
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`(FACS).
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`Binding activity can be tested in the methods using a cell surface expression
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`system comprising cells expressing a therapeutic protein, and a cell or cells can be
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`selected that bind to the target protein and a cell or cells can be selected that do not
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`bind to the target protein. The cell or cells that are selected that do not bind to the
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`target protein can be isolated and grown in a cell culture medium to generate a second
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`population of cells expressing the therapeutic protein on the surface. In some
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`examples binding activity is tested under condtions whereby cells from the second
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`population of cells are contacted with the target protein, and a cell or cells are
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`identified that bind to the target protein.
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`BRIEF DESCRIPTION OF THE FIGURES
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`FIGURE 1. Sequence of monoclonal antibody Erbitux®. Figure 1 depicts the
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`sequence of Erbitux® (SEQ ID NO:1 and 2). FIGURE 1A depicts the sequence of
`
`the heavy chain. FIGURE 1B depicts the sequence of the light chain. The variable
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`chains are underlined and the residues selected for modification are in boldface, italic
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`type.
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`DETAILED DESCRIPTION
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`30
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`Outline
`
`A.
`B.
`C.
`
`DEFINITIONS
`OVERVIEW OF METHOD
`In Vim: PHYSIOLOGIC SENSITIVE METHOD
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`1.
`2.
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`Support Containing Target Protein
`Contacting Binding Biomolecule Under Simulated Binding Conditions
`a.
`Generation of Variant Proteins
`b.
`Antibody Variants
`Exemplary Antibody Binding Molecule: anti-EGFR Antibody
`Detection and Identification of Tumor-Specific Binding Molecules
`3.
`METHODS OF EXPRESSING PROTEINS
`l.
`Vectors
`2.
`Cells and Expression Systems
`a.
`Prokaryotlc Expression
`b.
`Yeast
`c.
`Insects
`d.
`Mammalian Cells
`e.
`Plants
`Purification
`3.
`Examples
`
`D.
`
`E.
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`5
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`A.
`
`DEFINITIONS
`
`Unless defined otherwise, all technical and scientific terms used herein have
`
`the same meaning as is commonly understood by one of skill in the art to which the
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`invention(s) belongs/belong. All patents, patent applications, published applications
`
`and publications, Genbank sequences, databases, websites and other published
`
`materials referred to throughout the entire disclosure herein, unless noted otherwise,
`
`are incorporated by reference in their entirety. In the event that there are a plurality of
`
`definitions for terms herein, those in this section prevail. Where reference is made to
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`a URL or other such identifier or address, it is understood that such identifiers can
`
`change and particular information on the intemet can Come and go, but equivalent
`
`information can be found by searching the intemet. Reference thereto evidences the
`
`availability and public dissemination of such infomation.
`
`As used herein, a conditionally active protein is more active in one
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`environment, particularly one in vivo environment, compared to a second
`
`environment. For example, a conditionally active protein can be more active in a
`
`tumor environment than in a non-tumor environment, such as a non-tumor
`
`environment in the skin, GI tract or other non-tumor environment.
`
`As used herein, a therapeutic protein is a protein that has been used for therapy
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`to treat a subject having a disease or condition, can be used for therapy or is a
`
`candidate for therapy. For example, a candidate for therapy is a variant (e. g.
`
`containing amino acid modifications) of a therapeutic protein that has been used for
`
`therapy. For purposes herein, a therapeutic protein, including a protein that has been
`
`used for therapy, can be used for therapy or is a candidate for therapy, can be used in
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`the practice of the method herein as a test protein to identify therapeutic proteins that
`
`exhibit more activity under one set of conditions than another, and hence are
`
`conditionally active.
`
`‘ As used herein, a “test protein,” “tested protein,” “binding molecule,”
`
`“binding protein” or other variations thereof refer to molecules or proteins that are
`
`employed in the method herein. Any molecule or protein can be employed in the
`
`method herein to identify proteins that are conditionally active and exhibit activity
`
`under a condition or conditions that exist in a diseased microenvironment (e. g. tumor
`
`microenvironment) compared to a condition or conditions that exist in a non-diseased
`
`microenvironment. Exemplary of tested proteins are therapeutic proteins in order to
`
`evolve the therapeutic as conditionally active. Exemplary tested proteins are set forth
`
`in Table 3.
`
`As used herein, an antibody refers to immunoglobulins and immunoglobulin
`
`portions, whether natural or partially or wholly synthetic, such as recombinantly
`
`produced, including any portion thereof containing at least a portion of the variable
`
`region of the immunoglobulin molecule that is sufficient to form an antigen binding
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`site. Hence, an antibody or portion thereof includes any protein having a binding
`
`domain that is homologous or substantially homologous to an immunoglobulin
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`antigen binding site. For example, an antibody refers to an antibody that contains two
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`heavy chains (which