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  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
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  • A61K51/04—Organic compounds
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  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1027—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against receptors, cell-surface antigens or cell-surface determinants
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  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
  • A61K51/1051—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from breast, e.g. the antibody being herceptin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C259/00—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups
  • C07C259/04—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups without replacement of the other oxygen atom of the carboxyl group, e.g. hydroxamic acids
  • C07C259/06—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups without replacement of the other oxygen atom of the carboxyl group, e.g. hydroxamic acids having carbon atoms of hydroxamic groups bound to hydrogen atoms or to acyclic carbon atoms
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  • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
  • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
  • C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
  • C07C323/60—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton with the carbon atom of at least one of the carboxyl groups bound to nitrogen atoms
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  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
• • C—CHEMISTRY; METALLURGY
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  • C07K—PEPTIDES
  • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2267/00—Animals characterised by purpose
  • A01K2267/03—Animal model, e.g. for test or diseases
  • A01K2267/0331—Animal model for proliferative diseases
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  • C07C2601/00—Systems containing only non-condensed rings
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  • C07C2601/14—The ring being saturated
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  • C07K2317/55—Fab or Fab'
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  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/624—Disulfide-stabilized antibody (dsFv)

Definitions

• the invention relates generally to antibodies engineered with reactive cysteine residues and more specifically to antibodies with therapeutic or diagnostic applications.
• the cysteine engineered antibodies may be conjugated with chemotherapeutic drugs, toxins, affinity ligands such as biotin, and detection labels such as radioisotopes and fluorophores.
• the invention also relates to methods of using antibodies and antibody-drug conjugate compounds for in vitro, in situ, and in vivo diagnosis or treatment of mammalian cells, or associated pathological conditions.
• Immuno-positron emission tomography is a rapidly emerging method for tracking and quantifying monoclonal antibodies (mAbs) in vivo as it efficiently combines the high sensitivity of PET with the high specificity of mAbs.
• ImmunoPET aspires to be the clinical method of choice for non-invasive diagnosis providing "comprehensive immunohistochemical staining in vivo" (van Dongen GA, et al. "ImmunoPET: a navigator in monoclonal antibody development and applications" Oncologist
• PET Positron Emission Tomographic
• the isotopes are typically administered to a patient by injection of probe molecules that comprise a positron-emitting isotope, such as F-18, C-l 1, N-13, or 0-15, covalently attached to a molecule that is readily metabolized or localized in the body (e.g., glucose) or that chemically binds to receptor sites within the body.
• a positron-emitting isotope such as F-18, C-l 1, N-13, or 0-15
• covalently attached to a molecule that is readily metabolized or localized in the body e.g., glucose
• the isotope is administered to the patient as an ionic solution or by inhalation.
• Small immuno-PET imaging agents such as Fab antibody fragments (50 kDa) or diabodies, paired dimers of the covalently associated V H -V L region of Mab, 55 kDa (Shively et al (2007) J Nucl Med 48: 170- 2), may be particularly useful since they exhibit a short circulation half-life, high tissue permeability, and reach an optimal tumor to background ratio between two to four hours after
• Iodine 124 ( I) was coupled to antibody 3F9 and used to estimate the dosimetry for radioimmunotherapy of neuroblastoma (Larson SM, et al "PET scanning of iodine- 124-3 F9 as an approach to tumor dosimetry during treatment planning for radioimmunotherapy in a child with neuroblastoma" J Nucl Med 1992;33:2020-3). Later, as more sophisticated PET
• the complex decay scheme involves energetic positrons ( ⁇ + max. 1.5 and 2.1 MeV) which negatively affects the resolution of small animal microPET.
• a label such as a radioisotope, fluorescent dye, or drug moiety
• cytotoxic drugs have typically been conjugated to antibodies through the often-numerous lysine residues of an antibody, generating a heterogeneous antibody-drug conjugate mixture.
• the heterogeneous mixture typically contains a distribution of antibodies with from 0 to about 8, or more, attached drug moieties.
• Antibodies are large, complex and structurally diverse biomolecules, often with many reactive functional groups. Their reactivities with linker reagents and drug-linker
• Cysteine thiols are reactive at neutral pH, unlike most amines which are protonated and less nucleophilic near pH 7. Since free thiol (RSH, sulfhydryl) groups are relatively reactive, proteins with cysteine residues often exist in their oxidized form as disulfide-linked oligomers or have internally bridged disulfide groups. Extracellular proteins generally do not have free thiols (Garman, 1997, Non-Radioactive Labelling: A Practical Approach,
• the amount of free thiol in a protein may be estimated by the standard Ellman's assay.
• Immunoglobulin M is an example of a disulfide- linked pentamer
• immunoglobulin G is an example of a protein with internal disulfide bridges bonding the subunits together.
• DTT dithiothreitol
• selenol Singh et al (2002) Anal.
• Biochem. 304: 147-1566 is required to generate the reactive free thiol. This approach may result in loss of antibody tertiary structure and antigen binding specificity.
• Antibody cysteine thiol groups are generally more reactive, i.e. more nucleophilic, towards electrophilic conjugation reagents than antibody amine or hydroxyl groups.
• Cysteine residues have been introduced into proteins by genetic engineering techniques to form covalent attachments to ligands or to form new intramolecular disulfide bonds (Better et al (1994) J. Biol. Chem. 269(13):9644-9650; Bernhard et al (1994) Bioconjugate Chem. 5: 126- 132; Greenwood et al (1994) Therapeutic Immunology 1 :247-255; Tu et al (1999) Proc. Natl. Acad.
• cysteine thiol groups by the mutation of various amino acid residues of a protein to cysteine amino acids is potentially problematic, particularly in the case of unpaired (free Cys) residues or those which are relatively accessible for reaction or oxidation.
• Site-specific conjugation is preferred over random amino modification as it enables chemical modification of a site away from the binding site, promoting complete retention of biological activity and allowing control over the possible number of prosthetic groups added.
• Cysteine-engineered antibodies have been designed as FAB antibody fragments (thioFab) and expressed as full-length, IgG monoclonal (thioMab) antibodies. See: US 7521541; Junutula JR et al. "Rapid identification of reactive cysteine residues for site-specific labeling of antibody-Fabs" J Immunol Methods 2008;332:41-52; Junutula JR et al. "Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index” (2008) Nat Biotechnol.
• ThioFab and ThioMab antibodies have been conjugated through linkers at the newly introduced cysteine thiols with thiol-reactive linker reagents and drug-linker reagents to prepare cysteine- engineered antibody drug conjugates (Thio ADC) with anti-cancer properties, including anti- MUC16 (US 2008/0311134), anti-CD22 (US 2008/0050310), anti-ROB04 (US)
• the compounds of the invention include cysteine engineered antibodies where one or more amino acids of a parent antibody are replaced with a free cysteine amino acid.
• a cysteine engineered antibody comprises one or more free cysteine amino acids having a thiol reactivity value in the range of 0.6 to l .O.
• a free cysteine amino acid is a cysteine residue which has been engineered into the parent antibody and is not part of a disulfide bridge.
• Cysteine engineered antibodies may be useful in the diagnosis and treatment of cancer and include antibodies specific for cell surface and transmembrane receptors, and tumor-associated antigens (TAA). Such antibodies may be used as naked antibodies
• Embodiments of the methods for preparing and screening a cysteine engineered antibody include where the parent antibody is an antibody fragment, such as hu4D5Fabv8.
• the parent antibody may also be a fusion protein comprising an albumin-binding peptide sequence (ABP).
• the parent antibody may also be a humanized antibody selected from huMAb4D5-l, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (trastuzumab).
• Cysteine engineered antibodies of the invention may be site-specifically and efficiently coupled with a thiol-reactive reagent.
• the thiol-reactive reagent may be a radioisotope reagent, multifunctional linker reagent, a capture label reagent, a fluorophore reagent, or a drug-linker intermediate.
• the cysteine engineered antibody may be labeled with a detectable label, immobilized on a solid phase support and/or conjugated with a drug moiety.
• Another aspect of the invention is a zirconium-labelled, cysteine-engineered antibody comprising a cysteine engineered antibody (Ab) conjugated through a free cysteine amino acid to a linker (L) and a zirconium complex (Z), having Formula I: where p is 1 to 4.
• Ab cysteine engineered antibody
• L linker
• Z zirconium complex
• Another aspect of the invention is a desferrioxamine-labelled, cysteine-engineered antibody comprising a cysteine engineered antibody (Ab) conjugated through a free cysteine amino acid to a linker (L) and a desferoxamine moiety (Df), having Formula II:
• p 1 to 4.
• Another aspect of the invention is a method of making a desferrioxamine-labelled, cysteine-engineered antibody comprising comprising a cysteine engineered antibody (Ab) conjugated through a free cysteine amino acid to a linker (L) and a desferoxamine moiety (Df), having Formula II:
• L-Df is selected from:
• p 1 to 4.
• R is selected from:
• cysteine-engineered antibody having one or more free cysteine amino acids, whereby the desferrioxamine-labelled, cysteine-engineered antibody is formed.
• Another aspect of the invention is a method of making a zirconium-labelled, cysteine- engineered antibody comprising a cysteine engineered antibody (Ab) conjugated through a free cysteine amino acid to a linker (L) and a zirconium complex (Z), having Formula I: where p is 1 to 4;
• the method comprising complexing a zirconium reagent with a desferrioxamine- labelled, cysteine-engineered antibody comprising a cysteine engineered antibody (Ab) conjugated through a free cysteine amino acid to a linker (L) and a desferoxamine moiety (Df), having Formula II:
• p 1 to 4.
• Another aspect of the invention is a method of imaging comprising:
• zirconium-labelled, cysteine-engineered antibody comprises a cysteine engineered antibody (Ab) having one or more free cysteine amino acids conjugated with one or more zirconium complex (Z) through a linker (L), and having Formula I:
• Another aspect of the invention includes diagnostic uses for the compounds and compositions disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS
• Figure 1 A shows a three-dimensional representation of the hu4D5Fabv7 antibody fragment derived by X-ray crystal coordinates.
• the structure positions of the exemplary engineered Cys residues of the heavy and light chains are numbered (according to a sequential numbering system).
• Figure IB shows a sequential numbering scheme (top row), starting at the N-terminus in comparison with the Kabat numbering scheme (bottom row) for 4D5v7fabH. Kabat numbering insertions are noted by a,b,c.
• Figures 2 A and 2B show binding measurements with detection of absorbance at 450nm of hu4D5Fabv8 and hu4D5Fabv8 Cys mutant (ThioFab) phage variants: (A) non- biotinylated phage-hu4D5Fabv8 and (B) biotinylated phage-hu4D5Fabv8 (B) by the PHESELECTOR assay for interactions with BSA (open bar), HER2 (striped bar) or streptavidin (solid bar).
• FIG. 4A shows Fractional Surface Accessibility values of residues on wild type
• hu4D5Fabv8 Light chain sites are on the left side and heavy chain sites are on the right side.
• Figure 4B shows binding measurements with detection of absorbance at 450nm of
• Phage-hu4D5Fabv8 Cys variants were isolated and stored at 4 °C. Biotin conjugation was carried out either at day 2 or day 4 followed by PHESELECTOR analyses to monitor their interaction with Her2 and streptavidin as described in Example 2, and probe the stability of reactive thiol groups on engineered ThioFab variants.
• Figure 5 shows binding measurements with detection of absorbance at 450nm of biotin- maleimide conjugated-hu4D5Fabv8 (A121C) and non-biotinylated wild type hu4D5Fabv8 for binding to streptavidin and HER2. Each Fab was tested at 2 ng and 20 ng.
• Figure 6 shows ELISA analysis with detection of absorbance at 450nm of biotinylated ABP- hu4D5Fabv8 wild type (wt), and ABP-hu4D5Fabv8 cysteine mutants VI IOC and A121C for binding with rabbit albumin, streptavidin (SA), and HER2.
• Figure 7 shows ELISA analysis with detection of absorbance at 450nm of biotinylated ABP- hu4D5Fabv8 cysteine mutants (ThioFab variants): (left to right) single Cys variants ABP-V1 IOC, ABP-A121C, and double Cys variants ABP-V110C-A88C and ABP- VI 10C-A121C for binding with rabbit albumin, HER2 and streptavidin (SA), and probing with Fab-HRP or SA-HRP.
• ThioFab variants biotinylated ABP- hu4D5Fabv8 cysteine mutants
• Figure 8 shows binding of biotinylated ThioFab phage and an anti-phage HRP antibody to HER2 (top) and Streptavidin (bottom).
• Figure 13A shows a cartoon depiction of biotinylated antibody binding to immobilized HER2 with binding of HRP labeled secondary antibody for absorbance detection.
• Figure 13B shows binding measurements with detection of absorbance at 450nm of biotin- maleimide conjugated thio-trastuzumab variants and non-biotinylated wild type trastuzumab in binding to immobilized HER2. From left to right: VI IOC (single cys), A121C (single cys), VI 10C/A121C (double cys), and trastuzumab. Each thio IgG variant and trastuzumab was tested at 1, 10, and 100 ng.
• Figure 14A shows a cartoon depiction of biotinylated antibody binding to immobilized HER2 with binding of biotin to anti-IgG-HRP for absorbance detection.
• Figure 14B shows binding measurements with detection of absorbance at 450nm of biotin- maleimide conjugated-thio trastuzumab variants and non-biotinylated wild type trastuzumab in binding to immobilized streptavidin. From left to right: VI IOC (single cys), A121C (single cys), VI 10C/A121C (double cys), and trastuzumab. Each thio IgG variant and trastuzumab was tested at 1, 10, and 100 ng.
• Figure 15 shows the general process to prepare a cysteine engineered antibody (ThioMab) expressed from cell culture for conjugation.
• Figure 16 shows non-reducing (top) and reducing (bottom) denaturing polyacrylamide gel electrophoresis analysis of 2H9 ThioMab Fc variants (left to right, lanes 1-9): A339C; S337C; S324C; A287C; V284C; V282C; V279C; V273C, and 2H9 wild type after purification on immobilized Protein A.
• the lane on the right is a size marker ladder, indicating the intact proteins are about 150 kDa, heavy chain fragments about 50 kDa, and light chain fragments about 25 kDa.
• Figure 17A shows non-reducing (left) and reducing (+DTT) (right) denaturing polyacrylamide gel electrophoresis analysis of 2H9 ThioMab variants (left to right, lanes 1-4): L-V15C; S179C; S375C; S400C, after purification on immobilized Protein A.
• Figure 17B shows non-reducing (left) and reducing (+DTT) (right) denaturing
• Figure 18 shows western blot analysis of biotinylated Thio-IgG variants. 2H9 and 3A5
• ThioMab variants were analyzed on reduced denaturing polyacrylamide gel electrophoresis, the proteins were transferred to nitrocellulose membrane. The presence of antibody and conjugated biotin were probed with anti-IgG-HRP (top) and streptavidin-HRP (bottom), respectively. Lane 1 : 3A5 H-A121C. Lane 2: 3A5 L- VI IOC. Lane 3: 2H9 H-A121C. Lane 4: 2H9 L-V110C. Lane 5: 2H9 wild type.
• Figure 19 shows ELISA analysis for the binding of biotinylated 2H9 variants to streptavidin by probing with anti-IgG-HRP and measuring the absorbance at 450 nm of (top bar diagram).
• Bottom schematic diagram depicts the experimental design used in the ELISA analysis.
• Figure 20 shows bifunctional reagents for coupling chelator of 89 Zr desferoxamine B (Df, top) with proteins using amino reactive linkers, TFP-N-SucDf and Df-Bz-NCS (center) and thiol reactive linkers, Df-Chx-Mal, Df-Bac, and Df-lac (bottom).
• Figure 21 shows the preparation of Df-Chx-Mal, Df-Bac, Df-lac and conjugation to thio- trastuzumab via Cys residues incorporated into the heavy chain of Fab.
• Reaction conditions i. DIEA, DMF/H 2 0 (10: 1), RT, 0.5-1 h; ii. DIEA, DMF, 0°C, 4 h; iii. pH 7.5, RT, 1 h; iv. pH 9, RT, 5 h; v. pH 9, RT, 2 h.
• Figure 22 shows chelation of zirconium-89 oxalate with a desferrioxamine-labelled, cysteine- engineered antibody, such as variants of Df-linker-trastuzumab containing four linkers: N-Suc, Bz-SCN, Chx-maleimide (CHx-Mal), or acetyl (Ac).
• a desferrioxamine-labelled, cysteine- engineered antibody such as variants of Df-linker-trastuzumab containing four linkers: N-Suc, Bz-SCN, Chx-maleimide (CHx-Mal), or acetyl (Ac).
• Figure 23 shows mass spectrometry analysis of reduced antibodies showing separate signals from light and heavy chains.
• Figure 25 shows representative full-body images (maximum intensity projection) acquired 96 hours after the tail vein bolus injection of 100 ⁇ of Zr-Trasuzumab prepared using four different linkers (Bz-SCN, N-Suc, Chx-Mal, and Ac).
• Figure 26 shows In vivo uptake in selected tissues at 24, 96 and 144 h post injection as
• antibody herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity (Miller et al (2003) Jour, of Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species. An antibody is a protein generated by the immune system that is capable of recognizing and binding to a specific antigen. (Janeway, C, Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York).
• a target antigen generally has numerous binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody.
• An antibody includes a full-length immunoglobulin molecule or an immunologically active portion of a full-length
• immunoglobulin molecule i.e., a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease.
• the immunoglobulin disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and
• immunoglobulins can be derived from any species. In one aspect, however, the immunoglobulin is of human, murine, or rabbit origin.
• Antibody fragments comprise a portion of a full length antibody, generally the antigen binding or variable region thereof.
• Examples of antibody fragments include Fab, Fab, and fragments thereof.
• Fab', F(ab') 2 , and Fv fragments diabodies; linear antibodies; minibodies (Olafsen et al (2004) Protein Eng. Design & Sel. 17(4):315-323), fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR (complementary determining region), and epitope- binding fragments of any of the above which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
• the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies
• Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies.
• the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
• the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al (1975) Nature 256:495, or may be made by recombinant DNA methods (see for example: US 4816567; US 5807715).
• the monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al (1991) Nature, 352:624-628; Marks et al (1991) J. Mol. Biol, 222:581-597; for example.
• the monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (US 4816567; and Morrison et al (1984) Proc. Natl. Acad. Sci. USA, 81 :6851-6855).
• Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape etc) and human constant region sequences.
• an “intact antibody” herein is one comprising a VL and VH domains, as well as a light chain constant domain (CL) and heavy chain constant domains, CHI , CH2 and CH3.
• the constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variant thereof.
• the intact antibody may have one or more "effector functions" which refer to those biological activities attributable to the Fc constant region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include Clq binding;
• immunoglobulin antibodies can be assigned to different "classes.” There are five major classes of intact immunoglobulin antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into “subclasses” (isotypes), e.g., IgGl , IgG2, IgG3, IgG4, IgA, and IgA2.
• the heavy-chain constant domains that correspond to the different classes of antibodies are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
• the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
• Ig forms include hinge-modifications or hingeless forms (Roux et al (1998) J. Immunol. 161 :4083-4090; Lund et al (2000) Eur. J. Biochem. 267:7246-7256; US 2005/0048572; US 2004/0229310).
• An "ErbB receptor” is a receptor protein tyrosine kinase which belongs to the ErbB receptor family whose members are important mediators of cell growth, differentiation and survival.
• the ErbB receptor family includes four distinct members including epidermal growth factor receptor (EGFR, ErbBl, HER1), HER2 (ErbB2 or pl85neu), HER3 (ErbB3) and HER4 (ErbB4 or tyro2).
• a panel of anti-ErbB2 antibodies has been characterized using the human breast tumor cell line SKBR3 (Hudziak et al (1989) Mol. Cell. Biol. 9(3): 1165- 1172. Maximum inhibition was obtained with the antibody called 4D5 which inhibited cellular proliferation by 56%. Other antibodies in the panel reduced cellular proliferation to a lesser extent in this assay. The antibody 4D5 was further found to sensitize ErbB2- overexpressing breast tumor cell lines to the cytotoxic effects of TNF-a (US 5677171).
• the anti-ErbB2 antibodies discussed in Hudziak et al. are further characterized in Fendly et al
• the ErbB receptor will generally comprise an extracellular domain, which may bind an ErbB ligand; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a carboxyl-terminal signaling domain harboring several tyrosine residues which can be phosphorylated.
• the ErbB receptor may be a "native sequence” ErbB receptor or an "amino acid sequence variant" thereof.
• the ErbB receptor is native sequence human ErbB receptor. Accordingly, a "member of the ErbB receptor family" is EGFR (ErbBl), ErbB2, ErbB3, ErbB4 or any other ErbB receptor currently known or to be identified in the future.
• ErbBl "epidermal growth factor receptor", “EGFR” and “HER1” are used interchangeably herein and refer to EGFR as disclosed, for example, in Carpenter et al (1987) Ann. Rev. Biochem., 56:881-914, including naturally occurring mutant forms thereof (e.g., a deletion mutant EGFR as in Humphrey et al (1990) Proc. Nat. Acad. Sci. (USA) 87:4207-4211).
• the term erbBl refers to the gene encoding the EGFR protein product. Antibodies against HER1 are described, for example, in Murthy et al (1987) Arch. Biochem.
• ERRP epidermal growth factor receptor
• EGF epidermal growth factor receptor
• ErbB2 and "HER2” are used interchangeably herein and refer to human HER2 protein described, for example, in Semba et al (1985) Proc. Nat. Acad. Sci. (USA) 82:6497-6501 and Yamamoto et al (1986) Nature, 319:230-234 (Genebank accession number X03363).
• the term “erbB2” refers to the gene encoding human ErbB2 and "neu” refers to the gene encoding rat pl85neu.
• Preferred ErbB2 is native sequence human ErbB2.
• ErbB3 and "HER3” refer to the receptor polypeptide as disclosed, for example, in U.S. Patent Nos. 5183884 and 5480968 as well as Kraus et al (1989) Proc. Nat. Acad. Sci. (USA) 86:9193-9197.
• Antibodies against ErbB3 are known in the art and are described, for example, in U.S. Patent Nos. 5183884, 5480968 and in WO 97/35885.
• ErbB4 and HER4 herein refer to the receptor polypeptide as disclosed, for example, in EP Pat Application No 599,274; Plowman et al (1993) Proc. Natl. Acad. Sci. USA 90: 1746-1750; and Plowman et al (1993) Nature 366:473-475, including isoforms thereof, e.g., as disclosed in WO 99/19488.
• Antibodies against HER4 are described, for example, in WO 02/18444.
• Antibodies to ErbB receptors are available commercially from a number of sources, including, for example, Santa Cruz Biotechnology, Inc., California, USA.
• amino acid sequence variant refers to polypeptides having amino acid sequences that differ to some extent from a native sequence polypeptide. Ordinarily, amino acid sequence variants will possess at least about 70% sequence identity with at least one receptor binding domain of a native ErbB ligand or with at least one ligand binding domain of a native ErbB receptor, and preferably, they will be at least about 80%, more preferably, at least about 90%> homologous by sequence with such receptor or ligand binding domains. The amino acid sequence variants possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence of the native amino acid sequence. Amino acids are designated by the conventional names, one-letter and three-letter codes.
• Sequence identity is defined as the percentage of residues in the amino acid sequence variant that are identical after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Methods and computer programs for the alignment are well known in the art. One such computer program is "Align 2,” authored by Genentech, Inc., which was filed with user documentation in the United States Copyright Office, Washington, DC 20559, on December 10, 1991.
• Antibody-dependent cell-mediated cytotoxicity and “ADCC” refer to a cell- mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell.
• FcRs Fc receptors
• FcR expression on hematopoietic cells in summarized is Table 3 on page 464 of Ravetch and Kinet, (1991) "Annu. Rev. Immunol.” 9:457-92.
• ADCC activity of a molecule of interest may be assessed in vitro, such as that described in US 5500362 and US 5821337.
• useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
• PBMC peripheral blood mononuclear cells
• NK Natural Killer
• ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al (1998) PROC. NAT. ACAD. SCI. (USA) (USA) 95:652-656.
• Human effector cells are leukocytes which express one or more constant region receptors (FcRs) and perform effector functions. Preferably, the cells express at least FcyRIII and perform ADCC effector function. Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferred.
• the effector cells may be isolated from a native source thereof, e.g., from blood or PBMCs as described herein.
• Fc receptor or “FcR” are used to describe a receptor that binds to the Fc constant region of an antibody.
• the preferred FcR is a native sequence human FcR.
• a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and Fey RIII subclasses, including allelic variants and alternatively spliced forms of these receptors.
• FcyRII receptors include FcyRIIA (anadvant), FcyRIIB (anadvant), and othersadvant.
• activating receptor and FcyRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
• Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (IT AM) in its cytoplasmic domain.
• Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine - based inhibition motif (ITIM) in its cytoplasmic domain.
• ITIM immunoreceptor tyrosine - based inhibition motif
• FcR neonatal receptor
• “Complement dependent cytotoxicity” or “CDC” refers to the ability of a molecule to lyse a target in the presence of complement.
• the complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a molecule (e.g., an antibody) complexed with a cognate antigen.
• a CDC assay e.g., as described in Gazzano-Santoro et al J. Immunol. Methods, 202: 163 (1996), may be performed.
• “Native antibodies” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V H ) followed by a number of constant domains. Each light chain has a variable domain at one end (V L ) and a constant domain at its other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
• variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs).
• the variable domains of native heavy and light chains each comprise four FRs, largely adopting a ⁇ -sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
• the hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen- binding site of antibodies (see Kabat et al (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD).
• the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).
• hypervariable region when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding.
• the hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g., residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (HI), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al supra) and/or those residues from a "hypervariable loop” (e.g., residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (HI), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk (1987) J. Mol. Biol, 196:901-917).
• CDR complementarity determining region
• Framework Region or "FR” residues are those variable domain residues other than the hypervariable region residues as herein defined.
• Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual "Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.
• Fv is the minimum antibody fragment which contains a complete antigen- recognition and antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen- binding site on the surface of the V H -V L dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
• the Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain.
• Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region.
• Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear at least one free thiol group.
• F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
• the "light chains" of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequences of their constant domains.
• Single-chain Fv or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain.
• the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
• diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a variable heavy domain (VH) connected to a variable light domain (VL) in the same polypeptide chain (VH - VL).
• VH variable heavy domain
• VL variable light domain
• Diabodies are described more fully in, for example, EP 404,097; WO 93/11 161 ; and
• “Humanized” forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. Humanization is a method to transfer the murine antigen binding information to a non-immunogenic human antibody acceptor, and has resulted in many therapeutically useful drugs. The method of humanization generally begins by transferring all six murine complementarity determining regions (CDRs) onto a human antibody framework (Jones et al, (1986) Nature 321 :522-525). These CDR-grafted antibodies generally do not retain their original affinity for antigen binding, and in fact, affinity is often severely impaired. Besides the CDRs, select non-human antibody framework residues must also be incorporated to maintain proper CDR
• humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
• donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
• framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
• humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
• the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
• the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
• Fc immunoglobulin constant region
• a “free cysteine amino acid” refers to a cysteine amino acid residue which has been engineered into a parent antibody, has a thiol functional group (-SH), and is not paired as an intramolecular or intermolecular disulfide bridge.
• thiol reactivity value is a quantitative characterization of the reactivity of free cysteine amino acids.
• the thiol reactivity value is the percentage of a free cysteine amino acid in a cysteine engineered antibody which reacts with a thiol-reactive reagent, and converted to a maximum value of 1.
• a free cysteine amino acid on a cysteine engineered antibody which reacts in 100% yield with a thiol-reactive reagent, such as a biotin-maleimide reagent, to form a biotin-labelled antibody has a thiol reactivity value of 1.0.
• Another cysteine amino acid engineered into the same or different parent antibody which reacts in 80% yield with a thiol-reactive reagent has a thiol reactivity value of 0.8.
• Another cysteine amino acid engineered into the same or different parent antibody which fails totally to react with a thiol-reactive reagent has a thiol reactivity value of 0.
• Determination of the thiol reactivity value of a particular cysteine may be conducted by ELISA assay, mass spectroscopy, liquid chromatography, autoradiography, or other quantitative analytical tests.
• a “parent antibody” is an antibody comprising an amino acid sequence from which one or more amino acid residues are replaced by one or more cysteine residues.
• the parent antibody may comprise a native or wild type sequence.
• the parent antibody may have preexisting amino acid sequence modifications (such as additions, deletions and/or substitutions) relative to other native, wild type, or modified forms of an antibody.
• a parent antibody may be directed against a target antigen of interest, e.g. a biologically important polypeptide.
• Antibodies directed against nonpolypeptide antigens are also contemplated.
• Exemplary parent antibodies include antibodies having affinity and selectivity for cell surface and transmembrane receptors and tumor-associated antigens (TAA).
• TAA tumor-associated antigens
• exemplary parent antibodies include those selected from, and without limitation, anti-estrogen receptor antibody, anti-progesterone receptor antibody, anti-p53 antibody, anti-HER-2/neu antibody, anti-EGFR antibody, anti-cathepsin D antibody, anti- Bcl-2 antibody, anti-E-cadherin antibody, anti-CA125 antibody, anti-CA15-3 antibody, anti- CA19-9 antibody, anti-c-erbB-2 antibody, anti-P-glycoprotein antibody, anti-CEA antibody, anti-retinoblastoma protein antibody, anti-ras oncoprotein antibody, anti-Lewis X antibody, anti-Ki-67 antibody, anti-PCNA antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD9/p24 antibody, anti-CD 10 antibody, anti-CDl lc antibody, anti-CD 13 antibody, anti-CD 14 antibody, anti-CD 15 antibody, anti-CD 19 antibody, anti-CD20 antibody, anti
• an “isolated” antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or
• the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
• Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
• an antibody "which binds" a molecular target or an antigen of interest is one capable of binding that antigen with sufficient affinity such that the antibody is useful in targeting a cell expressing the antigen.
• the antibody is one which binds ErbB2
• it will usually preferentially bind ErbB2 as opposed to other ErbB receptors, and may be one which does not significantly cross-react with other proteins such as EGFR, ErbB3 or ErbB4.
• the extent of binding of the antibody to these non-ErbB2 proteins e.g., cell surface binding to endogenous receptor
• FACS fluorescence activated cell sorting
• RIA radioimmunoprecipitation
• CD proteins and their ligands such as, but not limited to: (i) CD3, CD4, CD8, CD19, CD20, CD22, CD34, CD40, CD79a (CD79a), and CD79p (CD79b); (ii) members of the ErbB receptor family such as the EGF receptor, HER2, HER3 or HER4 receptor; (iii) cell adhesion molecules such as LFA-1, Macl, pl50,95, VLA-4, ICAM-1, VCAM and ⁇ / ⁇ 3 integrin, including either alpha or beta subunits thereof (e.g.
• anti-CD 1 la, anti-CD 18 or anti-CD 1 lb antibodies growth factors such as VEGF; IgE; blood group antigens; flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; protein C, BR3, c-met, tissue factor, ⁇ 7 etc; and (v) cell surface and transmembrane tumor-associated antigens (TAA).
• growth factors such as VEGF; IgE; blood group antigens; flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; protein C, BR3, c-met, tissue factor, ⁇ 7 etc; and TAA).
• the term “monoclonal antibody 4D5" refers to an antibody that has antigen binding residues of, or derived from, the murine 4D5 antibody (ATCC CRL 10463).
• the monoclonal antibody 4D5 may be murine
• monoclonal antibody 4D5 or a variant thereof, such as a humanized 4D5.
• exemplary humanized 4D5 antibodies include huMAb4D5-l, huMAb4D5-2, huMAb4D5-3,
• huMAb4D5-4 huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (trastuzumab, HERCEPTIN®) as in US Patent No. 5821337.
• Phage display is a technique by which variant polypeptides are displayed as fusion proteins to a coat protein on the surface of phage, e.g., filamentous phage, particles.
• phage display One utility of phage display lies in the fact that large libraries of randomized protein variants can be rapidly and efficiently sorted for those sequences that bind to a target molecule with high affinity. Display of peptide and protein libraries on phage has been used for screening millions of polypeptides for ones with specific binding properties. Polyvalent phage display methods have been used for displaying small random peptides and small proteins, typically through fusions to either pill or pVIII of filamentous phage (Wells and Lowman, (1992) Curr. Opin. Struct.
• phagemid vectors are used, which simplify DNA manipulations. Lowman and Wells, Methods: A companion to Methods in Enzymology, 3:205-0216 (1991).
• Phage display includes techniques for producing antibody- like molecules (Janeway, C, Travers, P., Walport, M., Shlomchik (2001) Immunobiology, 5th Ed., Garland Publishing, New York, p627-628; Lee et al ).
• a "phagemid” is a plasmid vector having a bacterial origin of replication, e.g., ColEl, and a copy of an intergenic region of a bacteriophage.
• the phagemid may be used on any known bacteriophage, including filamentous bacteriophage and lambdoid bacteriophage.
• the plasmid will also generally contain a selectable marker for antibiotic resistance. Segments of DNA cloned into these vectors can be propagated as plasmids. When cells harboring these vectors are provided with all genes necessary for the production of phage particles, the mode of replication of the plasmid changes to rolling circle replication to generate copies of one strand of the plasmid DNA and package phage particles.
• the phagemid may form infectious or non-infectious phage particles.
• This term includes phagemids which contain a phage coat protein gene or fragment thereof linked to a heterologous polypeptide gene as a gene fusion such that the heterologous polypeptide is displayed on the surface of the phage particle.
• Linker means a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches an antibody to a drug moiety.
• a linker is specified as L.
• Linkers include a divalent radical such as an alkyldiyl, an arylene, a heteroarylene, moieties such as: -(CR 2 ) n O(CR 2 ) n -, repeating units of alkyloxy (e.g. polyethylenoxy, PEG, polymethyleneoxy) and alkylamino (e.g.
• polyethyleneamino, JeffamineTM polyethyleneamino, JeffamineTM
• diacid ester and amides including succinate, succinamide, diglycolate, malonate, and caproamide.
• label means any moiety which can be covalently attached to an antibody and that functions to: (i) provide a detectable signal; (ii) interact with a second label to modify the detectable signal provided by the first or second label, e.g. FRET (fluorescence resonance energy transfer); (iii) stabilize interactions or increase affinity of binding, with antigen or ligand; (iv) affect mobility, e.g. electrophoretic mobility, or cell-permeability, by charge, hydrophobicity, shape, or other physical parameters, or (v) provide a capture moiety, to modulate ligand affinity, antibody/antigen binding, or ionic complexation.
• FRET fluorescence resonance energy transfer
• d and 1 or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or 1 meaning that the compound is levorotatory.
• a compound prefixed with (+) or d is dextrorotatory.
• these stereoisomers are identical except that they are mirror images of one another.
• a specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture.
• a 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process.
• racemic mixture and racemate refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.
• organic or inorganic salts of an AZC include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, /?-toluenesulfonate, and pamoate (i.e., 1,1 ' -methyl ene-bis -(2-hydroxy-3- naphthoate)) salts.
• exemplary salts include, but are not limited
• a pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion.
• the counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound.
• a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterion.
• “Pharmaceutically acceptable solvate” refers to an association of one or more solvent molecules and an AZC.
• solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.
• BME beta-mercaptoethanol
• Boc N-(t-butoxycarbonyl)
• cit citrulline (2-amino-5-ureido pentanoic acid)
• dap is dolaproine
• DCC 1,3-dicyclohexylcarbodiimide
• DCM dichloromethane
• DEA diethylamine
• DEAD diethylazodicarboxylate
• DEPC diethylphosphorylcyanidate
• DIAD diisopropylazodicarboxylate
• DIEA N,N- diisopropylethylamine
• dil dolaisoleucine
• DMA is dimethylacetamide
• DMAP 4- dimethylaminopyridine
• DME is ethyleneglycol dimethyl ether (or 1 ,2-dimethoxyethane)
• DMF is N,N-dimethylformamide
• DMSO dimethylsulfoxide
• doe is
• DTT dithiothreitol
• EDCI l-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
• EEDQ 2-ethoxy-l- ethoxycarbonyl-l,2-dihydroquinoline
• ES-MS electrospray mass spectrometry
• EtOAc ethyl acetate
• Fmoc is N-(9-fluorenylmethoxycarbonyl)
• gly glycine
• HATU 0-(7- azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate
• HOBt 1- hydroxybenzotriazole
• HPLC high pressure liquid chromatography
• ile is isoleucine
• lys is lysine
• MeCN (CH 3 CN) is acetonitrile
• MeOH MeOH
• the compounds of the invention include cysteine engineered antibodies where one or more amino acids of a wild-type or parent antibody are replaced with a cysteine amino acid. Any form of antibody may be so engineered, i.e. mutated.
• a parent Fab antibody fragment may be engineered to form a cysteine engineered Fab, referred to herein as "ThioFab.”
• a parent monoclonal antibody may be engineered to form a
• ThioMab “ThioMab.” It should be noted that a single site mutation yields a single engineered cysteine residue in a ThioFab, while a single site mutation yields two engineered cysteine residues in a ThioMab, due to the dimeric nature of the IgG antibody. Mutants with replaced
• cysteine (Cys) residues are evaluated for the reactivity of the newly introduced, engineered cysteine thiol groups.
• the thiol reactivity value is a relative, numerical term in the range of 0 to 1.0 and can be measured for any cysteine engineered antibody.
• Thiol reactivity values of cysteine engineered antibodies of the invention are in the ranges of 0.6 to 1.0; 0.7 to 1.0; or 0.8 to 1.0.
• the design, selection, and preparation methods of the invention enable cysteine engineered antibodies which are reactive with electrophilic functionality. These methods further enable antibody conjugate compounds such as antibody-zirconium conjugate (AZC) compounds with zirconium atoms at designated, designed, selective sites. Reactive cysteine residues on an antibody surface allow specifically conjugating a zirconium moiety through a thiol reactive group such as maleimide or haloacetyl.
• a thiol reactive group such as maleimide or haloacetyl.
• the nucleophilic reactivity of the thiol functionality of a Cys residue to a maleimide group is about 1000 times higher compared to any other amino acid functionality in a protein, such as amino group of lysine residues or the N-terminal amino group.
• Thiol specific functionality in iodoacetyl and maleimide reagents may react with amine groups, but higher pH (>9.0) and longer reaction times are required (Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London).
• cysteine engineered antibodies of the invention preferably retain the antigen binding capability of their wild type, parent antibody counterparts.
• cysteine engineered antibodies are capable of binding, preferably specifically, to antigens.
• antigens include, for example, tumor-associated antigens (TAA), cell surface receptor proteins and other cell surface molecules, transmembrane proteins, signalling proteins, cell survival regulatory factors, cell proliferation regulatory factors, molecules associated with (for e.g., known or suspected to contribute functionally to) tissue development or differentiation, lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in vasculogenesis and molecules associated with (for e.g., known or suspected to contribute functionally to) angiogenesis.
• TAA tumor-associated antigens
• cell surface receptor proteins and other cell surface molecules include, for example, tumor-associated antigens (TAA), cell surface receptor proteins and other cell surface molecules, transmembrane proteins, signalling proteins, cell survival regulatory factors, cell proliferation regulatory factors, molecules associated with (for e.g., known or
• the tumor-associated antigen may be a cluster differentiation factor (i.e., a CD protein).
• An antigen to which a cysteine engineered antibody is capable of binding may be a member of a subset of one of the above-mentioned categories, wherein the other subset(s) of said category comprise other molecules/antigens that have a distinct characteristic (with respect to the antigen of interest).
• the parent antibody may also be a humanized antibody selected from huMAb4D5-l, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (Trastuzumab, HERCEPTIN®) as described in Table 3 of US 5821337, expressly incorporated herein by reference; humanized 520C9 (WO 93/21319) and humanized 2C4 antibodies as described herein.
• Cysteine engineered antibodies of the invention may be site-specifically and efficiently coupled with a thiol-reactive reagent.
• the thiol-reactive reagent may be a multifunctional linker reagent, a capture, i.e. affinity, label reagent (e.g. a biotin-linker reagent), a detection label (e.g. a fluorophore reagent), a solid phase immobilization reagent (e.g. SEPHAROSETM, polystyrene, or glass), or a zirconium-linker intermediate.
• label reagent e.g. a biotin-linker reagent
• detection label e.g. a fluorophore reagent
• solid phase immobilization reagent e.g. SEPHAROSETM, polystyrene, or glass
• zirconium-linker intermediate e.g. SEPHAROSETM, polystyrene, or glass
• reaction of a ThioFab with a biotin-linker reagent provides a biotinylated ThioFab by which the presence and reactivity of the engineered cysteine residue may be detected and measured.
• Reaction of a ThioFab with a multifunctional linker reagent provides a ThioFab with a functionalized linker which may be further reacted with a zirconium moiety reagent or other label.
• Reaction of a ThioFab with a zirconium-linker intermediate provides a ThioFab zirconium conjugate.
• the exemplary methods described here may be applied generally to the identification and production of antibodies, and more generally, to other proteins through application of the design and screening steps described herein.
• Such an approach may be applied to the conjugation of other thiol-reactive agents in which the reactive group is, for example, a maleimide, an iodoacetamide, a pyridyl disulfide, or other thiol-reactive conjugation partner (Haugland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London; Means (1990) Bioconjugate Chem. 1 :2; Hermanson, G. in
• the partner may be a cytotoxic agent (e.g. a toxin such as doxorubicin or pertussis toxin), a fluorophore such as a fluorescent dye like fluorescein or rhodamine, a chelating agent for an imaging or radiotherapeutic metal, a peptidyl or non-peptidyl label or detection tag, or a clearance-modifying agent such as various isomers of polyethylene glycol, a peptide that binds to a third component, or another carbohydrate or lipophilic agent.
• a cytotoxic agent e.g. a toxin such as doxorubicin or pertussis toxin
• a fluorophore such as a fluorescent dye like fluorescein or rhodamine
• a chelating agent for an imaging or radiotherapeutic metal e.g. a chelating agent for an imaging or radiotherapeutic metal
• a peptidyl or non-peptidyl label or detection tag
• the sites identified on the exemplary antibody fragment, hu4D5Fabv8, herein are primarily in the constant domain of an antibody which is well conserved across all species of antibodies. These sites should be broadly applicable to other antibodies, without further need of structural design or knowledge of specific antibody structures, and without interference in the antigen binding properties inherent to the variable domains of the antibody.
• Cysteine engineered antibodies which may be useful in the treatment of cancer include, but are not limited to, antibodies against cell surface receptors and tumor-associated antigens (TAA). Such antibodies may be used as naked antibodies (unconjugated to a label moiety) or as Formula I antibody-zirconium conjugates (AZC). Tumor-associated antigens are known in the art, and can prepared for use in generating antibodies using methods and information which are well known in the art. In attempts to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify transmembrane or otherwise tumor-associated polypeptides that are specifically expressed on the surface of one or more particular type(s) of cancer cell as compared to on one or more normal non- cancerous cell(s).
• tumor-associated polypeptides are more abundantly expressed on the surface of the cancer cells as compared to on the surface of the non-cancerous cells.
• the identification of such tumor-associated cell surface antigen polypeptides has given rise to the ability to specifically target cancer cells for destruction via antibody-based therapies.
• TAA examples include, but are not limited to, TAA (l)-(36) listed below.
• TAA examples include, but are not limited to, TAA (l)-(36) listed below.
• information relating to these antigens is listed below and includes names, alternative names, Genbank accession numbers and primary reference(s), following nucleic acid and protein sequence identification conventions of the National Center for Biotechnology Information (NCBI).
• NCBI National Center for Biotechnology Information
• Nucleic acid and protein sequences corresponding to TAA (l)-(36) are available in public databases such as GenBank.
• Tumor- associated antigens targeted by antibodies include all amino acid sequence variants and isoforms possessing at least about 70%, 80%>, 85%, 90%>, or 95% sequence identity relative to the sequences identified in the cited references, or which exhibit substantially the same biological properties or characteristics as a TAA having a sequence found in the cited references.
• a TAA having a variant sequence generally is able to bind specifically to an antibody that binds specifically to the TAA with the corresponding sequence listed.
• the sequences and disclosure in the reference specifically recited herein are expressly incorporated by reference.
• BMPR1B bone morphogenetic protein receptor-type IB, Genbank accession
• NP 001194 bone morphogenetic protein receptor, type IB /pid NP_001194.1 - Cross-references: MIM:603248; NP 001194.1; AY065994
• WO2004048938 (Example 2); WO2004032842 (Example IV); WO2003042661 (Claim 12); WO2003016475 (Claim 1); WO200278524 (Example 2); WO200299074 (Claim 19; Page 127-129); WO200286443 (Claim 27; Pages 222, 393); WO2003003906 (Claim 10; Page 293); WO200264798 (Claim 33; Page 93-95); WO200014228 (Claim 5; Page 133-136); US2003224454 (Fig 3); WO2003025138 (Claim 12; Page 150);
• member 5 /pid NP_003477.3 - Homo sapiens
• EP1394274 (Example 11); WO2004016225 (Claim 2); WO2003042661 (Claim 12);
• WO200289747 (Example 5; Page 618-619); WO2003022995 (Example 9; Fig 13A, Example 53; Page 173, Example 2; Fig 2A); NP 036581 six transmembrane epithelial antigen of the prostate
• WO200292836 (Claim 6; Fig 12); WO200283866 (Claim 15; Page 116-121);
• MPF MPF
• MSLN MSLN
• SMR megakaryocyte potentiating factor
• mesothelin mesothelin
• Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium
• Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1 -like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession no. AB040878) Nagase T., et al (2000) DNA Res. 7 (2): 143-150); WO2004000997 (Claim 1);
• WO2003003984 (Claim 1); WO200206339 (Claim 1; Page 50); WO200188133 (Claim 1; Page 41-43, 48-58); WO2003054152 (Claim 20); WO2003101400 (Claim 11);
• PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession no. AY358628); Ross et al (2002) Cancer Res. 62:2546-2553; US2003129192 (Claim 2); US2004044180 (Claim 12); US2004044179 (Claim 11); US2003096961 (Claim 11); US2003232056 (Example 5); WO2003105758 (Claim 12); US2003206918 (Example 5); EP1347046 (Claim 1); WO2003025148 (Claim 20);
• ETBR Endothelin type B receptor, Genbank accession no. AY275463
• WO2004048938 (Example 2); WO2004040000 (Claim 151); WO2003087768 (Claim 1); WO2003016475 (Claim 1); WO2003016475 (Claim 1); WO200261087 (Fig 1);
• WO2003016494 (Fig 6); WO2003025138 (Claim 12; Page 144); WO200198351 (Claim 1; Page 124-125); EP522868 (Claim 8; Fig 2); WO200177172 (Claim 1; Page 297-299);
• WO2003104275 (Claim 1); WO2004046342 (Example 2); WO2003042661 (Claim 12); WO2003083074 (Claim 14; Page 61); WO2003018621 (Claim 1); WO2003024392 (Claim 2; Fig 93); WO200166689 (Example 6);
• LocusID 54894; NP_060233.2; NM_017763_1
• STEAP2 (HGNC 8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1 , prostate cancer associated protein 1 , six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein, Genbank accession no.
• TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession no. NM 017636)
• WO200040614 (Claim 14; Page 100-103); WO200210382 (Claim 1; Fig 9A);
• WO2003042661 (Claim 12); WO200230268 (Claim 27; Page 391); US2003219806 (Claim 4); WO200162794 (Claim 14; Fig 1A-D);
• CRIPTO (CR, CRl, CRGF, CRIPTO, TDGFl, teratocarcinoma-derived growth factor, Genbank accession no. NP 003203 or NM 003212)
• WO200216413 (Claim 1; Page 94-95, 105); WO200222808 (Claim 2; Fig 1); US5854399 (Example 2; Col 17-18); US5792616 (Fig 2); Cross-references: MIM: 187395; NP_003203.1; NM_003212_1
• CD21 CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virus
• WO9102536 (Fig 9.1-9.9); WO2004020595 (Claim 1);
• CD79b (CD79B, CD79 , IGb (immunoglobulin-associated beta), B29, Genbank accession no. NM 000626 or 11038674)
• WO2003048202 (claim 1, pages 306 and 309); WO 99/558658, US6534482 (claim 13, Fig 17A/B); WO200055351 (claim 11, pages 1145-1146);
• FcRH2 (IFGP4, IRTA4, SPAPIA (SH2 domain containing phosphatase anchor protein la), SPAP1B, SPAP1C, Genbank accession no. NM 030764, AY358130)
• WO2003055439 (Claim 29; Fig 1A-B); WO2003025228 (Claim 37; Fig 5C);
• WO200222636 (Example 13; Page 95-107); WO200212341 (Claim 68; Fig 7);
• NCA (CEACAM6, Genbank accession no. M18728);
• WO200222153 (Page 45-47); US2002042366 (Page 20-21); WO200146261 (Page
• EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession no. NM 004442) Chan,J. and Watt, V.M., Oncogene 6 (6), 1057-1061 (1991) Oncogene 10 (5):897-905 (1995), Annu. Rev. Neurosci. 21 :309-345 (1998), Int. Rev. Cytol.
• WO2002102235 (Claim 13; Page 299); US2003091580 (Example 2); WO200210187 (Claim 6; Fig 10); WO200194641 (Claim 12; Fig 7b); WO200202624 (Claim 13; Fig 1A-1B); US2002034749 (Claim 54; Page 45-46); WO200206317 (Example 2; Page 320-321, Claim 34; Page 321-322); WO200271928 (Page 468-469); WO200202587 (Example 1; Fig 1); WO200140269 (Example 3; Pages 190-192); WO200036107 (Example 2; Page 205-207); WO2004053079 (Claim 12); WO2003004989 (Claim 1); WO200271928 (Page 233-234, 452-453); WO 0116318;
• PSCA Prostate stem cell antigen precursor, Genbank accession no. AJ297436
• WO2003003906 (Claim 10; Page 288); WO200140309 (Example 1; Fig 17);
• WO9851805 (Claim 17; Page 97); W09851824 (Claim 10; Page 94); WO9840403 (Claim 2; Fig IB);
• AAP14954 lipoma HMGIC fusion-partner-like protein /pid AAP14954.1 - Homo sapiens Species: Homo sapiens (human)
• WO2003054152 (Claim 20); WO2003000842 (Claim 1); WO2003023013 (Example 3,
• WO2004011611; WO2003045422 (Example; Page 32-33); WO2003014294 (Claim 35; Fig 6B); WO2003035846 (Claim 70; Page 615-616); WO200294852 (Col 136-137);
• WO200238766 (Claim 3; Page 133); WO200224909 (Example 3; Fig 3);
• CD22 B-cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814, Genbank accession No. AK026467); Wilson et al (1991) J. Exp. Med. 173: 137-146; WO2003072036 (Claim 1; Fig 1);
• CD79a (CD79A, CD79a, immunoglobulin-associated alpha, a B cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with Ig M molecules, transduces a signal involved in B-cell differentiation), pi: 4.84, MW: 25028 TM: 2 [P] Gene Chromosome: 19ql3.2, Genbank accession No. NP 001774.10)
• CXCR5 Burkitt's lymphoma receptor 1, a G protein-coupled receptor that is activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and perhaps development of AIDS, lymphoma, myeloma, and leukemia); 372 aa, pi: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome: l lq23.3, Genbank accession No. NP_001707.1)
• HLA-DOB Beta subunit of MHC class II molecule (la antigen) that binds peptides and presents them to CD4+ T lymphocytes); 273 aa, pi: 6.56 MW: 30820 TM: 1 [P] Gene Chromosome: 6p21.3, Genbank accession No. NP 002111.1)
• CD72 B-cell differentiation antigen CD72, Lyb-2) PROTEIN SEQUENCE Full maeaity...tafrfpd (1..359; 359 aa), pi: 8.66, MW: 40225 TM: 1 [P] Gene Chromosome: 9pl3.3, Genbank accession No. NP_001773.1)
• WO2004042346 (claim 65); WO2003026493 (pages 51-52, 57-58); WO200075655 (pages 105-106); Von Hoegen et al (1990) J. Immunol. 144(12):4870-4877; Strausberg et al (2002) Proc. Natl. Acad.
• LY64 Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine rich repeat (LRR) family, regulates B-cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosis); 661 aa, pi: 6.20, MW: 74147 TM: 1 [P] Gene Chromosome: 5ql2, Genbank accession No.
• FcRHl Fc receptor- like protein 1, a putative receptor for the immunoglobulin Fc domain that contains C2 type Ig-like and IT AM domains, may have a role in B-lymphocyte differentiation
• WO2003089624 (claim 7); (35) IRTA2 (Immunoglobulin superfamily receptor translocation associated 2, a putative immunoreceptor with possible roles in B cell development and lymphomagenesis;
• TENB2 tomoregulin, TPEF, HPP 1 , TR, putative transmembrane
• proteoglycan related to the EGF/heregulin family of growth factors and follistatin
• 374 aa NCBI Accession: AAD55776, AAF91397, AAG49451, NCBI RefSeq: NP 057276; NCBI Gene: 23671; OMIM: 605734; SwissProt Q9UIK5; Genbank accession No. AF179274; AY358907, CAF85723, CQ782436
• WO2004074320 (SEQ ID NO 810); JP2004113151 (SEQ ID NOS 2, 4, 8); WO2003042661 (SEQ ID NO 580); WO2003009814 (SEQ ID NO 411); EP1295944 (pages 69-70);
• WO200230268 page 329
• WO200190304 SEQ ID NO 2706
• US2004249130
• PMEL17 (silver homolog; SILV; D12S53E; PMEL17; (SI); (SIL); ME20; gplOO) BC001414; BT007202; M32295; M77348; NM 006928; McGlinchey,R.P. et al (2009) Proc. Natl. Acad. Sci. U.S.A. 106 (33), 13731-13736; Kummer,M.P. et al (2009) J. Biol. Chem. 284 (4), 2296-2306;
• TMEFF1 transmembrane protein with EGF-like and two follistatin-like domains 1; Tomoregulin- 1; H7365; C9orf2; C90RF2; U19878; X83961) NM 080655; NM 003692; Harms, P.W. (2003) Genes Dev. 17 (21), 2624-2629; Gery, S. et al (2003) Oncogene 22 (18):2723-2727;
• GDNF-Ral GDNF family receptor alpha 1 ; GFRA1; GDNFR; GDNFRA; RETL1; TRNR1; RET1L; GDNFR-alphal; GFR-ALPHA-1; U95847; BC014962; NM 145793)
• Ly6E lymphocyte antigen 6 complex, locus E; Ly67,RIG-E,SCA-2,TSA-l
• TMEM46 shisa homolog 2 (Xenopus laevis); SHISA2) NP 001007539.1;
• Ly6G6D lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT1
• NP_067079.2 NP_067079.2
• NM_021246.2 Mallya, M. et al (2002) Genomics 80 (1): 113-123; Ribas,G. et al (1999) J. Immunol. 163 (l):278-287;
• LGR5 leucme-rich repeat-containing G protein-coupled receptor 5; GPR49, GPR67
• NP 003658.1 NM 003667.2
• Salanti,G. et al (2009) Am. J. Epidemiol. 170 (5):537-545; Yamamoto,Y. et al (2003) Hepatology 37 (3):528-533
• RET ret proto-oncogene; MEN2A; HSCR1; MEN2B; MTC1; (PTC); CDHF12;
• LY6K lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348; FLJ35226) NP_059997.3; NM_017527.3; Ishikawa,N. et al (2007) Cancer Res. 67 (24): 11601-11611; de Nooij-van Dalen,A.G. et al (2003) Int. J. Cancer 103 (6):768-774; (46) GPR19 (G protein-coupled receptor 19; Mm.4787) NP_006134.1; NM_006143.2;
• Tyrosinase (TYR; OCAIA; OCAIA; tyrosinase; SHEP3) NP 000363.1; NM 000372.4; Bishop,D.T. et al (2009) Nat. Genet. 41 (8):920-925; Nan, H. et al (2009) Int. J. Cancer 125 (4):909-917;
• TMEM118 ring finger protein, transmembrane 2; RNFT2; FLJ14627
• GPR172A G protein-coupled receptor 172A; GPCR41; FLJ11856; D15Ertd747e
• the parent antibody may also be a fusion protein comprising an albumin-binding peptide (ABP) sequence (Dennis et al. (2002) “Albumin Binding As A General Strategy For Improving The Pharmacokinetics Of Proteins” J Biol Chem. 277:35035-35043; WO
• ABSP albumin-binding peptide
• Antibodies of the invention include fusion proteins with ABP sequences taught by: (i) Dennis et al (2002) J Biol Chem. 277:35035-35043 at Tables III and IV, page 35038; (ii) US 20040001827 at [0076] SEQ ID NOS: 9-22; and (iii) WO 01/45746 at pages 12-13, SEQ ID NOS: zl-zl4, and all of which are incorporated herein by reference.
• DNA encoding an amino acid sequence variant of the starting polypeptide is prepared by a variety of methods known in the art. These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared DNA encoding the polypeptide. Variants of recombinant antibodies may be constructed also by restriction fragment manipulation or by overlap extension PCR with synthetic oligonucleotides. Mutagenic primers encode the cysteine codon replacement(s). Standard mutagenesis techniques can be employed to generate DNA encoding such mutant cysteine engineered antibodies.
• Site-directed mutagenesis is one method for preparing substitution variants, i.e.
• mutant proteins This technique is well known in the art (see for example, Carter (1985) et al Nucleic Acids Res. 13:4431-4443; Ho et al (1989) Gene (Amst.) 77:51-59; and Kunkel et al (1987) Proc. Natl. Acad. Sci. USA 82:488). Briefly, in carrying out site-directed mutagenesis of DNA, the starting DNA is altered by first hybridizing an oligonucleotide encoding the desired mutation to a single strand of such starting DNA.
• a DNA polymerase is used to synthesize an entire second strand, using the hybridized oligonucleotide as a primer, and using the single strand of the starting DNA as a template.
• the oligonucleotide encoding the desired mutation is incorporated in the resulting double-stranded DNA.
• Site-directed mutagenesis may be carried out within the gene expressing the protein to be mutagenized in an expression plasmid and the resulting plasmid may be sequenced to confirm the introduction of the desired cysteine replacement mutations (Liu et al (1998) J. Biol. Chem. 273:20252-20260).
• Site-directed of protocols and formats including those commercially available, e.g. QuikChange® Multi Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA).
• PCR mutagenesis is also suitable for making amino acid sequence variants of the starting polypeptide. See Higuchi, (1990) in PCR Protocols, pp.177-183, Academic Press; Ito et al (1991) Gene 102:67-70; Bernhard et al (1994) Bioconjugate Chem. 5: 126-132; and Vallette et al (1989) Nuc. Acids Res. 17:723-733. Briefly, when small amounts of template DNA are used as starting material in a PCR, primers that differ slightly in sequence from the corresponding region in a template DNA can be used to generate relatively large quantities of a specific DNA fragment that differs from the template sequence only at the positions where the primers differ from the template.
• the starting material is the plasmid (or other vector) comprising the starting polypeptide DNA to be mutated.
• the codon(s) in the starting DNA to be mutated are identified. There must be a unique restriction endonuclease site on each side of the identified mutation site(s). If no such restriction sites exist, they may be generated using the above described oligonucleotide -mediated
• oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures, wherein the two strands of the oligonucleotide are synthesized separately and then hybridized together using standard techniques.
• This double-stranded oligonucleotide is referred to as the cassette.
• This cassette is designed to have 5' and 3' ends that are compatible with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid.
• This plasmid now contains the mutated DNA sequence. Mutant DNA containing the encoded cysteine replacements can be confirmed by DNA sequencing.
• Single mutations are also generated by oligonucleotide directed mutagenesis using double stranded plasmid DNA as template by PCR based mutagenesis (Sambrook and Russel, (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; Zoller et al (1983) Methods Enzymol. 100:468-500; Zoller, M.J. and Smith, M. (1982) Nucl. Acids Res.
• hu4D5Fabv8 displayed on M13 phage (Gerstner et al (2002) "Sequence Plasticity In The Antigen-Binding Site Of A Therapeutic Anti-HER2 Antibody", J Mol Biol. 321 : 851-62) was used for experiments as a model system. Cysteine mutations were introduced in hu4D5Fabv8-phage, hu4D5Fabv8, and ABP-hu4D5Fabv8 constructs. The hu4D5-ThioFab-Phage preps were carried out using the polyethylene glycol (PEG) precipitation method as described earlier (Lowman, Henry B. (1998) Methods in Molecular Biology (Totowa, New Jersey) 87 (Combinatorial Peptide Library Protocols) 249-264).
• PEG polyethylene glycol
• Oligonucleotides are prepared by the phosphoramidite synthesis method (US
• the phosphoramidite method entails cyclical addition of nucleotide monomer units with a reactive 3 ' phosphoramidite moiety to an oligonucleotide chain growing on a solid-support comprised of controlled-pore glass or highly crosslinked polystyrene, and most commonly in the 3' to 5' direction in which the 3' terminus nucleoside is attached to the solid-support at the beginning of synthesis (US 5047524; US 5262530).
• Oligonucleotides can be chemically labelled with non-isotopic moieties for detection, capture, stabilization, or other purposes (Andrus, A. "Chemical methods for 5' non-isotopic labelling of PCR probes and primers” (1995) in PCR 2: A Practical Approach, Oxford University Press, Oxford, pp. 39-54; Hermanson, G. in Bioconjugate Techniques (1996) Academic Press, San Diego, pp. 40-55, 643-671; Keller, G. and Manak, M. in DNA Probes Second Edition (1993), Stockton Press, New York, pp. 121-23).
• PHESELECTOR ASSAY Applied Biosystems, Foster City, CA.
• the PHESELECTOR Phage ELISA for Selection of Reactive Thiols assay allows for detection of reactive cysteine groups in antibodies in an ELISA phage format (US 7521541; Junutula JR et al. "Rapid identification of reactive cysteine residues for site- specific labeling of antibody-Fabs" J Immunol Methods 2008;332:41-52).
• the process of coating the protein (e.g. antibody) of interest on well surfaces, followed incubation with phage particles and then HRP labeled secondary antibody with absorbance detection is detailed in Example 2. Mutant proteins displayed on phage may be screened in a rapid, robust, and high-throughput manner. Libraries of cysteine engineered antibodies can be produced and subjected to binding selection using the same approach to identify
• FIG. 8 illustrates the PHESELECTOR Assay by a schematic representation depicting the binding of Fab or ThioFab to HER2 (top) and biotinylated ThioFab to streptavidin (bottom).
• DNA encoding the cysteine engineered antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
• the hybridoma cells serve as a source of such DNA.
• the DNA may be placed into expression vectors, which are then trans fected into host cells such as E.
• coli cells simian COS cells, Chinese Hamster Ovary (CHO) cells, or other mammalian host cells, such as myeloma cells (US 5807715; US 2005/0048572; US 2004/0229310) that do not otherwise produce the antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
• myeloma cells US 5807715; US 2005/0048572; US 2004/0229310
• the yields of hu4D5Fabv8 cysteine engineered antibodies were similar to wild type hu4D5Fabv8.
• Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al (1993) Curr. Opinion in Immunol. 5:256- 262 and Pluckthun (1992) Immunol. Revs. 130: 151-188.
• cysteine engineered antibodies e.g. ThioFabs, with highly reactive unpaired Cys residues
• a bacterial e.g. E. coli
• a mammalian cell culture system e.g. Chinese Hamster Ovary cells (CHO)
• CHO Chinese Hamster Ovary cells
• ThioFabs were expressed upon induction in 34B8, a non-suppressor E. coli strain (Baca et al (1997) Journal Biological Chemistry 272(16): 10678-84). See Example 3a.
• the harvested cell pellet was resuspended in PBS (phosphate buffered saline), total cell lysis was performed by passing through a microfluidizer and the ThioFabs were purified by affinity chromatography with protein G SEPHAROSETM (Amersham).
• ThioFabs were conjugated with biotin-PEO-maleimide as described above and the biotinylated-ThioFabs were further purified by Superdex-200TM (Amersham) gel filtration chromatography, which eliminated the free biotin-PEO-maleimide and the oligomeric fraction of ThioFabs.
• LC-ESI-MS Liquid chromatography electrospray ionization mass spectrometric analysis was employed for the accurate molecular weight determination of biotin conjugated Fab (Cole, R.B. Electro Spray Ionization Mass Spectrometry: Fundamentals, Instrumentation And Applications. (1997) Wiley, New York).
• the antibody Fab fragment hu4D5Fabv8 contains about 445 amino acid residues, including 10 Cys residues (five on the light and five on the heavy chain).
• the high-resolution structure of the humanized 4D5 variable fragment (Fv4D5) has been established, see:
• Figure 1 A shows a three-dimensional representation of the hu4D5Fabv8 antibody fragment derived by X-ray crystal coordinates.
• the structure positions of the engineered Cys residues of the heavy and light chains are numbered according to a sequential numbering system.
• This sequential numbering system is correlated to the Kabat numbering system (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD) for the 4d5v7fabH variant of trastuzumab according to Figure IB which shows the sequential numbering scheme (top row), starting at the N-terminus, differs from the Kabat numbering scheme (bottom row) by insertions noted by a,b,c.
• the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain.
• the cysteine engineered heavy chain variant sites are identified by the sequential numbering and Kabat numbering schemes in the following chart:
• Ml 3 phagemid-Cys mutant Fabs ( Figures 3 A and 3B) can be rapidly screened compared to Fab proteins.
• Phagemid-ThioFab binding to antigen and to streptavidin can be tested by coating HER2 and streptavidin, respectively, onto ELISA plates followed by probing with anti-Fab-HRP (Horse radish peroxidase) as described in Example 2 and depicted in Figure 8.
• This method allowed simultaneous monitoring of the effect on the antigen binding and the reactivity of the thiol group by the engineered Cys
• the method can be applied to screen the reactive thiol groups for any protein displayed on Ml 3 phage.
• Conjugated or unconjugated phagemid-ThioFabs are purified by simple PEG precipitation.
• FIG. 8 is a graphical representation of the PHESELECTOR assay, depicting binding of a biotinylated ThioFab phage and an anti-phage HRP antibody to HER2 (top) and Streptavidin (bottom).
• Biotin conjugated and unconjugated variants were tested for HER2 and streptavidin binding using an ELISA based PHESELECTOR assay ( Figure 8, Example 2) with an HRP (horseradish peroxidase)-conjugated anti-phage antibody.
• HRP horseradish peroxidase
• the ThioFabs-phage samples showed varying levels of streptavidin binding activity. From all the tested phage-ThioFabs, the A121C cysteine engineered antibody exhibited maximal thiol reactivity. Even though wild type hu4D5Fabv8- phage was incubated with the same amounts of biotin-maleimide, these phage had little streptavidin binding indicating that preexisting cysteine residues (involved in disulfide bond formation) from the hu4D5Fabv8 and Ml 3 phage coat proteins did not interfere with the site- specific conjugation of biotin-maleimide. These results demonstrate that the phage ELISA assay can be used successfully to screen reactive thiol groups on the Fab surface.
• the PHESELECTOR assay allows screening of reactive thiol groups in antibodies.
• a parameter, fractional surface accessibility was employed to identify and quantitate the accessibility of solvent to the amino acid residues in a polypeptide.
• the surface accessibility can be expressed as the surface area (A 2 ) that can be contacted by a solvent molecule, e.g. water.
• a solvent molecule e.g. water.
• the occupied space of water is approximated as a 1.4 A radius sphere.
• Software is freely available or licensable (Secretary to CCP4, Daresbury Laboratory, Warrington, WA4 4 AD, United Kingdom, Fax: (+44) 1925 603825, or by internet:
• AREAIMOL defines the solvent accessible surface of a protein as the locus of the centre of a probe sphere (representing a solvent molecule) as it rolls over the Van der Waals surface of the protein.
• AREAIMOL calculates the solvent accessible surface area by generating surface points on an extended sphere about each atom (at a distance from the atom centre equal to the sum of the atom and probe radii), and eliminating those that lie within equivalent spheres associated with neighboring atoms.
• AREAIMOL finds the solvent accessible area of atoms in a PDB coordinate file, and summarizes the accessible area by residue, by chain and for the whole molecule. Accessible areas (or area differences) for individual atoms can be written to a pseudo-PDB output file.
• AREAIMOL assumes a single radius for each element, and only recognizes a limited number of different elements. Unknown atom types (i.e. those not in AREAIMOL's internal database) will be assigned the default radius of 1.8 A. The list of recognized atoms is:
• AREAIMOL and SURFACE report absolute accessibilities, i.e. the number of square Angstroms (A).
• Fractional surface accessibility is calculated by reference to a standard state relevant for an amino acid within a polypeptide.
• the reference state is tripeptide Gly-X-Gly, where X is the amino acid of interest, and the reference state should be an 'extended' conformation, i.e. like those in beta-strands.
• the extended conformation maximizes the accessibility of X.
• a calculated accessible area is divided by the accessible area in a Gly-X- Gly tripeptide reference state and reports the quotient, which is the fractional accessibility. Percent accessibility is fractional accessibility multiplied by 100.
• Another exemplary algorithm for calculating surface accessibility is based on the SOLV module of the program xsae (Broger, C, F. Hoffman-LaRoche, Basel) which calculates fractional accessibility of an amino acid residue to a water sphere based on the X- ray coordinates of the polypeptide.
• Residues are sorted based on their role in functional and structural interactions of
• Thiol reactivity may be generalized to any antibody where substitution of amino acids with reactive cysteine amino acids may be made within the ranges in the light chain selected from: L-10 to L-20; L-38 to L-48; L-105 to L-l 15; L-139 to L-149; L-163 to L-173; and within the ranges in the heavy chain selected from: H-35 to H-45; H-83 to H-93; H-l 14 to H- 127; and H-l 70 to H-l 84, and in the Fc region within the ranges selected from H-268 to H- 291; H-319 to H-344; H-370 to H-380; and H-395 to H-405.
• Thiol reactivity may also be generalized to certain domains of an antibody, such as the light chain constant domain (CL) and heavy chain constant domains, CHI, CH2 and CH3. Cysteine replacements resulting in thiol reactivity values of 0.6 and higher may be made in the heavy chain constant domains ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ of intact antibodies: IgA, IgD, IgE, IgG, and IgM, respectively, including the IgG subclasses: IgGl, IgG2, IgG3, IgG4, IgA, and IgA2.
• Thiol reactivity data is shown in Figures 3A and 3B for amino acid residues of 4D5 ThioFab Cys mutants: (3 A) non-biotinylated (control) and (3B) biotinylated phage-ThioFabs. Reactive thiol groups on antibody/Fab surface were identified by PHESELECTOR assay analyses for the interaction of non-biotinylated phage-hu4D5Fabv8 (3 A) and biotinylated phage-hu4D5Fabv8 (3B) with BSA (open box), HER2 (grey box) or streptavidin (solid box). The assay was carried out as described in Example 2.
• Light chain variants are on the left side and heavy chain variants are on the right side.
• the binding of non-biotinylated 4D5 ThioFab Cys mutants is low as expected, but strong binding to HER2 is retained.
• the ratio of binding to streptavidin and to HER2 of the biotinylated 4D5 ThioFab Cys mutants gives the thiol reactivity values in Table 2.
• Background absorbance at 450 nm or small amounts of nonspecific protein binding of the biotinylated 4D5 ThioFab Cys mutants to BSA is also evident in Figure 3B. Fractional Surface Accessibility values of the selected amino acid residues that were replaced with a Cys residue are shown in Figure 4A.
• Amino acids at positions L-15, L-43, L-l 10, L-144, L-168, H-40, H-88, H-l 19, H- 121, H-l 22, H-l 75, and H-l 79 of an antibody may generally be mutated (replaced) with free cysteine amino acids. Ranges within about 5 amino acid residues on each side of these positions may also be replaced with free cysteine acids, i.e.
• L light chain
• H heavy chain
• A alanine
• S serine
• V valine
• C cysteine
• Thiol reactivity is measured as the ratio of OD 450 nm for streptavidin binding to OD 450 ancient for HER2 (antibody) binding (Example 2). Thiol reactivity value of 1 indicates complete biotinylation of the cysteine thiol.
• Fab preparation may require 2-3 days, depending on the scale of production. During this time, thiol groups may lose reactivity due to oxidation.
• stability of the thiol reactivity of phage-thioFabs was measured ( Figure 4B).
• ThioFab-phage purification on day 1, day 2 and day 4, all the samples were conjugated with biotin-PEO-maleimide and probed with phage ELISA assay (PHESELECTOR) to test HER2 and streptavidin binding.
• PESELECTOR phage ELISA assay
• the compounds of the invention include cysteine engineered antibodies where one or more amino acids of a parent antibody are replaced with a free cysteine amino acid.
• a cysteine engineered antibody comprises one or more free cysteine amino acids having a thiol reactivity value in the range of 0.6 to l .O.
• a free cysteine amino acid is a cysteine residue which has been engineered into the parent antibody and is not part of a disulfide bridge.
• cysteine engineered antibody is prepared by a process comprising:
• the cysteine engineered antibody may be more reactive than the parent antibody with the thiol-reactive reagent.
• the free cysteine amino acid residues may be located in the heavy or light chains, or in the constant or variable domains.
• Antibody fragments e.g. Fab, may also be engineered with one or more cysteine amino acids replacing amino acids of the antibody fragment, to form cysteine engineered antibody fragments.
• Another aspect of the invention provides a method of preparing (making) a cysteine engineered antibody, comprising:
• cysteine engineered antibody is more reactive than the parent antibody with the thiol-reactive reagent.
• Step (a) of the method of preparing a cysteine engineered antibody may comprise:
• Step (b) of the method of preparing a cysteine engineered antibody may comprise expressing the cysteine engineered antibody on a viral particle selected from a phage or a phagemid particle.
• Step (b) of the method of preparing a cysteine engineered antibody may also comprise:
• Another aspect of the invention is a method of screening cysteine engineered antibodies with highly reactive, unpaired cysteine amino acids for thiol reactivity
• Step (a) of the method of screening cysteine engineered antibodies may comprise:
• Step (b) of the method of screening cysteine engineered antibodies may comprise expressing the cysteine engineered antibody on a viral particle selected from a phage or a phagemid particle.
• Step (b) of the method of screening cysteine engineered antibodies may also comprise:
• the cysteine engineered antibodies of the invention may be conjugated with any label moiety which can be covalently attached to the antibody through a reactive cysteine thiol group (Singh et al (2002) Anal. Biochem. 304: 147-15; Harlow E. and Lane, D. (1999) Using Antibodies: A Laboratory Manual, Cold Springs Harbor Laboratory Press, Cold Spring Harbor, NY; Lundblad R.L. (1991) Chemical Reagents for Protein Modification, 2nd ed. CRC Press, Boca Raton, FL).
• the attached label may function to: (i) provide a detectable signal; (ii) interact with a second label to modify the detectable signal provided by the first or second label, e.g.
• FRET fluorescence resonance energy transfer
• Labelled cysteine engineered antibodies may be useful in diagnostic assays, e.g., for detecting expression of an antigen of interest in specific cells, tissues, or serum.
• the antibody will typically be labeled with a detectable moiety.
• Radioisotopes such as 3 H, n C, 14 C, 18 F, 32 P, 35 S, 64 Cu, 68 Ga,
• the antibody can be labeled with ligand reagents that bind, chelate or otherwise complex a radioisotope metal where the reagent is reactive with the engineered cysteine thiol of the antibody, using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al, Ed. Wiley- Interscience, New York, NY, Pubs. (1991).
• Chelating ligands which may complex a metal ion include DOT A, DOPA, DOTP, DOTMA, DTP A and TETA (Macrocyclics, Dallas, TX).
• Radionuclides can be targetted via complexation with cysteine-engineered antibodies as antibody-zirconium conjugates of the invention (Wu et al (2005) Nature Biotechnology 23(9): 1137-1146).
• Metal-chelate complexes suitable as antibody labels for imaging experiments are disclosed: US 5342606; US 5428155; US 5316757; US 5480990; US 5462725; US 5428139; US 5385893; US 5739294; US 5750660; US 5834456; Hnatowich et al (1983) J. Immunol. Methods 65: 147-157; Meares et al (1984) Anal. Biochem. 142:68-78; Mirzadeh et al (1990) Bioconjugate Chem. 1 :59-65; Meares et al (1990) J. Cancerl990, Suppl. 10:21-26; Izard et al (1992) Bioconjugate Chem.
• Fluorescent labels such as rare earth chelates (europium chelates), fluorescein types including FITC, 5-carboxyfluorescein, 6-carboxy fluorescein; rhodamine types including TAMRA; dansyl; Lissamine; cyanines; phycoerythrins; Texas Red; and analogs thereof.
• the fluorescent labels can be conjugated to antibodies using the techniques disclosed in Current Protocols in Immunology, supra, for example. Fluorescent dyes and fluorescent label reagents include those which are commercially available from
• the enzyme generally catalyzes a chemical alteration of a chromogenic substrate that can be measured using various techniques. For example, the enzyme may catalyze a color change in a substrate, which can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying a change in fluorescence are described above.
• the chemiluminescent substrate becomes electronically excited by a chemical reaction and may then emit light which can be measured (using a chemiluminometer, for example) or donates energy to a fluorescent acceptor.
• enzymatic labels include luciferases ⁇ e.g., firefly luciferase and bacterial luciferase; US 4737456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRP), alkaline phosphatase (AP), ⁇ - galactosidase, glucoamylase, lysozyme, saccharide oxidases ⁇ e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.
• luciferases ⁇ e.g., firefly luciferase and bacterial luciferase;
• enzyme-substrate combinations include, for example:
• HRP Horseradish peroxidase
• OPD orthophenylene diamine
• TMB 3,3',5,5'-tetramethylbenzidine hydrochloride
• ⁇ -D-galactosidase ( ⁇ -D-Gal) with a chromogenic substrate ⁇ e.g., p- nitrophenyl- -D-galactosidase) or fluorogenic substrate 4-methylumbelliferyl- -D- galactosidase.
• a chromogenic substrate ⁇ e.g., p- nitrophenyl- -D-galactosidase
• fluorogenic substrate 4-methylumbelliferyl- -D- galactosidase 4-methylumbelliferyl- -D- galactosidase.
• a label may be indirectly conjugated with a cysteine engineered antibody.
• the antibody can be conjugated with biotin and any of the three broad categories of labels mentioned above can be conjugated with avidin or streptavidin, or vice versa. Biotin binds selectively to streptavidin and thus, the label can be conjugated with the antibody in this indirect manner.
• the polypeptide variant is conjugated with a small hapten (e.g., digoxin) and one of the different types of labels mentioned above is conjugated with an anti-hapten polypeptide variant ⁇ e.g., anti-digoxin antibody).
• a small hapten e.g., digoxin
• an anti-hapten polypeptide variant e.g., anti-digoxin antibody
• polypeptide variant of the present invention may be employed in any known assay method, such as ELISA, competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Zola, (1987) Monoclonal Antibodies: A Manual of Techniques, pp.147-158, CRC Press, Inc.).
• a detection label may be useful for localizing, visualizing, and quantitating a binding or recognition event.
• the labelled antibodies of the invention can detect cell-surface receptors.
• Another use for detectably labelled antibodies is a method of bead-based immunocapture comprising conjugating a bead with a fluorescent labelled antibody and detecting a fluorescence signal upon binding of a ligand. Similar binding detection methodologies utilize the surface plasmon resonance (SPR) effect to measure and detect antibody-antigen interactions.
• SPR surface plasmon resonance
• Detection labels such as fluorescent dyes and chemiluminescent dyes (Briggs et al (1997) "Synthesis of Functionalised Fluorescent Dyes and Their Coupling to Amines and
• the labels preferably (iii) have good water-solubility to achieve effective conjugate concentration and detection sensitivity and (iv) are non-toxic to living cells so as not to disrupt the normal metabolic processes of the cells or cause premature cell death.
• fluorescently labelled events e.g. cell surface binding of peptide-dye conjugates may be conducted on an system (FMAT® 8100 HTS System, Applied Biosystems, Foster City, Calif.) that automates mix-and-read, non-radioactive assays with live cells or beads (Miraglia, "Homogeneous cell- and bead-based assays for high throughput screening using fluorometric microvolume assay technology", (1999) J. of Biomolecular Screening 4: 193-204).
• FMAT® 8100 HTS System Applied Biosystems, Foster City, Calif.
• labelled antibodies also include cell surface receptor binding assays, inmmunocapture assays, fluorescence linked immunosorbent assays (FLISA), caspase-cleavage (Zheng, "Caspase-3 controls both cytoplasmic and nuclear events associated with Fas-mediated apoptosis in vivo", (1998) Proc. Natl. Acad. Sci. USA 95:618-23; US 6372907), apoptosis (Vermes, "A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V" (1995) J. Immunol. Methods 184:39-51) and cytotoxicity assays. Fluorometric microvolume assay technology can be used to identify the up or down regulation by a molecule that is targeted to the cell surface
• Labelled cysteine engineered antibodies of the invention are useful as imaging biomarkers and probes by the various methods and techniques of biomedical and molecular imaging such as: (i) MRI (magnetic resonance imaging); (ii) MicroCT (computerized tomography); (iii) SPECT (single photon emission computed tomography); (iv) PET
• Imaging biomarkers may be objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention.
• Biomarkers may be of several types: Type 0 are natural history markers of a disease and correlate longitudinally with known clinical indices, e.g.
• Imaging biomarkers thus can provide pharmacodynamic (PD) therapeutic information about: (i) expression of a target protein, (ii) binding of a therapeutic to the target protein, i.e. selectivity, and (iii) clearance and half-life pharmacokinetic data.
• PD pharmacodynamic
• in vivo imaging biomarkers relative to lab-based biomarkers include: non-invasive treatment, quantifiable, whole body assessment, repetitive dosing and assessment, i.e. multiple time points, and potentially transferable effects from preclinical (small animal) to clinical (human) results. For some applications, bioimaging supplants or minimizes the number of animal experiments in preclinical studies.
• Radionuclide imaging labels include radionuclides such as H, C, C, F, P, S,
• radionuclide metal ion can be complexed with a chelating linker such as DOTA.
• Linker reagents such as DOTA-maleimide (4-maleimidobutyramidobenzyl-DOTA) can be prepared by the reaction of aminobenzyl-DOTA with 4-maleimidobutyric acid (Fluka) activated with isopropylchloro formate (Aldrich), following the procedure of Axworthy et al (2000) Proc. Natl. Acad. Sci. USA 97(4): 1802-1807). DOTA-maleimide reagents react with the free cysteine amino acids of the cysteine engineered antibodies and provide a metal complexing ligand on the antibody (Lewis et al (1998) Bioconj. Chem. 9:72-86).
• Chelating linker labelling reagents such as DOTA-NHS (l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid mono (N-hydroxysuccinimide ester) are commercially available (Macrocyclics, Dallas, TX).
• Receptor target imaging with radionuclide labelled antibodies can provide a marker of pathway activation by detection and quantitation of progressive accumulation of antibodies in tumor tissue (Albert et al (1998) Bioorg. Med. Chem. Lett. 8: 1207-1210).
• the conjugated radio-metals may remain intracellular following lysosomal degradation.
• FRET fluorescence resonance energy transfer
• Reporter groups are typically fluorescent dyes that are excited by light at a certain wavelength and transfer energy to an acceptor, or quencher, group, with the appropriate Stokes shift for emission at maximal brightness.
• Fluorescent dyes include molecules with extended aromaticity, such as fluorescein and rhodamine, and their derivatives.
• the fluorescent reporter may be partially or significantly quenched by the quencher moiety in an intact peptide. Upon cleavage of the peptide by a peptidase or protease, a detectable increase in fluorescence may be measured (Knight, C. (1995) "Fluorimetric Assays of Proteolytic Enzymes", Methods in Enzymology, Academic Press, 248: 18-34).
• the labelled antibodies of the invention may also be used as an affinity purification agent.
• the labelled antibody is immobilized on a solid phase such a Sephadex resin or filter paper, using methods well known in the art.
• the immobilized antibody is contacted with a sample containing the antigen to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the antigen to be purified, which is bound to the immobilized polypeptide variant. Finally, the support is washed with another suitable solvent, such as glycine buffer, pH 5.0, that will release the antigen from the polypeptide variant.
• Labelling reagents typically bear reactive functionality which may react (i) directly with a cysteine thiol of a cysteine engineered antibody to form the labelled antibody, (ii) with a linker reagent to form a linker-label intermediate, or (iii) with a linker antibody to form the labelled antibody.
• Reactive functionality of labelling reagents include: maleimide, haloacetyl, iodoacetamide succinimidyl ester (e.g.
• An exemplary reactive functional group is N-hydroxysuccinimidyl ester (NHS) of a carboxyl group substituent of a detectable label, e.g. biotin or a fluorescent dye.
• the NHS ester of the label may be preformed, isolated, purified, and/or characterized, or it may be formed in situ and reacted with a nucleophilic group of an antibody.
• the carboxyl form of the label is activated by reacting with some combination of a carbodiimide reagent, e.g. dicyclohexylcarbodiimide, diisopropylcarbodiimide, or a uranium reagent, e.g.
• TSTU (0-(N-Succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate
• HBTU O- benzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate
• HATU (0-(7- azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate
• an activator such as 1-hydroxybenzotriazole (HOBt)
• HOBt 1-hydroxybenzotriazole
• N-hydroxysuccinimide to give the NHS ester of the label.
• the label and the antibody may be coupled by in situ activation of the label and reaction with the antibody to form the label-antibody conjugate in one step.
• Other activating and coupling reagents include TBTU (2-(lH-benzotriazo-l-yl)-l-l,3,3- tetramethyluronium hexafluorophosphate), TFFH (N,N',N",N"'-tetramethyluronium 2- fluoro-hexafluorophosphate), PyBOP (benzotriazole- 1 -yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate, EEDQ (2-ethoxy-l-ethoxycarbonyl-l,2-dihydro-quinoline), DCC (dicyclohexylcarbodiimide); DIPCDI (diisopropylcarbodiimide), MSNT (l-(mesitylene-2- sulfonyl)-3-nitro
• ThioFab constructs were cloned into an expression vector under alkaline phosphatase promoter (Chang et al (1987) Gene 55: 189-196) and the ThioFab expression was induced by growing E. coli cells in the phosphate-free medium.
• ThioFabs were purified on a Protein G SEPHAROSETM column and analyzed on reducing and non-reducing SDS-PAGE gels.
• ThioFabs L-V15C, L-Vl IOC, H-A88C, and H-A121C were expressed and purified by Protein-G SEPHAROSETM column chromatography (see methods sections for details). Purified proteins were analyzed on SDS-PAGE gel in reducing (with DTT) and non-reducing (without DTT) conditions. Other reducing agents such as BME (beta-mercaptoethanol) can used in the gel to cleave interchain disulfide groups. It is evident from SDS-PAGE gel analysis that the major (-90%) fraction of ThioFab is in the monomeric form, while wild type hu4D5Fabv8 is essentially in the monomeric form (47 kDa).
• ThioFab (A121C) and wild type hu4D5Fabv8 were incubated with 100 fold excess of biotin-maleimide for 3 hours at room temperature and the biotinylated Fabs were loaded onto a Superdex-200TM gel filtration column. This purification step was useful in separating monomeric Fab from oligomeric Fab and also from excess free biotin-maleimide (or free zirconium reagent).
• FIG. 5 shows validation of the properties of ThioFab variants in the absence of the phage context.
• the proteins without phage fusion, hu4D5Fabv8 and hu4D5Fabv8-A121C (ThioFab-A121C) were expressed and purified using protein-G agarose beads followed by incubation with 100 fold molar excess of biotin-maleimide.
• Streptavidin and HER2 binding of a biotinylated cys engineered ThioFab and a non-biotinylated wild type Fab was compared.
• the extent of biotin conjugation (interaction with streptavidin) and their binding ability to HER2 were monitored by ELISA analyses. Each Fab was tested at 2ng and 20ng.
• Biotinylated A121C ThioFab retained comparable HER2 binding to that of wild type hu4D5Fabv8 (Figure 5).
• Wild type Fab and A121C-ThioFab were purified by gel filtration column chromatography. The two samples were tested for HER2 and streptavidin binding by ELISA using goat anti-Fab-HRP as secondary antibody. Both wild type (open box) and
• ThioFab (dotted box) have similar binding to HER2 but only ThioFab retained streptavidin binding. Only a background level of interaction with streptavidin was observed with non- biotinylated wild type hu4D5Fabv8 ( Figure 5).
• Plasma-protein binding can be an effective means of improving the pharmacokinetic properties of short lived molecules.
• Albumin is the most abundant protein in plasma.
• Serum albumin binding peptides can alter the pharmacodynamics of fused active domain proteins, including alteration of tissue uptake, penetration, and diffusion.
• albumin binding peptide sequence US 20040001827.
• a series of albumin binding peptides were identified by phage display screening (Dennis et al. (2002) “Albumin Binding As A General Strategy For Improving The Pharmacokinetics Of Proteins” J Biol Chem. 277:35035-35043; WO 01/45746).
• Compounds of the invention include ABP sequences taught by: (i) Dennis et al (2002) J Biol Chem.
• Albumin Binding (ABP)-Fabs were engineered by fusing an albumin binding peptide to the C-terminus of Fab heavy chain in 1 : 1 stoichiometric ratio (1 ABP / 1 Fab). It was shown that association of these ABP-Fabs with albumin increased their half life by more than 25 fold in rabbits and mice. The above described reactive Cys residues can therefore be introduced in these ABP-Fabs and used for site-specific conjugation with zirconium reagents followed by in vivo animal studies.
• Exemplary albumin binding peptide sequences include, but are not limited to the amino acid sequences listed in SEQ ID NOS: 1-5:
• the albumin binding peptide does not compete with ligands known to bind albumin and has a half life (T1 ⁇ 2) in rabbit of 2.3 hr.
• ABP-ThioFab proteins were purified on BSA-SEPHAROSETM followed by biotin-maleimide conjugation and purification on Superdex-S200 column chromatography as described in previous sections. Purified biotinylated proteins were homogeneous and devoid of any oligomeric forms (Example 4).
• FIG. 6 shows the properties of Albumin Binding Peptide (ABP)-ThioFab variants.
• ELISA analyses were carried out to test the binding ability of ABP-hu4D5Fabv8-wt, ABP- hu4D5Fabv8-Vl IOC and ABP-hu4D5Fabv8-A121C with rabbit albumin, streptavidin and HER2.
• Biotinylated ABP-ThioFabs are capable of binding to albumin and HER2 with similar affinity to that of wild type ABP-hu4D5Fabv8 as confirmed by ELISA ( Figure 6) and BIAcore binding kinetics analysis (Table 3).
• An ELISA plate was coated with albumin, HER2 and SA as described.
• Biotinylated ABP-ThioFabs were capable of binding to streptavidin compared to non biotinylated control ABP-hu4D5Fabv8-wt indicating that ABP- ThioFabs were conjugated with biotin maleimide like ThioFabs in a site specific manner as the same Cys mutants were used for both the variants (Figure 6).
• Table 3 BIAcore kinetic analysis for HER2 and rabbit albumin binding to biotinylated
• ABP albumin binding peptide
• an albumin-binding peptide may be linked to the antibody by covalent attachment through a linker moiety.
• HER2/Fab, SA/Fab and SA/SA indicate that their interactions were monitored by anti-Fab-HRP, SA- HRP, respectively.
• SA/Fab monitors the presence of single biotin per Fab and more than one biotin per Fab is monitored by SA/SA analysis. Binding of HER2 with double cys mutants is similar to that of single Cys variants ( Figure 7). However the extent of biotinylation on double Cys mutants was higher compared to single Cys variants due to more than one free thiol group per Fab molecule ( Figure 7).
• the single cys mutants H-A88C, H- A121C and L-Vl IOC of trastuzumab, and double cys mutants VI 10C-A121C and VI 10C- A121C of trastuzumab were expressed in CHO (Chinese Hamster Ovary) cells by transient fermentation in media containing 1 mM cysteine.
• the A88C mutant heavy chain sequence (450 aa) is SEQ ID NO:6.
• the A121C mutant heavy chain sequence (450 aa) is SEQ ID NO:7.
• the VI IOC mutant light chain sequence (214 aa) is SEQ ID NO:8.
• the cysteine engineered thio-trastuzumab antibodies comprise one or more of the following variable region heavy chain sequences with a free cysteine amino acid (SEQ ID NOS: 9-16). Mutant Sequence SEQ ID NO
• cysteine engineered thio-trastuzumab antibodies comprise one or more of the following variable region light chain sequences with a free cysteine amino acid (SEQ ID NOS: 17-27).
• FIG. 13 A shows a cartoon depiction of biotinylated antibody binding to immobilized HER2 and HRP labeled secondary antibody for absorbance detection.
• Figure 13B shows binding measurements to immobilized HER2 with detection of absorbance at 450 nm of (left to right): non-biotinylated wild type trastuzumab (Wt), biotin- maleimide conjugated thio-trastuzumab variants VI IOC (single cys), A121C (single cys), and VI 10C-A121C (double cys).
• Wt non-biotinylated wild type trastuzumab
• VI IOC sino cys
• A121C single cys
• VI 10C-A121C double cys
• Figure 14A shows a cartoon depiction of a biotinylated antibody binding to immobilized HER2 with binding of biotin to anti-IgG-HRP for absorbance detection.
• Figure 14B shows binding measurements with detection of absorbance at 450 nm of biotin- maleimide conjugated thio-trastuzumab variants and non-biotinylated wild type trastuzumab in binding to streptavidin. From left to right: VI IOC (single cys), A121C (single cys), VI 10C/A121C (double cys), and trastuzumab. Each thio IgG trastuzumab variant and parent trastuzumab was tested at 1, 10, and 100 ng. The measurements show that the HER2
• ThioMabs have high thiol reactivity.
• Cysteine was introduced into the full-length 2H9 anti-EphB2R antibody at certain residues.
• the single cys mutant H-A121C of 2H9 was expressed in CHO (Chinese Hamster Ovary) cells by transient fermentation in media containing 1 mM cysteine.
• the A121C 2H9 mutant heavy chain sequence (450 aa) is SEQ ID NO:28.
• Cysteine engineered thio-2H9 antibodies comprise the following Fc constant region heavy chain sequences with a free cysteine amino acid (SEQ ID NOS: 29-38).
• Figure 16 shows non-reducing (top) and reducing (bottom) denaturing SDS-PAGE (polyacrylamide gel electrophoresis) analysis of 2H9 ThioMab Fc variants (left to right, lanes 1-9): A339C; S337C; S324C; A287C; V284C; V282C; V279C; and V273C, with 2H9 wild type, after purification on immobilized Protein A.
• the lane on the right is a size marker ladder, indicating the intact proteins are about 150 kDa, heavy chain fragments about 50 kDa, and light chain fragments about 25 kDa.
• Figure 17A shows non-reducing (left) and reducing (right) denaturing polyacrylamide gel electrophoresis analysis of 2H9 ThioMab variants (left to right, lanes 1-4): L-V15C; S179C; S375C; S400C, after purification on immobilized Protein A.
• Figure 17B shows non-reducing (left) and reducing (+DTT) (right) denaturing polyacrylamide gel electrophoresis analysis of additional 2H9 and 3A5 ThioMab variants after purification on immobilized Protein A.
• the 2H9 ThioMab variants (in the Fab as well as Fc region) were expressed and purified as described.
• all the proteins are homogenous on SDS-PAGE followed by the reduction and oxidation procedure of Example 11 to prepare reactive ThioMabs for conjugation (Example 12).
• Cysteine was introduced into the full-length 3A5 anti-MUC16 antibody at certain residues.
• the single cys mutant H-A121C of 3A5 was expressed in CHO (Chinese Hamster Ovary) cells by transient fermentation in media containing 1 mM cysteine.
• the A121C 3A5 mutant heavy chain sequence (446 aa) comprises SEQ ID NO:39.
• Cysteine engineered thio-3A5 anti-MUC16 antibodies comprise the following variable region heavy chain sequences with a free cysteine amino acid (SEQ ID NOS: 40-44). Mutant Sequence SEQ ID NO:
• Cysteine engineered thio-3A5 anti-MUC16 antibodies comprise the following variable region light chain sequences with a free cysteine amino acid (SEQ ID NOS: 45-49).
• the thiol reactivity of full length, IgG cysteine engineered antibodies was measured by biotinylation and streptavidin binding.
• a western blot assay was set up to screen the ThioMab that is specifically conjugated with biotin-maleimide.
• the antibodies are analyzed on reducing SDS-PAGE and the presence of Biotin is specifically probed by incubating with streptavidin-HRP.
• streptavidin-HRP interaction is either observed in heavy chain or light chain depending on which engineered cys variant is being used and no interaction is seen with wild type, indicating that ThioMab variants specifically conjugated the biotin at engineered Cys residue.
• Figure 18 shows denaturing gel analysis of reduced, biotinylated Thio-IgG variants after capture on immobilized anti-IgG-HRP (top gel) and streptavidin-HRP (bottom gel).
• Lane 1 3A5 H- A121C.
• Lane 2 3A5 L-V110C.
• Lane 3 2H9 H-A121C.
• Lane 4 2H9 L-V110C.
• Lane 5 anti-EphB2R 2H9 parent, wild type.
• Each mutant (lanes 1-4) was captured by anti-IgG with HRP detection (top) indicating that selectivity and affinity were retained.
• Capture by immobilized streptavidin with HRP detection (bottom) confirmed the location of biotin on heavy and light chains.
• the location of cysteine mutation on the cysteine engineered antibodies in lanes 1 and 3 is the heavy chain.
• the location of cysteine mutation on the cysteine engineered antibodies in lanes 2 and 4 is the light chain.
• the cysteine mutation site undergoes conjugation with the biotin-maleimide reagent.
• Analysis of the ThioMab cysteine engineered antibodies of Figure 18 and a 2H9 V15C variant by LC/MS gave quantitative indication of thiol reactivity (Table 5).
• Cysteine engineering was conducted in the constant domain, i.e. Fc region, of IgG antibodies. A variety of amino acid sites were converted to cysteine sites and the expressed mutants, i.e. cysteine engineered antibodies, were assessed for their thiol reactivity.
• FIG. 19 schematic diagram, the streptavidin-biotin interaction is monitored by probing with anti-IgG-HRP followed by measuring absorbance at 450 nm.
• These results confirmed 2H9- ThioFc variants V282C, A287C, A339C, S375C and S400C had moderate to highest Thiol reactivity.
• the extent of biotin conjugation of 2H9 ThioMab Fc variants was quantitated by LS/MS analysis as reported in Table 6.
• the LS/MS analysis confirmed that the A282C, S375C and S400C variants had 100% biotin conjugation and V284C and A339C had 50% conjugation, indicating the presence of a reactive cysteine thiol group.
• the other ThioFc variants, and the parent, wild type 2H9 had either very little biotinylation or none.
• Desferoxamine B ( ⁇ '- ⁇ 5-[acetyl(hydroxy)amino]pentyl ⁇ -N-[5-( ⁇ 4-[(5- aminopentyl)(hydroxy)amino]-4-oxobutanoyl ⁇ amino)pentyl]-N-hydroxysuccinamide (CAS Reg. No. 70-51-9); and also known as Deferoxamine, desferoxamine B, DFO-B, DFOA, DFB or desferal) is a bacterial siderophore produced by the actinobacter Streptomyces pilosus ( Figure 20 top). Desferoxamine B has medical applications as a chelating agent used to remove excess iron from the body (Miller, Marvin J.
• TFP-N-Suc-Df Figure 20 center
• mAb-N-Suc-Df was chelated with 89 Zr.
• Embodiments of zirconium complexes also include zirconium-binding (chelating) ligands such as DTP A (CAS Reg. No. 67-43-6), DOP A(l, 4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid) (Liu, Shuang (2008) Advanced Drug Delivery Reviews 60(12): 1347-1370), cyclopentadienyl, and allyl groups (Erker, G. (1991) Pure and Applied Chemistry 63(6):797-806; Erker, G. (1990) Jour, of Organometallic Chem. 400(1-2): 185- 203), each of which are incorporated by reference herein.
• DTP A CAS Reg. No. 67-43-6
• DOP A(l, 4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid) Liu, Shuang (2008) Advanced Drug Delivery Reviews 60(12): 1347-1370
• Zirconium complexes (Z) and other radionuclides may be conjugated to antibodies (Ab), including monoclonal antibodies (mAbs) through ⁇ -amino group in lysine side chain or through thiol group of cysteine. Since approximately 40 lysine side chains (Wang L et al "Structural characterization of the maytansinoid-monoclonal antibody immunoconjugate, huN901-DMl, by mass spectrometry” (2005) Protein Sci. 14:2436-46) or 8 cysteines (Hamblett KJ et al. "Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate" (2004) Clin Cancer Res.
• THlOMABs site-specifically radiolabeled cysteine- engineered antibodies
• One aspect of the present invention is a method for site-specific radiolabeling of THIOMABs using novel Df-based thiol reactive bifunctional reagents
• trastuzumab trastuzumab
• Zr zinc-reacted calcium
• One metastable isomer of zirconium is 89 Zr with a half-life of 78.4 hours with decay modes of beta (electron emission), positron (beta plus), and gamma radiation.
• radioisotope or other labels may be incorporated in the conjugate in known ways (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57; "Monoclonal Antibodies in Immunoscintigraphy” Chatal, CRC Press 1989).
• Carbon- 14-labeled 1-isothiocyanatobenzyl- 3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of a radionuclide to the antibody (WO 94/11026).
• a “Linker” (L) is a bifunctional or multifunctional moiety which can be used to link one or more zirconium complex moieties (Z) and an antibody unit (Ab) to form antibody- zirconium conjugates (AZC) of Formula I.
• Antibody-zirconium conjugates (AZC) can be conveniently prepared using a Linker having reactive functionality for binding to zirconium and to the Antibody.
• a cysteine thiol of a cysteine engineered antibody (Ab) can form a bond with a functional group of a linker reagent, a zirconium label moiety or zirconium- linker intermediate.
• a Linker has a reactive site which has an electrophilic group that is reactive to a nucleophilic cysteine present on an antibody.
• the cysteine thiol of the antibody is reactive with an electrophilic group on a Linker and forms a covalent bond to a Linker.
• Useful electrophilic groups include, but are not limited to, maleimide and haloacetamide groups.
• Cysteine engineered antibodies may react with linker reagents or zirconium-linker intermediates, with electrophilic functional groups such as maleimide or a-halo carbonyl, according to the conjugation method at page 766 of Klussman, et al (2004), Bioconjugate Chemistry 15(4):765-773, and according to the protocols of Examples 17-19.
• the Z moieties are the same.
• Z moieties are different.
• Exemplary embodiments of the Formula I antibody-zirconium conjugate (AZC) compounds include:
• R is independently H or Ci-C 6 alkyl; and n is 1 to 12.
• a Linker has a reactive functional group which has a nucleophilic group that is reactive to an electrophilic group present on an antibody.
• Useful electrophilic groups on an antibody include, but are not limited to, aldehyde and ketone carbonyl groups.
• the heteroatom of a nucleophilic group of a Linker can react with an electrophilic group on an antibody and form a covalent bond to an antibody unit.
• Useful nucleophilic groups on a Linker include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.
• the electrophilic group on an antibody provides a convenient site for attachment to a Linker.
• the Linker may be substituted with groups which modulated solubility or reactivity.
• a charged substituent such as sulfonate (-SO 3 ) or ammonium, may increase water solubility of the reagent and facilitate the coupling reaction of the linker reagent with the antibody or the zirconium moiety, or facilitate the coupling reaction of Ab-L (antibody-linker intermediate) with Z, or Z-L (zirconium-linker
• the compounds of the invention expressly contemplate, but are not limited to, AZC prepared with linker reagents: BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4- vinylsulfone)benzoate), and including bis-maleimide reagents: DTME, BMB, BMDB, BMH, BMOE, BM(PEO) 2 , and BM(PEO) 4 3, which are commercially available from Pierce
• Bis-maleimide reagents allow the attachment of the thiol group of a cysteine engineered antibody to a thiol-containing zirconium moiety, label, or linker intermediate, in a sequential or concurrent fashion.
• Other functional groups besides maleimide, which are reactive with a thiol group of a cysteine engineered antibody, zirconium moiety, label, or linker intermediate include iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyl disulfide isocyanate, and isothiocyanate.
• linker reagents can also be obtained via other commercial sources, such as Molecular Biosciences Inc. (Boulder, CO), or synthesized in accordance with procedures described in Toki et al (2002) J. Org. Chem. 67: 1866-1872; Walker, M.A. (1995) J. Org. Chem. 60:5352-5355; Frisch et al (1996) Bioconjugate Chem. 7: 180-186; US 6214345; WO 02/088172; US 2003130189; US2003096743; WO 03/026577; WO 03/043583; and WO 04/032828.
• Exemplary linker reagents include:
• n is an integer ranging from 1-10 and T is -H or -S0 3 Na;
• n is an integer ranging from 0-3;
• linker L may be a dendritic type linker for covalent attachment of more than one zirconium moiety through a branching, multifunctional linker moiety to an antibody (Sun et al (2002) Bioorganic & Medicinal Chemistry Letters 12:2213- 2215; Sun et al (2003) Bioorganic & Medicinal Chemistry 11 : 1761-1768).
• Dendritic linkers can increase the molar ratio of zirconium to antibody, i.e. loading of the AZC.
• a cysteine engineered antibody bears only one reactive cysteine thiol group, a multitude of zirconium moieties may be attached through a dendritic linker.
• branched, dendritic linkers include those with self-immolative 2,6-bis(hydroxymethyl)-p-cresol and 2,4,6-tris(hydroxymethyl)-phenol dendrimer units (WO 2004/01993; Szalai et al (2003) J. Amer. Chem. Soc. 125: 15688-15689; Shamis et al (2004) J. Amer. Chem. Soc. 126: 1726-1731; Amir et al (2003) Angew. Chem. Int. Ed. 42:4494- 4499).
• An aspect of the invention is a desferrioxamine-labelled, cysteine-engineered antibody comprising a cysteine engineered antibody (Ab) conjugated through a free cysteine amino acid to a linker (L) and a desferoxamine moiety (Df), having Formula II:
• L-Df is selected from:
• p 1 to 4.
• the antibody-zirconium conjugates (AZC) of Formula I may be prepared by several routes, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of a cysteine group of a cysteine engineered antibody with a linker reagent, to form antibody-linker intermediate Ab-L, via a covalent bond, followed by reaction with an activated zirconium label moiety Z; and (2) reaction of a nucleophilic group of a zirconium moiety with a linker reagent, to form zirconium label- linker intermediate Z-L, via a covalent bond, followed by reaction with a cysteine group of a cysteine engineered antibody. Conjugation methods (1) and (2) may be employed with a variety of cysteine engineered antibodies, zirconium label moieties, and linkers to prepare the antibody-zirconium conjugates of Formula I.
• Antibody cysteine thiol groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker reagents and zirconium-linker
• intermediates including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides, such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups; and (iv) disulfides, including pyridyl disulfides, via sulfide exchange.
• active esters such as NHS esters, HOBt esters, haloformates, and acid halides
• alkyl and benzyl halides such as haloacetamides
• aldehydes ketones
• carboxyl and maleimide groups
• disulfides including pyridyl disulfides, via sulfide exchange.
• Nucleophilic groups on a zirconium lable moiety include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents.
• the cysteine engineered antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (Cleland's reagent, dithiothreitol) or TCEP (tris(2-carboxyethyl)phosphine hydrochloride; Getz et al (1999) Anal. Biochem. Vol 273:73-80; Soltec Ventures, Beverly, MA).
• a reducing agent such as DTT (Cleland's reagent, dithiothreitol) or TCEP (tris(2-carboxyethyl)phosphine hydrochloride; Getz et al (1999) Anal. Biochem. Vol 273:73-80; Soltec Ventures, Beverly, MA).
• a reducing agent such as DTT (Cleland's reagent, dithiothreitol) or TCEP (tris(2-carboxyethyl)phosphine hydroch
• the reduced ThioMab was diluted and loaded onto HiTrap S column in 10 mM sodium acetate, pH 5, and eluted with PBS containing 0.3M sodium chloride. Disulfide bonds were reestablished between cysteine residues present in the parent Mab with dilute (200 nM) aqueous copper sulfate (CuS0 4 ) at room temperature, overnight.
• Other oxidants i.e.
• oxidizing agents and oxidizing conditions, which are known in the art may be used.
• Ambient air oxidation is also effective. This mild, partial reoxidation step forms intrachain disulfides efficiently with high fidelity.
• An approximate 10 fold excess of zirconium-linker intermediate is added, mixed, and let stand for about an hour at room temperature to effect conjugation and form the ThioMab antibody-zirconium conjugate.
• the conjugation mixture is gel filtered and loaded and eluted through a HiTrap S column to remove excess zirconium- linker intermediate and other impurities.
• Figure 15 shows the general process to prepare a cysteine engineered antibody expressed from cell culture for conjugation.
• Cysteine adducts presumably along with various interchain disulfide bonds, are reductively cleaved to give a reduced form of the antibody.
• the interchain disulfide bonds between paired cysteine residues are reformed under partial oxidation conditions, such as exposure to ambient oxygen.
• the newly introduced, engineered, and unpaired cysteine residues remain available for reaction with linker reagents or zirconium-linker intermediates to form the antibody conjugates of the invention.
• the ThioMabs expressed in mammalian cell lines result in externally conjugated Cys adduct to an engineered Cys through -S-S- bond formation. Hence the purified
• ThioMabs have to be treated with reduction and oxidation procedures as described in Example 11 to produce reactive ThioMabs. These ThioMabs are used to conjugate with maleimide containing radiolabels, cytotoxic drugs, fluorophores, and other labels.
• TFP-N-SucDf-Fe The protected active ester TFP-N-SucDf-Fe was prepared according to the previously described procedure (Verel I et al "89Zr Immuno-PET: Comprehensive Procedures For The Production Of 89Zr-Labeled Monoclonal Antibodies” (2003) J Nucl Med 44: 1271-81) and conjugated to trastuzumab using a 5-fold molar excess of TFP-N-SucDf-Fe to yield N-SucDf- trastuzumab with an average of 1.6 molecules of desferoxamine (Table 8).
• Df-Bz-SCN- trastuzumab was obtained by coupling an 8-fold molar excess of Df-Bz-SCN at pH 8.5 (Perk LR, et al. "Facile radiolabeling of monoclonal antibodies and other proteins with zirconium- 89 or gallium-68 for PET Imaging using p-isothiocyanatobenzyl-desferrioxamine” (2008) Nature Protocols; published online:DOI: 10.1038/nprot.2008.22).
• the reaction provided Df- Bz-SCN-trastuzumab decorated in average with 2.4 molecules of desferoxamine (Table 1).
• novel maleimide based thiol reactive bifunctional linker Df-Chx-Mal was prepared from equimolar amounts of desferoxamine mesylate and SMCC ( Figure 21,
• Example 13 The reaction was complete within 30 min at room temperature and the product was isolated by precipitation upon addition of water in 45% yield and more than 95% purity.
• the reaction of an 8.5-fold molar excess of Df-Chx-Mal with freshly prepared thio- trastuzumab (Figure 21 Example 17) provided Df-Chx-Mal-thio-trastuzumab conjugate with exactly 2 molecules of desferoxamine in 1 h (Table 1, Figure 21). Bromoacetyl
• BDf-Bac desferoxamine
• Df-Iac desferoxamine mesylate and bromoacetyl bromide at 0°C
• the product was obtained in 14% yield after HPLC purification.
• the alkylation of freshly prepared thio- trastuzumab ( Figure 21, Example 16) with a 12-fold molar excess of Df-Bac provided the conjugate (Df-Ac-thio-trastuzumab) with 1.8 molecules of Df per antibody within 5 h (Table 8, Figure 21, Example 18).
• the low reactivity of bromide prompted us to explore the more reactive iodoacetyl derivative (Df-Iac).
• Df-Iac was prepared in 53% yield by the reaction of desferoxamine mesylate with a slight excess of N-hydroxysuccinimidyl iodoacetate ( Figure 21, Example 15). The product was obtained in more than 95% purity by precipitation from the reaction mixture. The subsequent reaction of an 11 -fold excess of Df-Iac provided Df-Ac- thio-trastuzumab decorated with 1.8 molecules of Df within 2 h (Table 1, Figure 21, Example 19). Based on our experience, Df-Chx-Mal is the preferred reagent of the three compounds investigated.
• the Zr was chelated as 89-zirconium oxalatewith all four variants of Df- trastuzumab using previously described experimental procedure (Verel I et al "89Zr immuno- PET: comprehensive procedures for the production of 89Zr-labeled monoclonal antibodies” (2003) J Nucl Med. 44: 1271-81).
• the radiolabeled proteins were purified on a desalting column and the final solution was concentrated to the required volume by membrane
• the yield, purity, and final specific activity of the Zr conjugates are summarized in Table 9.
• the chelation yield was over 80% with the exception of the Df-N-Suc linker obtained in lower yield presumably due to the lower amount of Df per antibody molecule and/or incomplete removal of Fe(III) used to protect the chelator during activation and conjugation.
• the product purity was over 90% with a small amount (1-6%) of high molecular weight
• Df-Chx-Mal-thio-trastuzumab provided the Zr complex in 99% purity (Table 9) as opposed to Df-Ac conjugate which was contaminated with approximately 8% of a low molecular weight impurity and 2% of high molecular weight aggregates.
• the contaminant resisted removal using NAP- 10 column but the removal was possible using repeated buffer exchange on Amicon filter.
• the obtained K D values were compared to non-modified trastuzumab (0.91 ⁇ 0.20 nM).
• the K D for the thio-trastuzumab conjugate containing Chx-Mal linker was 0.93 ⁇ 0.15 nM and the values for the conjugates containing Ac linker were 1.22 ⁇ 0.22 nM for conjugate prepared using Df-Bac and 0.87 ⁇ 0.15 nM prepared using Df-Iac.
• the results of the biological activity analyses indicate that the modification of thio-trastuzumab did not affect the binding affinity of the antibody to HER2.
• Fig. 3 The Zr-trastuzumab uptake in selected tissues is summarized in Fig 4.
• the images at 1 h (not shown) were dominated by the high blood pool uptake with the exception of Df-Bac where rapid hepatobiliary excretion of the lipophilic impurity resulted in an elevated uptake in intestine.
• the impurity was totally cleared within the first 24 h and elevated small and large intestine uptake was not detected at 24 h or later time after tracer injection.
• the tissue uptake of Df-Ac conjugate was resultantly slightly (-8%) lower, the tumor to blood ratios (Table 10) were not affected by the loss of injected radioactivity.
• the images at 96 h were dominated by the high tumor uptake
• BT474 (3+ expression level of HER2) xenografts exhibited lower absolute uptake of the tracer (15 %ID/g) than measured previously by Dijkers et al in SKOV3 (3+ expression level of HER2) 33.4 ⁇ 7.7%ID/g (Dijkers EC, et al. "Development and Characterization of Clinical-Grade 89Zr-Trastuzumab for HER2/neu ImmunoPET Imaging" (2009) J Nucl Med 50(6):974-981).
• the tumor to blood ratio of 5.7-7.1 (Table 10) is comparable to the value obtained with SKOV3 (tumor to blood of 7.6).
• the difference in tumor uptake may be attributed to the tumor model and total dose of trastuzumab.
• a material with higher specific activity was used hence so significantly less antibody was injected (35 ⁇ g, 1.4 mg/kg) compared to the Dijkers et al study with SKOV3 (100 ⁇ g, 4 mg/kg).
• the difference in trastuzumab was attributed to the tumor model and total dose of trastuzumab.
• the bone uptake may originate from the breakdown of the Zr-
• thiol specific reagents Three thiol specific reagents are exemplified herein for the chemoselective conjugation of desferrioxamine (Df) to monoclonal antibodies through the thiol group of cysteine of cysteine-engineered antibodies.
• the thiol-specific Df-reagents were obtained by the acylation of the amino group of desferrioxamine B in 14% (Df-Bac), 53% (Df-Iac) and 45% (Df-Chx-Mal) yields and conjugated to thio-trastuzumab resulting in site-specific modification on both engineered cysteines within 1-5 h.
• the binding activities of site-specific thio-trastuzumab conjugates to HER2 were identical to the activity of non-modified trastuzumab.
• the Df-modified thio-trastuzumabs (Df-Ac-thio-trastuzumab and Df-Chx-Mal-
• Df-Chx-Mal is a useful reagent for conjugation of Df to antibodies through cysteine side chain and showed several advantages over Df-Bac and Df-Iac.
• moderate pH 7.5 was required for complete conjugation of Df-Chx-Mal within 1 h as compared to pH 9 and 2 or 5 h required for Df-Bac
• the site specifically Zr labeled engineered THIOMAB conjugates can be used similarly as 18 F labeled THIOFAB conjugates (Gill HS, et al. "A modular platform for the rapid site-specific radiolabeling of proteins with 18F exemplified by quantitative positron emission tomography of human epidermal growth factor receptor 2" (2009) Jour, of Med. Chem. 52:5816-25) as valuable tools for PET imaging applications in biomedical research.
• the antibody-zirconium conjugates (AZC) of the invention may be administered by any route appropriate to the condition to be treated.
• the AZC will typically be administered parenterally, i.e. infusion, subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural.
• compositions of diagnostic antibody-zirconium conjugates (AZC) of the invention are typically prepared for parenteral administration, i.e. bolus, intravenous, intratumor injection with a pharmaceutically acceptable parenteral vehicle and in a unit dosage injectable form.
• An antibody-zirconium conjugate (AZC) having the desired degree of purity is optionally mixed with pharmaceutically acceptable diluents, carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.), in the form of a lyophilized formulation or an aqueous solution.
• Acceptable diluents, carriers, excipients, and stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparag
• Zn-protein complexes Zn-protein complexes
• non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG).
• TWEENTM TWEENTM
• PLURONICSTM polyethylene glycol
• PEG polyethylene glycol
• the active pharmaceutical ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in
• sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semi permeable matrices of solid hydrophobic polymers containing the AZC, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
• sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or polyvinyl alcohol)), polylactides (US 3773919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.
• polyesters for example, poly(2-hydroxyethyl-methacrylate), or polyvinyl alcohol
• polylactides US 3773919
• copolymers of L-glutamic acid and gamma-ethyl-L-glutamate non-degradable ethylene-vinyl acetate
• the formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

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