HK1171679B - Zirconium-radiolabeled, cysteine engineered antibody conjugates - Google Patents

Description

Zirconium radiolabeled, cysteine engineered antibody conjugates

This application claims priority to U.S. serial No. 12/612,912, applied on day 11/5 2009, which is a continuation of U.S. serial No. 12/399,241, applied on day 3/6 2009, which is U.S. serial No. 11/233,258, applied on day 22/9/2005, a continuation of U.S. serial No.7,521,541, now granted on day 21/4/2009, and also claims priority to U.S. provisional application serial No. 60/612,468, applied on day 23/9/2004 and U.S. provisional application serial No. 60/696,353, applied on day 30/6/2005, in accordance with 35 USC § 119(e), each of which is incorporated herein by reference in its entirety.

Technical Field

The present invention relates generally to antibodies engineered with reactive cysteine residues, and more particularly to antibodies having therapeutic or diagnostic applications. Cysteine engineered antibodies may be conjugated with chemotherapeutic drugs, toxins, affinity ligands (e.g., biotin), and detection labels such as radioisotopes and fluorophores. The invention also relates to methods of using the antibodies and antibody-drug conjugate compounds for in vitro, in situ, and in vivo diagnosis or treatment of mammalian cells or associated pathological conditions.

Background

Molecular imaging is an important tool for the development and evaluation of new drugs. Immuno-positron emission tomography (immuno-PET) is an emerging method for in vivo tracking and quantification of monoclonal antibodies (mabs) because it effectively combines the high sensitivity of PET with the high specificity of mabs. Immuno-PET is expected to be a clinically-alternative for non-invasive diagnosis, providing "comprehensive in vivo immunohistochemical staining" (van Dongen GA, et al, "Immuno-PET: an navigator in monoclonal antibody detection and applications" Oncoloist 2007;12: 1379-89). Because immunoPET requires a positron-emitting radioisotope attached to a target-specific molecule, it is important to match The biological half-life of The molecule to that of The radionuclide (Verel I, et al, "The reagent of immuno-PET in radioimmunotherapy" J Nucl Med 2005;46Suppl 1: 164S-71S). Although antibodies (-150 kDa) have plasma half-lives ranging from days to weeks, imaging typically provides the maximum target-to-background ratio (target-to-background ratio) 2-6 days after administration of the antibody-based tracer, requiring the use of, for example, half a dayThe decay period is 3.3 days and 4.2 days respectively⁸⁹Zr and¹²⁴a radioisotope of I. Unfortunately, existing⁶⁴The half-life of Cu (12.7h) is too short to provide images with good contrast in this time frame.

Positron Emission Tomography (PET) imaging agents (immunoPET) developed from Mab (monoclonal antibody) templates hold promise as tools for locating and quantifying molecular targets, and may improve non-invasive clinical diagnosis of pathological conditions (vanDongen et al (2007) Oncoloist 12;1379-89; Williams et a (2001) Cancer Biotherm radiopharmam 16:25-35; Holliger et al (2005) Nat Biotechnol 23: 1126-36). PET is a molecular imaging technology that is increasingly being used for disease detection. PET imaging systems produce images based on the distribution of positron-emitting isotopes in patient tissue. The isotope is typically administered to the patient by injecting a probe molecule containing a positron-emitting isotope, such as F-18, C-11, N-13 or O-15, that is covalently linked to a molecule that is readily metabolized or localized in the body (e.g., glucose) or to a molecule that chemically binds to a receptor site in the body. Sometimes, the isotope is administered to the patient as an ionic solution or by inhalation. Small immunopet imaging agents, e.g. Fab antibody fragments (50 kDa) or diabodies, covalently linked V of mabs_H-V_LThe regiodoublet, 55kDa (Shively et al (2007) J Nucl Med 48:170-2), may be particularly useful because it exhibits a short circulating half-life, high tissue permeability, and achieves an optimal tumor background ratio between 2-4 hours post-injection, facilitating the use of short half-life isotopes, such as widely used¹⁸F (109.8 min).

Iodine 124(¹²⁴I) Linked to antibody 3F 9and used to evaluate the dosimetry of radioimmunotherapy of neuroblastoma (Larson SM, et al, "PET scanning of iodine-124-3F9as an early to clinical therapy with multiple treatment planning for radioimmunotherapy in a child with neuroblastoma" J Nucl Med1992;33: 2020-3). Since the advent of more advanced PET test equipment and improved radioiodination techniques,¹²⁴i was used for a number of immuno-PET studies (Verel I, et al, "High-quality I-lamellar monoclonal antibodies for use as PET sizing agents pro 131I-radial imaging of PET with the proviso that 31:1645-52, Lee FT et al, "Immuno-PET of human colloidal fibrous-fibrous BALB/c-fibrous using 124I-CDR-fibrous-A33-monoclonal antibodies" J Nucl medium 2001;42:764-9; Sundaresg G, et al, "124I-lamellar fibrous-media Nuodies and fibrous-polymer mixture, anti-particulate-fibrous-binder J2003, et al," PET 19I-lamellar fibrous-nucleic acids, PET 2J 2-fibrous-binder J52; PET J2-fibrous-binder J52; PET J2-fibrous-polymeric-binder J52, anti-fibrous binder J2, PET 2-fibrous binder J2, and fibrous binder J2. A-fibrous binder J2. A Med 2004, 45:1237-44, Robinson MK, et al, "Quantitative image-positional image imaging of HER 2-positional image with an index-124 label anti-HER2 diabody" Cancer Res2005, 65:1471-8, Jayson GC, et al, "Molecular imaging and biological evaluation of HuMV833 anti-VEGF antibody: images for tertiary design of antigenic agents" J Natl Cancer Inst 2002;94:1484-93; Divgi CR, et al, "predictive characterization of Molecular diagnostic-cell diagnostic-124-Molecular CR, et al," PET 250: 3: map I-33 (PET-8) and "Molecular analysis of Molecular diagnostic-PCR). Although used for connection¹²⁴The relatively simple radioiodination techniques of I to mAb are available, with significant limitations delaying widespread preclinical use of this radionuclide, especially the complex decay pattern (complex decay scheme) involving energetic positrons (β)⁺max.1.5 and 2.1MeV) that negatively impact the resolution of small animal microscopic PET. In addition, internalized iodinated proteins undergo enzymatic deiodination, free iodide is rapidly cleared from the target cell, rendering the PET map provided incapable of reflecting actual mAb uptake (Perera RM et al, "Internalization, intracellular transfection, and biodiversity of monoclonal antibodies 806: a novel anti-epithelial growth factor receptor anthe tibody "Neoplasia (New York, N.Y 2007;9: 1099-110). Use of⁸⁹Zr overcomes these disadvantages because⁸⁹Positron emitted in Zr decay (β)⁺max.897keV) provides¹⁸F and¹¹c equivalent microscopic PET resolution (about 1 mm). Additionally, internalized⁸⁹Metabolites of Zr-mAbs are trapped lysosomally in cells, providing a better correlation of actual mAb uptake with PET imaging (van Dongen GA, et al, "Immuno-PET: a promoter in monoclonal antibody levels and associations" Oncologyst 2007;12: 1379-89).

Conventional attachment means, i.e., attaching a label, such as a radioisotope, fluorochrome or drug moiety, to an antibody via a covalent bond, typically results in a heterogeneous mixture of molecules in which the label moiety is attached to many sites of the antibody. For example, cytotoxic drugs are typically conjugated to antibodies through the usually large number of lysine residues of the antibody, resulting in a heterogeneous antibody-drug conjugate mixture. Depending on the reaction conditions, the heterogeneous mixture generally comprises a distribution of antibodies having from 0 to about 8 or more drug moieties attached. In addition, there is a possible heterogeneous mixture in each subgroup of conjugates with a specific integer ratio of drug moiety to antibody, where the drug moiety is attached to a different site of the antibody. The analytical and preparative methods are insufficient to separate and characterize the antibody-drug conjugate species molecules in the heterogeneous mixture resulting from the conjugation reaction. Antibodies are large, complex and structurally diverse biomolecules, often with many reactive functional groups. Its reactivity with linker reagents and drug linker intermediates depends on factors such as pH, concentration, salt concentration, and co-solvents. Furthermore, the multi-step conjugation process may not be reproducible because of difficulties in controlling reaction conditions and characterizing reactants and intermediates.

Unlike most amino groups, which are protonated and less nucleophilic near pH7, cysteine thiols are reactive at neutral pH. Proteins with cysteine residues are usually present in their oxidized form, e.g. disulfide-linked oligomers, or have internally bridged disulfide groups, due to the relatively strong reactivity of the free thiol (RSH, sulfhydryl) groups. Extracellular proteins generally do not have free thiols (Garman,1997, Non-Radioactive Labelling: A practical approach, Academic Press, London, page 55). The amount of free thiol of a protein can be estimated by standard Ellman assay. Immunoglobulin M is an example of a disulfide-linked pentamer, while immunoglobulin G is an example of a protein with internal disulfide bonds that bond subunits together. In such proteins, it is necessary to reduce the disulfide bond using a reducing agent such as Dithiothreitol (DTT) or selenol (selenol) (Singh et al (2002) anal. biochem.304:147-156) to generate a reactive free thiol. This approach can result in loss of antibody tertiary structure and antigen binding specificity.

Antibody cysteine thiol groups are generally more reactive, i.e. more nucleophilic, than antibody amino or hydroxyl groups towards electrophilic conjugation reagents. Cysteine residues have been introduced into proteins by genetic engineering techniques to form covalent linkages to ligands or to form new intramolecular disulfide bonds (Better et al (1994) J. biol. chem.269(13): 9644-. However, designing cysteine thiol groups by mutating different amino acid residues of proteins to cysteine amino acids can be problematic, particularly in the case of unpaired (free Cys) residues or residues that are relatively easy to react or oxidize. In concentrated solutions of proteins, whether in the periplasm of E.coli, culture supernatants, or partially or fully purified proteins, unpaired Cys residues on the protein surface can pair and oxidize to form intermolecular disulfides, and thus protein dimers or multimers. Disulfide dimer formation deprives the new Cys of reactivity for conjugation to a drug, ligand, or other label. Furthermore, if the protein is oxidized to form an intramolecular disulfide bond between the newly engineered Cys and the existing Cys residue, none of the 2 Cys groups are available for active site participation and interaction. In addition, proteins can become inactive or non-specific through misfolding or loss of tertiary structure (Zhang et al (2002) anal. biochem.311: 1-9).

Site-specific conjugation is preferred over random amino modification because it enables chemical modification at sites remote from the binding site, facilitating intact retention of biological activity and allowing control of the possible number of prosthetic groups added. Cysteine engineered antibodies in the form of FAB antibody fragments (ThioFab) have been designed and expressed as full-length, IgG monoclonal (ThioMab) antibodies. See: US 7521541, Junutula JR et al, "Rapid identification of reactive cysteines for Site-specific examination of antibodies-Fabs," J Immunol Methods 2008;332:41-52; Junutula JR et al, "Site-specific conjugation of a cytoxic drug to antibodies improvesthes the thermal index" (2008) Nat Biotechnology.26: 925-32, the contents of which are incorporated by reference. ThioFab and ThioMab antibodies have been conjugated via a linker to a thiol-reactive linker reagent and a drug linker reagent on the newly introduced cysteine thiol to prepare cysteine engineered antibody drug conjugates (Thio ADCs) with anti-cancer properties, including anti-MUC 16(US 2008/0311134), anti-CD 22(US 2008/0050310), anti-ROBO 4(US2008/0247951), anti-TENB 2(US 2009/0117100), anti-CD 79B (US2009/0028856; US 2009/0068202) Thio ADCs.

SUMMARY

The compounds of the invention include cysteine engineered antibodies in which one or more amino acids of the parent antibody are replaced with free cysteine amino acids. The cysteine engineered antibody comprises one or more free cysteine amino acids having a thiol reactivity value in the range of 0.6-1.0. The free cysteine amino acids are cysteine residues that have been engineered into the parent antibody and are not part of a disulfide bond.

Cysteine engineered antibodies are useful for the diagnosis and treatment of cancer and include antibodies specific for cell surface and transmembrane receptors, and Tumor Associated Antigens (TAAs). Such antibodies can be used as naked antibodies (unconjugated to a drug or labeling moiety) or as antibody-zirconium conjugates (AZC).

An embodiment of the method of making and screening cysteine engineered antibodies includes where the parent antibody is an antibody fragment, such as hu4D5Fabv 8. 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 the group consisting of huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (trastuzumab).

The cysteine engineered antibodies of the invention may be site-specific and operatively linked to a thiol-reactive reagent. The thiol-reactive reagent may be a radioisotope reagent, a 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 support, and/or conjugated to a drug moiety.

Another aspect of the invention is a zirconium-labeled, 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 the general formula I:

Ab-(L-Z)p I

wherein p is 1 to 4.

Another aspect of the invention is a desferrioxamine (desferrioxamine) -labeled, cysteine engineered antibody comprising a cysteine engineered antibody (Ab) conjugated through a free cysteine amino acid to a linker (L) and a desferrioxamine moiety (Df), having the general formula II:

Ab-(L-Df)p II

wherein L-Df is selected from:

wherein the wavy line indicates attachment to the antibody (Ab); and

p is 1 to 4.

Another aspect of the invention is a desferrioxamine labelling reagent selected from the structures:

wherein R is selected from:

another aspect of the invention is a method of making a desferrioxamine-labeled, cysteine engineered antibody comprising a cysteine engineered antibody (Ab) conjugated through a free cysteine amino acid to a linker (L) and a desferrioxamine moiety (Df), having the general formula II:

Ab-(L-Df)p II

wherein L-Df is selected from:

wherein the wavy line indicates attachment to the antibody (Ab); and

p is 1 to 4;

the method comprises reacting a composition selected from the group consisting of the following structures with a cysteine engineered antibody having one or more free cysteine amino acids:

wherein R is selected from:

thereby forming a desferrioxamine-labelled, cysteine engineered antibody.

Another aspect of the invention is a method of making a zirconium-labeled, 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 the general formula I:

Ab-(L-Z)p I

wherein p is 1 to 4;

the method comprises complexing a zirconium reagent with a desferrioxamine-labeled, cysteine engineered antibody comprising a cysteine engineered antibody (Ab) conjugated through a free cysteine amino acid to a linker (L) and a desferrioxamine moiety (Df), having the general formula II:

Ab-(L-Df)p II

wherein L-Df is selected from:

wherein the wavy line indicates attachment to the antibody (Ab); and

p is 1 to 4;

thereby forming a desferrioxamine-labelled, cysteine engineered antibody.

Another aspect of the invention is an imaging method comprising:

administering a zirconium-labeled, cysteine engineered antibody to the animal; and

detecting the presence of the zirconium-labeled, cysteine engineered antibody in vivo by imaging,

wherein the zirconium-labeled, cysteine-engineered antibody comprises a cysteine-engineered antibody (Ab) having one or more free cysteine amino acids conjugated to one or more zirconium complexes (Z) via a linker (L), and has the general formula I:

Ab-(L-Z)p I

wherein p is 1 to 4.

Another aspect of the invention includes the diagnostic use of the compounds and compositions disclosed herein.

Brief Description of Drawings

Fig. 1A shows a three-dimensional plot of hu4D5Fabv7 antibody fragment as deduced from X-ray crystal coordinates. The structural positions of exemplary engineered Cys residues of heavy and light chains are numbered (according to the sequential numbering system).

Fig. 1B shows the sequential numbering scheme (top row), starting from the N-terminus compared to the Kabat numbering scheme of 4D5v7fabH (bottom row). The Kabat numbering insertions are annotated with a, b, c.

FIGS. 2A and 2B show the measurement of binding (detected using absorbance at 450 nm) of hu4D5Fabv8 and hu4D5Fabv8Cys mutant (ThioFab) phage variants as determined by PHESELECTOR: (A) non-biotinylated phage-hu 4D5Fabv8 and (B) biotinylated phage-hu 4D5Fabv8 to detect interaction with BSA (open bars), HER2 (striped bars) or streptavidin (solid bars).

FIGS. 3A and 3B show the measurement of binding (measured using absorbance at 450 nm) of hu4D5Fabv8 (left) and hu4D5Fabv8Cys mutant (ThioFab) variants as determined by PHESELECTOR: (A) non-biotinylated phage-hu 4D5Fabv8 and (B) organismsThe phage-hu 4D5Fabv8 was biotinylated to detect interaction with BSA (open bars), HER2 (striped bars), or streptavidin (solid bars). The light chain variant is on the left and the heavy chain variant is on the right. Thiol reactivity = streptavidin-bound OD_450nmHER2 (antibody) bound OD_450nm

FIG. 4A shows fractional surface Accessibility values (Fractionalsurface Accessibility values) of residues on wild-type hu4D5Fabv 8. The light chain site is on the left and the heavy chain site is on the right.

Figure 4B shows binding measurements (detected using absorbance at 450 nm) of biotinylated hu4D5Fabv8 (left) and hu4D5Fabv8Cys mutant (ThioFab) variants to detect interaction with HER2 (day 2), streptavidin (DA) (day 2), HER2 (day 4), and SA (day 4). The phage-hu 4D5Fabv8Cys variant was isolated and stored at 4 ℃. Biotin conjugation was performed on day 2 or day 4 followed by pheselect analysis to monitor its interaction with Her2 and streptavidin and to detect the stability of the reactive thiol group on the engineered ThioFab variants as described in example 2.

Figure 5 shows binding measurements (detected using absorbance at 450 nm) of biotin-maleimide conjugated hu4D5Fabv8(a121C) and non-biotinylated wild type hu4D5Fabv8 to detect binding to streptavidin and HER 2. Each Fab was tested at 2ng and 20 ng.

FIG. 6 shows an ELISA assay (measured using absorbance at 450 nm) of biotinylated ABP-hu4D5Fabv8 wild type (wt) and ABP-hu4D5Fabv8 cysteine mutants V110C and A121C to detect binding to rabbit albumin, Streptavidin (SA), and HER 2.

FIG. 7 shows an ELISA analysis (measured using absorbance at 450 nm) of biotinylated ABP-hu4D5Fabv8 cysteine mutant (ThioFab variant): single Cys variants ABP-V110C, ABP-a121C and bis Cys variants ABP-V110C-a88C and ABP-V110C-a121C (left to right) were tested for binding to rabbit albumin, HER2 and Streptavidin (SA) and detected with Fab-HRP or SA-HRP.

Figure 8 shows the binding of biotinylated ThioFab phage and anti-phage HRP antibodies to HER2 (top) and streptavidin (bottom).

Figure 13A shows a graphical depiction of biotinylated antibody binding to immobilized HER2, using binding of HRP-labeled secondary antibody for absorbance detection.

Figure 13B shows the binding measurements (detected using absorbance at 450 nm) of biotin-maleimide conjugated thiotrastuzumab variants and non-biotinylated wild-type trastuzumab bound to immobilized HER 2. From left to right: V110C (single cys), A121C (single cys), V110C/A121C (double cys) and trastuzumab. Each thioIgG variant and trastuzumab was detected at1, 10 and 100 ng.

Figure 14A shows a pictorial depiction of biotinylated antibody bound to immobilized HER2, using biotin in combination with anti-IgG-HRP for absorbance detection.

Figure 14B shows the binding measurements (detected using absorbance at 450 nm) of biotin-maleimide conjugated thiotrastuzumab variants and non-biotinylated wild-type trastuzumab bound to immobilized streptavidin. From left to right: V110C (single cys), A121C (single cys), V110C/A121C (double cys) and trastuzumab. Each thioIgG variant and trastuzumab was detected at1, 10 and 100 ng.

Fig. 15 shows a general method for preparing cysteine engineered antibodies (thiomabs) for conjugation expressed by cell culture.

FIG. 16 shows non-reducing (top) and reducing (bottom) denaturing polyacrylamide gel electrophoresis analysis of the 2H9ThioMab Fc variant (left to right, lanes 1-9): A339C, S337C, S324C, A287C, V284C, V282C, V279C, V273C, and 2H9 wild-type after purification with immobilized protein A. The lanes on the right are size marker steps indicating that the intact protein is about 150kDa, the heavy chain fragment is about 50kDa, and the light chain fragment is about 25 kDa.

FIG. 17A shows non-reducing (left) and reducing (+ DTT) (right) denaturing polyacrylamide gel electrophoresis analysis of 2H9ThioMab variants (left to right, lanes 1-4): L-V15C, S179C, S375C, S400C after purification with immobilized protein A.

FIG. 17B shows non-reducing (left) and reducing (+ DTT) (right) denaturing polyacrylamide gel electrophoresis analysis of 2H 9and 3A5ThioMab variants after purification with immobilized protein A.

FIG. 18 shows a Western blot analysis of biotinylated thioIgG variants. The 2H 9and 3A5ThioMab variants were analyzed on reductive denaturing polyacrylamide gel electrophoresis and the proteins were transferred to nitrocellulose membranes. The presence of antibody and conjugated biotin was detected using anti-IgG-HRP (top) and streptavidin-HRP (bottom), respectively. Lane 1: 3A5H-A121C, lane 2:3A5L-V110C, lane 3:2H9H-A121C, lane 4:2H9L-V110C, lane 5:2H9 wild type.

FIG. 19 shows an ELISA assay of biotinylated 2H9 variant bound to streptavidin, detected using anti-IgG-HRP and measured for absorbance at 450nm (top bar graph). The bottom schematic depicts the experimental design used in the ELISA assay.

FIG. 20 shows a coupling for⁸⁹Bifunctional reagents for Zr desferrioxamine B chelator (Df, top) and protein, using amino-reactive linkers, TFP-N-SucDf and Df-Bz-NCS (intermediate) and thiol-reactive linkers, Df-Chx-Mal, Df-Bac, and Df-lac (bottom).

FIG. 21 shows the preparation of Df-Chx-Mal, Df-Bac, Df-lac and conjugation to thiotrastuzumab by incorporation of Cys residues in the Fab heavy chain. Reaction conditions are as follows: DIEA, DMF/H₂O(10:1),RT,0.5-1h;ii.DIEA,DMF,0℃,4h;iii.pH7.5,RT,1h;iv.pH9,RT,5h;v.pH9,RT,2h。

Figure 22 shows zirconium oxalate-89 conjugated to a desferrioxamine-labelled, cysteine engineered antibody, for example comprising 4 linkers: chelation of Df-linker-trastuzumab by N-Suc, Bz-SCN, Chx-maleimide (CHx-Mal), or acetyl (Ac).

Figure 23 shows mass spectrometry analysis of reduced antibodies, showing signals from light and heavy chains separately. A: thiotrastuzumab, B: Df-Ac-thio trastuzumab (using Df-Bac), and C: Df-Ac-thio trastuzumab (using Df-Iac), and D: Df-Chx-Mal-thio trastuzumab.

FIG. 24 shows⁸⁹Zr-Chx-Mal-thio trastuzumab (open circle) and⁸⁹stability of Zr-Df-Ac-thio trastuzumab (filled circle) in mouse serum at 37 ℃ (n = 3).

FIG. 25 shows 100 μ Ci bolus injections in tail vein prepared using 4 different linkers (Bz-SCN, N-Suc, Chx-Mal and Ac)⁸⁹Representative whole body images (maximum intensity projection) obtained 96 hours after Zr-trastuzumab.

Figure 26 shows in vivo uptake in selected tissues at 24, 96 and 144h post injection as measured by PET.

Detailed description of exemplary embodiments

Reference will now be made in detail to specific embodiments of the invention, examples of which are illustrated in the accompanying structures and formulae. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that they are not intended to limit the invention to these embodiments. On the contrary, the invention is intended to cover all alternatives, modifications and equivalents, which may be included within the scope of the invention as defined by the appended claims.

Those skilled in the art will recognize a variety of methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

Unless defined otherwise, technical and scientific terms used herein and as commonly understood by one of ordinary skill in the art to which this invention belongs have the same meaning and are in accordance with Singleton et al (1994) Dictionary of Microbiology and molecular Biology, second edition, J.Wiley & Sons, New York, NY, and Janeway, C.A., transitions, P.A., Walport, M.A., Shlomchik (2001) immunology, fifth edition, Garland Publishing, New York.

Definition of

Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings:

when trade names are used herein, applicants intend to include the active pharmaceutical ingredient of the trade name product formulation, the imitation drug (genericdrug) and the trade name product independently.

The term "antibody" is used herein in the broadest sense and specifically includes 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) journal. of Immunology 170: 4854-4861). The antibody may be murine, human, humanized, chimeric, or derived from other species. Antibodies are proteins produced by the immune system that are capable of recognizing and binding to a particular antigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, fifth edition, Garland Publishing, New York). The target antigen typically has multiple binding sites, also known as epitopes, that are recognized by CDRs on various antibodies. Each antibody that specifically binds a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. Antibodies include full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules or portions thereof that comprise an antigen that immunospecifically binds to a target of interest, such targets including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein may be of any class (e.g., IgG, IgE, IgM, IgD and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA 2) or subclass of immunoglobulin molecule. The immunoglobulin may be derived from any species. In one aspect, however, the immunoglobulin is of human, murine or rabbit origin.

An "antibody fragment" comprises a portion of a full-length antibody, typically comprising the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments; a diabody; a linear antibody; minibodies (Olafsen et al (2004) Protein Eng. Des ign)&Sel.17(4): 315-; and multispecific antibodies formed from antibody fragments.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that contain different antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody is directed against a single antigenic site on the antigen. In addition to its specificity, monoclonal antibodies have the advantage that their synthesis may not be contaminated by other antibodies. The modifier "monoclonal" indicates that the characteristics of the antibody are obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be prepared by the hybridoma method first described by Kohler et al (1975) Nature 256:495, or by recombinant DNA methods (see, e.g., US 4816567; US 5807715). Monoclonal antibodies can also be analyzed from phage antibody libraries using techniques such as those described in Clackson et al (1991) Nature,352: 624-.

Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical 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 is identical 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-. 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 an antibody comprising VL and VH domains, as well as a light chain constant domain (CL) and a heavy chain constant domain, CH1, CH2, and CH 3. The constant domain may be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. An intact antibody may have one or more "effector functions," which refer to biological activities arising from the Fc constant region (either the native sequence Fc or the amino acid sequence variant Fc region) of the antibody. Examples of antibody effector functions include C1q binding; complement-dependent cytotoxicity; fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down-regulation of cell surface receptors such as B cell receptors and BCR.

Depending on the amino acid sequence of its heavy chain constant domain, whole antibodies can be divided into different "classes". There are 5 major classes of intact immunoglobulin antibodies: IgA, IgD, IgE, IgG and IgM, and some of them can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA and IgA 2. The heavy chain constant domains corresponding to different antibody species are referred to as α, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Ig forms include hinge-modified or hingeless forms (Roux et al (1998) J.Immunol.161:4083-4090; Lund et al (2000) Eur.J.biochem.267:7246-7256; US2005/0048572; US 2004/0229310).

An "ErbB receptor" is a receptor protein tyrosine kinase belonging to the ErbB receptor family, the members of which are important mediators of cell growth, differentiation and survival. The ErbB receptor family includes 4 different members, namely including epidermal growth factor receptor (EGFR, ErbB1, HER1), HER2(ErbB2 or p185neu), HER3(ErbB3) and HER4(ErbB4 or tyro 2). A panel of anti-ErbB antibodies has been characterized using the human breast tumor cell line SKBR3 (Hudziak et al (1989) mol. cell. biol.9(3): 1165-1172). Maximal inhibition was obtained with an antibody named 4D5, which inhibited cell proliferation by 56%. In this assay, other antibodies in the group reduced cell proliferation to a lesser extent. The antibody 4D5 was also found to sensitize breast tumor cell lines overexpressing ErbB2 to the cytotoxic effects of TNF-a (US 5677171). The anti-ErbB 2 antibodies discussed In Hudziak et al are characterized In Fendly et al (1990) Cancer Research 50:1550-1558; Kotts et al (1990) In Vitro 26(3):59A; Sarup et al (1991) Growth Regulation1:72-82; Shepard et al J. (1991) Clin.Immunol.11(3):117-127; Kumar et al (1991) mol.cell.biol.11(2):979-986; Lewis et al (1993) Cancer Immunol.37: 255-263; Pietras et al (1994) cogene 9: 9-1838; Schitta et al (1994) Cancer Research54:5301 w.: 5309; Schizor et al (1997) 1985; Nature J.1465) 1985: 14615: 1985; Nature et al (1996) 19815: 14615: 1985) and J.14615. 1985).

ErbB receptors generally comprise an extracellular domain that binds ErbB ligands; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a carboxy-terminal signaling domain comprising several tyrosine residues that can be phosphorylated. The ErbB receptor may be a "native sequence" ErbB receptor or an "amino acid sequence variant" thereof. Preferably, the ErbB receptor is a native sequence human ErbB receptor. Accordingly, an "ErbB receptor family member" is EGFR (ErbB1), ErbB2, ErbB3, ErbB4 or any other ErbB receptor now known or later identified.

The terms "ErbB 1", "epidermal growth factor receptor", "EGFR", and "HER 1" are used interchangeably herein and refer to EGFR as disclosed, for example, in Carpenter et al (1987) Ann.Rev.biochem.,56:881-914, and include naturally occurring mutant forms thereof (e.g., deletion mutant EGFR in Humphrey et al (1990) Proc. Nat.Acad. Sci. (USA)87: 4207-4211). The term erbB1 refers to a gene that encodes the EGFR protein product. Antibodies against HER1 are described, for example, in Murthy et al (1987) Arch.biochem.Biophys.,252: 549-plies 560 and WO 95/25167.

The terms "ERRP", "EGF receptor-related protein", "EGFR-related protein", and "epidermal growth factor receptor-related protein" are used interchangeably herein and refer to ERRPs as disclosed, for example, in US 6399743 and US publication No. 2003/0096373.

The expressions "ErbB 2" and "HER 2" are used interchangeably herein and refer to, for example, the human HER2 protein (Genebank accession number X03363) described in Semba et al (1985) Proc. Nat. Acad. Sci. (USA)82: 6497-. The term "erbB 2" refers to the gene encoding human erbB2, and "neu" refers to the gene encoding rat p185 neu. Preferred ErbB2 is native sequence human ErbB 2.

"ErbB 3" and "HER 3" refer to receptor polypeptides such as those disclosed in U.S. Pat. Nos. 5183884 and 5480968 and Kraus et al (1989) Proc. Nat. Acad. Sci. (USA)86: 9193-. Antibodies directed against ErbB3 are known in the art and described, for example, in U.S. patent No. 5183884,5480968 and WO 97/35885.

The terms "ErbB 4" and "HER 4" herein refer to receptor polypeptides such as those described in EP patent 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, and include isoforms thereof such as those disclosed in WO 99/19488. Antibodies against HER4 are described, for example, in WO 02/18444.

Antibodies to ErbB receptors are commercially available from a variety of sources, including, for example, Santa cruz biotechnology, inc.

The term "amino acid sequence variant" refers to a polypeptide having an amino acid sequence that differs from a native sequence polypeptide by some amount. Typically, amino acid sequence variants will have at least about 70% sequence identity to at least one receptor binding domain of a native ErbB ligand or to at least one ligand binding domain of a native ErbB receptor, and preferably, the variants will have at least about 80%, more preferably, at least about 90% sequence homology to such a receptor or ligand binding domain. Amino acid sequence variants have substitutions, deletions and/or insertions at specific positions within the amino acid sequence of the native amino acid sequence. Amino acids are designated by conventional names, single and three letter codes.

"sequence identity" is defined as the percentage of residues that are identical in amino acid sequence variants after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Methods and computer programs for alignment are well known in the art. One such computer program is "Align 2" manufactured by Genentech, inc., which is archived with user documentation at the United States Copyright Office (United States Copyright Office), Washington, DC20559, 12, 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 antibodies bound to target cells and subsequently cause lysis of the target cells. The main cells mediating ADCC, NK cells, express Fc γ RIII only, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. FcR expression on hematopoietic cells is summarized in table 3 on page 464 of ravatch and Kinet (1991) "annu. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed, for example as described in US 5500362 and US 5821337. Effector cells that can be used in such assays include Peripheral Blood Mononuclear Cells (PBMCs) and Natural Killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest may be assessed in vivo, for example in an animal model as disclosed in Clynes et al (1998) proc.nat.acad.sci. (USA)95: 652-.

"human effector cells" are leukocytes which express one or more constant region receptors (fcrs) and perform effector functions. Preferably, the cells express at least Fc γ RIII and function ADCC effector function. Examples of human leukocytes that mediate ADCC include Peripheral Blood Mononuclear Cells (PBMCs), Natural Killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; PBMC and NK cells are preferred. The effector cells may be isolated from their natural source, e.g., from blood, or may be PBMCs as described herein.

The term "Fc receptor" or "FcR" is used to describe a receptor that binds the Fc constant region of an antibody. A preferred FcR is a native sequence human FcR. In addition, a preferred FcR is one that binds an IgG antibody receptor (gamma receptor) and includes the Fc γ RI, Fc γ RII and Fc γ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors. Fc γ RII receptors include Fc γ RIIA ("activating receptor") and Fc γ RIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor Fc γ RIIA comprises an Immunoreceptor Tyrosine Activation Motif (ITAM) in its cytoplasmic domain. The inhibitory receptor Fc γ RIIB contains an Immunoreceptor Tyrosine Inhibitory Motif (ITIM) in its cytoplasmic domain. (see"annu.rev.immuno |" 15: 203-. In ravatch and Kinet, "annu. 457-92 (1991); capel et al (1994) immunoassays 4: 25-34; and deHaas et al (1995) J.Lab.Clin.Med.126: FcRs are reviewed in 330-41. Other fcrs, including those that will be identified later, are included within the term "FcR" herein. This term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al (1976) j.immunol., 117: 587 and Kim et al (1994) j.immunol.24: 249).

"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) that complexes a cognate antigen. To assess complement activation, a method such as that described in Gazzano-Santoro et al j.immunol.methods, 202: 163 (1996).

"Natural antibodies" are generallyIs a heterotetrameric glycoprotein of about 150,000 daltons, consisting of 2 identical light (L) chains and 2 identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain at one end (V)_H) Followed by several 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 variable domain of the light chain is aligned with the variable domain of the heavy chain. Specific amino acid residues are believed to form the interface between the light and heavy chain variable domains.

The term "variable" refers to the fact that specific portions of variable domains vary widely in sequence among antibodies and are used for the binding and specificity of each specific antibody for its specific antigen. However, the variability is not evenly distributed throughout the variable domains of the antibody. It is concentrated in 3 hypervariable region-named segments in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called Framework Regions (FR). The variable domains of native heavy and light chains each comprise 4 FRs, predominantly in the β -sheet configuration, connected by 3 hypervariable regions which form loops connecting, or sometimes forming part of, the β -sheet structure. The hypervariable regions in each chain are maintained together by the FRs, in close proximity to each other, and together with the hypervariable regions from the other chain contribute to the formation of the antigen-binding site of the antibody (see Kabat et al (1991) sequencing of Proteins of Immunological Interest, fifth edition Public Health Service, national institutes of Health, Bethesda, Md.). The constant domains are not directly involved in binding of the antibody to the antigen, but exhibit a variety of effector functions, such as participation of the antibody in antibody-dependent cell-mediated cytotoxicity (ADCC).

The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. Hypervariable regions typically comprise amino acid residues from the "complementarity determining regions" or "CDRs" (e.g.residues 24-34(L1),50-56(L2) and 89-97(L3) in the light chain variable domain and 31-35(H1),50-65(H2) and 95-102(H3) in the heavy chain variable domain; Kabat et al, supra) and/or residues from the "hypervariable loops" (e.g.residues 26-32(L1),50-52(L2) and 91-96(L3) in the light chain variable domain and 26-32(H1),53-55(H2) and 96-101(H3) in the heavy chain variable domain; Chothia and Lesk (1987) J.mol.biol.,196:901 917). "framework region" or "FR" residues are variable domain residues other than the hypervariable region residues defined herein.

Papain digestion of antibodies produces 2 identical antigen-binding fragments, called "Fab" fragments, each having a single antigen-binding site, and a remaining "Fc" fragment, the name of which reflects its ability to crystallize readily. Pepsin treatment produced F (ab') 2 fragments with 2 antigen binding sites and still capable of cross-linking the antigen.

"Fv" is the smallest antibody fragment that contains the entire antigen recognition and antigen binding site. This region consists of a dimer of one heavy and one light chain variable domain in close, non-covalent linkage. In this configuration, the 3 hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Together, the 6 hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only 3 antigen-specific hypervariable regions) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments by the addition of a small number of residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab '-SH is referred to herein as Fab' in which the cysteine residues of the constant domains have at least one free thiol group. F (ab ') 2antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical linkages of antibody fragments are known.

The "light chains" of antibodies from any vertebrate species can be classified into one of two distinct types, named κ and λ, based on the amino acid sequences of their constant domains.

"Single chain Fv" or "scFv" antibody fragments comprise the V of an antibody_HAnd V_LFor an overview of scFv, see Pl ü ckthun, described in The Pharmacology of monoclonal Antibodies, vol.113, Rosenburg and Moore eds, Springer-Verlag, New York, pp.269-315(1994), scFv fragments of anti-ErbB 2 Antibodies in WO 93/16185, U.S. Pat. No. 5571894, and 5587458.

The term "diabodies" refers to small antibody fragments having 2 antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing of the two domains on the same chain, the domains are forced to pair with the complementary domains of the other chain to create 2 antigen binding sites. Diabodies are described more fully in, for example, EP 404,097, WO 93/11161, and Hollinger et al (1993) Proc. nat. l.Acad. Sci. USA 90: 6444. sup. 6448.

"humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal non-human immunoglobulin-derived sequences. Humanization is a method of transferring murine antigen binding information to a non-immunogenic human antibody recipient, and many therapeutically useful drugs have been obtained. Humanization methods generally begin by transferring all 6 murine Complementarity Determining Regions (CDRs) to 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 compromised. In addition to the CDRs, selected non-human antibody framework residues must also be added to maintain the correct CDR configuration (Chothia et al (1989) Nature 342: 877). It has been shown that transfer of key mouse framework residues to human recipients restores antigen binding and affinity in order to support the structural conformation of the grafted CDRs (Riechmann et al (1992) J.mol.biol.224,487-499; Foote and Winter (1992) J.mol.biol.224:487-499; Pres et al (1993) J.Immunol.151,2623-2632; Werther et al (1996) J.Immunol.methods 157:4986-4995; and Presta et al (2001) Thromb.Haemost.85: 379-389). In most cases, 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 non-human primate having the desired specificity, affinity, and capacity (capacity). In some cases, Framework Region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications were made to further improve antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically 2, 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 will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see US 6407213, Jones et al (1986) Nature,321:522-525, Riechmann et al (1988) Nature 332:323-329, and Presta (1992) curr. Op. struct. biol.,2: 593-596.

"free cysteine amino acids" refer to cysteine amino acid residues that have been engineered into a parent antibody, have thiol functional groups (-SH), and are not paired as intramolecular or intermolecular disulfide bonds.

The term "thiol reactivity value" is a quantitative representation of the reactivity of a free cysteine amino acid. The thiol reactivity value is the percentage of free cysteine amino acids that react with the thiol-reactive agent in the cysteine engineered antibody and can reach a maximum of 1. For example, the free cysteine amino acid on the cysteine engineered antibody that yields 100% yield in the formation of a biotin-labeled antibody upon reaction with a thiol-reactive reagent, such as a biotin maleimide reagent, has a thiol reactivity value of 1.0. Another cysteine amino acid engineered into the same or a different parent antibody that reacted with a thiol-reactive reagent to give an 80% yield has a thiol reactivity value of 0.8. Another cysteine amino acid engineered into the same or a different parent antibody that is completely non-reactive with a thiol-reactive agent has a thiol reactivity value of 0. Determination of the thiol reactivity value for a particular cysteine can be performed by ELISA assay, mass spectrometry, liquid chromatography, autoradiography, or other quantitative analytical tests.

A "parent antibody" is an antibody comprising an amino acid sequence in 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. A parent antibody may have amino acid sequence modifications (e.g., additions, deletions, and/or substitutions) already present with respect to other native, wild-type, or modified forms of the antibody. The parent antibody may be directed against a target antigen of interest, such as a biologically important polypeptide. Antibodies directed against non-polypeptide antigens (e.g., tumor-associated glycolipid antigens; see US 5091178) are also contemplated.

Exemplary parent antibodies include antibodies with affinity and selectivity for cell surface and transmembrane receptors and Tumor Associated Antigens (TAAs).

Other exemplary parent antibodies include those selected from, but are not limited to, anti-estrogen receptor antibodies, anti-progesterone receptor antibodies, anti-P53 antibodies, anti-HER-2/neu antibodies, anti-EGFR antibodies, anti-cathepsin D antibodies, anti-Bcl-2 antibodies, anti-E-cadherin (cadherin) antibodies, anti-CA 125 antibodies, anti-CA 15-3 antibodies, anti-CA 19-9 antibodies, anti-c-erbB-2 antibodies, anti-P-glycoprotein antibodies, anti-CEA antibodies, anti-retinoblastoma protein antibodies, anti-ras tumor protein antibodies, anti-Lewis X antibodies, anti-Ki-67 antibodies, anti-PCNA antibodies, anti-CD 3 antibodies, anti-CD 4 antibodies, anti-CD 5 antibodies, anti-CD 7 antibodies, anti-CD 8 antibodies, anti-CD 9/P24 antibodies, anti-CD 10 antibodies, anti-CD 11c antibodies, anti-CD 13 antibodies, anti-CD 14 antibodies, anti-CD 15 antibodies, anti-CD 19 antibodies, anti-CD 20 antibodies, anti-CD 23 antibody, anti-CD 30 antibody, anti-CD 31 antibody, anti-CD 33 antibody, anti-CD 34 antibody, anti-CD 35 antibody, anti-CD 38 antibody, anti-CD 41 antibody, anti-LCA/CD 45 antibody, anti-CD 45RO antibody, anti-CD 45RA antibody, anti-CD 39 antibody, anti-CD 100 antibody, anti-CD 95/Fas antibody, anti-CD 99 antibody, anti-CD 106 antibody, anti-ubiquitin antibody, anti-CD 71 antibody, anti-c-myc antibody, anti-cytokeratin antibody, anti-vimentin antibody, anti-HPV protein antibody, anti-kappa light chain antibody, anti-lambda light chain antibody, anti-melanosome antibody, anti-prostate specific antigen antibody, anti-S-100 antibody, anti-tau antigen antibody, anti-fibrin antibody, anti-keratin antibody, and anti-Tn-antigen antibody.

An "isolated" antibody is one that has been identified and isolated and/or recovered from a component of its natural environment. Contaminant components of their natural environment are materials that interfere with diagnostic or therapeutic uses for antibodies, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to an extent greater than 95% by weight of the antibody, and most preferably greater than 99% by weight as determined by the Lowry method, (2) to an extent sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues by use of a rotary cup sequencer, or (3) homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie Brilliant blue or preferably using silver staining. Isolated antibodies include antibodies in situ within recombinant cells, as at least one component of the antibody's natural environment is not present. However, in general, the isolated antibody will be prepared by at least one purification step.

An antibody that "binds" to a molecular target or antigen of interest (e.g., the ErbB2 antigen) is an antibody that is capable of binding to the antigen with sufficient affinity such that the antibody can be used to target cells expressing the antigen. When the antibody is an antibody that binds ErbB2, it typically preferentially binds ErbB2 (as compared to other ErbB receptors) and may be an antibody that does not significantly cross-react with other proteins such as EGFR, ErbB3, or ErbB 4. In such embodiments, the extent of binding of antibodies to these non-ErbB 2 proteins (e.g., to the cell surface of the endogenous receptor) will be less than 10% as determined by Fluorescence Activated Cell Sorting (FACS) analysis or Radioimmunoprecipitation (RIA). Occasionally, anti-ErbB 2 antibodies will not significantly cross-react with rat neu protein, as described, for example, in Schecter et al (1984) Nature 312:513 and Drebin et al (1984) Nature 312: 545-548.

Molecular targets for antibodies encompassed by the invention include CD proteins and their ligands, such as, but not limited to: (i) CD3, CD4, CD8, CD19, CD20, CD22, CD34, CD40, CD79 α (CD79a), and CD79 β (CD79 b); (ii) ErbB receptor family members such as EGF receptor, HER2, HER3 or HER4 receptor; (iii) cell adhesion molecules, such as LFA-1, Mac1, p150,95, VLA-4, ICAM-1, VCAM and α v/β 3 integrins, including the α or β subunit thereof (e.g., anti-CD 11a, anti-CD 18 or anti-CD 11b antibodies); (iv) growth factors such as VEGF, IgE, blood group antigens, flk2/flt3 receptor; obesity (OB) receptors; mpl receptor, CTLA-4, protein C, BR3, C-met, tissue factor, beta 7, etc.; and (v) cell surface and transmembrane Tumor Associated Antigens (TAAs).

Unless otherwise indicated, the term "monoclonal antibody 4D 5" refers to an antibody having or derived from the antigen binding residues of murine 4D5 antibody (ATCC CRL 10463). For example, monoclonal antibody 4D5 may be murine monoclonal antibody 4D5 or a variant thereof, such as humanized 4D 5. Exemplary humanized 4D5 includes huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (trastuzumab,)。

"phage display" is a technique by which variant polypeptides are displayed on the surface of phage, e.g., filamentous phage, as fusion proteins with coat proteins. One application of phage display is based on the fact that large libraries of randomized protein variants can be rapidly and efficiently sorted to obtain sequences that bind target molecules with high affinity. The display of peptide and protein libraries on phage has been used to screen millions of polypeptides to obtain polypeptides with specific binding properties. Multivalent phage display methods have been used to display small random peptides and small proteins, generally by fusion to pIII or pVIII of filamentous phage (Wells and Lowman, (1992) curr. Opin. struct. biol.,3:355-362, and references cited therein). In monovalent phage display, a protein or peptide library is fused to a phage coat protein or portion thereof and expressed at low levels in the presence of the wild-type protein. The affinity effect is reduced for multivalent phages, so sorting is based on the inherent ligand affinity and uses phagemid vectors that simplify DNA manipulation. Lowman and Wells, Methods: acompanion to Methods in Enzymology, 3: 205-. Phage display includes techniques for generating antibody-like molecules (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) immunology, fifth edition, Garland Publishing, New York, p627-628; Lee et al).

A "phagemid" is a plasmid vector having a bacterial origin of replication (e.g., Co1E 1) and one copy of the phage intergenic region. The phagemid can be used with any known bacteriophage, including filamentous phage and lambda-shaped phage. Plasmids also typically include selectable markers for antibiotic resistance. The DNA segments cloned into these vectors can be propagated as plasmids. When cells containing these vectors are provided with all the genes required for the production of phage particles, the mode of replication of the plasmid is changed to rolling circle replication to produce single-stranded copies of plasmid DNA and package the phage particles. Phagemids can form infectious or non-infectious phage particles. The term includes phagemids comprising a phage coat protein or fragment thereof linked to a heterologous polypeptide gene in gene fusion such that the heterologous polypeptide is displayed on the surface of the phage particle.

"linker", "linker unit" or "link" refers to a chemical moiety comprising a covalent bond or chain of atoms that covalently links an antibody to a drug moiety. In various embodiments, the linker is designated as L. Linkers include divalent radicals such as alkyl substituents (alkyldiyl), arylene, heteroarylene, such as: - (CR)₂)_nO(CR₂)_nAlkoxy (e.g. polyethylene oxide, PEG, polymethyl methacrylate)Oxyalkylene) and alkylamino (e.g. polyvinylamine, Jeffamine)^TM) The repeating unit portion of (a); and diacid esters and amides, including succinate, succinamide, diglycolate (diglycolate), malonate, and caproamide.

The term "label" refers to any moiety that can be covalently linked to an antibody and that serves the following functions: (i) providing a detectable signal; (ii) interaction with a second label to modify a detectable signal provided by the first or second label, e.g., FRET (fluorescence resonance energy transfer); (iii) stabilizing the interaction with or increasing the affinity for binding to the antigen or ligand; (iv) (iv) influencing mobility by charge, hydrophobicity, shape or other physical parameters, such as electrophoretic mobility or cell permeability, or (v) providing a capture moiety to modulate ligand affinity, antibody/antigen binding, or ion complexation.

The stereochemical definitions and conventions used herein generally follow S.P. Parker, eds, McGraw-HillDirectionbearing of Chemical Terms (1984) McGraw-Hill Book Company, New York, and Eliel, E.and Wilen, S.A., Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., New York. Many organic compounds exist in optically active form, i.e., they have the ability to rotate the plane of plane polarized light. In describing optically active compounds, the prefixes D and L, or R and S, are used to designate the absolute configuration of the molecule with respect to its chiral center. The prefixes d and l or (+) and (-) denote the sign of the compound rotating plane-polarized light, and (-) or1 denotes that the compound is left-handed. Compounds with (+) or d are dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. Particular stereoisomers may also be referred to as enantiomers, and mixtures of such isomers are often referred to as enantiomeric mixtures. 50: an enantiomeric mixture of 50 is referred to as a racemic mixture or racemate and occurs when there is no stereoselectivity or stereospecificity in the chemical reaction or process. The term "racemic mixture" or "racemate" refers to an equimolar mixture of 2 enantiomeric species, which is not optically active.

The phrase "pharmaceutically acceptable salt" as used herein refers to pharmaceutically acceptable organic or inorganic salts of AZC. Exemplary salts 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, methylsulfonate, ethylsulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1, 1' -methylenebis (2-hydroxy-3-naphthoic) salt). A pharmaceutically acceptable salt may involve the inclusion of another molecule, such as an acetate ion, a succinate ion, or other counterion. The counterion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, a pharmaceutically acceptable salt may have more than one charged atom in its structure. There may be multiple counterions where the multiple charged atoms are part of a pharmaceutically acceptable salt. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and/or one or more counterions.

"pharmaceutically acceptable solvate" refers to the association of one or more solvent molecules with AZC. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

The following abbreviations are used herein and have the indicated definitions: BME is β -mercaptoethanol, Boc is N- (tert-butoxycarbonyl), cit is citrulline (2-amino-5-ureidovaleric acid), dap is dolaproine, DCC is 1, 3-dicyclohexylcarbodiimide, DCM is dichloromethane, DEA is diethylamine, DEAD is diethyl azodicarboxylate, DEPC is diethyl cyanophosphate, DIAD is diisopropyl azodicarboxylate, DIEA is N, N-diisopropylethylamine, dil is dolaisoleucine, DMA is dimethylacetamide, DMAP is 4-dimethylaminopyridine, DME is ethylene glycol dimethyl ether (or 1, 2-dimethoxyethane), DMF is N, N-dimethylformamide, DMSO is dimethyl sulfoxide, doe is dolaphenine, dov is N, N-dimethylvaline, DTNB is 5, 5' -dithiobis (2-nitrobenzoic acid), DTPA is diethylenetriaminepentaacetic acid, DTT is dithiothreitol, EDCI is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, EEDQ is 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline, ES-MS is electrospray mass spectrometry, EtOAc is ethyl acetate, Fmoc is N- (9-fluorenylmethoxycarbonyl), gly is glycine, HATU is 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate, HOBt is 1-hydroxybenzotriazole, HPLC is high pressure liquid chromatography, ile is isoleucine, lys is lysine, MeCN (CH3CN) is acetonitrile, MeOH is methanol, Mtr is 4-anisyldiphenylmethyl (or 4-methoxytrityl), nor is (1S, 2R) - (+) -ephedrine, PAB is p-aminophenylcarbamoyl, PBS is phosphate buffer (pH 7), PEG is polyethylene glycol, Ph is phenyl, Pnp is p-nitrophenyl, MC is 6-maleimidocaproyl, phe is L-phenylalanine, PyBrop is tripyrrolidinyl hexafluorophosphate, SEC is size exclusion chromatography, Su is succinimide, TFA is trifluoroacetic acid, TLC is thin layer chromatography, UV is ultraviolet light, and val is valine.

Cysteine engineered antibodies

The compounds of the invention include cysteine engineered antibodies in which one or more amino acids of the wild type or parent antibody are replaced with cysteine amino acids. Any antibody format can be engineered so that it is mutated. For example, a parent Fab antibody fragment may be engineered to form a cysteine engineered Fab, referred to herein as a "ThioFab. Similarly, a parent monoclonal antibody can be engineered to form a "ThioMab". It should be noted that single-site mutations result in a single engineered cysteine residue in ThioFab, whereas single-site mutations result in 2 engineered cysteine residues in ThioMab due to the dimeric nature of IgG antibodies. Mutants with a substituted ("engineered") cysteine (Cys) residue were evaluated for reactivity with newly introduced, engineered cysteine thiol groups. Thiol reactivity values are relative numerical terms in the range of 0 to 1.0 and can be measured on any cysteine engineered antibody. The cysteine engineered antibodies of the invention have a thiol reactivity value in the range 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 present invention result in cysteine engineered antibodies that react with electrophilic functional groups (functionalities). These methods also result in antibody conjugate compounds, such as antibody-zirconium conjugate (AZC) compounds having a zirconium atom at a designated, designed, selective site. Reactive cysteine residues on the antibody surface allow specific conjugation of zirconium moieties through thiol reactive groups such as maleimide or haloacetyl. The nucleophilic reactivity of the thiol function of a Cys residue to a maleimide group is 1000 times greater than any other amino acid function in the protein (e.g., the amino group of a lysine residue or the N-terminal amino group). Thiol-specific functional groups in iodoacetyl and maleimide reagents can react with amino groups but require higher pH (>9.0) and longer reaction times (Garman,1997, Non-radioactive labelling: A Practical Approach, Academic Press, London).

The cysteine engineered antibodies of the invention preferably retain the antigen binding ability of their wild-type, parent antibody counterparts. Thus, the cysteine engineered antibody is capable of binding, preferably specifically binding, to an antigen. Such antigens include, for example, Tumor Associated Antigens (TAAs), cell surface receptor proteins and other cell surface molecules, transmembrane proteins, signal transduction proteins, cell survival regulatory factors, cell proliferation regulatory factors, molecules associated with (e.g., known or suspected to be functionally involved in) tissue development or differentiation, lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis and molecules associated with (e.g., known or suspected to be functionally involved in angiogenesis). The tumor associated antigen may be a population of differentiation factors (i.e., CD proteins). An antigen capable of binding a cysteine engineered antibody may be a member of a subset of one of the above classes, wherein other subsets of the classes comprise other molecules/antigens (relative to the antigen of interest) with different characteristics.

The parent antibody may also be a humanized antibody selected from the group consisting of huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (trastuzumab,) (ii) a Humanized 520C9(WO 93/21319) and humanized antibodies of the humanized 2C4 antibody described herein.

The cysteine engineered antibodies of the invention can be site specifically and efficiently linked to a thiol-reactive reagent. The thiol-reactive reagent can be a multifunctional linker reagent, a capture reagent, i.e., an affinity, label reagent (e.g., biotin linker reagent), a detection label (e.g., fluorophore reagent), a solid phase immobilization reagent (e.g., SEPHAROSE reagent)^TMPolystyrene, or glass), or a zirconium linker intermediate. An example of a thiol-reactive reagent is N-ethylmaleimide (NEM). In exemplary embodiments, the reaction of a ThioFab with a biotin linker reagent provides a biotinylated ThioFab by which the presence and reactivity of the engineered cysteine residues can be detected and measured. Reaction of a ThioFab with a multifunctional linker reagent provides a ThioFab with a functionalized linker, which can 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.

By applying the design and screening steps described herein, the exemplary methods described herein can be applied generally to the identification and production of antibodies, and more generally to other proteins.

Such methods are applicable to the conjugation of other thiol-reactive reagents, where the reactive groups are, for example, maleimide, iodoacetamide, dithiopyridine (pyridylal), or other thiol-reactive conjugation partners (Haughand, 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 application, Academic Press, London; Means (1990) bioconjugate.1: 2; Hermanson, G.in Bioconjugate Techniques (1996) Academic Press, Sanego, Sanpp.40-55, 643). The partner may be a cytotoxic agent (e.g. a toxin such as doxorubicin or pertussis toxin), a fluorophore such as a fluorescent dye, e.g. fluorescein or rhodamine, a chelator for imaging or radiotherapy metals, a peptidyl or non-peptidyl label or detection tag, or a clearance modifier such as various isomers of polyethylene glycol, a peptide conjugated to a third component, or another carbohydrate or lipophilic agent.

The sites identified on the exemplary antibody fragment hu4D5Fabv8 herein are primarily in the constant regions of the antibody, which are highly conserved among antibodies of all species. These sites should be broadly applicable to other antibodies, require no further structural design or information on the structure of the particular antibody, and do not interfere with the inherent antigen binding properties of the variable domains of the antibody.

Cysteine engineered antibodies useful for the treatment of cancer include, but are not limited to, antibodies directed against cell surface receptors and Tumor Associated Antigens (TAAs). Such antibodies can be used as naked antibodies (unconjugated to a label moiety) or antibody-zirconium conjugates of general formula I (AZC). Tumor-associated antigens are well known in the art and can be prepared for use in generating antibodies using methods and information well known in the art. In order to find effective cellular targets for cancer diagnosis and treatment, researchers have attempted to identify transmembrane or tumor-associated polypeptides that are specifically expressed on the surface of one or more specific types of cancer cells as compared to one or more normal non-cancer cells. Typically, such tumor-associated polypeptides are expressed in greater amounts on the surface of cancer cells than on the surface of non-cancer cells. The identification of such tumor-associated cell surface antigen polypeptides has led to the ability to specifically target the destruction of cancer cells by antibody-based therapies.

Examples of TAAs include, but are not limited to, TAAs (1) - (36) listed below. For convenience, information relating to these antigens is set forth below and is known in the art, including names, alias names, Genbank accession numbers and primary references, following nucleic acid and protein sequence identification conventions by the National Center for Biotechnology Information (NCBI). Nucleic acid and protein sequences corresponding to TAAs (1) - (36) are available in public databases such as GenBank. Tumor-associated antigens targeted by the antibodies include all amino acid sequence variants and isoforms having at least about 70%,80%,85%,90%, or 95% sequence identity to the sequences identified in the cited references, or antigens that exhibit substantially the same biological properties or characteristics as TAAs having the sequences in the applied references. For example, a TAA having a variant sequence is generally capable of specifically binding to an antibody that specifically binds to a TAA having the corresponding listed sequence. The sequences and disclosures of the references specifically enumerated herein are expressly incorporated by reference.

Tumor associated antigens (1) - (36):

(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbank accession No. NM-001203)

ten Dijke, P., et al Science 264(5155):101-

NP-001194 bone morphogenetic protein receptor IB type/pid = NP-001194.1-

Cross-reference MIM 603248, NP 001194.1, AY065994

(2) E16(LAT1, SLC7A5, Genbank accession No. NM-003486) biochem. Biophys. Res. Commun.255(2), 283-49 (1999), Nature 395(6699):288-291(1998), Gaugitsch, H.W., et al (1992) J.biol. chem.267(16):11267-11273), WO2004048938 (example 2), WO2004032842 (example IV), WO2003042661 (claim 12), WO2003016475 (claim 1), WO200278524 (example 2), WO 200902974 (claim 19; page 127-129), WO 200642843 (claim 27; page 222,393), WO2003003906 (claim 10; page 293), WO200264798 (claim 33; page 93-95; page 302228; WO 2004435; page 322133; WO 2004435; page 133; WO 2004435);

NP _003477 solute carrier family 7 (cationic amino acid transporter, y + system), member 5/pid = NP _ 003477.3-homo sapiens

Cross-referencing MIM 600182, NP 003477.3, NM 015923, NM 003486-1

(3) STEAP1 (prostate six transmembrane epithelial antigen, Genbank accession No. NM _012449)

Cancer Res.61(15),5857 and 5860(2001), Hubert, R.S., et al (1999) Proc. Natl.Acad.Sci.U.S.A.96(25):14523 and 14528), WO2004065577 (claim 6), WO2004027049 (FIG. 1L), EP1394274 (example 11), WO2004016225 (claim 2), WO2003042661 (claim 12), US2003157089 (example 5), US2003185830 (example 5), US2003064397 (FIG. 2), WO200289747 (example 5; page 619 of eye 618), WO2003022995 (example 9; FIG. 13A, example 53; page 173, example 2; FIG. 2A);

NP 036581 six transmembrane epithelial antigen of prostate

Cross-reference MIM 604415, NP-036581.1, NM-012449-1

(4)0772P (CA125, MUC16, Genbank accession No. AF361486) J. biol. chem.276(29):27371-

(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiator, mesothelin (mesothelin), Genbank accession No. NM-005823) Yamaguchi, N.et al biol. chem.269(2),805-808(1994), Proc. Natl. Acad. Sci. U.S.A.96(20):11531-11536(1999), Proc. Natl. Acad. Sci. U.S.A.93(1):136-140(1996), J.biol. chem.270(37):21984-21990 (1995); WO2003101283 (claim 14); WO 2002105 (claim 13; 287-288), WO2002101075 (claim 4; 308-309; WO200271928 (320-321); WO 0312 (NM 52-94157; NM-601051; MIM-005814.2; WO 2006335; 31-366335; WO 308-309; WO 2; 31-601051; MIM-005814.2; WO 2; 31-36 005823; WO 2; 35; WO 2; 35; No.

(6) Napi3B (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3B, Genbank accession No. NM-006424)

Biol. chem.277(22): 19665-;

cross-referencing MIM 604217, NP 006415.1, NM 006424-1

(7) Sema5B (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin (Semaphorin) 5B Hlog, Sema domain, 7 thrombospondin (thrombospondin) repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (Semaphorin) 5B, Genbank accession number AB040878)

Nagase T, et al (2000) DNA Res.7(2): 143-;

registration Q9P283, EMBL, AB040878, BAA95969.1.Genew, HGNC:10737, (8) PSCA hlg (2700050C12Rik, C530008016Rik, RIKEN cDNA2700050C12, RIKEN cDNA2700050C12 gene, Genbank accession number AY358628), Ross et al (2002) Cancer Res.62: 2546-;

cross-reference GI 37182378, AAQ88991.1, AY358628_1

(9) ETBR (Endothelin B receptor, Genbank accession number AY275463), Nakamuta M.et al biochem. Biophys.Res.Commun.56, 34-39,1991, Ogawa Y.et al biochem.Biophys.Res.Commun.178,248-255,1991; Arai H.et al Jpn.Circ.J.56,1303-1307,1992; Arai H.et al J.biol.Chem.268,3463-3470,1993; Sakamoto A.yanagawa.M.et al biochem.Biophys.Res.178, 656-663,1991; Elshurbagy N.A.et al J.biol.Chem.3-3879,1993; Hawth.Res.K.K.K.K.K.K.K.K.K.K.K.No. 92, 656, Gene J.37, Gene J.37.7, J.05-85, Gene J.J.05, Gene J.85, Gene J.7, Gene J.85, Gene J.120. 2000, Gene J.7, Gene J.85, Gene J.7, Gene J.85, Gene J.103, Gene J.52.240, Gene J.J.103, Gene J.240, Gene J.7.7.103, Gene J.52.J.52.7, Gene J.J.7, Gene J.7, Gene J.J.103, Gene J.J.52, et al. Et al mol.Med.7,115-124,2001, Pingault V.et al (2002) hum.Genet.111,198-206, WO2004045516 (claim 1), WO2004048938 (example 2), WO2004040000 (claim 151), WO2003087768 (claim 1), WO2003016475 (claim 1), WO200261087 (figure 1), WO 20030194 (figure 6), WO 200302645138 (claim 12; page 144), WO200198351 (claim 1; page 124 and 125), EP522868 (claim 8; figure 2), WO200177172 (claim 1; page 297 and 299), US2003109676, US6518404 (figure 3), US5773223 (claim 1a; Col31-34);

(10) MSG783(RNF124, hypothetical protein FLJ20315, Genbank accession No. NM — 017763);

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);

cross-reference LocusID 54894, NP-060233.2, NM-017763-1

(11) STEAP2 (HGNC-8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene1, prostate cancer associated protein 1, prostate 6 transmembrane epithelial antigen 2,6 transmembrane prostate protein, Genbank accession number AF455138)

Lab.Inves t.82(11):1573-1582(2002)), WO2003087306, US2003064397 (claim 1; FIG. 1), WO200272596 (claim 13; pages 54-55), WO200172962 (claim 1; FIG. 4B), WO2003104270 (claim 11), WO2003104270 (claim 16), US2004005598 (claim 22), WO2003042661 (claim 12), US2003060612 (claim 12; FIG. 10), WO200226822 (claim 23; FIG. 2), WO 20021621612 (claim 12; FIG. 10);

cross-reference GI 22655488, AAN04080.1, AF455138_1

(12) TrpM4(BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession No. NM-017636)

Xu, X.Z., et al Proc. Natl. Acad. Sci. U.S.A.98(19):10692-10697(2001), Cell109(3):397-407(2002), J.biol. chem.278(33):30813-30820(2003)); US2003143557 (claim 4); 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; FIGS. 1A-D);

cross-reference MIM 606936, NP 060106.2, NM 017636_1

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, Genbank accession number NP-003203 or NM-003212)

Ciccodiola, A., et al EMBO J.8(7): 1987-;

WO2003083041 (example 1), WO2003034984 (claim 12), WO200288170 (claim 2; pages 52-53), WO2003024392 (claim 2; FIG. 58);

WO200216413 (claim 1; pp 94-95,105), WO200222808 (claim 2; FIG. 1), US5854399 (example 2; Col 17-18), US5792616 (FIG. 2);

cross-referencing MIM 187395, NP 003203.1, NM 003212-1

(14) CD21(CR2 (complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs.73792Genbank accession number M26004)

Fujisaku et al (1989) J.biol.chem.264(4): 2118. 2125), WeisJ.J., et al J.exp.Med.167,1047-1066,1988, Moore M.et al Proc.Natl.Acad.Sci.U.S.A.84,9194-9198,1987, Barel M.et al mol.35, 1025-1031,1998, Weis J.J., et al Proc.Natl.Acad.Sci.U.S.A.83,5639-5643,1986, Sinha S.K., et al (1993) J.Immunol.150,5311-5320, WO2004045520 (example 4), US2004005538 (example 1), WO2003062401 (claim 9), WO 200404591520 (example 4), WO 20040291520 (example 4) and WO 2004024595 (FIG. 051);

registration P20023, Q13866, Q14212, EMBL, M26004, AAA35786.1.

(15) CD79B (CD79B, CD79 beta, IGb (immunoglobulin associated beta), B29, Genbank accession No. NM-000626 or 11038674)

Natl.Acad.Sci.U.S.A. (2003)100(7):4126-4131, Blood (2002)100(9):3068-3076, Muller et al (1992) Eur.J.Immunol.22(6):1621-1625), WO2004016225 (claim 2, FIG. 140), WO2003087768, US2004101874 (claim 1, page 102), WO 2003062402401 (claim 9), WO200278524 (example 2), US2002150573 (claim 5, page 15), US 5644044033, WO 2008202 (claim 1, page 306 and 309), WO 99/558658, US6534482 (claim 13, FIG. 17A/B), WO 200351 (claim 11, page 1145-1146);

cross-referencing MIM 147245, NP 000617.1, NM 000626-1

(16) FcRH2(IFGP4, IRTA4, SPAP1A (phosphatase-anchored protein 1a comprising SH2 domain), SPAP1B, SPAP1C, Genbank accession No. NM _030764, AY358130) Genome Res.13(10):2265-2270(2003), Immunogenet ics 54(2):87-95(2002), Blood 99(8):2662-2669(2002), Proc. Natl.Acad.Sci.U.S.A.98(17):9772-9777(2001), Xu, M.J., et al (2001) biochem.Biophys.Res.Scu.280 (3):768-775; WO2004016225 (2): WO2003077836; WO 200gp 865; WO 18D-Res.30803-2008923; WO 2009723: 493-30803; WO 2009723: 493-3073; WO 2009723: 8925; WO 2009723: 3073; WO 2009725; WO 2009723: 3073: WO 3: 3073: 3023; WO 200973: 3073; WO 3: WO 2009725; WO 3: 3073: WO 3: 3073; WO 3: WO 2009725; WO 3: 3097

(17) HER2(ErbB2, Genbank accession number M11730)

Coissens L., et al Science (1985)230(4730):1132-1139);

yamamoto T, et al Nature 319,230-

Proc.Natl.Acad.Sci.U.S.A.82,6497-6501,1985;Swiercz

J.M., et al J.cell biol.165,869-880,2004, Kuhns J.J., et al J.biol.chem.274,36422-36427,1999, Cho H-S, et al Nature 421,756-760,2003, Ehsani A, et al (1993) Genomics 15,426-429, WO2004048938 (example 2), WO2004027049 (FIG. 1I), WO2004009622, WO2003081210, WO2003089904 (claim 9), WO2003016475 (claim 1), US2003118592, WO2003008537 (claim 1), WO2003055439 (claim 29; FIG. 1A-B), WO2003025228 (claim 37; FIG. 5C), WO200222636 (example 13; pp.95-107), WO 20040341; WO 20040847; WO 200935843847 (WO 20021497,979; WO 20043849,979; WO 200435460,979; WO 200435445; WO 20043 Example 3; FIG. 4);

register P04626, EMBL, M11767, AAA35808.1 EMBL, M11761, AAA35808.1.

(18) NCA (CEACAM6, Genbank accession number M18728);

barnet T, et al Genomics3, 59-66,1988, Tawaragi Y, et al Biochem.

Biophys.Res.Commun.150,89-96,1988, Strausberg R.L., et al Proc.Natl.Acad.Sci.U.S.A.99: 16899-169903,2002, WO2004063709, EP1439393 (claim 7), WO2004044178 (example 4), WO2004031238, WO2003042661 (claim 12), WO200278524 (example 2), WO200286443 (claim 27; page 427), WO200260317 (claim 2);

registration P40199, Q14920, EMBL, M29541, AAA59915.1 EMBL, M18728;

(19) MDP (DPEP1, Genbank accession BC017023)

Proc.Natl.Acad.Sci.U.S.A.99(26):16899-16903(2002));

WO2003016475 (claim 1), WO200264798 (claim 33; pages 85-87), JP05003790 (FIGS. 6-8), WO9946284 (FIG. 9);

cross-reference MIM 179780, AAH17023.1, BC017023_1

(20) IL20R α (IL20Ra, ZCYTOR7, Genbank accession No. AF184971);

clark H.F., et al Genome Res.13,2265-2270,2003, Mungall A.J., et al Nature425,805-811,2003, Blumberg H., et al Cell 104,9-19,2001, Dumoutier L., et al J.Immunol.167,3545-3549,2001, Parrish-Novak J., et al J.biol.Chem.277,47517-47523,2002, Pletnev S., et al (2003) Biochemistry 42: 12617-;

the registrations Q9UHF4, Q6UWA9, Q96SH8, EMBL, AF184971, AAF01320.1.

(21) Brevican (BCAN, BEHAB, Genbank accession number AF229053) Gary s.c., et al Gene 256,139-147,2000, Clark h.f., et al Genome res.13,2265-2270,2003, straussbergr.l., et al proc.natl.acad.sci.u.s.a.99,16899-16903,2002, US2003186372 (claim 11), US2003186373 (claim 11), US2003119131 (claim 1; fig. 52), US2003119122 (claim 1; fig. 52), US 2009126 (claim 1), US 2009121 (claim 1; fig. 52), US2003119129 (claim 1), US2003119130 (claim 1), US2003119128 (claim 1; fig. 52), US2003119125 (claim 3012003019125), WO 2003012021 (claim 1);

(22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession No. NM _004442) Chan, J. and Watt, V.M., Oncogene 6(6), 1057-;

cross-referencing MIM 600997, NP 004433.2, NM 004442-1

(23) ASLG659(B7h, Genbank accession number AX092328)

US20040101899 (claim 2), WO2003104399 (claim 11), WO2004000221 (FIG. 3), US2003165504 (claim 1), US2003124140 (example 2), US2003065143 (FIG. 60), WO2002102235 (claim 13; page 299), US2003091580 (example 2), WO200210187 (claim 6; FIG. 10), WO200194641 (claim 12; FIG. 7B), WO 20020220220213 (claim 13; FIG. 1A-1B), US 2002104749 (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; page 190-63192), WO 200610-322 (page 200400326; page 207-49207-4979; WO 20027147234-200-34079; page 27147234; WO 200203200-200-47234; page 27147234; WO 200203200-340989; WO 20035479; WO 200340989;

(24) PSCA (prostate stem cell antigen precursor, Genbank accession number AJ297436) Reiter R.E., et al Proc. Natl.Acad.Sci.U.S. A.95,1735-1740,1998; Gu Z., et al Oncogene 19,1288-1296,2000; biochem. Biophys. Res. Commun. (2000)275(3):783-788; WO2004022709; EP1394274 (example 11); US2004018553 (claim 17); WO2003008537 (claim 1); WO200281646 (claim 1; page 164); WO2003003906 (claim 10; page 288); WO200140309 (example 1; FIG. 17); US2001055751 (example 1; FIG. 1B); WO 20020030098752 (claim 18; FIG. 1; WO 4039098403; WO 519894; WO 200989894; WO 98982);

registers 043653, EMBL, AF043498, AAC39607.1.

(25) GEDA (Genbank accession number AY260763);

AAP14954 lipoma HMGIC fusion partner-like protein/pid = AAP 14954.1-homo sapiens species homo sapiens (human)

WO2003054152 (claim 20), WO2003000842 (claim 1), WO2003023013 (example 3, claim 20), US2003194704 (claim 45);

cross-reference GI 30102449, AAP14954.1, AY260763_1

(26) BAFF-R (B cell activating factor receptor, BLyS receptor 3, BR3, Genbank accession number AF116456), BAFF receptor/pid = NP-443177.1-homo sapiens

Thompson, J.S., et al Science 293(5537), 2108-;

cross-reference MIM 606269, NP 443177.1, NM 052945-1, AF132600

(27) 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);

cross-referencing MIM 107266, NP 001762.1, NM 001771-1

(28) CD79a (CD79A, CD79a, immunoglobulin associated alpha, B cell specific protein covalently interacting with Ig beta (CD79B) and forming a complex with IgM molecules on the surface, transducing a signal involved in B cell differentiation), pI 4.84, MW 25028TM 2[ P ] gene chromosome 19q13.2, Genbank accession number NP-001774.10)

WO2003088808, US20030228319, WO2003062401 (claim 9), US2002150573 (claim 4, pages 13-14), WO9958658 (claim 13, FIG. 16), WO9207574 (FIG. 1), US5644033, Ha et al (1992) J.Immunol.148(5): 1526-;

(29) CXCR5(Burkitt lymphoma receptor 1, a G protein-coupled receptor activated by CXCL13 chemokines, plays a role in lymphocyte migration and humoral defense, in HIV-2 infection and possibly in the pathogenesis of AIDS, lymphoma, myeloma and leukemia) 372aa, pI:8.54MW:41959TM:7[ P ] Gene chromosome: 11q23.3, Genbank accession No. NP _001707.1) WO2004040000; WO2004015426; US2003105292 (example 2); US6555339 (example 2); WO200261087 (fig. 1); WO200157188 (claim 20, page 269); WO200172830 (pages 12-13); WO200022129 (example 1, page 152-153, example 2, page 254-256); WO9928468 (claim 1, page 38); US5440021 (example 2, col 49-52); WO9428931 (pages 56 to 58); WO9217497 (claims 7, fig. 5); dobner et al (1992) Eur.J.Immunol.22:2795-2799; barella et al (1995) biochem.J.309:773-779;

(30) HLA-DOB (the beta subunit of MHC class II molecules (Ia antigen) that binds to and presents the peptide to CD4+ T lymphocytes), 273aa, pI:6.56MW:30820TM:1[ P ] gene chromosome: 6P21.3, Genbank accession number NP-002111.1)

Tonnelle et al (1985) EMBO J.4(11) 2839-2847, Jonsson et al (1989) Immunogenetics 29(6) 411-413, Beck et al (1992) J.mol. biol.228: 433-;

(31) P2X5 (purine receptor P2X ligand-gated ion channel 5, ion channel gated by extracellular ATP, possibly involved in synaptic transmission and neurogenesis, defects of which may contribute to the pathophysiology of spontaneous detrusor instability), 422aa), pI 7.63, MW 47206TM:1[ P ] gene chromosome 17P13.3, Genbank accession number NP-002552.2)

Le et al (1997) FEBS Lett.418(1-2):195-199; WO2004047749; WO2003072035 (claim 10); Touchman et al (2000) Genome Res.10:165-173; WO200222660 (claim 20); WO2003093444 (claim 1); WO2003087768 (claim 1); WO2003029277 (page 82);

(32) CD72(B cell differentiation antigen CD72, Lyb-2) protein complete sequence maease.. tafrfpd (1..359;359aa), pI:8.66, MW:40225TM:1[ P ] gene chromosome 9P13.3, Genbank accession NP-001773.1)

WO2004042346 (claim 65), WO2003026493 (pages 51-52, 57-58), WO200075655 (page 105-106), Von Hoegen et al (1990) J.Immunol.144(12): 4870-;

(33) LY64 (lymphocyte antigen 64(RP105), type I membrane protein of the leucine-rich repeat (LRR) family, regulation of B-cell activation and apoptosis, loss of function associated with increased disease activity in systemic lupus erythematosus patients), 661aa, pI:6.20, MW:74147 TM:1[ P ] gene chromosome 5q12, Genbank accession NP-005573.1)

US2002193567, WO9707198 (claim 11, pages 39-42), Miura et al (1996) Genomics38(3): 299-;

(34) FcRH1(Fc receptor-like protein 1, putative receptor of immunoglobulin Fc domain comprising Ig-like and ITAM domains of type C2, likely to play a role in B-lymphocyte differentiation), 429aa, pI:5.28, MW:46925TM:1[ P ] gene chromosome 1q21-1q22, Genbank accession number NP-443170.1), WO2003077836, WO200138490 (claim 6, FIG. 18E-1-18-E-2), Davis et al (2001) Proc. Natl. Acad. Sci USA 98(17): 9772-;

(35) IRTA2 (immunoglobulin superfamily receptor translocation related 2, putative immunoreceptor that may play a role in B cell development and lymphopoiesis; deregulation of this gene by translocation in some B cell malignancies); 977aa, pI:6.88MW:106468TM:1[ P ] gene chromosome 1q21, Genbank accession No. human AF343662, AF343663, AF343664, AF343665, AF369794, AF 39453, AK090423, AK 090090090, AL 418387, AY358085; Mouse: AK089756, AY 090158, AY506558; NP-112571.1 WO2003024392 (claim 2, FIG. 97); Nakayama et AL (2000) biochem. Biophys. Res. Commun.277(1):124 30127; WO 20030490; WO 20020077490; claim 3, WO 20013818B-836);

(36) TENB2(TMEFF2, tomorgulin, TPEF, HPP1, TR, putative transmembrane proteoglycans, related to the EGF/regulin family of growth factors and follistatin), 374aa, NCBI accession No. AAD55776, AAF91397, AAG49451, NCBI RefSeq: NP-057276, NCBI gene: 23671, OMIM:605734, SwissProtQ9UIK5, Genbank accession No. AF179274, AY358907, CAF85723, CQ782436

WO2004074320(SEQ ID NO 810), JP2004113151(SEQ ID NO 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, US2004022727, WO2004063355, US2004197325, US2003232350, US2004005563, US2003124579, Horie et al (2000) Genomics 67: 146-;

(37) PMEL17 (silver homolog; SILV; D12S53E; PMEL17; (SI); (SIL); ME20; gp100) 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;

(38) TMEF 1 (transmembrane protein with EGF-like and 2 follistatin-like domains 1; Tomoregulin-1; H7365; C9ORF2; C9ORF2; U19878; X83961) NM-080655; NM-003692; Harms, P.W. (2003) GenesDev.17(21), 2624-;

(39) GDNF-Ra 1(GDNF family receptor 1; GFRA1; GDNFR; GDNFRA; RETL1; TRNR1; RET1L; GDNFR-ALPHA 1; GFR-ALPHA-1; U95847; BC014962; NM _145793) NM _005264; Kim, M.H. et al (2009) mol.cell.biol.29(8), 2264. minus 2277; Treano, J.J. et al (1996) Nature 382(6586):80-83;

(40) ly6E (lymphocyte antigen 6 complex, locus E; Ly67, RIG-E, SCA-2, TSA-1) NP-002337.1; NM-002346.2; de Nooij-van Dalen, A.G. et al (2003) int.J.cancer103(6), 768-and 774; Zammit, D.J. et al (2002) mol.cell.biol.22(3): 946-and 952;

(41) TMEM46(SHISA homolog 2 (Xenopus laevis); SHISA2) NP-001007539.1; NM-001007538.1; Furushima, K.et al (2007) Dev.biol.306(2), 480-cok 492; Clark, H.F. et al (2003) Genome Res.13(10): 2265-cok 2270;

(42) ly6G6D (lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT1) NP-067079.2; NM-021246.2; Mallya, M. et al (2002) Genomics 80(1): 113-287 123; Ribas, G. et al (1999) J.Immunol.163(1):278-287;

(43) LGR5 (containing leucine rich repeats of G protein-coupled receptor 5; GPR49, GPR67) NP-003658.1; NM-003667.2; Salant i, G. et al (2009) am. J. epidemol.170 (5):537-545; Yamamoto, Y. et al (2003) Hepatology 37(3) 528-533;

(44) RET (RET proto-Oncogene; MEN2A; HSCR1; MEN2B; MTC1; (PTC), CDHF12; Hs.168114; RET51; RET-ELE1) NP-066124.1; NM-020975.4; Tsukamoto, H. et al (2009) Cancer Sci.100(10): 1895-;

(45) 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-;

(46) GPR19(G protein-coupled receptor 19; Mm.4787) NP-006134.1, NM-006143.2, Montpetit, A. and Sinnett, D. (1999) hum. Genet.105(1-2):162-164; O' Down, B.F. et al (1996) FEBSLett.394(3): 325-329);

(47) GPR54(KISS1 receptor; KISS1R; GPR54; HOT7T175; AXOR12) NP-115940.2; NM-032551.4; Nanvenot, J.M. et al (2009) mol. Pharmacol.75(6):1300-1306; Hata, K. et al (2009) Anticancer Res.29(2):617-623;

(48) ASPHD1 (comprising aspartate beta-hydroxylase domain 1; LOC253982) NP-859069.2; NM-181718.3; Gerhard, D.S. et al (2004) Genome Res.14(10B): 2121-;

(49) tyrosinase (TYR; OCAIA; OCA1A; tyrosinase; SHEP3) NP-000363.1; NM-000372.4; Bishop, D.T. et al (2009) nat. Genet.41(8): 920-;

(50) TMEM118 (cyclic finger protein, transmembrane 2; RNFT2; FLJ14627) NP-001103373.1; NM-001109903.1; Clark, H.F. et al (2003) Genome Res.13(10):2265-

(51) GPR172A (G protein-coupled receptor 172A; GPCR41; FLJ11856; D15Ertd747e) NP-078807.1; NM-024531.3; Ericsson, T.A. et al (2003) Proc. Natl. Acad. Sci. U.S.A.100(11):6759-6764; Takeda, S. et al (2002) FEBSLett.520(1-3): 97-101).

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 Stratagene For Improving The pharmacological Of Proteins" J Biol chem.277:35035-35043; WO 01/45746). The antibodies of the invention include fusion proteins having the ABP sequences taught in (i) Dennis et al (2002) J Biol chem.277:35035 and 35043, pages 35038, (ii) US20040001827 [0076] SEQ ID NOS:9-22, and (III) WO01/45746, pages 12-13, SEQ ID NOS: z1-z14, which are all incorporated herein by reference.

Mutagenesis

DNA encoding amino acid sequence variants 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 previously prepared DNA encoding the polypeptide. Variants of recombinant antibodies can also be constructed by restriction enzyme fragment manipulation or by overlap extension PCR using synthetic oligonucleotides. The mutagenic primer encodes a cysteine codon substitution. DNA encoding such mutated cysteine engineered antibodies can be generated using standard mutagenesis techniques. General guidance can be found in Sambrook et al Molecular Cloning, Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989, and Ausubel et al Current Protocols in Molecular Biology, Greene publishing and Wiley-Interscience, New York, N.Y., 1993.

Site-directed mutagenesis is a method of making substitution variants, i.e., muteins. This technique is well known in the art (see, e.g., 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 performing site-directed mutagenesis of DNA, such starting DNA is altered by first hybridizing an oligonucleotide encoding the desired mutation to a single strand of the starting DNA. After hybridization, the entire second strand is synthesized using a DNA polymerase, using the hybridized oligonucleotide as a primer and the single strand of the starting DNA as a template. Thus, an oligonucleotide encoding the desired mutation is incorporated into the resulting double-stranded DNA. Site-directed mutagenesis may be performed in an expression plasmid within the gene expressing the protein to be mutagenized, and the resulting plasmid may be sequenced to confirm the introduction of the desired cysteine substitution mutation (Liu et al (1998) J.biol.chem.273:20252-20260). Fixed-point protocols and formats include those that are commercially available, e.g.Multi-Site-Directed Mutagenesis (Multi Site-Directed Mutagenesis) kit (Stratagene, La Jolla, Calif.).

PCR mutagenesis is also suitable for generating amino acid sequence variants of the starting polypeptide. See, Higuchi, (1990) in PCRProtocols, pp.177-183, Academic Press, Ito et al (1991) Gene102:67-70, Bernhard et al (1994) Bioconjugate chem.5:126-132, and Vallette et al (1989) Nuc. acids Res.17: 723-733. Briefly, when a small amount of template DNA is used as starting material in PCR, primers that differ slightly from the corresponding region sequences in the template DNA can be used to generate a relatively large number of specific DNA fragments that differ from the template sequence only in the positions where the primers differ from the template.

Another method for preparing variants, cassette mutagenesis, is based on the technique described by Wells et al (1985) Gene34: 315-. The starting material is a plasmid (or other vector) containing the starting polypeptide DNA to be mutated. The codons to be mutated in the starting DNA are identified. Unique restriction enzyme sites must be located on each side of the identified mutation site. If such restriction sites are not present, the oligonucleotide-mediated mutagenesis method described above can be used to generate restriction sites to introduce them into the appropriate locations in the starting polypeptide DNA. Plasmid DNA was cut at these sites to linearize the plasmid. Double-stranded oligonucleotides encoding the DNA sequence between the restriction sites but containing the desired mutation are synthesized using standard procedures, wherein 2 strands of the oligonucleotide are synthesized separately and then hybridized together using standard techniques. This double-stranded oligonucleotide is called a cassette. This cassette was designed to have 5 'and 3' ends compatible with the ends of the linearized plasmid, so that it could be directly ligated to the plasmid. This plasmid now contains the mutated DNA sequence. Mutant DNA comprising the encoded cysteine substitution can be confirmed by DNA sequencing.

Single mutations can also be generated by PCR-based mutagenesis using double-stranded plasmid DNA as template by oligonucleotide-directed mutagenesis (Sambrook and Russel, (2001) Molecular Cloning: A Laboratory Manual, 3 rd edition; Zoller et al (1983) Methods enzymol.100: 468-.

In The present invention, hu4D5Fabv8(Gerstner et al (2002) "Sequence specificity In The antibiotic-Binding Site Of A Therapeutic Anti-HER2 Antibody", J Mol biol.321:851-62) displayed on M13 phage was used as a model system for The experiment. Cysteine mutations were introduced in the hu4D5Fabv 8-phage, hu4D5Fabv8, and ABP-hu4D5Fabv8 constructs. The preparation of hu4D 5-ThioFab-phages was carried out using the polyethylene glycol (PEG) precipitation method (Lowman, Henry B. (1998) Methods in Molecular Biology (Totowa, New Jersey)87(Combinatorial Peptide Library Protocols)249-264) described previously.

Oligonucleotides were prepared by phosphoramidite synthesis (US 4415732; US 4458066; Beaucage, S. and Iyer, R. (1992) "Advances in the synthesis of oligonucleotides by the phosphoramidite approach", Tetrahedron48: 2223-2311). The phosphoramidite method requires the cyclic addition of a nucleotide monomer unit having a reactive 3 'phosphoramidite moiety to an oligonucleotide chain extending on a solid support composed of controlled pore glass or highly cross-linked polystyrene, most commonly in the 3' to 5 'direction, with the 3' terminal nucleoside attached to the solid support at the start of synthesis (US 5047524; US 5262530). This method is typically performed using an automated, commercially available synthesizer (Applied Biosystems, Foster City, CA). Non-isotopically partially chemically labeled oligonucleotides can be used for detection, capture, stabilization or other purposes (Andrus, A. "Chemical methods for 5' non-interactive labeling 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), StockPress, New York, pp.121-23).

PHESELECTOR assay

The PHESELECTOR (phage ELISA for selection of reactive thiols) assay allows the detection of reactive cysteine groups in antibodies in the form of ELISA phage (US 7521541; Junutula JR et al, "Rapid diagnosis of reactive cysteine residues for site-specific labeling of antibodies-Fabs" J Immunol Methods 2008;332: 41-52). The process of coating the protein of interest (e.g., antibody) on the well surface, followed by incubation with phage particles and then with HRP-labeled secondary antibody, and detection with absorbance is described in detail in example 2. Mutant proteins displayed on phage can be screened in a rapid, efficient and high throughput manner. Cysteine engineered antibody libraries can be generated and binding selection performed using the same method to identify appropriate reactive sites for free Cys incorporation from random protein-phage libraries of antibodies or other proteins. This technique involves reacting the cysteine muteins displayed on the phage with an affinity reagent or reporter group that is also thiol-reactive. FIG. 8 illustrates schematically the PHESELECTOR assay in which the binding of Fab or ThioFab to HER2 (top) and biotinylated ThioFab to streptavidin (bottom) is depicted.

Protein expression and purification

DNA encoding the cysteine engineered antibody can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of a murine antibody). Hybridoma cells serve as a source of such DNA. Once isolated, the DNA may be placed into an expression vector, which is then transfected into a host cell, such as an E.coli cell, simian COS cell, Chinese Hamster Ovary (CHO) cell, or other mammalian host cell, such as a myeloma cell that does not otherwise produce antibody proteins (US 5807715; US2005/0048572; US 2004/0229310), to obtain synthesis of monoclonal antibodies in the recombinant host cell. The yield of hu4D5Fabv8 cysteine engineered antibody was similar to that of wild-type hu4D5Fabv 8. A review article on recombinant expression of DNA encoding an antibody in bacteria includes Skerra et al (1993) Curr. opinion in Immunol.5: 256-188 and Pl ü ckthun (1992) Immunol.Revs.130: 151-188.

After design and selection, cysteine engineered antibodies, such as ThioFab with highly reactive unpaired Cys residues, can be generated by: (i) expression in bacteria, such as the E.coli system or mammalian cell culture system (WO 01/00245), such as Chinese Hamster Ovary (CHO); and (ii) purification using conventional protein purification techniques (Lowman et al (1991) J. biol. chem.266(17): 10982-.

ThioFab was expressed after induction in non-inhibitory E.coli strain 34B8(Baca et al (1997) Journal biological chemistry 272(16): 10678-84). See example 3 a. The harvested cell pellet was resuspended in PBS (phosphate buffered saline), whole cell lysis (total cells) was performed by passing through a microfluidizer (microfluidizer), and ThioFab was purified by affinity chromatography using protein G sepharose (Amersham). ThioFab was conjugated to biotin-PEO-maleimide as described above and passed through Superdex-200^TM(Amersham) gel filtration chromatography further purified the biotinylated ThioFab, which removed free biotin-PEO-maleimide and the oligomeric portion of the ThioFab.

Mass spectrometric analysis

The exact molecular weight of the biotin-conjugated Fab was determined by liquid chromatography electrospray Ionization Mass Spectrometry (LC-ESI-MS) analysis (Cole, R.B. electro Spray Ionization Mass Spectrometry: Fundamentals, Instrumentation And Applications, 1997, Wiley, New York). The amino acid sequence of the biotinylated hu4D5Fabv8(a121C) peptide was determined by trypsinization followed by LC-ESI-tandem MS analysis (table 4, example 3 b).

The antibody Fab fragment hu4D5Fabv8 contains about 445 amino acid residues, including 10 Cys residues (5 on the light chain and 5 on the heavy chain). The high resolution structure of the humanized 4D5 variable fragment (Fv4D5) has been determined, see: eigenbrot et al, "X-Ray Structures Of The Antibody-Binding Domains From Three Variants Of Humanized Anti-P185her 2Antibody 4D 5And company With Molecular Modeling" (1993) J Mol biol.229: 969-995). All Cys residues are present as disulfide bonds, so these residues do not have any reactive thiol groups to conjugate with zirconium-maleimide (unless treated with a reducing agent). Thus, the newly engineered Cys residue may remain unpaired and capable of reacting, i.e., conjugating, with an electrophilic linker reagent or a zirconium linker intermediate, such as zirconium-maleimide. Fig. 1A shows a three-dimensional plot of hu4D5Fabv8 antibody fragment as deduced from X-ray crystal coordinates. The structural positions of the engineered Cys residues of the heavy and light chains were numbered according to the serial numbering system. This sequential numbering system is associated with the Kabat numbering system for the 4d5v7fabH variant of trastuzumab according to fig. 1B (Kabat et al, (1991) Sequences of proteins of Immunological Interest, fifth edition public Health Service, national institutes of Health, Bethesda, MD), which shows a sequential numbering scheme (top row), starting from the N-terminus, and the difference of the Kabat numbering system (bottom row) is the insertion marked with a, B, c. Using the Kabat numbering system, the actual linear amino acid sequence may comprise fewer or additional shortened or inserted amino acids corresponding to the FRs or CDRs of the variable domain. The cysteine engineered heavy chain variant sites are identified in the following table by sequential numbering and the Kabat numbering scheme.

4D5Fab heavy chain variants	Serial number	Kabat numbering
			A40C	Ala-40	Ala-40
A88C	Ala-88	Ala-84
			S119C	Ser-119	Ser-112
S120C	Ser-120	Ser-113
			A121C	Ala-121	Ala-114
S122C	Ser-122	Ser-115
			A175C	Ala-175	Ala-168

Compared to Fab proteins, M13 phagemid-Cys mutant fabs (fig. 3A and 3B) could be rapidly screened. The binding of phagemid-ThioFab to antigen and streptavidin can be detected by coating HER2 and streptavidin, respectively, on an ELISA plate followed by detection with anti-Fab-HRP (horseradish peroxidase), as described in example 2 and depicted in fig. 8. This approach allows simultaneous monitoring of the effect on antigen binding and reactivity of thiol groups by engineered Cys residues/conjugated biotin molecules. This method can also be applied to screen for reactive thiol groups of any protein displayed on the M13 phage. The conjugated or unconjugated phagemid-ThioFab was purified by simple PEG precipitation.

The antigen-binding fragment of humanized 4D5(hu4D5Fab) was well expressed in E.coli and displayed on phage (Garrrard et al (1993) Gene 128: 103-. The antibody Fab fragment hu4D5Fabv8 was displayed on M13 phage as a model system in an ELISA-based assay for detection of thiol reactivity. FIG. 8 is a schematic representation of the PHESELECTOR assay depicting binding of biotinylated ThioFab phage and anti-phage HRP antibodies to HER2 (top) and streptavidin (bottom). Since it is remote from the antigen-binding surface (Eigenbrot et al (1993) J Mol biol.229: 969. sup. 995), 5 amino acid residues (L-Ala43, H-Ala40, H-Ser119, H-Ala121 and H-Ser122) were initially selected from the crystal structure information. The protein database X-ray crystal structure was named 1 FVC. Cys residues were engineered at these positions by site-directed mutagenesis. ThioFab-phage preparations were isolated and reacted with biotinylation reagents.

HER2 and streptavidin binding of biotin-conjugated and unconjugated variants were detected using an ELISA-based PHESELECTOR assay (fig. 8, example 2) using HRP (horseradish peroxidase) conjugated anti-phage antibodies. The interaction of non-biotinylated phage-hu 4D5Fabv8 (fig. 2A) and biotinylated phage-hu 4D5Fabv8 (fig. 2B) with BSA (open box), HER2 (grey box) or streptavidin (solid box) was monitored by anti-M13 horseradish peroxidase (HRP) antibody by performing a standard HRP reaction and measuring the absorbance at 450 nm. The absorbance generated by the conversion of the colorimetric substrate (turnover) was measured at 450 nm. Reactivity of ThioFab with HER2 measures antigen binding. Reactivity of ThioFab with streptavidin measures the degree of biotinylation. Reactivity of ThioFab with BSA is a negative control for non-specific interaction. As shown in fig. 2A, all ThioFab-phage variants have similar binding to HER2 compared to the wild-type hu4D5Fabv 8-phage. Furthermore, conjugation to biotin did not interfere with ThioFab binding to HER2 (fig. 2B).

Surprisingly and unexpectedly, the ThioFab-phage samples showed varying levels of streptavidin binding activity. Of all phage-ThioFab tested, the a121C cysteine engineered antibody exhibited the greatest thiol reactivity. Although wild-type hu4D5Fabv 8-phage were incubated with the same amount of biotin-maleimide, these phage had little streptavidin binding, indicating that the preexisting cysteine residues (involved in disulfide bond formation) from the hu4D5Fabv8 and M13 phage coat protein did not interfere with the site-specific conjugation of biotin-maleimide. These results demonstrate that phage ELISA assays can be successfully used to screen for reactive thiol groups on Fab surfaces.

The pheselect assay allows for the screening of reactive thiol groups in antibodies. The a121C variant identified by this method is exemplary. The entire Fab molecule can be efficiently searched to identify more ThioFab variants with reactive thiol groups. The accessibility of solvents to amino acid residues in polypeptides is identified and quantified using the parametric score surface accessibility (fractional accessibility). Surface accessibility can be expressed as the surface area accessible to solvent molecules (e.g., water)The water occupies about the spaceA sphere of radius. The relevant software is freely available or licensable (Secretariy to CCP4, Daresbury Laboratory, Warrington, WA 44 AD, United Kingdom, Fax: (+44)1925603825, or by The Internet: www.ccp4.ac.uk/dist/html/INDEX. html), for example The CCP4 program group of Crystallography Programs that apply algorithms to calculate The surface accessibility of each amino acid of a Protein using known X-ray Crystallography derived coordinates ("The CCP4 Suite: Programs for Protein Crystallography" (1994) acta. Crystal.D50: 760-. The 2 exemplary software modules for performing the SURFACE reachability calculations are "AREAIMOL" and "SURFACCE" based on the algorithms of B.Lee and F.M.Richards (1971) J.mol.biol.55: 379-. AREAIMOL defines the solvent accessible surface of a protein as the locus of rolling of the center of a probe sphere (representing a solvent molecule) on the van der Waals surface of the protein. AREAIMOL is created by creating curved points on an extended sphere about each atom (equal to the sum of atoms at a distance from the center of the atom)Radius of the probe and distance) and excluding those points located in the equivalent sphere associated with adjacent atoms (associated with the solvent) to calculate the surface area accessible to the solvent. AREAIMOL finds the solvent accessible area of atoms in the PDB coordinate file and summarizes the accessible area for the entire molecule by residue, by chain. The reachable area (or area difference) of each atom may be recorded as a pseudo-PDB output file. AREAIMOL assumes that each element is a single radius and only identifies a limited number of different elements. Unknown atom types (i.e., those not in the AREAIMOL internal database) will be designated asIs determined. The list of atoms identified is:

AREAIMOL and SURFACCE report absolute reachability, namely angstromsThe reference state is the tripeptide Gly-X-Gly, where X is the amino acid of interest, and the reference state should be the "extended" conformation, i.e., the conformation in the β -chain, for example.

Another exemplary algorithm for calculating surface reachability is the SOLV module based on the program xsae (Broger, c., f.hoffman-LaRoche, Basel), which calculates the fractional reachability of amino acid residues to a water sphere based on the X-ray coordinates of the polypeptide.

Fractional surface accessibility of each amino acid in hu4D5Fabv7 was calculated using the crystal structure information (Eigenbrot et al (1993) J Mol biol.229: 969-995). Fractional surface accessibility (fracacc) values for amino acids of the light and heavy chains of hu4D5Fabv7 are shown in descending order in table 1.

TABLE 1

hu4D5Fabv 7-light chain

hu4D5Fabv 7-heavy chain

The following 2 criteria were used to identify residues that can replace the engineered hu4D5Fabv8 with Cys residues:

1. amino acid residues that are completely buried (i.e., less than 10% fractional surface accessibility) are excluded. Table 1 shows that the 134 (light chain) and 151 (heavy chain) residues of hu4D5Fabv8 are more than 10% accessible (fractional surface accessibility). The first 10 most accessible Ser, Ala and Val residues were selected because they are more similar in structure to Cys than other amino acids, allowing the newly engineered Cys to introduce only minimal structural limitations in the antibody. Other cysteine substitution sites can also be screened and used for conjugation.

2. Residues are classified based on their role in functional and structural interactions of Fab. Residues not involved in antigen interactions and remote from existing disulfide bonds are further selected. The newly engineered Cys residues should be different from the antigen binding residues and not interfere with antigen binding and not mismatch with the cysteines involved in disulfide bond formation.

The following hu4D5Fabv8 residues meet the criteria described above and were selected as replacements with Cys: L-V15, L-A43, L-V110, L-A144, L-S168, H-A88, H-A121, H-S122, H-A175 and H-S179 (shown in FIG. 1).

Thiol reactivity can be generalized to any antibody, among which can be in a sequence selected from: l-10 to L-20, L-38 to L-48, L-105 to L-115, L-139 to L-149, L-163 to L-173; and in a group selected from: h-35 to H-45, H-83 to H-93, H-114 to H-127, and H-170 to H-184; and amino acid substitutions with reactive cysteine amino acids in the Fc region selected from the range of H-268 to H-291, H-319 to H-344, H-370 to H-380, and H-395 to H-405.

Thiol reactivity can also be generalized to specific domains of antibodies, such as the light chain constant domain (CL) and the heavy chain constant domains CH1, CH2, and CH 3. The antibody may be expressed in whole antibody: IgA, IgD, IgE, IgG, and IgM, including the IgG subclasses: cysteine substitutions resulting in thiol reactivity values of 0.6 and higher were made in the heavy chain constant domains α,, γ, and μ of IgG1, IgG2, IgG3, IgG4, IgA, and IgA 2.

It is evident from the crystal structure data that the 10 Cys mutants were selected to be distant from the antigen binding site, e.g. in this case the interface with HER 2. The indirect effect of these mutants on functional interactions can be experimentally tested. The thiol reactivity of all Cys Fab variants was measured and calculated as described in examples 1 and 2 and is listed in table 2. The residues L-V15C, L-V110C, H-A88C and H-A121C have reactive and stable thiol groups (FIGS. 3A and 3B). Mutants V15C, V110C, a144C, S168C are light chain Cys variants. Mutants a88C, a121C, a175C, S179C are heavy chain Cys variants. Surprisingly and unexpectedly, sites with a high fraction of surface accessibility do not have the highest thiol reactivity as calculated by the pheselect assay (table 2). In other words, fractional surface accessibility (table 1, 2) is not related to thiol reactivity (table 2). Indeed, engineered Cys residues on sites with a moderate surface accessibility of 20% to 80% (fig. 4A, table 1), or partially exposed sites such as Ala or Val residues, exhibit higher thiol reactivity, i.e., >0.6 (fig. 3B, table 2), than Cys introduced at Ser residues, so the pheselect assay must be used in screening for thiol reactive sites, since the crystal structure information alone is not sufficient to select these sites (fig. 3B and 4A).

The 4D5ThioFab Cys mutant is shown in fig. 3A and 3B: (3A) thiol reactivity data for amino acid residues of non-biotinylated (control) and (3B) biotinylated phage-ThioFab. Reactive thiol groups on the antibody/Fab surface were identified by PHESELECTOR assay to detect the interaction of non-biotinylated phage-hu 4D5Fabv8 (3A) and biotinylated phage-hu 4D5Fabv8 (3B) with BSA (open bars), HER2 (grey bars) or streptavidin (solid bars). The assay was performed as described in example 2. The light chain variant is on the left and the heavy chain variant is on the right. The binding of the non-biotinylated 4D5ThioFab Cys variant was low as expected, but retained strong binding to HER 2. The ratio of the binding of biotinylated 4D5 ThioFabCys mutant to streptavidin and to HER2 provided the thiol reactivity values in table 2. Also evident in fig. 3B is the background absorbance at 450nm or the binding of biotinylated 4D5ThioFab Cys mutants to a small amount of unspecific protein of BSA. Fractional surface accessibility values for selected amino acid residues substituted with Cys residues are shown in figure 4A. Fractional surface accessibility was calculated from the available hu4D5Fabv7 structure and is shown in Table 1 (Eigenbrot et al (1993) J Mol biol.229: 969-. The conformational parameters of the hu4D5Fabv7 and hu4D5Fabv8 structures are highly consistent and allow determination of any correlation between fractional surface accessibility calculations for hu4D5Fabv7 and thiol reactivity of hu4D5Fabv8 cysteine mutants. The measured thiol reactivity of the Cys residues of the phage ThioFab introduced at the partially exposed residues (Ala or Val) was higher than the thiol reactivity of the Cys residues introduced at the Ser residues (table 2). As can be seen from the ThioFabCys mutants of table 2, there was little or no correlation between the thiol reactivity values and the fractional surface accessibility.

The amino acids at positions L-15, L-43, L-110, L-144, L-168, H-40, H-88, H-119, H-121, H-122, H-175, and H-179 of the antibody can generally be mutated (replaced) to a free cysteine amino acid. The free cysteine amino acids may also be substituted for the cysteine engineered antibodies of the invention within the range of about 5 amino acid residues on each side of these positions, i.e., L-10 to L-20, L-38 to L-48, L-105 to L-115, L-139 to L-149, L-163 to L-173, H-35 to H-45, H-83 to H-93, H-114 to H-127, and H-170 to H-184, and within the range of Fc regions selected from H-268 to H-291, H-319 to H-344, H-370 to H-380, and H-395 to H-405.

TABLE 2Thiol reactivity of phage-ThioFab

L = light chain, H = heavy chain, a = alanine, S = serine, V = valine, C = cysteine

^*By OD bound to streptavidin_450nmOD binding to HER2 (antibody)_450nmMeasuring thiolReactivity (example 2). A thiol reactivity value of 1 indicates complete biotinylation of the cysteine thiol.

The 2 Cys variants of the light chain (L-V15C and L-V110C) and the 2 Cys variants of the heavy chain (H-A88C and H-A121C) were selected for further analysis, as these variants showed the highest thiol reactivity (Table 2).

Unlike phage purification, Fab production may take 2-3 days, depending on the scale of production. During this time, the thiol group may lose reactivity due to oxidation. To examine the stability of the thiol group on hu4D5Fabv 8-phage, the stability of the thiol reactivity of phage-ThioFab was measured (fig. 4B). After ThioFab-phage purification, on days 1,2 and 4, all samples were conjugated with biotin-PEO-maleimide and detected with phage ELISA assay (PHESELECTOR) to test HER2 and streptavidin binding. L-V15C, L-V110C, H-A88C and H-A121C retained a significant amount of thiol reactivity compared to other ThioFab variants (FIG. 4B).

Methods of making cysteine engineered antibodies

The compounds of the invention include cysteine engineered antibodies in which one or more amino acids of a parent antibody are replaced with a free cysteine amino acid. The cysteine engineered antibody comprises one or more free cysteine amino acids having a thiol reactivity value in the range of 0.6 to 1.0. The free cysteine amino acids are cysteine residues that have been engineered into the parent antibody and are not part of a disulfide bond.

In one aspect, a cysteine engineered antibody is prepared by a method comprising:

(a) replacing one or more amino acid residues of a parent antibody with cysteine; and

(b) the thiol reactivity of the cysteine engineered antibody is determined by reacting the cysteine engineered antibody with a thiol reactive reagent.

The cysteine engineered antibody may be more reactive with a thiol-reactive reagent than the parent antibody.

The free cysteine amino acid residues may be located in the heavy or light chain, or in the constant or variable domain. An antibody fragment, such as a Fab, may also be engineered with one or more cysteine amino acids in place of the amino acids of the antibody fragment to form a cysteine engineered antibody fragment.

Another aspect of the invention provides a method of making (producing) a cysteine engineered antibody comprising:

a) introducing one or more cysteine amino acids in a parent antibody to produce a cysteine engineered antibody; and

b) determining the thiol reactivity of the cysteine engineered antibody using a thiol-reactive reagent;

wherein the cysteine engineered antibody is more reactive with a thiol-reactive reagent than the parent antibody.

Step (a) of the method of making a cysteine engineered antibody may comprise:

(i) mutagenizing a nucleic acid sequence encoding a cysteine engineered antibody;

(ii) expressing the cysteine engineered antibody; and

(iii) isolating and purifying the cysteine engineered antibody.

Step (b) of the method of making a cysteine engineered antibody may comprise expressing said cysteine engineered antibody on a viral particle selected from a bacteriophage or a phagemid particle.

Step (b) of the method of making a cysteine engineered antibody may further comprise:

(i) reacting the cysteine engineered antibody with a thiol-reactive affinity reagent to produce an affinity labeled, cysteine engineered antibody; and

(ii) measuring binding of the affinity-labeled, cysteine engineered antibody to a capture medium.

Another aspect of the invention is a method of screening for thiol reactivity of a cysteine engineered antibody having highly reactive, unpaired cysteine amino acids comprising:

a) introducing one or more cysteine amino acids in a parent antibody to produce a cysteine engineered antibody;

b) reacting the cysteine engineered antibody with a thiol-reactive affinity reagent to produce an affinity labeled, cysteine engineered antibody; and

c) measuring binding of the affinity-labeled, cysteine engineered antibody to a capture medium; and

d) the thiol reactivity of the cysteine engineered antibody is determined with a thiol reactive reagent.

Step (a) of the method of screening for cysteine engineered antibodies may comprise:

(i) mutagenizing a nucleic acid sequence encoding a cysteine engineered antibody;

(ii) expressing the cysteine engineered antibody; and

(iii) isolating and purifying the cysteine engineered antibody.

Step (b) of the method of screening for cysteine engineered antibodies may comprise expressing said cysteine engineered antibodies on viral particles selected from phage or phagemid particles.

Step (b) of the method of screening for a cysteine engineered antibody may further comprise:

(i) reacting the cysteine engineered antibody with a thiol-reactive affinity reagent to produce an affinity labeled, cysteine engineered antibody; and

(ii) measuring binding of the affinity-labeled, cysteine engineered antibody to a capture medium.

Labeled cysteine engineered antibodies

Cysteine engineered Antibodies of the invention may be conjugated to any label moiety covalently linked to the antibody through a reactive cysteine thiol group to 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 Springs Harbor, NY; Lundblad r.l. (1991) Chemical Reagents for protein modification, second edition CRC Press, Boca Raton, FL). The attached label may perform the following functions: (i) providing a detectable signal; (ii) interaction with a second label to modify a detectable signal provided by the first or second label, e.g., FRET (fluorescence resonance energy transfer); (iii) stabilizing the interaction with or increasing the affinity for binding to the antigen or ligand; (iv) (iv) influencing mobility by charge, hydrophobicity, shape or other physical parameters, such as electrophoretic mobility or cell permeability, or (v) providing a capture moiety to modulate ligand affinity, antibody/antigen binding, or ion complexation.

The labeled cysteine engineered antibodies may be used in diagnostic assays, for example, to detect expression of an antigen of interest in a particular cell, tissue, or serum. For diagnostic applications, the antibody will generally be labeled with a detectable moiety. A large number of markers can be used, which can be generally classified into the following categories:

a) radioisotopes (radionuclides), e.g.³H,¹¹C,¹⁴C,¹⁸F,³²P,³⁵S,⁶⁴Cu,⁶⁸Ga,⁸⁶Y,⁸⁹Zr,⁹⁹Tc,¹¹¹In,¹²³I,¹²⁴I,¹²⁵I,¹³¹I,¹³³Xe,¹⁷⁷Lu,²¹¹At, or²¹³And (4) Bi. The radioisotope labeled antibodies are useful in receptor targeted imaging experiments. Can use the combination,A ligand reagent that chelates or complexes a radioisotope metal, wherein the reagent reacts with an engineered cysteine thiol group of an antibody, is labeled with an antibody using the techniques described in Current Protocols in Immunology, Vol.1 and 2, Coligen et al, Wiley-Interscience, New York, NY, Pubs. (1991). Chelating ligands that can complex metal ions include DOTA, DOPA, DOTP, DOTMA, DTPA and TETA (Macrocyclics, Dallas, TX). The radionuclide can be targeted by complexation with cysteine engineered antibodies in the form of antibody-zirconium conjugates of the invention (Wu et al (2005) Nature Biotechnology 23(9): 1137-.

In 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.cancer1990, Sup.10: 21-26, Izard et al (1992) Bioconjugate chem.3:346-350, Nikula et al (1995) Merge.biol.22: 387-90, Camera et al (1993) Merge.Nucl.22: 387-90, Merge.19851: 19851.20: Biocal et al [ 1985 ] 19851: 19851; German et al [ 10 ] J.19851: 19851; German.19851: 19851; Ver) J.15; Verafe.19851: 19851; Ver.19851: 19851) J.19851; Ver; Ver.19851: 19851: 10) J.19851: 19851; Verproximate to biomedical) J.19851: 19851: 10) J.19851; Verproximate to 2483-90, Blend et al (2003) Cancer Biotherapy & Radiopharmaceuticals 18:355-363, Nikula et al (1999) J.Nucl.Med.40:166-76, Kobayashi et al (1998) J.Nucl.Med.39:829-36, Mardirossian et al (1993) Nucl.Med.biol.20:65-74, Roselli et al (1999) Cancer Biotherapy & Radiopharmaceuticals,14:209-20, disclose metal chelate complexes suitable for use as antibody markers for imaging experiments.

(b) Fluorescent labels, such as rare earth chelates (europium chelates), fluorescein types including FITC, 5-carboxyfluorescein, 6-carboxyfluorescein; rhodamine types include TAMRA; dansyl; lissamine; a cyanine pigment; phycoerythrin; texas Red; and the like. Fluorescent labels can be conjugated to antibodies using techniques such as those disclosed in Current Protocols in Immunology (supra). Fluorescent dyes and fluorescent marker reagents include those commercially available from Invitrogen/molecular probes (Eugene, OR) and Pierce Biotechnology, Inc. (Rockford, IL).

(c) Various enzyme-substrate markers are available or published (US 4275149). Enzymes generally catalyze chemical changes in chromogenic substrates, which can be measured using a variety of techniques. For example, the enzyme may catalyze a color change in the substrate, which can be measured by a spectrophotometer. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying changes in fluorescence are described above. The chemiluminescent substrate may be excited by chemically reacting its electrons and may then emit measurable light (e.g., using a chemiluminescent meter) or provide energy to a fluorescent acceptor. Examples of enzyme labels include luciferase (e.g., firefly luciferase and bacterial luciferase; US 4737456), luciferin, 2, 3-dihydrophthalazinedione, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRP), Alkaline Phosphatase (AP), β -galactosidase, glucoamylase, lysozyme, saccharide oxidase (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidase (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes to antibodies are described in O' Sullivan et al (1981) "Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme immumunassay", Methods in Enzyme.

Examples of enzyme-substrate combinations include, for example:

(i) horseradish peroxidase (HRP) and catalase (as substrates), wherein the catalase oxidizes a dye precursor (e.g., o-phenylenediamine (OPD) or 3,3 ', 5, 5' -tetramethylbenzidine hydrochloride (TMB));

(ii) alkaline Phosphatase (AP) and p-nitrophenyl phosphate (as chromogenic substrate); and

(iii) beta-D-galactosidase (beta-D-Gal) and a chromogenic substrate (e.g., p-nitrophenyl-beta-D-galactosidase) or fluorogenic substrate 4-methylumbelliferyl-beta-D-galactosidase.

A large number of other enzyme-substrate combinations can be used by those skilled in the art. For a general review see US 4275149 and US 4318980.

The label may be indirectly conjugated to the cysteine engineered antibody. For example, the antibody may be conjugated to biotin and any of the 3 broad classes of labels described above may be conjugated to avidin or streptavidin, or vice versa. Biotin binds selectively to streptavidin and thus the label can be conjugated to the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the label to the polypeptide variant, the polypeptide variant is conjugated to a small hapten (e.g., digoxin), and one of the different types of labels described above is conjugated to a polypeptide variant that is anti-hapten (e.g., anti-digoxin antibody). Thus, indirect conjugation of a marker to a polypeptide variant can be achieved (Hermanson, G. (1996) in Bioconjugate Techniques Academic Press, San Diego).

The polypeptide variants of the invention may be used 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 Pres, Inc.).

The detection marker can be used to locate, visualize and quantify the binding or recognition event. The labeled antibodies of the invention can detect cell surface receptors. Another use of detectably labeled antibodies is bead-based immunocapture methods that involve conjugating a bead to a fluorescently labeled antibody and detecting a fluorescent signal upon ligand binding. Similar binding detection methods utilize the Surface Plasmon Resonance (SPR) effect to measure and detect antibody-antigen interactions.

Detection markers such as Fluorescent Dyes and chemiluminescent Dyes (Briggs et al (1997) "Synthesis of functionalized Fluorescent Dyes and Their Coupling to Amines and amino acids," J.chem.Soc., Perkin-Trans.1:1051-1058) provide detectable signals and are generally useful for labeling antibodies, preferably with the following properties: (i) the labeled antibody should produce a very high signal and low background so that small amounts of antibody can be sensitively detected in both cell-free and cell-based assays; and (ii) the labeled antibody should be light-resistant so that a fluorescent signal can be observed, monitored and recorded without significant photobleaching. For applications involving binding of labeled antibodies to membranes or cell surfaces, particularly cell surfaces of living cells, the label preferably (iii) has good water solubility to obtain effective conjugate concentrations and detection sensitivity and (iv) is not toxic to living cells, so as not to disrupt normal metabolic processes of the cells or cause premature cell death.

Systems capable of automated mixing and reading of non-radioactive assays using living cells or beads (8100HTS System, Applied Biosystems, Foster City, Calif.) direct quantification of cell fluorescence intensity and calculation of fluorescence labeling events, such as cell surface binding of peptide-dye conjugates (Miragia, "Homogeneous cells-and bead-based assays for high throughput Screening using fluorescent assays technology", (1999) J.of Biomolecular Screening4: 193-. Uses for labeled antibodies also include cell surface receptor binding assays, immunocapture assays, fluorescence-linked immunosorbent assays (FLISA), Caspase-cleavage (Zheng, "Caspase-3controls bone cytoplasma and nuclear events associated with Fas-mediated apoptosis in vivo", (1998) Proc. Natl. Acad. Sci. USA 95:618-23; US6372907), apoptosis (Vermes, "A novel ay for apoptosis. flow cytometric detection of photodynamic expression on epithelial cells using fluorescence in sandwich Anxin V" (1995) J. Immunol. Methods: 39-51) and Fine 184And (4) determining cytotoxicity. Up-or down-regulation of molecules that target the cell surface can be identified using fluorescence micro-volume assay techniques (Swartzman, "A homogenetic and multiplexed immunological assay for high-throughput screening using fluorometric microbial assay technology" (1999) anal. biochem.271: 143-51).

The labeled cysteine engineered antibodies of the invention are useful as a variety of biomedical and molecular imaging methods and techniques, for example: (i) MRI (magnetic resonance imaging); (ii) micro CT (computed tomography); (iii) SPECT (single photon emission computed tomography); (iv) PET (positron emission tomography) Chen et al (2004) Bioconjugate chem.15: 41-49; (v) bioluminescence; (vi) fluorescence; and (vii) imaging biomarkers and probes for ultrasound. Immunoscintigraphy is an imaging procedure in which an animal or human patient is administered a radioactive substance-labeled antibody and photographed at an in vivo site where the antibody is concentrated (US 6528624). Imaging biomarkers can be objectively measured and evaluated as indicators of normal biological processes, pathological processes, or pharmacological responses to therapeutic interventions. Biomarkers can be of several types: type 0 is a natural history (naturalhistory) marker of the disease and is longitudinally correlated with known clinical indicators, such as MRI assessment of joint (synovial) inflammation in rheumatoid arthritis; type I markers capture the effect of intervention according to a mechanism of action, although the mechanism may not be correlated with clinical effect; the I I-type marker serves as a surrogate end point (surrogate end point) where alteration of the biomarker or signal prediction "confirms" the clinical benefit of the targeted response, such as bone erosion as measured by CT in rheumatoid arthritis. Imaging biomarkers can thus provide information about: (i) expression of the target protein, (ii) binding of the therapeutic agent to the target protein, i.e., selectivity, and (iii) Pharmacodynamic (PD) therapeutic information for clearance and half-life pharmacokinetic data. Advantages of in vivo imaging biomarkers over laboratory-based biomarkers include: non-invasive treatment, quantifiable, systemic evaluation, repeated dosing and evaluation, i.e. multiple time points, and potentially transferable effects from preclinical (small animals) to clinical (human) results. In some applications, bioimaging replaces or minimizes the number of animal experiments in preclinical studies.

Radionuclide imaging markers include radionuclides such as³H,¹¹C,¹⁴C,¹⁸F,³²P,³⁵S,⁶⁴Cu,⁶⁸Ga,⁸⁶Y,⁸⁹Zr,⁹⁹Tc,¹¹¹In,¹²³I,¹²⁴I,¹²⁵I,¹³¹I,¹³³Xe,¹⁷⁷Lu,²¹¹At, or²¹³And (4) Bi. The radionuclide metal ion can be complexed with a chelating linker such as DOTA. Linker reagents such as DOTA-maleimide (4-maleimidobutyrylaminobenzyl-DOTA) can be prepared by reaction of aminobenzyl-DOTA with 4-maleimidobutyric acid (Fluka) activated with isopropyl chloroformate (Aldrich) following the procedure of Axworthy et al (2000) Proc.Natl.Acad.Sci.USA97(4): 1802-. The DOTA-maleimide reagent reacts with the free cysteine amino acid of the cysteine engineered antibody and provides a metal complexing ligand on the antibody (Lewis et al (1998) bioconj. chem.9: 72-86). Chelating linker labeling reagents such as DOTA-NHS (1,4,7, 10-tetrathionopurine cyclododecane-1, 4,7, 10-tetraacetic acid mono (N-hydroxysuccinimide ester) are commercially available (macrocycles, Dallas, TX) receptor target imaging using radionuclide-labeled antibodies can provide pathway activating labels (Albert et al (1998) bioorg.med.chem.lett.8: 7-1210) conjugated radiometals can be retained intracellularly after lysosomal degradation.

Peptide labeling methods are well known. See Haughland, 2003, Molecular Probes Handbook of fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; Brinkley,1992, Bioconjugate chem.3:2; Garman, (1997) Non-Radioactive laboratory: a prechecal apparatus, Academic Press, London, Means (1990) Bioconjugate chem.1:2; Glazer et al (1975) Chemical Modification of Proteins. laboratory Techniques in biochemistry and Molecular Biology (T.S. Work and E.Work, eds.), American Elsevier Publishing Co., New York, Lundblad, R.L. and Noyes, C.M, (1984) Chemical Reagents for Protein Modification, volume I and II, Press, New York; Pfleiderer, G.2004) "Modification of Proteins", model Chemistry, H.S. Press, Waldon et al (1985) Biochemical Modification of Chemistry, Walker et al, (Becton et al: Biochemical Modification of company, J.23: 14: Biochemical Modification of Chemical Modification, publication, J.23: 14: Biochemical Modification chem et al (J.12; Biochemical Modification of Chemical Modification, 14: Biochemical Modification of Chemical Modification, J.23: 14; German, Biochemical Modification of Chemical Modification, Biochemical Modification, J.21: 14: Le300, publication No. 12; German, Biochemical Modification of Chemical Modification, Biochemical Modification, J.23: 14, German, and Chemical Modification, Release et al, publication No. 12; German, publication No. 2; Chemical Modification, German, publication No. 12; Chemical Modification, publication No. 2; Chemical Modification, publication No. 2; Chemical Modification, publication No. 2; Chemical Modification.

Peptides and proteins labeled with 2 moieties (fluorescent reporter and quencher) undergo Fluorescence Resonance Energy Transfer (FRET) when brought into sufficient proximity. The reporter group is typically a fluorescent dye that is excited by light of a particular wavelength and transfers energy to an acceptor, or quencher group, emitting at maximum brightness with an appropriate Stokes shift. Fluorescent dyes include molecules with extended aromaticity, such as fluorescein and rhodamine, and their derivatives. The fluorescent reporter can be partially or significantly quenched by a quencher moiety in the intact peptide. The increase in fluorescence which can be detected is measured by cleavage of the peptide by peptidases or proteases (Knight, C. (1995) "Fluorimetric Assays of Proteolytic Enzymes", Methods in Enzymology, Academic Press,248: 18-34).

The labeled antibodies of the present invention may also be used as affinity purifiers. In this method, the labeled antibody is immobilized on a solid phase such as Sephadex resin or filter paper using methods well known in the art. The immobilized antibody is contacted with a sample comprising the antigen to be purified, and the support is then washed with a suitable solvent that will remove substantially all of the material in the sample except the antigen to be purified that is bound to the immobilized polypeptide variant. Finally, the support is washed with another suitable solvent, such as glycine buffer, ph5.0, which releases the antigen from the polypeptide variant.

The labeling reagent typically has a reactive functional group that can react (i) directly with the cysteine thiol of the cysteine engineered antibody to form a labeled antibody, (ii) with a linker reagent to form a linker-label intermediate, or (iii) with a linker antibody to form a labeled antibody. The reactive functional groups of the labeling reagent include: maleimides, haloacetyls, iodoacetamide succinimidyl esters (e.g., NHS, N-hydroxysuccinimide), isothiocyanates, sulfonyl chlorides, 2, 6-dichlorotriazinyl, pentafluorophenyl, and phosphoramidites, although other functional groups may also be used.

Exemplary reactive functional groups are detectable labels such as N-hydroxysuccinimide ester (NHS) of the carboxyl substituent of biotin or a fluorescent dye. The NHS ester of the label may be preformed, isolated, purified and/or characterized, or formed in situ and reacted with a nucleophilic group of an antibody. Typically, the carboxy form of the label is activated by reaction with a carbodiimide reagent, such as dicyclohexylcarbodiimide, diisopropylcarbodiimide, or a uronium reagent, such as TSTU (O- (N-succinimide) -N, N, N ', N' -tetramethyluronium tetrafluoroborate, HBTU (O-benzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate), or HATU (O- (7-azobenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate), an activator, such as 1-hydroxybenzotriazole (HOBt) and N-hydroxysuccinimide, in a combination to form an NHS ester of the label. In some cases, the label and antibody can be linked by activating the label in situ and reacting with the antibody in a one-step reaction to form a label-antibody conjugate. Other activating and linking reagents include TBTU (2- (1H-benzotriazol-1-yl) -1-1,3, 3-tetramethyluronium hexafluorophosphate), TFFH (N, N ', N ", N'" -tetramethyluronium 2-fluoro-hexafluorophosphate), PyBOP (benzotriazol-1-yl-oxy-tripyrrolidinophosphonium hexafluorophosphate, EEDQ (2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline), DCC (dicyclohexylcarbodiimide); DIPCDI (diisopropylcarbodiimide), MSNT (1- (mesitylene-2-sulfonyl) -3-nitro-1H-1, 2, 4-triazole, and arylsulfonyl halides such as triisopropylbenzenesulfonyl chloride.

Conjugation of biotin-maleimide to ThioFab

The above ThioFab performance is determined in the presence of phage because the fusion of Fab with the phage coat protein is partialChanging the accessibility or reactivity of the Cys thiol. Thus, the thioFab construct was cloned into an expression vector under the control of the alkaline phosphatase promoter (Chang et al (1987) Gene55:189-196) and the thioFab was induced to be expressed by culturing E.coli cells in phosphate-free medium. In protein G SEPHAROSE^TMThioFab was purified on a column and analyzed on reducing and non-reducing SDS-PAGE gels. These analyses allow to assess whether the ThioFab retains its reactive thiol group or fails by forming intramolecular or intermolecular disulfide bonds. Expresses ThioFabsL-V15C, L-V110C, H-A88C and H-A121C and passes through the protein G SEPHAROSE^TMColumn purification (details see methods section.) analysis of purified proteins on SDS-PAGE gels under reducing (containing DTT) and non-reducing (not containing DTT) conditions other reducing agents such as BME (β -mercaptoethanol) can be used in the gel to cleave interchain disulfide bonds it is evident from the SDS-PAGE gel analysis that the major part of the ThioFab (-90%) is in monomeric form, whereas the wild-type hu4D5Fabv8 is essentially in monomeric form (47 kDa).

ThioFab (A121C) and wild-type hu4D5Fabv8 were incubated with a 100-fold excess of biotin-maleimide for 3 hours at room temperature, and biotinylated Fab was loaded onto Superdex-200^TMAnd (4) gel filtration column. This purification step was used to separate monomeric Fab from oligomeric Fab as well as excess free biotin-maleimide (or free zirconium reagent).

Figure 5 shows verification of performance of ThioFab variants in the absence of phage. The proteins hu4D5Fabv8 and hu4D5Fabv8-A121C (ThioFab-A121C) that were not fused to phage were expressed and purified using protein-G agarose beads followed by incubation with a 100-fold molar excess of biotin-maleimide. Streptavidin and HER2 binding was compared for biotinylated Cys engineered ThioFab and non-biotinylated wild type Fab. The extent of biotin conjugation (interaction with streptavidin) and its binding capacity to HER2 was monitored by ELISA analysis. Each Fab was tested at 2ng and 20 ng.

Biotinylated a121C ThioFab retained HER2 binding comparable to wild-type hu4D5Fabv8 (fig. 5). The wild-type Fab and A121C-ThioFab were purified by gel filtration column chromatography. Binding of 2 samples to HER2 and streptavidin was detected by ELISA using goat anti-Fab-HRP as the secondary antibody. Both wild type (open box) and ThioFab (dotted box) have similar binding to HER2, but only the ThioFab retains streptavidin binding. Only interaction with background levels of streptavidin was observed for non-biotinylated wild-type hu4D5Fabv8 (fig. 5). Mass spectrometric (LC-ESI-MS) analysis of biotinylated-ThioFab (a121C) resulted in a main peak with 48294.5 daltons compared to wild-type hu4D5Fabv8(47737 daltons). The 537.5 dalton difference between the 2 molecules corresponds exactly to the single biotin-maleimide conjugated to the ThioFab. Mass spectrometry protein sequencing (LC-ESI-tandem mass spectrometry) results further confirmed that the conjugated biotin molecule was located on the newly engineered Cys residue (table 4, example 3).

Site-specific conjugation of biotin-maleimide to Albumin Binding Peptide (ABP) -ThioFab

Plasma protein binding can be an effective way to improve the pharmacokinetic properties of short-lived molecules. Albumin is the largest amount of protein in plasma. Serum Albumin Binding Peptides (ABPs) can alter the pharmacodynamics of the fused active domain proteins, including alterations in tissue uptake, penetration and diffusion. These pharmacodynamic parameters can be modulated by the specific selection of the appropriate serum 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 Stratagene For Improving the pharmacological Of Proteins" J Biol chem.277:35035-35043; WO 01/45746). The compounds of the invention include (i) the ABP sequences taught in tables III and IV of Dennis et al (2002) J Biol chem.277:35035-35043, page 35038, (ii) SEQ ID NOS:9-22 of [0076] US20040001827, and (III) WO01/45746, pages 12-13, SEQ ID NO: z1-z14, which are incorporated herein by reference in their entirety.

Albumin Binding (ABP) -Fab was constructed by fusing albumin binding peptide to the C-terminus of the Fab heavy chain in a stoichiometric ratio of 1: 1(1 ABP/1 Fab). The results show that binding of these ABP-fabs to albumin increases their half-life by more than 25-fold in rabbits and mice. Such reactive Cys residues can thus be introduced into 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 set forth in SEQ ID NOS: 1-5:

the Albumin Binding Peptide (ABP) sequence binds albumin of various species (mouse, rat, rabbit, bovine, rhesus (rhesus), baboon, and human) with a Kd (rabbit) =0.3 μ M. The albumin binding peptide does not compete with known albumin binding ligands and has a half-life of 2.3 hours in rabbits (T1/2). As described in the previous section, in BSA-Sepharose^TMThe ABP-ThioFab protein was purified up, then conjugated with biotin-maleimide and purified on Superdex-S200 column chromatography. The purified biotinylated protein was homogeneous and did not contain any oligomeric forms (example 4).

Figure 6 shows the performance of Albumin Binding Peptide (ABP) -ThioFab variants. ELISA assays were performed to examine the binding ability of ABP-hu4D5Fabv8-wt, ABP-hu4D5Fabv8-V110C and ABP-hu4D5Fabv8-A121C to rabbit albumin, streptavidin and HER 2. Biotinylated ABP-ThioFab was able to bind albumin and HER2 with similar affinity as wild-type ABP-hu4D5Fabv8, as verified by ELISA (fig. 6) and BIAcore binding kinetics analysis (table 3). ELISA plates were coated with albumin, HER2 and SA as described. Binding of biotinylated ABP-ThioFab to albumin, HER2 and SA was detected with anti-Fab HRP. Compared to the non-biotinylated control ABP-hu4D5Fabv8-wt, biotinylated ABP-ThioFab was able to bind streptavidin, indicating that ABP-ThioFab was conjugated to biotin-maleimide in a site-specific manner similar to ThioFab, since the same Cys mutant was used in both variants (fig. 6).

TABLE 3BIAcore kinetic analysis of HER2 and Rabbit albumin binding biotinylated ABP-hu4D5Fabv8 wild type and ThioFab

Antibodies	kon(M-1s-1)	koff(s-1)	Kd(nM)
				HER2 binding
Wild type	4.57x105	4.19x10-5	0.0917
				V110C	4.18x105	4.05x10-5	0.097
A121C	3.91x105	4.15x10-5	0.106
				Rabbit albumin binding
Wild type	1.66x105	0.0206	124
				V110C	2.43x105	0.0331	136
A121C	1.70x105	0.0238	140

ABP = albumin binding peptide

Alternatively, the albumin binding peptide may be linked to the antibody via covalent linkage through a linker moiety.

Construction of ABP-ThioFab with 2 free thiol groups per Fab

The above results indicate that all 4 (L-V15C, L-V110C, H-A88C, and H-A121C) ThioFab (cysteine engineered Fab antibody) variants have a reactive thiol group that can be used for site-specific conjugation with a label reagent, a linker reagent, or a zirconium linker intermediate. L-V15C can be expressed and purified, but in relatively low yields. The expression and purification yields of the L-V110C, H-A88C, and H-A121C variants were similar to hu4D5Fabv 8. These mutants can therefore be used for further analysis and recombination to yield more than 1 thiol group per Fab. For this purpose, one thiol group was constructed on the light chain and one on the heavy chain to obtain 2 thiol groups per Fab molecule (L-V110C/H-A88C and L-V110C/H-A121C). The two bis-Cys variants were expressed in an e.coli expression system and purified. Purified biotinylated ABP-ThioFab was found to be homogeneous similar to that of the single Cys variant.

The effect of constructing 2 reactive Cys residues on each Fab was investigated (fig. 7). The presence of a second biotin was detected by detecting the binding of biotinylated ABP-ThioFab to SA using streptavidin-HRP (fig. 7). For HER2/Fab analysis, ELISA plates were coated with HER2 and detected with anti-Fab HRP. For SA/Fab analysis, ELISA plates were coated with SA and detected with anti-Fab HRP. For SA/SA analysis, ELISA plates were coated with SA and detected with SA HRP. Fig. 7. The biotinylated ABP-hu4D5Fabv8cys variants were analyzed by ELISA for interaction with HER2, Streptavidin (SA). HER2/Fab, SA/Fab and SA/SA indicate that their interaction is monitored by anti-Fab-HRP, SA-HRP, respectively. SA/Fab monitored the presence of a single biotin on each Fab, and SA/SA analysis monitored the presence of more than one biotin on each Fab. HER2 binding to the double Cys mutant was similar to that of the single Cys variant (fig. 7). However, due to the presence of more than one free thiol group on each Fab molecule, biotinylation was higher on the double Cys mutant compared to the single Cys variant (fig. 7).

Construction of ThioIgG variants of trastuzumab

Introduction of cysteine into the full-Length monoclonal antibody Trtuzumab: (Genentech Inc.). Expression of Single cys mutants of trastuzumab H-A88C, H-A121C and L-V110C, and the double cys mutant of trastuzumab V110C-A121 in CHO (Chinese hamster ovary) cells by transient fermentation in media containing 1mM cysteineC and V110C-A121C. The A88C mutant heavy chain sequence (450 aa) is SEQ ID NO 6. A21C mutant heavy chain sequence (450)

aa) is SEQ ID NO 7. The V110C mutant light chain sequence (214 aa) is SEQ ID NO: 8. EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRCEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:6

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSCSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:7

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTCAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO:8

According to one embodiment, the cysteine engineered thio-trastuzumab antibody comprises one or more of the following variable region heavy chain sequences with free cysteine amino acids (SEQ ID NOS: 9-16).

Mutants	Sequence of	SEQ ID NO:
			A40C	WVRQCPGKGL	SEQ ID NO:9
A88C	NSLRCEDTAV	SEQ ID NO:10
			S119C	LVTVCSASTKGPS	SEQ ID NO:11
S120C	LVTVSCASTKGPS	SEQ ID NO:12
			A121C	LVTVSSCSTKGPS	SEQ ID NO:13
S122C	LVTVSSACTKGPS	SEQ ID NO:14
			A175C	HTFPCVLQSSGLYS	SEQ ID NO:15
S179C	HTFPAVLQCSGLYS	SEQ ID NO:16

According to another embodiment, the cysteine engineered thio-trastuzumab antibody comprises one or more of the following variable region light chain sequences with free cysteine amino acids (SEQ ID NOS: 17-27).

Mutants	Sequence of	SEQ ID NO:
			V15C	SLSASCGDRVT	SEQID NO:17
A43C	QKPGKCPKLLI	SEQ ID NO:18
			V110C	EIKRTCAAPSV	SEQ ID NO:19
S114C	TCAAPCVFIFPP	SEQ ID NO:20
			S121C	FIFPPCDEQLK	SEQ ID NO:21
S127C	DEQLKCGTASV	SEQ ID NO:22
			A144C	FYPRECKVQWK	SEQ ID NO:23
A153C	WKVDNCLQSGN	SEQ ID NO:24
			N158C	ALQSGCSQESV	SEQ ID NO:25
S168C	VTEQDCKDSTY	SEQ ID NO:26
			V205C	GLSSPCTKSFN	SEQ ID NO:27

The thiol reactivity and HER2 binding activity of the resulting full-length thio-trastuzumab IgG variants were determined. Fig. 13A shows a schematic of biotinylated antibody bound to immobilized HER2 and HRP labeled secondary antibody for absorbance detection. Figure 13B shows binding measurements with immobilized HER2 (measured using absorbance at 450 nm) (left to right): non-biotinylated wild-type trastuzumab (Wt), biotin-maleimide conjugated thio-trastuzumab variants V110C (single cys), a121C (single cys), and V110C/a121C (double cys). Each thio IgG variant and trastuzumab were detected at1, 10 and 100 ng. Measurements showed that biotinylated anti-HER 2ThioMab retained HER2 binding activity.

Figure 14A shows a schematic of biotinylated antibody binding to immobilized HER2 using biotin binding to anti-IgG-HRP for absorbance detection. Figure 14B shows the binding measurement (detected using absorbance at 450 nm) of biotin-maleimide conjugated thiotrastuzumab variants and non-biotinylated wild-type trastuzumab bound to streptavidin. From left to right: V110C (single cys), A121C (single cys), V110C/A121C (double cys) and trastuzumab. Each thioIgG trastuzumab variant and parent trastuzumab was tested at1, 10 and 100 ng. Measurements showed that HER2ThioMab had high thiol reactivity.

Cysteine was introduced into specific residues of the full-length 2H9 anti-EphB 2R antibody. The single cys mutant H-a121C of 2H9 was expressed in CHO (chinese hamster ovary) cells by transient fermentation in medium containing 1mM cysteine. The A121C 2H9 mutant heavy chain sequence (450 aa) is SEQ ID NO: 28.

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMHWVRQAPGKGLEWVGFINPSTGYTDYNQKFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCTRRPKIPRHANVFWGQGTLVTVSSCSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:28

The cysteine engineered thio-2H9 antibody comprises the following Fc constant region heavy chain sequence (SEQ ID NOS: 29-38) with free cysteine amino acids.

Mutants	Sequence of	SEQ ID NO:
			V273C	HEDPECKFNWYVDGVEVHNAKTKPR	SEQ ID NO:29
V279C	HEDPEVKFNWYCDGVEVHNAKTKPR	SEQ ID NO:30
			V282C	HEDPEVKFNWYVDGCEVHNAKTKPR	SEQ ID NO:31
V284C	HEDPEVKFNWYVDGVECHNAKTKPR	SEQ ID NO:32
			A287C	HEDPEVKFNWYVDGVEVHNCKTKPR	SEQ ID NO:33
S324C	YKCKVCNKALP	SEQ ID NO:34
			S337C	IEKTICKAKGQPR	SEQ ID NO:35
A339C	IEKTISKCKGQPR	SEQ ID NO:36
			S375C	KGFYPCDIAVE	SEQ ID NO:37
S400C	PPVLDCDGSFF	SEQ ID NO:38

FIG. 16 shows non-reducing (top) and reducing (bottom) denaturing SDS-PAGE (polyacrylamide gel electrophoresis) analysis of the 2H9ThioMab Fc variant (left to right, lanes 1-9): A339C, S337C, S324C, A287C, V284C, V282C, V279C, V273C, and 2H9 wild-type after purification with immobilized protein A. The lanes on the right are size marker steps indicating that the intact protein is about 150kDa, the heavy chain fragment is about 50kDa, and the light chain fragment is about 25 kDa. FIG. 17A shows non-reducing (left) and reducing (right) denaturing polyacrylamide gel electrophoresis analysis of 2H9Thiomab variants (left to right, lanes 1-4): L-V15C, S179C, S375C, S400C after purification with immobilized protein A. FIG. 17B shows non-reducing (left) and reducing (+ DTT) (right) denaturing polyacrylamide gel electrophoresis analysis of additional 2H 9and 3A5ThioMab variants after purification with immobilized protein A. The 2H9ThioMab variants (in Fab and Fc regions) were expressed and purified as described. As seen in fig. 16, 17A and 17B, all proteins were homogeneous on SDS-PAGE, followed by preparation of reactive ThioMab for conjugation by the reduction and oxidation procedure of example 11 (example 12).

Cysteine was introduced into specific residues of the full-length 3a5 anti-MUC 16 antibody. The single cys mutant H-a121C of 3a5 was expressed in CHO (chinese hamster ovary) cells by transient fermentation in medium containing 1mM cysteine. The A121C 3A5 mutant heavy chain sequence (446 aa) comprises SEQ ID NO: 39.

DVQLQESGPGLVNPSQSLSLTCTVTGYSITNDYAWNWIRQFPGNKLEWMGYINYSGYTTYNPSLKSRISITRDTSKNQFFLHLNSVTTEDTATYYCARWDGGLTYWGQGTLVTVSACSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:39

Cysteine engineered thio-3A5 anti-MUC 16 antibodies comprise the following variable region heavy chain sequence (SEQ ID NOS: 40-44) with free cysteine amino acids.

Mutants	Sequence of	SEQ ID NO:
			F45C	NWIRQCPGNK	SEQ ID NO:40
A90C	LNSCTTEDTAT	SEQ ID NO:41
			A121C	GQGTLVTVSACSTKGPSVFPL	SEQ ID NO:42
A175C	HTFPCVLQSSGLYS	SEQ ID NO:43
			V176C	HTFPACLQSSGLYS	SEQ ID NO:44

Cysteine engineered thio-3A5 anti-MUC 16 antibodies comprise the following variable region light chain sequence (SEQ ID NOS: 45-49) with free cysteine amino acids.

Mutants	Sequence of	SEQ ID NO:
			L15C	FLSVSCGGRVT	SEQ ID NO:45
A43C	QKPGNCPRLLI	SEQ ID NO:46
			V110C	EIKRTCAAPSV	SEQ ID NO:47
A144C	FYPRECKVQWK	SEQ ID NO:48
			S168C	VTEQDCKDSTY	SEQ ID NO:49

Thiol reactivity of Thiomab

The thiol reactivity of the full-length IgG cysteine engineered antibody (ThioMab) was measured by biotinylation and streptavidin binding. Western blot assay was performed to screen for thiomabs specifically conjugated to biotin-maleimide. In this assay, antibodies were analyzed on reducing SDS-PAGE and the presence of biotin was specifically detected by incubation with streptavidin-HRP. As seen in fig. 18, depending on the engineered Cys variant used, streptavidin-HRP interaction was observed on the heavy or light chain, and no interaction was seen in the wild type, indicating that the ThioMab variant specifically conjugated biotin on the engineered Cys residue. FIG. 18 shows a 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: 3A5H-A121C, lane 2:3A5L-V110C, lane 3:2H9H-A121C, lane 4:2H9L-V110C, lane 5: anti-EphB 2R 2H9 parent, wild type. Each mutant was captured by using HRP-detected anti-IgG (top) (lanes 1-4), indicating that selectivity and affinity were retained. The positions of the biotin on the heavy and light chains were verified by immobilized streptavidin capture (bottom) using HRP detection. The location of the cysteine mutation on the cysteine engineered antibody in lanes 1 and 3 is on the heavy chain. The location of the cysteine mutation on the cysteine engineered antibody in lanes 2 and 4 is on the light chain. Conjugation to biotin-maleimide reagents was performed at the cysteine mutation site.

Analysis of the ThioMab cysteine engineered antibody and the 2H9V15C variant of fig. 18 by LC/MS provided a quantitative indication of thiol reactivity (table 5).

TABLE 5Biotinylated LC/MS quantification of ThioFab-thiol reactivity

Thiomab variants	Number of biotins per Thiomab
		2H9wt	0.0
2H9 L-V15C	0.6
		2H9 L-V110C	0.5
2H9 H-A121C	2.0
		3A5 L-V110C	1.0
3A5 H-A121C	2.0

Cysteine engineering was performed in the constant domain of IgG antibodies, the Fc region. Multiple amino acid sites were converted to cysteine sites and the thiol reactivity of the expressed mutants, i.e. cysteine engineered antibodies, was evaluated. The thiol reactivity of biotinylated 2H9ThioMab Fc variants was quantitatively assessed by HRP by capture on immobilized streptavidin in an ELISA assay (fig. 19). An ELISA assay was set up to rapidly screen Cys residues with reactive thiol groups. As depicted in the schematic diagram of fig. 19, streptavidin-biotin interactions were monitored by using anti-IgG-HRP detection followed by measurement of absorbance at 450 nm. These results demonstrate that 2H9-ThioFc variant V282C, a287C, a339C, S375C and S400C have moderate to maximal thiol reactivity. The extent of biotin conjugation of the 2H9ThioFab Fc variants was quantified by LS/MS analysis, as shown in table 6. LS/MS analysis demonstrated that the a282C, S375C and S400C variants had 100% biotin conjugation and V284C and a339C had 50% conjugation, indicating the presence of reactive cysteine thiol groups. Other ThioFc variants and the parent wild-type 2H9 have very low or no biotinylation.

TABLE 62H9Fc ThioMab biotinylated LC/MS quantitation

2H9ThioMab Fc variants	% biotinylation
		V273C	0
V279C	31
		V282C	100
V284C	50
		A287C	0
S324C	71
		S337C	0
A339C	54
		S375C	100
S400C	100
		(wild type 2H9)	0

Thiol reactivity of Thio-4D5Fab light chain variants

Screening of multiple cysteine engineered light chain variant fabs against ErbB 2antibody 4D5 provided some variants with thiol reactivity values of 0.6 and higher (table 7), as measured by the pheselect assay of figure 8. The thiol reactivity values of table 7 were normalized to the heavy chain 4D5ThioFab variant (HC-a121C), which was set to 100%, assuming that the HC-a121C variant was fully biotinylated and expressed as a percentage value.

TABLE 7Percent thiol reactivity value for 4D5ThioFab light chain variants

4D5ThioFab variants	Thiol reactivity value (%)
		V15C	100
V110C	95
		S114C	78
S121C	75
		S127C	75
A153C	82
		N158C	77
V205C	78
		(HC-A121C)	100
(4D5 wild type)	25

Zirconium labeling reagent

Using the exemplary bifunctional reagent based on desferrioxamine B (Desferrioxamine B) (Df)⁸⁹Zr is complexed to antibodies, including monoclonal antibodies (mabs). Desferrioxamine B (N' - {5- [ acetyl (hydroxy) amino group)]Pentyl } -N- [5- ({4- [ (5-aminopentyl) (hydroxy) amino group]-4-oxobutanoyl } amino) pentyl]-N-hydroxysuccinamide (CAS reg.no. 70-51-9); also known as Deferoxamine (Deferoxamine), Deferoxamine B (Deferoxamine B), DFO-B, DFOA, DFB or desferal (desferal) is a bacterial siderophore produced by Streptomyces pilosus of the phylum actinomycetemcomitans (Streptomyces pilosus) (top of fig. 20). Desferrioxamine B has medical applications, for example as a chelator for removing excess iron in the body (Miller, Marvin J. "Synthesis and therapeutic potential of Hydroxamic acid based minerals and analytes" (1989) Chemical Reviews 89(7): 1563-. The mesylate salt of DFO-B is commercially available. The initial experiment used N- (S-acetyl) thioacetyl-Df (SATA-Df) and mAb, 4- [ N-maleimidomethyl ] modified with a maleimido group attached to the amino group of the lysine side chain]Cyclohexane-1-carboxylate (mAb-SMCC) was performed (Meijs WE et al, "Zirconium-lamellar monoclonal antibodies and the arrangement in vivo-bearing nuclear microorganism" (1997) J.Nucl. Med.38:112-8; Meijs WE et al, "A facial method for the labeling of proteins with Zirconium isomers" (1996) Nucl. Med. biol.23: 439-48). However, the resulting thioether conjugate (mAb-SMCC-SATA-Df) is unstable in human serum at 37 ℃ (Verel I et al "89 Zrimmno-PET: complex process for the production of89Zr-labeled monoclonal antibodies" (2003) J Nucl Med 44: 1271-81). Another exemplary amino-reactive bifunctional chelating agent based on Df modified with succinic anhydride (Suc) was used to convert the amino group of Df to a carboxylic acid and then activated to 2,3,5, 6-tetrafluorophenyl ester (TFP). TFP-N-Suc-Df (middle in FIG. 20) was linked to the lysine-amino group of mAb and purified mAb-N-Suc-Df was linked to⁸⁹Zr is chelated. Obtained⁸⁹Zr-mAb-N-Suc-Df was stable under physiological conditions and compared its biodistribution in mice with mAb-SMCC-SATA-Df (Verel I et al "89 znimuno-PET:(2003) J Nucl Med.44: 1271-81). However, the preparation of TFP-N-Suc-Df requires protection of the hydroxyamide group in the form of an Fe (III) complex. In and with⁸⁹Removal of iron by treatment with EDTA before Zr chelation, but the multi-step process is too tedious and carries the risk of incomplete removal of iron from the deferoxamine and/or incomplete removal of EDTA from the conjugation buffer, which may negatively affect⁸⁹Yield of Zr-chelation. Thus, a heterobifunctional amino-reactive reagent, p-isothiocyanatobenzene-deferoxamine (Df-Bz-NCS), which incorporates Df into proteins via thiourea linkages, was recently developed, FIG. 20, in the middle (Perk LR et al, "factory Radioactive Of monoclonal antibodies And other proteins with quaternary ammonium-89 or gallium-68for PET Imaging using p-isothiocyanatocyanzyl-desferrioxamine" (2008) Nature Protocols, published on-line: DOI:10.1038/nprot.2008.22; Perk LR et al, "p-isothiocyanatocyanzyl-desferrioxamine: a new biochemical ligand binding Of monoclonal antibodies-biological samples-PET 2009). Antibody conjugates prepared using Df-Bz-NCS showed comparable stability and imaging performance to reference conjugates prepared using TFP-N-Suc-Df. Since the development of a peptide via lysine-amino group⁸⁹Reliable method for Zr binding to antibodies, use⁸⁹The number of reported preclinical and clinical immuno-PET studies for Zr-labeled antibodies has increased rapidly (Verel I, et al "Long-driven position antibodies zirconia-89 and iodine-124for screening of the underlying radioactive conjugates with PET" (2003) Cancer biotherapeutic.18: 655-61; Nagengate WB, et al "In vivo VEGF imaging with radioactive nanoparticles In human underlying ligand magnetic ligand graft" (2007) J Nucl Med.48:1313-9; Perk LR, et al "(89) Zr as PET surface ligand for screening of the reactive conjugates of the therapeutic antigens with PET surface ligand J (90) Y (177) nuclear binding protein J-1 ligand J.8; Q-expressing binding of the therapeutic conjugates of the therapeutic antigens with Met ligand J.906: 8; Q-1 binding protein J.8; Q.8. for screening of the therapeutic conjugates of the non-grafted antibodies (90) and the reagent J.Environmental impact DN30 "(2008) European Journal Of nucleic acid And Molecular Imaging35:1857-67; Perk LR et al" Preparation And evaluation Of (89) Zr-Zevalin for monitoring Of (90) Y-Zevalin biological monitoring with Molecular Imaging "(2006) European Journal Of nucleic acid And Molecular Imaging 33:1337-45; Borj. PK et al" Performance Of analysis-Molecular Imaging with Molecular Imaging 2006-latex Of nucleic acid And Molecular Imaging U6389-latex Imaging 2009: EGFR J-3 "(2009: EGFR-3) (US 2009-3: EGFR J-3): 974-981).

Embodiments of zirconium complexes also include zirconium binding (chelating) ligands such as DTPA (CAS Reg. No.67-43-6), DOPA (1,4,7, 10-Tetraazathioprine cyclododecane-1, 4,7, 10-tetraacetic acid) (Liu, Shuang (2008) advanced drug Delivery Reviews 60(12): 1347-.

Zirconium complexes (Z) and other radionuclides can be conjugated to antibodies (Ab), including monoclonal antibodies (mAb), through the-amino group in the lysine side chain or the thiol group of cysteine. Since there are approximately 40 lysine side chains in the mAb (Wang L et al, "Structural conjugation of the monoclonal antibody, hu N901-DM1, by mass spectrometry" (2005) Protein Sci.14:2436-46) or 8 cysteines (Hamblett KJ et al, "Effects of drug delivery on the antibody activity of a monoclonal antibody drug conjugate" (2004) Clin cancer Res.10:7063-70) available for conjugation, both methods provide heterogeneity in terms of mAb conjugation ratio and conjugation site. Modification of a lysine residue within the binding site may reduce the biological activity of the conjugate (Cai W et al "PET imaging of the biological cancer in xenogenic-bearing microorganism by use of an 18F-labeledt84.66anti-pathological-biological antibody" (2007) J Nucl Med.48:304-10; Shiity JE. "18F labeling for imaging-PET: where we space and conjugate media" (2007) J Nucl Med.48:170-2; Taiit JF et al "imaging detection of cell surface in vivo with a New biological sample by site 2004-specific methods" (Sch) J Nucl.47: 1546-53; engineering EA et al "imaging of the biological cancer with a New biological sample V radial ligand by use of a biological sample J" (2003) biological activity of the biological cancer in blood sample J.187. biological antibody J.5) (which this modification of the biological cancer biological antibody J.5) results in a reduction of the biological half-life of the biological cancer protein of the biological cancer sample J. Clin Cancer Res.10: 7063-70). These limitations can be avoided by using a biochemical assay for rapid identification of preferred amino acids for cysteine mutation in antibodies, PHESELECTOR (US 7521541; Junutula JR et al, "Rapid identification of reactive cysteine residues for site-specific labeling of antibodies" J immunological Methods 2008;332:41-52), using mAbs containing selectively placed cysteines engineered for site-specific conjugation purposes. The resulting antibody (ThiomaB) is then chemoselectively and Site-specifically conjugated to a cytotoxic drug without loss of binding affinity and without adverse effect on antibody scaffold stability (Junutula JR et al, "Site-specific conjugation of an extracellular drug to an antibody improves the therapeutic index" (2008) Natbiotechnol.26: 925-32).

From an imaging perspective, high target affinity and minimal non-specific uptake are required to obtain optimal image quality. Thus, a site-specific radiolabeled cysteine engineered antibody (THIOMAB) can provide a tracer with invariant binding affinity and scaffold stability, which can minimize non-specific uptake of metabolites outside of the target tissue. One aspect of the present invention is the use of the novel Df-based thiol-reactive bifunctional reagent maleimide cyclohexyldesferrioxamine (Df-Chx-Mal), bromoacetyl-acetylMethods for site-specific radiolabeling of Thiomab with deferoxamine (Df-Bac) and iodoacetyldeferoxamine (Df-Iac) (FIG. 20). Exemplary embodiments include those wherein the agents are site-specifically conjugated to trastuzumab Thiomab (thio trastuzumab)⁸⁹Zr sequestered and evaluated in vitro and in vivo.

A metastable isoenerogen of zirconium is⁸⁹Zr, with a half-life of 78.4 hours, has a decay pattern of β (electron emission), positrons (β +) and gamma radiation.

Radioisotopes or other labels can be incorporated into the conjugate in known manner (Fraker et al (1978) biochem. Biophys. Res. Commun.80:49-57; Monoclonal Antibodies in immunoscintigraphy "Chatal, CRC Press 1989). Carbon-14 labeled 1-isothiocyanatobenzene-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelator for conjugating radionuclides to antibodies (WO 94/11026).

Joint

A "linker" (L) is a bifunctional or multifunctional moiety that can be used to link one or more zirconium complex moieties (Z) and an antibody unit (Ab) to form an antibody-zirconium conjugate of general formula I (AZC). Antibody-zirconium conjugates (AZC) can be conveniently prepared using a linker having reactive functional groups that bind zirconium and bind antibodies. The cysteine thiol of the cysteine engineered antibody (Ab) may form a bond with a functional group of a linker reagent, a zirconium label moiety, or a zirconium-linker intermediate.

In one aspect, the linker has a reactive site with an electrophilic group that reacts with a nucleophilic cysteine present on the antibody. The cysteine thiol of the antibody reacts with an electrophilic group on the linker and forms a covalent bond with the linker. Useful electrophilic groups include, but are not limited to, maleimide and haloacetamide groups.

The cysteine engineered antibody may be reacted with a linker reagent or zirconium-linker intermediate, with an electrophilic functional group such as maleimide or alpha-halocarbonyl according to the conjugation method of Klussman, et al (2004), Bioconjugate Chemistry 15(4) p 766 of 765-773 and the protocol according to examples 17-19.

In another embodiment, the Z moieties are the same.

In another embodiment, the Z moieties are different.

Exemplary embodiments of antibody-zirconium conjugated (AZC) compounds of formula I include:

wherein X is:

y is:

r is independently H or C₁-C₆An alkyl group; n is 1 to 12.

In another embodiment, the linker has a reactive functional group having a nucleophilic group that reacts with an electrophilic group present on the antibody. Electrophilic groups on antibodies that may be used include, but are not limited to, the carbonyl groups of aldehydes and ketones. The heteroatoms of the nucleophilic group of the linker can react with electrophilic groups on the antibody and form covalent bonds with antibody units. Useful nucleophilic groups on the linker include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazide, hydrazine carboxylate, and aroylhydrazide. Electrophilic groups on the antibody provide convenient sites for attachment to linkers.

In another embodiment, the linker may be substituted with groups that modulate solubility or reactivity. For example, charged substituents such as sulfonate (-SO)₃ ^-) Or ammonium may increase the water solubility of the reagent and facilitate the linking reaction of the linker reagent to the antibody or zirconium moiety, or the linking reaction of Ab-L (antibody-linker intermediate) to Z, or Z-L (zirconium-linker intermediate) to Ab (depending on the synthetic route used to make AZC).

The compounds of the present invention specifically contemplate, but are not limited to, AZC: 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 (succinimide- (4-vinylsulfone) benzoate), and including bismaleimide reagents: DTME, BMB, BMDB, BMH, BMOE, BM (PEO)₂And BM (PEO)₄₃These reagents are commercially available from Pierce Biotechnology, inc, Customer Service Department, p.o. box 117, Rockford, il.61105u.s.a. The bismaleimide reagent allows the thiol group of the cysteine engineered antibody to be attached to the zirconium moiety comprising a thiol, a label, or a linker intermediate in a sequential or simultaneous fashion. Other functional groups besides maleimide that react with the thiol group, zirconium moiety, label or linker intermediate of the cysteine engineered antibody include iodoacetamide, bromoacetamide, vinylpyridine, disulfide, pyridyldisulfide, isocyanate and isothiocyanate.

Useful linker reagents are available from other commercial sources, such as Molecular Biosciences Inc. (Boulder, CO), or synthesized according to the 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; US6214345; WO 02/088172; US 2003130189; US2003096743; WO 03/026577; WO 03/043583; and WO 04/032828.

Exemplary linker reagents include:

wherein n is an integer ranging from 1 to 10 and T is-H or-SO₃Na；

Wherein n is an integer ranging from 0 to 3;

in another embodiment, the linker L may be a dendritic linker for covalently linking more than one zirconium moiety to an antibody via a branched multifunctional linker moiety (Sun et al (2002) Bioorganic & Medicinal Chemistry letters 12: 2213-. The dendritic linker can increase the molar ratio of zirconium to antibody, i.e. AZC loading. Thus, when the cysteine engineered antibody has only one reactive cysteine thiol group, multiple zirconium moieties can be linked by a dendritic linker.

The following exemplary embodiments of the dendritic linker reagent allow conjugation of up to 9 nucleophilic zirconium moiety reagents by reaction with the chloroethyl mustard functional group:

other embodiments of branched dendritic linkers include linkers with self-depleting (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-.

Desferrioxamine-labelled cysteine engineered antibodies

One 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 desferrioxamine moiety (Df), having the general formula II:

Ab-(L-Df)p II

wherein L-Df is selected from:

wherein the wavy line indicates attachment to the antibody (Ab); and

p is 1 to 4;

preparation of antibody-zirconium conjugates

Antibody-zirconium conjugates of general formula I (AZC) can be prepared by several routes, using organic chemical reactions, conditions and reagents known to those skilled in the art, including: (1) reaction of the cysteine group of the cysteine engineered antibody with a linker reagent to form an antibody-linker intermediate Ab-L by covalent bond, followed by reaction with an activated zirconium label moiety Z; and (2) reaction of the nucleophilic group of the zirconium moiety with a linker reagent to form a zirconium label-linker intermediate by covalent bond, followed by reaction with the cysteine group of the cysteine engineered antibody. Conjugation methods (1) and (2) can be used with a variety of cysteine engineered antibodies, zirconium label moieties, and linkers to prepare antibody-zirconium conjugates of general formula I.

The antibody cysteine thiol group is nucleophilic and capable of reacting with electrophilic groups on the linker reagent and zirconium-linker intermediate to form a covalent bond, said electrophilic groups comprising: (i) active esters, such as NHS esters, HOBt esters, haloformates and acid halides; (ii) alkyl and benzyl halides, such as halogenated acetamides; (iii) aldehydes, ketones, carboxyl and maleimide groups; and (iv) disulfides, including pyridine disulfide, by sulfide exchange. Nucleophilic groups on the zirconium label moiety include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arohydrazide groups capable of reacting with electrophilic groups on linker moieties and linker reagents to form covalent bonds.

Cysteine engineered antibodies can be rendered reactive for conjugation to linker reagents by treatment with reducing agents such as DTT (Cleland reagent, dithiothreitol) or TCEP (tris (2-carboxyethyl) hydrophosphate; Getz et al (1999) anal. biochem. Vol 273:73-80; SoltecVents, Beverly, Mass.) under specific conditions. The full-length, cysteine engineered monoclonal antibody (ThioMab) expressed in CHO cells was reduced with about a 50-fold excess of TCEP at 37 ℃ for 3 hours to reduce disulfide bonds that may have formed between newly introduced cysteine residues and cysteines present in the media. The reduced ThioMab was diluted and loaded onto a HiTrap S column at 10mM sodium acetate, pH 5and eluted with PBS containing 0.3M sodium chloride. Diluted (200nM) aqueous copper sulfate (CuSO) at room temperature₄) Overnight, the double bonds between cysteine residues present in the parent Mab were reestablished. Other oxidizing agents known in the art, i.e., oxidizing agents and oxidizing conditions, can be used. Ambient air oxidation is also effective. This mild partial reoxidation step is effective in forming intrachain disulfides with high fidelity. An about 10-fold excess of zirconium-linker intermediate was added, mixed and left at room temperature for about 1 hour for conjugation to occur and for the formation of ThioMab antibody-zirconium conjugates. The conjugate mixture was gel filtered, then loaded onto a HiTrap S column and eluted to remove excess zirconium-linker intermediate and other impurities.

Figure 15 shows a general process for preparing cysteine engineered antibodies for conjugated cell culture expression. Cysteine adducts, presumably reduced cleaved with multiple interchain disulfide bonds to provide reduced forms of the antibodies. Interchain disulfide bonds between paired cysteine residues are reformed under conditions of partial oxidation, such as exposure to ambient oxygen. The newly introduced, engineered and unpaired cysteine residues retain the ability to react with a linker reagent or zirconium-linker intermediate to form an antibody conjugate of the invention. ThioMab expressed in mammalian cell lines results in an externally conjugated Cys adduct formed with an engineered Cys through an-S-S-bond. The purified ThioMab must therefore be treated with reduction and oxidation procedures as described in example 11 to produce a reactive ThioMab. These thiomabs are used for conjugation with maleimide-containing radiolabels, cytotoxic drugs, fluorophores, and other labels.

⁸⁹Preparation and analysis of Zr-Df-trastuzumab conjugate

The protected activated ester TFP-N-SucDf-Fe was prepared according to The procedure described previously (Vere lI et al "89 Zr Immuno-PET: comprehensive procedures For The Production Of89 Zr-laboratory Monoclonal Antibodies" (2003) JNucl Med 44:1271-81) and conjugated to trastuzumab using a 5-fold molar excess Of TFP-N-SucDf-Fe to give N-SucDf-trastuzumab with an average Of 1.6 molecules Of deferoxamine (Table 8). Df-Bz-SCN-trastuzumab (Perk LR, et al, "simple radio labeling of monoclonal antibodies and other proteins with zirconium-89or gallium-68for PET Imaging using p-isothiocyanato zyl-desferrioxamine" (2008) Nature protocols; published oncoline: DOI:10.1038/nprot.2008.22) was obtained by ligation to Df-Bz-SCN in 8-fold molar excess at pH 8.5. The reaction provided Df-Bz-SCN-trastuzumab modified with an average of 2.4 molecules of deferoxamine (table 1).

A novel maleimide-based thiol-reactive bifunctional linker Df-Chx-Mal was prepared from equimolar amounts of deferoxamine mesylate and SMCC (FIG. 21, example 13). The reaction was completed within 30 minutes at room temperature and the product was isolated by precipitation after addition of water to give a yield of 45% and a purity of greater than 95%. Reaction of a 8.5-fold molar excess of Df-Chx-Mal with freshly prepared thio-trastuzumab (fig. 21, example 17) provided Df-Chx-Mal-thio-trastuzumab conjugated with exactly 2 molecules of deferoxamine within 1 hour (table 1, fig. 21). Bromoacetyldeferoxamine (BDf-Bac) was prepared by reacting equimolar amounts of deferoxamine mesylate and bromoacetyl bromide at 0 deg.C (example 14). The product was obtained in 14% yield after HPLC purification. Alkylation of freshly prepared thio-trastuzumab with a 12-fold molar excess of Df-Bac provided a conjugate of 1.8 molecules of Df (Df-Ac-thio-trastuzumab) per antibody within 5 hours (table 8, fig. 21, example 18). The low reactivity of bromide prompted us to try the more reactive iodoacetyl derivative (Df-Iac). Df-Iac was prepared by reaction of deferoxamine mesylate with a slight excess of iodoacetic acid N-hydroxysuccinimide ester in 53% yield (fig. 21, example 15). The product was obtained in greater than 95% purity by precipitation from the reaction mixture. The subsequent reaction of 11-fold excess of Df-Iac provided Df-Ac-thio-trastuzumab with 1.8 molecules of Df modification within 2 hours (table 1, fig. 21, example 19). Based on our experience, Df-Chx-Mal is the preferred agent among the 3 compounds studied. In particular, the Df-Chx-Mal reaction is completed at a mild pH and within 1 hour, in contrast to the higher pH and longer reaction time required for the alkylation of thiol groups using Df-Bac and Df-Iac. In addition, the low reactivity of the haloacetamides may have resulted in incomplete loading of the 2 available cysteines of thio-trastuzumab.

TABLE 8 reaction conditions and yields of Df-linker-trastuzumab conjugates prepared using different reagents

Using the experimental procedure described previously will⁸⁹Zr chelated with all 4 Df-trastuzumab variants as 89-zirconium oxalate (Verel I et al, "89 Zr immuno-PET: complex procedure for the production of89Zr-labeled monoclonal antibodies" (2003) J Nucl Med.44: 1271-81). The radiolabeled protein was purified on a desalting column and the final solution was filtered through a membraneConcentrate to the desired volume. In Table 9, summarize⁸⁹Yield, purity and final specific activity of Zr conjugates. In general, the chelation yield was greater than 80%, except for the Df-N-Suc linker obtained in lower yields, which may be due to lower Df amount per antibody, and/or incomplete removal of fe (iii) used to protect the chelator during activation and conjugation. After purification of the Df-trastuzumab variant using a desalting column, the product purity was greater than 90% with small amounts (1-6%) of high molecular weight aggregates detected in each sample. Df-Chx-Mal-thi o-trastuzumab provided 99% purity⁸⁹Zr complexes (table 9), in contrast, the Df-Ac conjugates had contamination of about 8% low molecular weight impurities and 2% high molecular weight aggregates. The contaminants were not removed using the NAP-10 column, but were removed by repeated buffer exchanges on an Amicon filter.

TABLE 9 radiolabelling⁸⁹Yield, specific activity and purity of Zr-Df-linker-trastuzumab

⁸⁹Biological Activity of Zr-Df-trastuzumab conjugates

The biological activity of the newly prepared site-specific Df-linker-thio-trastuzumab conjugate was determined by Scatchard analysis using a binding assay with the BT474 breast cancer cell line. The obtained K_DValues were compared to unmodified trastuzumab (0.91 ± 0.20 nM). K of thio-trastuzumab conjugates comprising Chx-Mal linker_DIs 0.93 + -0.15 nM, and the values for the conjugate containing the Ac linker are 1.22 + -0.22 nM (conjugate prepared using Df-Bac) and 0.87 + -0.15 nM (prepared using Df-Iac). The results of the biological activity assay indicate that the modification of thio-trastuzumab did not affect the binding affinity of the antibody to HER 2.

In vitro serum stability

Having previously reported bonds containing amide or thioureaDf-antibody conjugates Of N-Suc and Bz-SCN linkers are stable in vitro (in serum at 37 ℃ for more than 6 days) (Verel I et al "89 Zrimmno-PET: comprehensive procedures for the production Of89 Zr-layered monoclonal antibodies" J NuclMed 2003;44:1271-81; Perk LR et al (2009) European Journal Of nucleic acids and molecular Imaging35 (1857) 1867). Having Chx-Mal and Ac linkers was determined in mouse serum at 37 deg.C⁸⁹Stability of the Zr-thio-trastuzumab conjugate. No significant antibody binding was observed over a5 day period⁸⁹Loss of Zr. Both thio conjugates are stable, antibody-binding⁸⁹The average loss of Zr is 1.8% (R) ((R))⁸⁹Zr-Df-Chx-Mal-thio-trastuzumab) and 1.4% ((1.4%) per day⁸⁹Zr-Df-Ac-thio-trastuzumab) (fig. 24). Slow formation of high molecular weight species (possibly aggregates) was observed in both the Df-Chx-Mal and Df-Ac linkers.

In vivo microscopic PET imaging

For 20 xenografts with subcutaneous BT474M1 (size 200 mm)³) Animals (5 animals per group) were injected intravenously⁸⁹Zr-trastuzumab. The amount of antibody injected per animal was 1.4. + -. 0.29 mg/kg. The maximum intensity shadowgraph image (96 hours post injection) representative of the animal is shown in figure 3. In FIG. 4, the results in selected tissues are summarized⁸⁹Zr-trastuzumab uptake. The image at1 hour (not shown) was occupied by a hypercoagulant uptake, with the exception of Df-Bac, where rapid hepatobiliary excretion of lipophilic impurities resulted in increased uptake in the intestine. The impurities were completely cleared within the first 24 hours and no increased small and large intestinal uptake was detected at 24 hours or later after tracer injection. Although the tissue uptake of the Df-Ac conjugate was thus slightly reduced (-8%), the tumor to blood ratio (table 10) was not affected by the loss of radioactivity injected. Images at 96 hours were occupied by high tumor uptake, at 4 different sites⁸⁹Minor differences were observed in the Zr-trastuzumab variant (fig. 25). Tumor uptake was identical for each tracer, reaching a maximum at 24 hours post-injection and a maximum at 144 hoursTumor blood ratio (due to blood clearance) (table 10). Thiol-based conjugates compared to amine-based conjugates (Df-Bz-SCN and Df-N-Suc) at 96 and 144 hours post-injection⁸⁹Zr-Df-Chx-Mal-thio-trastuzumab exhibits increased bone uptake (P)<0.05). Bone uptake of Df-Ac-thio-trastuzumab was not significantly improved (P =0.20) compared to Df-Bz-SCN and Df-N-Suc linker, but could become significant when correcting for 8% of the radioactivity loss in the first 24 hours. As expected, the renal uptake of each tracer was low because of the antibody-based tracer (fig. 26), but⁸⁹Zr-Df-Chx-Mal-thio-trastuzumab was slightly higher (P) at 24, 96 and 144 hours than other linkers<0.05）。

TABLE 10 mean tumor blood ratios at 24, 96 and 144 hours post injection

BT474 (HER 2 expression level 3 +) xenografts showed lower absolute uptake of tracer (15% ID/g) than Dijkers et al measured in SKOV3 (HER 2 expression level 3 +) (Dijkers EC, et al "Development and Characterization of clinical-Grade 89Zr-Trastuzumab for HER2/neu ImmunoPET Imaging" (2009) J NuclMed 50(6): 974. sup. 981). However, the tumor blood ratios of 5.7-7.1 (Table 10) were comparable to the value obtained in SKOV3 (tumor blood ratio of 7.6). The difference in tumor uptake may be due to the tumor model and the total dose of trastuzumab. Materials with higher specific activities were used, and therefore significantly less antibody (35 μ g,1.4mg/kg) was injected than studies using SKOV3 (100 μ g,4mg/kg) by Dijkers et al. Differences in specific activity may also result in lower free of 2-3% ID/g in the experiments herein compared to the SKOV3 model (5-10% ID/g)⁸⁹Bone uptake of Zr. Unfortunately, Dijkers et al do not provide relevant improvementsThe bone uptake of (1). Zirconium binding plasma proteins are known (Mealey J, Jr. "Turn-over of carrier-free zirconia-89 in man" (1957) Nature 179:673-4 and then deposited in mineral bone (Fletcher CR. "The radio local scaffolds of zirconia-95 and niobium-95" (1969) Health Phys.16:209-20; Shiraishi Y and dIchikawa R. "adsorption and coverage of 144Ce and 95Zr-95Nb in newborn, jeven and adult. rates" (1972) Health Phys.22:373-8) because The injected material does not contain free plasma proteins⁸⁹Zr, bone uptake may be derived from⁸⁹Degradation of Zr-antibodies or non-specific attachment to antibodies⁸⁹Zr, in comparison with Df⁸⁹Zr, said free⁸⁹Zr can then be transchelated with plasma proteins.

Exemplified herein are 3 thiol-specific reagents for conjugating deferoxamine (Df) to monoclonal antibodies through thiol chemistry of the cysteine engineered antibody. Thiol-specific Df reagents of 14% (Df-Bac),53% (Df-Iac) and 45% (Df-Chx-Ma l) yield were obtained by aminoacylation of desferrioxamine B and conjugated to thio-trastuzumab to obtain site-specific modifications on 2 engineered cysteines in 1-5 hours. The binding activity of the site-specific thio-trastuzumab conjugate to HER2 was identical to that of unmodified trastuzumab. Df-modified thio-trastuzumab (Df-Ac-thio-trastuzumab and Df-Chx-Mal-thio-trastuzumab) with⁸⁹Zr chelation (FIG. 22) gave yields of greater than 80% in 1 hour, which is comparable to lysine conjugates prepared using the Df-Bz-SCN and Df-N-Suc linkers described previously.⁸⁹Zr-Df-Ac-thio-trastuzumab and⁸⁹both Zr-Df-Chx-Mal-thio-trastuzumab showed comparable stability in mouse sera. 2 compounds also showed PET imaging capabilities comparable to lysine conjugates in the BT474M1 breast cancer model, reaching tumor uptake of 10-15% ID/g and tumor blood ratios in the range of 6.1-7.1. Overall, the new reagents are readily available, show good reactivity with thiol groups of proteins, and demonstrate reactivity with thiol groups of proteins⁸⁹Very good chelating properties of Zr.⁸⁹The Zr-labeled antibody was stable in serum and showedExcellent PET imaging performance. Df-Chx-Mal is an effective agent for conjugating Df to antibodies through cysteine side chains, and shows several advantages over Df-Bac and Df-Iac. First, full conjugation of Df-Chx-Mal within 1 hour required a neutral pH of 7.5, in contrast to Df-Bac and Df-Iac which required pH 9and 2 or 5 hours. In addition, site specificity⁸⁹The Zr-labeled engineered Thiomab conjugates can be as¹⁸F-labeled ThiOMAB conjugates (Gill HS, et al, "A modular platform for the rapid site-specific radio Radioactive testing of proteins with 18F explicit by quantitative positive examination of the tomogry of human epidermal growth factor receiver 2" (2009) journal.of Med.chem.52:5816-25) are similarly useful as valuable tools for PET imaging applications in biomedical research.

Administration of antibody-zirconium conjugates

The antibody-zirconium conjugate (AZC) of the invention may be administered by any route suitable for the condition to be treated. AZC will generally be administered parenterally, i.e. infusion, subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural.

Pharmaceutical preparation

Pharmaceutical formulations of the diagnostic antibody-zirconium conjugates (AZC) of the invention are generally prepared for parenteral administration, i.e. by bolus injection, intravenous, intratumoral injection with a pharmaceutically acceptable parenteral carrier, and in a unit dose injectable form. The antibody-zirconium conjugate (AZC) of the desired purity is optionally mixed with pharmaceutically acceptable diluents, carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences (1980) 16 th edition, Osol, a.

Useful diluents, carriers, excipients, and stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphoric acid, citric acid, and other organic acids; antioxidants include ascorbic acid and methionine; preservatives (e.g. octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkylbenzylsAcid esters such as methyl or propyl benzoate; 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, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants such as TWEEN^TM,PLURONICS^TMOr polyethylene glycol (PEG). For example, lyophilized anti-ErbB 2antibody formulations are described in WO97/04801, which is expressly incorporated herein by reference.

The active pharmaceutical ingredient may also be encapsulated (entrap) in microcapsules prepared, for example by coacervation techniques or by interfacial polymerization, for example hydroxymethylcellulose or gelatin-and polymethylmethacrylate microcapsules, respectively, in colloidal drug delivery systems (for example liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions (macroemulsions). Such techniques are disclosed in Remington's Pharmaceutical Sciences 16 th edition, Osol, A. eds (1980).

Can be prepared into sustained release preparation. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers comprising AZC, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g. poly-2-hydroxymethyl methacrylate), or polyvinyl alcohol), polylactide (US 3773919), copolymers of L-glutamic acid and λ -ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT^TM(injectable microspheres consisting of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly D- (-) -3-hydroxybutyric acid.

Formulations for in vivo administration must be sterile, which can be readily accomplished by filtration through sterile filtration membranes.

The formulations include those suitable for the above routes of administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Related techniques and formulations are generally found in Remington's Pharmaceutical Sciences (Mack Publishing co., Easton, PA). Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. Formulations are generally prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Aqueous suspensions of the invention contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents, for example sodium carboxymethylcellulose, croscarmellose, povidone, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth and acacia, and dispersing or wetting agents, for example naturally-occurring phosphatides (for example lecithin), condensation products of an alkylene oxide and a fatty acid (for example stearic acid polyoxyethylene), condensation products of ethylene oxide and a long chain aliphatic alcohol (for example heptadecaethylene oxycetanol), condensation products of ethylene oxide and a partial ester derived from a fatty acid and a hexitol anhydride (for example polyoxyethylene sorbitan monooleate). Aqueous suspensions may also contain one or more preservatives, for example ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

Pharmaceutical compositions of AZC may be in the form of sterile injectable preparations, for example sterile injectable aqueous or oleaginous suspensions. Such suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents as described above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1, 3-butanediol, or as a lyophilized powder. Among the useful carriers and solvents that may be employed are water, ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils may be conventionally employed as a solvent or suspending medium. Any bland fixed oil may be employed for such purpose including synthetic mono-or diglycerides. In addition, fatty acids, such as oleic acid, may likewise be used in the preparation of injectables.

The amount of active ingredient combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, an aqueous solution intended for intravenous infusion may contain 3-500 μ g of active ingredient per mL of solution so that an appropriate volume of infusion at a rate of about 30 mL/hour may be made.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may contain suspending agents and thickening agents.

Although oral administration of protein therapy is disfavored due to hydrolysis or denaturation in the intestine, formulations of AZC suitable for oral administration, for example capsules, cachets or tablets, each containing a predetermined amount of AZC, may be prepared in discrete units.

The formulations may be packaged in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind described above. Preferred unit dose formulations are those containing a daily dose or unit daily sub-dose, as set out herein above, or a suitable fraction thereof, of the active ingredient.

The invention also provides a veterinary composition comprising at least one active ingredient as defined above and a corresponding veterinary carrier. Veterinary carriers are materials that are effective for the purpose of administering the composition and can be solid, liquid, or gaseous materials that are inert or useful in the veterinary art and are compatible with the active ingredient. These veterinary compositions may be administered parenterally, orally or by other desired routes.

Method for imaging labeled antibody

In another embodiment of the invention, the cysteine engineered antibody may be labeled with a radionuclide, a fluorescent dye, a bioluminescent trigger substrate moiety, a chemiluminescent trigger substrate moiety, an enzyme and other detection markers for imaging experiments with diagnostic, pharmacodynamic and therapeutic applications via cysteine thiol. In general, labeled cysteine engineered antibodies, i.e., "biomarkers" or "probes," are administered by injection, perfusion, or oral ingestion to perfused organ or tissue samples of living organisms, such as humans, rodents, or other small animals. The distribution of the probes is detected over a period of time and presented by an image.

Examples

Unless otherwise stated, solvent and chemical formulations were purchased from Aldrich (Milwaukee, Wis.) the products were analyzed and purified using a reverse phase HPLC system, System A: Phenomenex BioSep-SEC-S3000 (300 × 4.60.60 mM,5 μm), 50mM PBS 0.5 mL/min, equipped with ultraviolet light absorption and radioactivity detector (PMT), System B: Altima C-18(100 × 22.0.0 mM,5 μm)0.05% TFA +10-50% acetonitrile, 0-30 min, 24 mL/min, equipped with an ultraviolet light detector₁₈Mass spectrometry analysis of low molecular weight products was performed on PE Sciex API 150EX LCMS system of columns. LCMS analysis of proteins was performed on a TSQ quantum triple quadrupole mass spectrometer (Thermo Electron, Thermo fisher scientific inc., USA) with an extended mass range. The protein samples for LCMS analysis were reduced by treatment with 20mM Dithiothreitol (DTT) at 37 ℃ for 1 hour to separate the heavy and light chains. PRLP-S1000 at 75 ℃ heatingMicro inner diameter column (50mm × 2.1)mm, Polymer Laboratories, Varian Inc., USA). A linear gradient from 30-40% B (solvent A, 0.05% TFA in water; solvent B, 0.04% TFA in acetonitrile) was used and the eluent was directly ionized using an electrospray source. Data were collected by the Xcalibur data system and deconvoluted (deconvolution) using promas software (Novatia, Monmouth Junction, NJ). NMR spectra were recorded at 298K on a Bruker Avance II400 spectrometer and chemical shifts relative to TMS were reported. The protein concentration was measured at 280nm using an Eppendorf Biophotometer (Westbury, NY). With oxalic acid in a 1M oxalic acid solution⁸⁹Forms of Zr (IV) obtained from the central organization of cancer by biological Sloan-Kettering (New York, NY) with a specific activity of 470-1195Ci/mmol⁸⁹Zr (Holland JP, et al (2009) "stabilized methods for the production of high specificity zirconium-89" Nuclear Med biol.36: 729-39). Heterobifunctional linker succinimide 4- [ N-maleimidomethyl ] purchased from Pierce (Rockford, IL)]Cyclohexane-1-carboxylate (SMCC), N-hydroxysuccinimide iodoacetate, was purchased from Indofine Chemical Company (Hillsborough, N.J.). NAP-10 column is available from (GEHealthcare, USA), and Amicon Ultra-4 centrifugal filter (10,000MWCO) is available from Millipore (Billerica, MA). Df-Bz-SCN was purchased from Macrocyclics (Dallas, TX).

Example 1Preparation of biotinylated ThioFab phage

ThioFab-phage (5X 10)¹²Phage particles) was reacted with a 150-fold excess of biotin-PEO-maleimide ((+) -biotin-3-maleimidopropionamidine-3, 6-dioxaoctanediamine, Oda et al (2001) Nature Biotechnology 19:379-382, Pierce Biotechnology, Inc.) at room temperature for 3 hours. Excess biotin-PEO-maleimide was removed from the biotin-conjugated phage by repeated PEG precipitation (3-4 times). Other commercially available biotinylation reagents having electrophilic groups that react with cysteine thiol groups can be used, including biotin-BMCC, PEO-iodoacetylbiotin, iodoacetyl-LC-biotin, and biotin-HPDP (Pierce Biotechnology, Inc.), and N^α- (3-maleimidopropionyl)Biocytin (MPB, Molecular Probes, Eugene, OR). Other commercial sources of biotinylated, bifunctional, and multifunctional linker reagents include Molecular Probes, Eugene, OR, and Sigma, st.

Biotin-PEO-Maleimide

Example 2PHESELECTOR assay

Bovine Serum Albumin (BSA), erbB2 extracellular domain (HER 2) and streptavidin (2. mu.g/ml, 100. mu.l) were coated onto Maxi sorp 96-well plates, respectively. After using 0.5% Tween-20

After blocking (in PBS), the cells were incubated with biotinylated and non-biotinylated hu4D5Fabv 8-ThioFab-phage (2 x1010 phage particles) for 1 hour at room temperature, followed by incubation with horseradish peroxidase (HRP) -labeled secondary antibodies (anti-M13 phage coat protein, pVIII protein antibody). FIG. 8 illustrates schematically the PHESELECTOR assay, which describes the binding of Fab or ThioFab to HER2 (top) and the binding of biotinylated ThioFab to streptavidin (bottom).

A standard HRP reaction was performed and the absorbance measured at 450 nm. By calculating the OD of streptavidin₄₅₀And OD of HER2₄₅₀The ratio of (a) to (b) measures the thiol reactivity. A thiol reactivity value of 1 indicates complete biotinylation of the cysteine thiol. In the case of Fab protein binding measurements, hu4D5Fabv8(2-20ng) was used, followed by incubation with HRP-labeled goat polyclonal anti-Fab antibody.

Example 3aExpression and purification of ThioFab

After induction, in the non-inhibitory E.coli strain 34B8(Baca et al (1997) Journal BiologicalChemi)string 272(16) 10678-84) express ThioFab. Resuspending the harvested cell pellet in PBS (phosphate buffered saline), performing whole cell lysis by passing through a microfluidizer, and using the protein GSEPHAROSE^TMAffinity chromatography of (Amersham) purified ThioFab.

Express ThioFab L-V15C, L-V110C, H-A88C, and H-A121C and pass through the protein GSEPHAROSE^TMAnd (5) performing column chromatography purification. Oligomeric Fab is present in fractions 26-30, while most monomeric forms are in fractions 31-34. Fractions consisting of the monomeric form were pooled and analyzed by SDS-PAGE together with wild-type hu4D5Fabv8 on reducing (containing DTT or BME) or non-reducing (not containing DTT or BME) SDS-PAGE gels. The gel-filtered fraction of A121C-ThioFab was analyzed on non-reducing SDS-PAGE.

ThioFab was conjugated to biotin-PEO-maleimide as described above and passed through Superdex-200^TM(Amersham) gel filtration chromatography further purified the biotinylated ThioFab to remove free biotin-PEO-maleimide and oligomeric portions of the ThioFab. Wild-type hu4D5Fabv8 and hu4D5Fabv8 a121C-ThioFab (0.5 mg amount) were each incubated separately with a 100-fold molar excess of biotin-PEO-maleimide at room temperature for 3 hours and loaded onto a Superdex-200 gel filtration column to separate free biotin as well as oligomeric Fab from monomeric form.

Example 3bAnalysis of ThioFab

The enzymatically digested fragments of biotinylated hu4D5Fabv8(A121C) ThioFab and wild-type hu4D5Fabv8 were analyzed by liquid chromatography electrospray ionization mass spectrometry (LS-ESI-MS). The difference between the basic mass of 48294.5 for biotinylated hu4D5Fabv8(a121C) and the basic mass of 47737.0 for wild-type hu4D5Fabv8 was 557.5 mass units. This fragment indicates a single biotin-PEO-maleimide (C)₂₃H₃₆N₅O₇S₂) The presence of the moiety. Table 4 shows the assignment of fragmentation values, which validate the sequence.

Table 4.Of biotinylated hu4D5Fabv8ThioFab A121C after Trypsin digestion

LC-ESI-Mass Spectrometry

Amino acids	b fragment	y fragment
			A (alanine)	72
M (methionine)	203	2505
			D (aspartic acid)	318	2374
Y (tyrosine)	481	2259
			W (Tryptophan)	667	2096
G (Glycine)	724	1910
			Q (glutamic acid)	852	1853
G (Glycine)	909	1725
			T (threonine)	1010	1668
L (leucine)	1123	1567
			V (valine)	1222	1454
T (threonine)	1323	1355
			V (valine)	1422	1254
S (serine)	1509	1155
			S (serine)	1596	1068
C (cysteine) + biotin	2242	981
			S (serine)	2329	335
T (threonine)	2430	248
			K (lysine)		175

SDS-PAGE gel analysis with or without DTT or BME reduction of biotinylated ABP-hu4D5Fabv8-A121C, biotinylated ABP-hu4D5Fabv8-V110C, biotinylated bis-Cys ABP-hu4D5Fabv8- (V110C-A88C), and biotinylated bis-Cys ABP-hu4D5Fabv8- (V110C-A121C) was performed before and after Superdex-200 gel filtration.

Mass Spectrometry (MS/MS) of hu4D5Fabv8- (V110C) -BMPEO-DM1 (after Superdex-200 gel filtration purification): fab + 151607.5, Fab 50515.5. This data shows 91.2% conjugation. MS/MS analysis of hu4D5Fabv8- (V110C) -BMPEO-DM1 (reduced): LC 23447.2, LC +124537.3, hc (fab) 27072.5. This data shows that all DM1 was conjugated to the Fab light chain.

Example 11Reduction/oxidation Thiomab for conjugation

Reduction at 37 ℃ with a 50-fold excess of TCEP (tris (2-carboxyethyl) hydrophosphate; Getz et al (1999) anal. biochem. Vol 273:73-80; Soltec vents, Beverly, Mass.) in CHO cellsFull-length, cysteine engineered monoclonal antibody (ThioMab) was achieved for 3 hours. The reduced ThioMab (fig. 15) was diluted and loaded onto a HiTrap S column at 10mM sodium acetate, pH5 and eluted with PBS containing 0.3M sodium chloride. 200nM aqueous copper sulfate (CuSO) at room temperature₄) The eluted reduced ThioMab was treated overnight. Ambient air oxidation is also effective.

Example 13N- [4- (N-Maleimidomethyl) cyclohexane-1-carboxy]Deferoxamine (Df-Chx-Mal)

Dissolving 4- [ N-Maleimidomethyl ] in a mixture of DMF (2.0mL) and 0.2mL of water]Cyclohexane-1-carboxylate (SMCC,40mg,0.12mmol), deferoxamine mesylate (78mg,0.12 mmol) and N, N-diisopropylethylamine (22. mu.L, 0.13mmol) (FIG. 21). The resulting cloudy solution was stirred at room temperature for 30 minutes. Water (8 mL) was added and the precipitated product was isolated by filtration, washed with water, and dried under reduced pressure to give 42mg (45%) of N- [4- (N-maleimidomethyl) cyclohexane-1-carboxy as a white solid]Deferoxamine (Df-Chx-Mal) (bottom of FIG. 20).¹H NMR(400MHz,d₆-DMSO)0.87-0.90(M,2H),1.20-1.26(M,8H),1.35-1.41(M,6H),1.45-1.55(M,8H),1.60-1.70(M,4H),1.97(s,3H, acetyl), 2.26-2.29(M,4H),2.56-2.60(M,4H),2.95-3.05(M,6H),3.24(d, J =7.0Hz,2H),3.44-3.48(M,6H),7.00(s,2H, maleimide), 7.62(t, J =5.4Hz,1H, amide), 7.75(M,2H, amide), 9.59(s,2H, hydroxyl), 9.64(s,1H, hydroxyl): MS (M/z) [ M + H ]/(M, z)]⁺C₃₇H₆₂N₇O₁₁Calculated as 780.44 and found 780.6.

Example 14N-bromoacetyl deferoxamine (Df-Bac)

A solution of bromoacetyl bromide (27. mu.L, 0.30mmol) in DMF (1mL) was added dropwise over 5 minutes to a cooled (0 ℃ C.) mixture of deferoxamine mesylate (200mg,0.30mmol) and N, N-diisopropylethylamine (106. mu.L, 0.60mmol) in DNF (5 mL), and the reaction mixture was stirred at 0 ℃ for 4 hours (FIG. 21). Water (10 mL) was added and the product was isolated using HPLC (system B, retention time 7.5 min) to give 29mg (14%) of N-bromoacetyl deferoxamine (Df-Bac) as a white solid (bottom of fig. 20).¹H NMR(400MHz,d₆-DMSO)1.18-1.26(M,6H),1.35-1.42(M,6H),1.45-1.55(M,6H),1.97(s,3H, acetyl), 2.24-2.30(M,4H),2.54-2.59(M,4H),2.96-3.07(M,6H),3.44-3.47(M,6H),3.82(s,2H, bromoacetyl), 7.74(M,2H, amide), 8.21(t,1H, amide), 9.59(s,2H, hydroxyl), 9.63(s,1H, hydroxyl)]⁺C₂₇H₅₀BrN₆O₉Calculated as 681.27,683.27, found 681.1,683.0

Example 15N-iodoacetyl deferoxamine (Df-Iac)

Deferoxamine mesylate (200mg,0.30mmol) and N, N-diisopropylethylamine (53 μ L,0.30mmol) were mixed in DMF (4mL) and water (0.4 mL). N-hydroxysuccinimide iodoacetate (93mg,0.33mmol) was added and the resulting mixture was stirred at room temperature for 1 hour (FIG. 21). Water (8 mL) was added and the precipitated product was isolated, washed with water and dried under reduced pressure to give 115mg (53%) N-iodoacetyldesferrioxamine (Df-Iac) as a white solid (bottom of FIG. 20).¹H NMR(400MHz,d₆-DMSO)1.20-1.25(M,6H),1.35-1.42(M,6H),1.47-1.54(M,6H),1.97(s,3H, acetyl), 2.25-2.29(M,4H),2.56-2.59(M,4H),2.98-3.03(M,6H),3.45(M,6H),3.61(s,2H, iodoacetyl), 7.75(M,2H, amide), 8.17(t,1H, amide), 9.57(s,2H, hydroxyl), 9.61(s,1H, hydroxyl)]⁺C₂₇H₅₀IN₆O₉729.26 is calculated, 729.1 is measured.

Example 16Thio-trastuzumab

Ala in the heavy chain has been previously described¹¹⁴Construction, expression and purification of Thiomab having a Cys substitution (Junutula JR, et al, "Site-specific conjugation of a cytoxic drug to antibody improves the therapeutic index" (2008) Nat Biotechnol 26: 925-32). Preparation of isolated thio-trastuzumab for conjugation by reduction and reoxidation procedure to remove Cys¹¹⁴A bound disulfide adduct. First, the protein was reduced for 24 hours by treatment with 40-fold molar excess of DTT and 2mM EDTA in 88mM Tris buffer, pH 7.5. To remove DTT prior to reoxidation, the thio-trastuzumab solution was adjusted to pH5 by the addition of 10mM sodium succinate buffer. The solution was then loaded onto an ion exchange column (HiTrap SP FF, GE Healthcare) that had been sterilized and equilibrated with 10mM sodium succinate buffer pH 5. The column was washed with 10mM sodium succinate buffer (10 mL) and then the thio-trastuzumab eluted with 3mM Tris,150mM NaCl buffer, pH 7.5. Reoxidation of thio-trastuzumab was achieved by treatment with 25-fold molar excess of dehydroascorbic acid (100 mM in N, N-Dimethylacetamide (DMA)) in 75mM Tris,150mM NaCl pH7.5 buffer for 3.5 hours at 25 ℃. After reoxidation, the thio-trastuzumab was conjugated with deferoxamine without further purification. MS ESI (m/z) found light chain 23440.0, heavy chain 50627.3.

Example 17Df-Chx-Mal-thio-trastuzumab

By applying a voltage at 1: 1 (1mL) Df-Chx-Mal (1.5mg, 2. mu. mol) was dissolved in a mixture of DMF and DMA and heated to 44 ℃ for 30 min to prepare a 2mM stock of bifunctional chelating agent. The stock was then divided into aliquots and stored at-80 deg.C (FIG. 21). An aliquot of the stock solution (220. mu.L, 0.440. mu. mol) was then added to a solution of thio-trastuzumab (7.5mg,52nmol) in 50mM Tris,150mM NaCl buffer pH7.5(1.5mL) and incubated at room temperature for 1 hour. The buffer of the solution was then replaced with 0.25M sodium acetate buffer on an AmiconUltra-4 filter to give 1mL of Df-Chx-Mal-thio-trastuzumab conjugate solution at a concentration of 6 mg/mL. MS ESI (m/z) found light chain 23440.2, heavy chain 51407.3 (FIG. 23, D).

Example 18Df-Ac-thio-trastuzumab using Df-Bac

A12 mM stock of bifunctional chelating agent was prepared by dissolving Df-Bac (8mg, 12. mu. mol) in 1mL of DMA (FIG. 21). The stock solution was then divided into small portions and stored at-80 ℃. The buffer of reoxidized thio-trastuzumab was replaced on the Amicon Ultra-4 filter with 0.05M sodium borate buffer pH 9. An aliquot of Df-Bac stock solution (35. mu.L, 0.410. mu. mol) was added to a solution of thio-trastuzumab (4.9mg,34nmol) in 0.05M sodium borate buffer pH9(1mL) and incubated for 5 hours at room temperature. The reaction mixture was loaded onto a NAP-10 column and Df-Ac-thio-trastuzumab eluted with 1.5mL of 0.25M sodium acetate buffer to give a concentration of 3.2mg/mL product. MS ESI (m/z) found light chain 23440.1, heavy chain 51228.1 (FIG. 23, B).

Example 19Df-Ac-thio-trastuzumab using Df-Iac

A11 mM stock of bifunctional chelating agent was prepared by dissolving Df-Iac (8mg, 11. mu. mol) in DMSO (1mL), and then the stock was divided into aliquots and stored at-80 deg.C (FIG. 21). The thio-trastuzumab solution (3.2mL) was adjusted to pH9 by the addition of 0.5mL of 0.1M sodium carbonate. An aliquot of the stock solution (110 μ L,1.20 μmol) was then added to a solution of thio-trastuzumab (16mg,110nmol) in 50mM Tris,150mM NaCl,0.0125M sodium carbonate buffer pH9(4mL) and incubated at room temperature for 2 hours. The buffer of the solution was then replaced with 0.25M sodium acetate buffer on an Amicon Ultra-4 filter to give 1mL of Df-Ac-thio-trastuzumab conjugate solution at a concentration of 8 mg/mL. MS ESI (m/z) found light chain 23440.1, heavy chain 51228.3 (FIG. 23, C).

Example 20Preparation of⁸⁹General procedure for Zr chelate complexes

Oxalic acid in 1M oxalic acid⁸⁹Zr (IV) solution (2-4mCi, 100. mu.L) with 2M Na₂CO₃The solutions (45 μ L) were mixed and incubated at room temperature for 3 minutes, followed by the addition of 0.5M HEPES buffer (0.15mL) (FIG. 22). Df-thio-trastuzumab conjugate (1mg,7nmol) was diluted with 0.25M sodium acetate/0.5% gentisic acid to a final volume of 0.356mL and added⁸⁹And (3) Zr solution. Finally, a second portion of HEPES buffer (0.350mL) was added to give a total volume of 1 mL. The mixture was incubated at room temperature for 1 hour. In order to remove freed⁸⁹Zr, radiolabeled protein was purified using NAP-10 desalting column. The NAP-10 column was equilibrated with 20mL of 0.25M sodium acetate/0.5% gentisic acid. The reaction mixture was loaded onto a NAP-10 column and eluted with 1.5mL of 0.25M sodium acetate/0.5% gentisic acid buffer (1.5mL)⁸⁹Zr-Df-thio-trastuzumab. If necessary, 89 Zr-Df-thio-trastuzumab was concentrated to the desired volume using an Amicon Ultra-4 filter. The product was analyzed by SEC HPLC (system a).

Example 21In vitro serum stability

In 0.25M sodium acetate/0.5% gentisic acid buffer (0.1 mL)⁸⁹Zr-Df-thio-trastuzumab conjugate 0.5-1.5mCi (1mg) solution plusFresh mouse serum (0.9mL) was added and incubated at 37 ℃ for 0-96 hours. Serum solution samples (20 μ L) were analyzed using SECHPCs (System A) and the results are shown in FIG. 24.

Example 22Animal model

Mild XID nude mice, 6-8 weeks old, were obtained from Harlan Sprague Dawley (Livermore, Calif.) 3 days prior to cell inoculation, mice were subcutaneously implanted 3 days on the left flank with 0.36mg of a 60 day slow release 17 β -estradiol tablet (Innovative research of America) to maintain serum estrogen levels.5 5 × 10 in 50% phenol-free red matrix gel was seeded in the mammary fat pads of the mice⁶BT474M1 cells. BT474M1 is a subclone of human breast tumor cell line BT474 purchased from Pacific Medical Center, California. Animal Care and handling followed the protocol approved by Genentech's institutional danimal Care and Use Committee, which was certified by the experimental animal assessment and approval Committee (AAALAC).

Example 23Microscopic PET imaging

Mice were anesthetized with about 3% sevoflurane to effect and injected intravenously via the tail vein with about 0.1mCi in isotonic solution (100-⁸⁹After injection of the tracer for 1,24,96 and 144 hours, PET imaging was performed on an Inveon PET/CT as follows (fig. 25). mice anesthetized with sevoflurane were placed on a scanning bed in the first, prone position and a static scan was acquired for 15 or 30 minutes.

Statistical analysis: the graph of FIG. 26 was constructed using R software version 2.4.1(R Foundation for Statistical Computing, Vienna, Austria). Statistical significance was determined using the two-tailed Student t-test or ANOVA, and p-values less than 0.05 were considered significant; data are presented as mean ± standard deviation, if not otherwise stated.

The present invention is not to be limited in scope by the specific embodiments disclosed in the examples which are intended as illustrations of some aspects of the invention, and any functionally equivalent embodiments are within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art and are intended to fall within the scope of the appended claims.

All patents, patent applications, and references cited throughout this specification are expressly incorporated by reference.

Claims (10)

1. Use of a zirconium-labeled, 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) in the preparation of an imaging agent, having the general formula I:

Ab-(L-Z)p I

wherein p is 1-4, wherein Z comprises zirconium complexed with desferrioxamine B (Df), and wherein L-Df is selected from:

wherein the wavy line indicates attachment to the antibody (Ab); and is

Wherein the cysteine engineered antibody comprises the sequence of SEQ ID NO 13 in the heavy chain:

LVTVSSCSTKGPS SEQ ID NO:13

wherein the cysteine in SEQ ID NO 13 is a free cysteine amino acid; or

Wherein the cysteine engineered antibody comprises the sequence of SEQ ID NO 19 in the light chain:

EIKRTCAAPSV (SEQ ID NO:19)

wherein the cysteine in SEQ ID NO 19 is a free cysteine amino acid.

2. The zirconium-labeled, cysteine-engineered antibody of claim 1 wherein p is 2.

3. The use of claim 1, wherein the cysteine engineered antibody is prepared by a method comprising the steps of:

(i) mutagenizing a nucleic acid sequence encoding a cysteine engineered antibody;

(ii) expressing the cysteine engineered antibody; and

(iii) isolating and purifying the cysteine engineered antibody.

4. The use of claim 1, wherein the cysteine engineered antibody is selected from the group consisting of a monoclonal antibody, a bispecific antibody, a chimeric antibody, a human antibody, a humanized antibody, and a Fab fragment.

5. The use of claim 1, wherein the cysteine engineered antibody is a121C thio-trastuzumab.

6. The use of claim 1, wherein the cysteine engineered antibody is prepared by a method comprising replacing one or more amino acid residues of a parent antibody with one or more free cysteine amino acids, wherein the parent antibody selectively binds to an antigen and the cysteine engineered antibody selectively binds to the same antigen as the parent antibody.

7. The use of claim 1, wherein⁸⁹Zirconium is complexed with the following structure:

wherein the wavy line indicates the connection to the joint (L).

8. Use of a zirconium-labeled, cysteine engineered antibody for the preparation of a pharmaceutical formulation for imaging comprising:

administering a zirconium-labeled, cysteine engineered antibody to the animal; and

detecting the presence of the zirconium-labeled, cysteine engineered antibody in vivo by imaging,

wherein the zirconium-labeled, cysteine engineered antibody is as defined in any one of claims 1-6.

9. The use of claim 8, wherein the zirconium-labeled, cysteine engineered antibody binds an antigen.

10. The use of claim 8, wherein the animal is a tumor xenograft mouse model.