MX2007003404A - Cysteine engineered antibodies and conjugates - Google Patents

Abstract

Antibodies are engineered by replacing one or more amino acids of a parent antibody with non cross-linked, highly reactive cysteine amino acids. Antibody fragments may also be engineered with one or more cysteine amino acids to form cysteine engineered antibody fragments (ThioFab). Methods of design, preparation, screening, and selection of the cysteine engineered antibodies are provided. Cysteine engineered antibodies (Ab), optionally with an albumin-binding peptide (ABP) sequence, are conjugated with one or more drug moieties (D) through a linker (L) to form cysteine engineered antibody-drug conjugates having Formula I:Ab-(L-D)pI where p is 1 to 4. Diagnostic and therapeutic uses for cysteine engineered antibody drug compounds and compositions are disclosed. The present invention is directed to cyclopropyl piperidine compounds that inhibit the glycine transporter GlyT1 and which are useful in the treatment of neurological and psychiatric disorders associated with glycinergic or glutamatergic neurotransmission dysfunction and diseases in which the glycine transporter GlyT1 is involved. Antibodies are engineered by replacing one or more amino acids of a parent antibody with non cross-linked, highly reactive cysteine amino acids. Antibody fragments may also be engineered with one or more cysteine amino acids to form cysteine engineered antibody fragments (ThioFab). Methods of design, preparation, screening, and selection of the cysteine engineered antibodies are provided. Cysteine engineered antibodies (Ab), optionally with an albumin-binding peptide (ABP) sequence, are conjugated with one or more drug moieties (D) through a linker (L) to form cysteine engineered antibody-drug conjugates having Formula I:Ab-(L-D)pI where p is 1 to 4. Diagnostic and therapeutic uses for cysteine engineered antibody drug compounds and compositions are disclosed.

Description

ANTIBODIES AND CONJUGATES DESIGNED FROM CYSTEINE This non-provisional application filed under 37 CFR §1.53 (b), claims the benefit under 35 USC §119 (c) of the US Provisional Application. Ser. No. 60 / 612,468 filed on September 23, 2004 and the Provisional Application of E.U. Ser. No. 60 / 696,353 filed on June 30, 2005, each of which is incorporated herein by reference in its entirety. FIELD OF THE INVENTION The invention relates in general to antibodies designed with reactive cysteine residues and more specifically to antibodies with therapeutic or diagnostic applications. The designed antibodies of cysteine can be conjugated with chemotherapeutic drugs, toxins, affinity ligands such as biotin, and detection labels such as fluorophores. The invention also relates to methods for using antibodies and antibody-drug conjugates, for in vitro, in situ and in vivo diagnosis or treatment of mammalian cells or associated pathological conditions. BACKGROUND OF THE INVENTION Antibody therapy has been established for the targeted treatment of patients with cancer, immunological and angiogenic disorders. In attempts to discover effective cellular targets for diagnosis and therapy of cancer with antibodies, researchers have sought to identify transmembrane or other tumor-associated polypeptides, which are specifically expressed on the surface of cancer cells compared to normal, non-cancerous cells. The identification of such tumor-associated cell surface antigen polypeptides, i.e., tumor-associated antigens (TAA), has resulted in the ability to specifically target cancer cells for destruction through antibody-based therapies. The use of antibody-drug conjugates (ADCs), ie, immunoconjugates, for the local delivery of cytotoxic or cytostatic agents, ie, drugs to destroy or inhibit tumor cells in the treatment of cancer (Lambert, J., (2005 ) Curr Opinion in Pharmacology 5: 543-549; u et al., (2005) Nature Biotechnology 23 (9): 1137-1146; Payne G., (2003) Cancer Cell 3: 207-212; Syrigos and Epenetos ( 1999) Anticancer Research 19: 605-614; Niculescu-Duvaz and Springer (1997) Adv. Drug Del. Rev., 26: 151-172; US 4975278) theoretically allows targeted delivery of the drug residue to tumors, and accumulation intracellular therein, wherein the systemic administration of these unconjugated drug agents can result in unacceptable levels of toxicity to normal cells as well as to the tumor cells to be eliminated (Baldwin et al., (1986) Lancet p. (Mar. 15, 1986): 603-05; Thorpe (1985) "Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological and Clinical Applications. A. Pinchera et al., (Eds.) Pp. 475-506). Maximum efficacy is sought with minimal toxicity through it. Efforts to design and refine ADC have focused on the selectivity of monoclonal antibodies (mAbs) as well as on drug binding and drug release properties (Lambert J., (2005) Curr. Opinion in Pharmacology 5: 543-549 Both polyclonal antibodies and monoclonal antibodies have been reported to be useful in these strategies (Rowland et al., (1986) Cancer Immunol. Immunother., 21: 183-87) .The drugs used in these methods include daunomycin, doxorubicin, methotrexate. and vindesine (Rowland et al., (1986) supra.) Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (Mandler et al. , (2000) J. del Nat. Cancer Inst. 92 (19): 1573-1581; Mandler et al., (2000 = Bioorganic &Med. Chem. Letters 10: 1025-1028; Mandler et al., (2002 ) Bioconjugate Chem., 13: 786-791), maytansinoids (EP 139121 3; Liu et al., (1996) Proc. Nati Acad. Sci. , USA 93: 8618-8623), and calicheamicin (Lode et al., (1998) Cancer Res. 58: 2928; Hinman et al., (1993) Cancer Res., 53: 3336- 3342. The toxins can effect their cytotoxic and cytostatic effects by mechanisms that include tubulin binding, DNA binding or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands. An antibody-radioisotope conjugate has been approved. ZEVALIN® (ibritumomab tiuxetan, Biogen / Idec) is composed of a murine monoclonal IgGl kappa antibody directed against the CD20 antigen found on the surface of normal and malignant B-cells and the radioisotope li: LIn or 90Y linked by a detiourea-chelator binding ( Wiseman et al., (2000) Eur. J. Nucí, Med. 27 (7): 766-77; Wiseman et al., (2002) Blood 99 (12): 4336-42; Witzig et al., (2002) J. Clin Oncol., 20 (10): 2453-63; Witzig et al., (2002) J. Clin. Oncol., 20 (15) -.3262-69). Although ZEVALIN® has activity against non B-cell Hodgkin lymphoma, administration results in severe and prolonged cytopenias in the majority of patients. MYLOTARG ™ (gentuzumab ozogamicin, Wyeth Pharmaceuticals), an antibody-drug conjugate composed of a hu CD33 antibody bound to calicheamicin, was approved in 2000 for the treatment of acute myeloid leukemia by injection (Drugs of the Future (2000) 25 ( 7): 686; U.S. Patent Nos. 4970198; 5079233; 5585089; 5606040; 5693762; 5739116; 5767285; 5773001). Cantuzumab Mertansine (Immunogen, Inc.), an antibody-drug conjugate composed of the huC242 antibody bound through the SPP disulfide bond to the maytansinoid drug residue, DM1 (Xie et al., (2004) J. of Pharm. Ther., 308 (3): 1073-1082), advances in Phase II trials for the treatment of cancers that express CanAg, such as colon, pancreatic, gastric and others. MLN-2704 (Millennium Pharm, BZL Biologics, Immunogen Inc.), an antibody-drug conjugate composed of the specific anti-prostate membrane antigen monoclonal antibody (PSMA) bound to the maytansinoid drug residue, DM1, is in development for the potential treatment of prostate tumors. The auristatin, auristatin E (AE) and monometillauristatin (MMAE) peptides, synthetic analogs of dolastatin (WO 02/088172) have been conjugated to: (i) cBR96 monoclonal chimeric antibodies (specific for Lewis Y in carcinomas); (ii) cACLO that is specific for CD30 in hematological malignancies (Klussman et al., (2004), Bioconjugate Chemistry 15 (4): 765-773, Doronina et al., (2003) Nature Biotechnology 21 (7): 778- 784; Francisco et al., (2003) Blood 102 (4): 1458-1465; EU 2004/0018194; (üi) anti-CD20 antibodies such as rituxan (WO 04/032828) for the treatment of cancers expressing CD20 and immune disorders; (iv) anti-EphB2 2H9 and anti-IL-8 antibodies for the treatment of colorectal cancer (Mao, et al., (2004) Cancer Research 64 (3): 781-788); (v) E-selectin antibody (Bhaskar et al., (2003) Cancer Res., 63: 6387-6394); and (vi) other anti-CD30 antibodies (WO 03/043583). The variants of auristatin E are described in US 5767237 and US 6124431. Monomethyl auristatin E conjugated to monoclonal antibodies is described in Senter et al. , Proceedings of the American Association for Cancer Research, Volume 45, Excerpt Number 623, filed March 28, 2004. Analogues of auristatin MMAE and MMAF have been conjugated to various antibodies (WO 2005/081711). Conventional means for attaching, i.e., binding by covalent linkages, a drug residue to an antibody, generally leads to a heterogeneous mixture of molecules wherein the drug residues are bound at a number of sites in the antibody. For example, cytotoxic drugs have been conjugated to antibodies through the frequently numerous lysine residues of the antibody, generating a heterogeneous mixture of antibody-drug conjugate. Depending on the reaction conditions, the heterogeneous mixture typically contains an antibody distribution with from 0 to about 8, or more, bound drug residues. Furthermore, within each subgroup of conjugates with a particular integer ratio of drug to antibody residues, it is a potentially heterogeneous mixture in which the drug residue is bound at several sites in the antibody. The analytical and preparative methods are inadequate to separate and characterize the antibody-drug conjugate species molecules within the heterogeneous mixture resulting from a conjugation reaction. Antibodies are large, complex, and structurally diverse biomolecules, often with many reactive functional groups. Their reactivities with binding reagents and drug-linker intermediates depend on factors such as pH, concentration, salt concentration, and co-solvents. In addition, the multi-step conjugation process may be non-reproducible due to the difficulties in controlling the reaction conditions and characterizing the reagents and intermediates. Cysteine thiols are reactive at a neutral pH, unlike most amines that are protonated and less nucleophilic at about pH 7. Since the free thiol groups (RSH, sulfhydryl) are relatively reactive, the proteins with cysteine residues often exist in their oxidized form as disulphide linking oligomers or have internally bridged disulfide groups. Extracellular proteins generally do not have free thiols (Garman, 1997, Non-Radioactive Labeling: A Practical Approach, Academic Press, London, page 55). The amount of free thiol in the protein can be estimated by standard Ellman analysis. Immunoglobulin M is an example of a disulfide bonding pentamer, while immunoglobulin G is an example of a protein with internal disulfide bonds that bind the subunits together. In proteins such as these, reduction of the disulfide linkages with a reagent such as dithiothreitol (DTT) or selenol (Singh et al., (2002) Anal. Biochem., 304: 147-156) is required to generate the reactive free thiol. . This procedure can result in the loss of the tertiary structure of the antibody and the specificity of antigen binding. The cysteine thiol antibody groups are generally more reactive, i.e., more nucleophilic, towards electrophilic conjugation reagents, than the amine or hydroxyl antibody groups. Cysteine residues have been introduced into proteins by genetic manufacturing techniques to form covalent linkages to ligands or to form new intramolecular disulfide linkages (Better et al., (1994) J. Biol. Chem., 13: 9644-9650; Bernhard et al. al., (1994), Bioconjugate Chem., 5: 126-132, Greenwood et al., (1994) Therapeutic Immunology 1: 247-255; Tu et al., (1999) Proc. Nati. Acad. Sci. 96: 4862-4867; Kanno et al., (2000) J of Biotechnology, 76: 207-214; Chmura et al., (2001) Proc. Nati. Acad. Sci., USA 98 (15): 8480-8484; US 6248564). However, the design in thiol groups of cysteine by mutation of several amino acid residues of a protein to Cysteine amino acid is potentially problematic, particularly in the case of unpaired residues (free Cys) or in those that are relatively accessible for reaction or oxidation. In concentrated solutions of the protein, either in the periplasm of E. coli, culture supernatants, or partially or completely purified protein, unpaired Cys residues on the surface of the protein can pair and oxidize to form intermolecular disulfides, and therefore, dimers or protein multimers. The formation of the disulfide dimer produces the new non-reactive Cys for conjugation to a drug, ligand or other brand. In addition, if the protein oxidatively forms an intramolecular disulfide bond between the newly manufactured Cys and an existing Cys residue, both Cys groups are not available for active participation and interactions at the site. In addition, the protein can become inactive or non-specific due to an error in folding or a loss of tertiary structure (Zhang et al., (2002) Anal. Biochem., 311: 1-9). SUMMARY Compounds of the invention include designed cysteine antibodies wherein one or more amino acids of an original antibody are replaced with a free cysteine amino acid. A designed cysteine antibody comprises one or more free cysteine amino acids that have a value of thiol reactivity in the range of 0.6 to 1.0. A free cysteine amino acid is a cysteine residue that has been manufactured within the original antibody and is not part of a disulfide bridge. In one aspect, the designed cysteine antibody is prepared by a process comprising: (a) the replacement of one or more amino acid residues of an original antibody with cysteine; and (b) determining the thiol reactivity of the designed cysteine antibody by reacting the designed cysteine antibody with a thiol-reactive reagent. The designed cysteine antibody may be more reactive than the original antibody with the thiol reactive reagent. The amino acid residues of free cysteine can be located in the heavy or light chains, or in the constant or variable domains. Antibody fragments, e.g., Fab, can also be designed with one or more amino acids of cysteine by replacing the amino acids of the antibody fragment, to form antibody fragments designed of cysteine. Another aspect of the invention provides a method for the preparation (production) of a designed cysteine antibody, comprising: (a) the introduction of one or more amino acids into an original antibody in order to generate the designed cysteine antibody; and (b) determining the reactivity of the thiol of the designed cysteine antibody with a thiol-reactive reagent; wherein the designed cysteine antibody is more reactive than the original antibody with the thiol reactive reagent. Step (a) of the method of preparing a designed cysteine antibody can comprise: (i) mutagenesis of a nucleic acid sequence encoding the designed cysteine antibody; (ii) the expression of the designed cysteine antibody; and (iii) the isolation and purification of the designed cysteine antibody. Step (b) of the method of preparing a designed cysteine antibody can comprise the expression of the designed cysteine antibody in a viral particle selected from a phage particle or a phagemid. Step (b) of the method of preparing a designed cysteine antibody can also comprise: (i) reacting the designed antibody of cysteine with a thiol-reactive affinity reagent to generate an affinity-labeled designed cysteine antibody; and (ii) measuring the binding of the designed affinity tagged cysteine antibody to a capture medium. One aspect of the invention is a method for selecting designed cysteine antibodies with unreacted, highly-reactive cysteine amino acids by thiol reactivity, comprising: (a) introducing one or more amino acids of cysteine into an original antibody in order to generate a designed cysteine antibody: (b) reacting the designed cysteine antibody with a thiol-reactive affinity reagent to generate an affinity-labeled cysteine-labeled antibody; and (c) measuring the binding of the designed affinity tagged cysteine antibody to a capture medium; and (d) determining the reactivity of the thiol of the designed cysteine antibody with the thiol reactive reagent. Step (a) of the method of selecting a designed cysteine antibody may comprise: (i) the mutagenesis of an acid sequence nucleic acid coding for the designed cysteine antibody; (ii) the expression of the designed cysteine antibody; and (iii) the isolation and purification of the designed cysteine antibody. Step (b) of the method of selecting a designed cysteine antibody can comprise the expression of the designed cysteine antibody in a viral particle selected from a phage particle or a phagemid. Step (b) of the method of selecting a designed cysteine antibody can also comprise: (i) reacting the designed cysteine antibody with a thiol-reactive affinity reagent to generate a designed affinity-labeled cysteine antibody; and (ii) measuring the binding of the designed affinity tagged cysteine antibody to a capture medium. The designed antibodies of cysteine may be useful in the treatment of cancer and include antibodies specific for cell surface and transmembrane receptors, and tumor associated antigens (TAA). Such antibodies can be used as naked antibodies (not conjugated to a drug or brand residue) or as conjugates antibody-drug (ADC) of Formula I. Modes of methods of preparing and visualizing a designed cysteine antibody include when the original antibody is an antibody fragment, such as hu4D5Fabv8. The original antibody can also be a fusion protein comprising an albumin binding peptide (ABP) sequence. The original antibody can also be a humanized antibody selected from huMAb4D5-l, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, hu Ab4D5-5, huMAb4D5-6, hu Ab4D5-7, and huMAb4D5-8 (trastuzumab). The designed cysteine antibodies of the invention can be coupled site-specifically and efficiently with a thiol-reactive reagent. The thiol-reactive reagent can be a multifunctional linker reagent, a capture tag reagent, a fluorophore reagent or a drug binding intermediate. The designed cysteine antibody can be labeled with a detectable label, immobilized on a solid phase support and / or conjugated with a drug residue. Another aspect of the invention is an antibody-drug conjugate compound comprising a designed antibody of cysteine (Ab), and a drug residue (D), wherein the designed cysteine antibody is linked through one or more amino acids of free cysteine by a linker residue (L) to D; having the compound the Formula I.

Ab- (L-D) p I where p is 1, 2, 3 or 4; and wherein the designed cysteine antibody is prepared by a process comprising the replacement of one or more amino acid residues of an original antibody by one or more free cysteine amino acids. Drug residues include, but are not limited to, a maytansinoid, an auristatin, a dolastatin, a tricothecene, CC1065, a calicheamicin and other enedin antibiotics, a taxane, an anthracycline and stereoisomers, isoesters, their analogues or derivatives. Exemplary drug residues include DM1, MMAE and MMAF. The antibody-drug conjugate of Formula I may further comprise an albumin binding peptide (ABP) sequence; the composition having the Formula: ABP-Ab- (LD) p The other aspect of the invention is a composition comprising an antibody designed from cysteine or an antibody-drug conjugate designed from cysteine and a physiologically or pharmaceutically acceptable carrier or diluent. . This composition for therapeutic use is sterile and can be lyophilized. Another aspect of the invention includes diagnostic and therapeutic uses for the compounds and compositions described herein. The pharmaceutical compositions include combinations of compounds of Formula I and one or more chemotherapeutic agents. Another aspect of the invention is a method for destroying or inhibiting the proliferation of tumor cells or cancer cells comprising treating the cells with an amount of an antibody-drug conjugate of the invention, or a pharmaceutically acceptable salt or solvate of the invention. same, being effective for the destruction or inhibition of the proliferation of tumor cells or cancer cells. Other aspects of the invention include methods for the treatment of cancer, an autoimmune disease or an infectious disease, comprising administering to a patient in need thereof, an effective amount of the antibody-drug conjugate compound of the invention, or a salt thereof. or pharmaceutically acceptable solvate thereof. Another aspect of the invention is a method for the treatment of cancer in a mammal, wherein the cancer is characterized by overexpression of an ErbB receptor. Optionally the mammal does not respond or responds weakly to treatment with a non-conjugated anti-ErbB antibody. The method comprises administering to the mammal a therapeutically effective amount of an antibody-drug conjugate compound of the invention.

Another aspect of the invention is a method for inhibiting the growth of tumor cells that overexpress a growth factor receptor selected from the group consisting of the HER2 receptor and the EGF receptor., comprising administering to a patient an antibody-drug conjugate compound that specifically binds to said growth factor receptor, and a chemotherapeutic agent, wherein said antibody-drug conjugate and said chemotherapeutic agent are each administered at effective amounts to inhibit the growth of tumor cells in the patient. Another aspect of the invention is a method for the treatment of a human patient susceptible to or diagnosed with a disorder characterized by overexpression of the ErbB2 receptor, comprising administering an effective amount of a combination of an antibody-drug conjugate compound and a chemotherapeutic agent. Another aspect of the invention is a method of analysis for the detection of cancer cells, comprising: the exposure of the cells to an antibody-drug conjugate compound, and the determination of the degree of binding of the antibody-drug conjugate compound to the cells Another aspect of the invention is an article of manufacture comprising an antibody-drug conjugate compound, a package, and a packaging insert or label indicating that the compound can be used for the treatment of cancer. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A shows a three-dimensional representation of the antibody fragment hu4D5Fabv7 derived by X-ray crystal coordinates. The structural positions of the exemplary Cys residues of the heavy and light chains are numbered (according to a system of sequential numbering). Figure IB shows a sequential numbering scheme (upper row), starting at the N ending compared to the Kabat numbering scheme (lower row) for 4D5v7fabH. The Kabat numbering inserts are noted as a, b, c. Figures 2A and 2B show binding calculations with absorbance detection at 450 nm of phage variants of hu4D5Fabv8 and Cys mutant hu4D5Fabv8 (ThioFab) (A): non-biotinylated phage-hu4D5Fabv8 and (B) biotinylated phage-hu4D5Fabv8 by PHESELECTOR analysis for interactions with BSA (open bar), HER2 (bar listed) or streptavidin (solid bar). Figures 3A and 3B show the union calculations with absorbance detection at 450 nm of variants of hu4D5Fabv8 (left) and mutant Cys hu4D5Fabv8 (ThioFab) (A): non-biotinylated phage-hu4D5Fabv8 and (B) biotinylated phage-hu4D5Fabv8 by PHESELECTOR analysis for interactions with BSA (open bar) , HER2 (bar listed) or streptavidin (solid bar). The light chain variants are on the left side and the heavy chain variants are on the right side. Reactivity thiol = OD450 nm for binding of streptavidin ÷ OD450 nm for binding of HER2 (antibody). Figure 4A shows the Fractional Surface Accessibility values of the residues in wild type hu4D5Fabv8. The light chain sites are on the left side and the heavy chain sites are on the right side. Figure 4B shows binding calculations with absorbance detection at 450 nm of biotinylated hu4D5Fabv8 variants (left) and Cys mutant hu4D5Fabv8 (ThioFab) for interactions with HER2 (day 2), streptavidin (SA) (day 2), HER2 ( day 4), and SA (day 4). The Cys phage-hu4D5Fabv8 variants were isolated and stored at 4 ° C. The biotin conjugation was carried out either on day 2 or on day 4 followed by PHESELECTOR analysis to monitor its interaction with HER2 and streptavidin as described in Example 2, and test the stability of thiol groups reagents in manufactured ThioFab variants. Figure 5 shows binding calculations with absorbance detection at 450 nm of biotin-maleimide conjugate hu4D5Fabv8 (A121C) and non-biotinylated wild-type hu4D5Fabv8 for binding to streptavidin and HER2. Each Fab was tested at 2 ng and 20 ng. Figure 6 shows ELISA analysis with absorbance detection at 450 nm of biotinylated wild-type (wt) ABP-hu4D5Fabv8, and cysteine mutants ABP-hu4D5Fabv8 V110C and A121C for binding with rabbit albumin, streptavidin (SA) and HER2. Figure 7 shows ELISA analysis with absorbance detection at 450 nm of biotinylated ABP-hu4D5Fabv8 cysteine mutants (ThioFab variants); (from left to right) unique Cys variants ABP-V110C, ABP-A121C, and double Cys variants ABP-V110C-A88C and ABP-V110C-A121C for binding with rabbit albumin, HER2 and streptavidin (SA), and testing with Fab-HRP or SA-HRP. Figure 8 shows the binding of biotinylated ThioFab phage and an anti-phage HRP antibody for HER2 (upper part) and streptavidin (lower part). Figure 9 shows an exemplary representation of an ABP-ThioFab fusion protein drug conjugate that binds to a HER2 receptor antigen. ABP = albumin binding protein.

Figure 10 shows an in vitro cell proliferation analysis of SK-BR-3 cells treated with - · - trastuzumab; -A- trastuzumab-SMCC-DMl; and -? - cysteine mutant hu4D5Fabv8 (A121C) -BMPE0-DM1. Figure 11 shows an in vitro cell proliferation analysis of SK-BR-3 cells treated with: -o-trastuzumab; - · - trastuzumab-SMCC-DMl; and -? - cysteine mutant hu4D5Fabv8 (V110C) -BMPEO-DM1. Figure 12 shows the change in mean tumor volume over time in athymic mice with mammary tumor allografts MMTV-HER2 Fo5, dosed on day 0 with: † vehicle (buffer); - | - cysteine mutant ABP-hu4D5Fabv8 (V110C light chain) -DM1; and - · - ABP-cysteine mutant hu4D5Fabv8 (A121C heavy chain) -DM1. Figure 13A shows an illustration of the binding of immobilized biotinylated antibody to HER2 with secondary antibody binding labeled with HRP for absorbance detection. Figure 13B shows binding calculations with absorbance detection at 450 nm of thio-trastuzumab variants of biot ina-maleimide conjugate and non-biotinylated wild-type trastuzumab at immobilized HER2 binding. From left to right: V110C (single cys), A121C (single cys), V110C / A121C (double cys), and trastuzumab. Each variant thio IgG and trastuzumab was tested at l, 10 and 100 ng. Figure 14A shows an illustration of the binding of the biotinylated antibody to immobilized HER2 with the binding of biotin to anti-IgG-HRP for absorbance detection. Figure 14B shows binding calculations with absorbance detection at 450 nm of thio trastuzumab variants of biotin-maleimide conjugate and non-biotinylated wild-type trastuzumab at immobilized streptavidin binding. From left to right: V110C (single cys), A121C (single cys), V110C / A121C (double cys) and trastuzumab. Each thio IgG variant and trastuzumab was tested at l, 10 and 100 ng. Figure 15 shows the general process for preparing a designed cysteine antibody (ThioMab) expressed from a cell culture for conjugation. Figure 16 shows analysis of denatured polyacrylamide gel electrophoresis of non-reduction (top) and reduction (bottom) of 2H9 ThioMab Fe variants (from left to right, fields 1-9); A339C; S337C; A287C; V284C; V282C; V279C; V273C and wild-type 2H9 after purification on immobilized Protein A. The field on the right is a dimension marker scale, indicating that intact proteins are approximately 150 kDa, heavy chain fragments approximately 50 kDa, and chain fragments light of approximately 25 kDa. Figure 17A shows denatured polyacrylamide gel electrophoresis analysis of nonreduction (left) and reduction (+ DTT) (right) of 2H9 ThioMab variants (from left to right, fields 1-4): L-V15C; S179C; S375C; S400C, after purification in Protein A immobilized. Figure 17B shows denatured polyacrylamide gel electrophoresis analysis of nonreduction (left) and reduction (+ DTT) (right) of 2H9 and 3A5 ThioMab Fe variants after purification on immobilized Protein A. Figure 18 shows western immunoassay of biotinylated Thio-IgG variants. The 2H9 and 3A5 variants were analyzed in reduced denatured polyacrylamide gel electrophoresis, the proteins were transformed into nitrocellulose membrane. The presence of antibody and biotin conjugate were tested with anti-IgG-HRP (upper part) and streptavidin-HRP (lower part), respectively. Field 1: 3A5 H-A121C. Field 2: 3A5 L-V110C. Field 3: 2H9 H-A121C. Field 4: 2H9 L-V110C. Field 5: 2H9 wild type. Figure 19 shows ELISA analysis for the binding of biotinylated 2H9 variants to streptavidin by testing with anti-IgG-HRP and calculating the absorbance at 450 nm (diagram in the upper bar). The lower schematic diagram illustrates the experimental design used in the ELISA analysis. Figure 20 shows an analysis of cell proliferation of SK-BR-3 cells treated with: - · - trastuzumab; -A-trastuzumab-SMCC-DMl with a drug loading of 3.4 DMl / Ab; and -? - thio-trastuzumab (A121C) -BMPE0-DM1 with a drug loading of 1.6 DMl / Ab. Figure 21A shows an in vitro cell proliferation assay of HT 1080EphB2 cells treated with: -0-2H9 anti-EphB2R of origin; and -? - thio 2H9 (A121C) -BMPE0-DM1. Figure 21B shows an in vitro cell proliferation assay of BT 474 cells treated with: -0- 2H9 anti-EphB2R of origin; and -? - thio 2H9 (A121C) -BMPE0-DM1. Figure 22 shows an in vitro cell proliferation assay for PC3 / neo cells treated with: -? - 3A5 anti-MUC16-SMCC-DM1; and -j-thio 3A5 (A121C) BMPE0-DM1. Figure 23 shows an in vitro cell proliferation assay for PC3 / MUC16 cells treated with: -? - 3A5 anti-MUC16-SMCC-DMl; and -j-thio 3A5 (A121C) BMPE0-DM1. Figure 24 shows an in vitro cell proliferation assay for OVCAR-3 cells treated with: -? - 3A5 anti-MUC16-SMCC-DMl; and - | - thio 3A5 (A121C) BMPE0-DM1. Figure 25 shows the change in mean tumor volume during 21 days in athymic mice with allografts of mammary tumor MMTV-HER2 Fo5, after a single dose on day 0 with: † vehicle (shock absorber); - · - trastuzumab-SMCC-DM1 10 mg / kg with a drug loading of 3.4 DMl / Ab; - thio-trastuzumab (A121C) -SMCC-DM1 21 mg / kg with a drug loading of 1.6 DMl / Ab; and - thio trastuzumab (A121C) -SMCC-DM1 10 mg / kg with a drug loading of 1.6 DMl / Ab. DETAILED DESCRIPTION OF THE EXEMPLARY MODALITIES Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying structures and formulas. Although the invention will be described in conjunction with the embodiments listed, 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 present invention as defined by the claims. The person skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which may be used in the practice of the present invention. The present invention is not limited in any way to the methods and materials described. Unless defined otherwise, the technical and scientific terms used herein have the same meaning commonly understood by that of ordinary experience in the art to which this invention, and are consistent with: Singleton et al., (1994) Dictionary of Microbiology and Molecular Biology, 2nd edition, J. iley & Sons, New York, NY; and Janeway, C, Travers, P., alport, M., Shlomchik (2001) Immunobiology, 5th edition, Garland Publishing, New York. DEFINITIONS Unless otherwise determined, the following terms and phrases as used herein are intended to have the following meanings: When using trade names herein, applicants intend to independently include the product formulation of the trademark, the generic drug and the active pharmaceutical ingredient (s) of the trademark product. The term "antibody" is used herein in the broadest sense and specifically covers, monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (eg, bispecific antibodies) and antibody fragments while exhibiting the biological or desired activity ( Miller et al., (2003) Hour of Immunology 170: 4854-4861). The antibodies can be murine, human, humanized, chimeric or derived from other species. An antibody is a protein generated by the immune system, which is able to recognize and bind to a specific antigen. (Janeway C, Travers P., Walport M., Shlomchik (2001) Immuno Biology, 5th edition, Garland Publishing, New York). A target antigen generally has numerous binding sites, also called epitopes, recognized by CDRs in multiple antibodies. Each antibody that binds specifically to a different epitope has a different structure. In this way, an antigen can have more than one corresponding antibody. An antibody includes a full length immunoglobulin molecule or an immunologically active portion of a full length immunoglobulin molecule, ie, a molecule that contains an antigen binding site that immunospecifically binds to an antigen of a target of interest or part of the same, including such targets, but not limited to cancer cells or cells that produce immune antibodies associated with an autoimmune disease. The immunoglobulin treated herein can be of any type (e.g., IgG, IgE, IgM, IgD and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. Immunoglobulins can be derived from any species. However, in one aspect, the immunoglobulin is of human, murine or rabbit origin. The "antibody fragments" comprise a portion of a full length antibody, generally the antigen binding or the variable region thereof. Examples of antibody fragments include Fab, Fab 'fragments, F (ab ') 2 and Fv fragments; diabodies; linear antibodies; minibodies (Olafsen et al., (2004) Protein Eng. Design &Sel., 17 (4): 315-323), fragments produced by a Fab expression library, anti-idiotypic antibodies (anti-Id), CDR (complementarity determining region), and epitope binding fragments of any of the foregoing that immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for naturally occurring mutations that may occur naturally. present in smaller quantities. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In addition, in contrast to the preparations of the polyclonal antibody that includes different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant in the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can synthesized without being contaminated by other antibodies. The "monoclonal" modifier indicates the character of the antibody obtained from a substantially homogenous population of antibodies, and is not to be understood as requiring the production of the antibody by any particular method. For example, monoclonal antibodies that are 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 can be prepared by recombinant DNA methods (see for example: US 4816567; US 5807715). The "monoclonal antibodies" can also be isolated from libraries of phage antibodies using the techniques described in Clackson et al., (1991) Nature, 352: 624-628; arks et al., (1991) J. Mol. Biol. , 222: 581-597, for example. Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequences in antibodies derived from particular species or belonging to a particular class or subclass of antibody , while the rest of the chain (s) is identical or homologous to the corresponding sequences in antibodies derived from other species or belonging to another class or subclass of antibody, as well as fragments of such antibodies, provided they exhibit the desired biological activity (US 4816567; and Morrison et al., Proc. Nati. Acad. Sci. USA, 81: 6851-6855). Chimeric antibodies of interest herein include "primatized" antibodies that comprise variable domain antigen binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape, etc.) and human constant region sequences. An "intact" antibody herein is one that comprises VL and VH domains, as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2, and CH3. The constant domains may be constant domains of natural sequence (e.g., constant domains of human natural sequence) or variants of amino acid sequence thereof. The intact antibody may have one or more "effector functions," which refer to those biological activities attributable to the Fe constant region (a Fe region of natural sequence or Fe region variant amino acid sequence) of an antibody. Examples of antibody effector functions include Clq binding; Complement-dependent cytotoxicity; Fe receptor binding; antibody-mediated cell-mediated cytotoxicity (ADCC); phagocytosis and sub-regulation of cell surface receptors such as the B cell receptor and BCR. Depending on the amino acid sequence of constant dominance of their heavy chains, intact antibodies can be assigned to different "classes". There are five major classes of immunoglobulin antibodies: IgA, IgD, IgE, IgG and IgM, and several of these can further be divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains that correspond to different classes of antibodies are called a, d, e,? and μ respectively. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Ig forms include joint modifications or non-articulated forms (Roux et al., (1998) J. Immunol., 161: 4083-4090; Lund et al., (2000) Eur. J. Biochem., 267: 7246- 7256; US 2005/0048572; US 2004/0229310). An "ErbB receptor" is a receptor protein tyrosine kinase that belongs to the ErbB receptor family whose members are important mediators of cell growth, differentiation and survival. The ErbB receptor family includes four distinct members including the epidermal growth factor receptor (EGFR, ErbBl, HERI), HER2 (ErbB2 or pl85neu), HER3 (ErbB3) and HER4 (ErbB4 or tyro2). A panel of anti-Erb2 antibodies was characterized using the human breast tumor cell line (Hudziak et al., (1989) Mol. Cell. Biol. 9 (3): 1165-1172. antibody called 4D5 that inhibited cell proliferation by 56%. Other antibodies in the panel reduced cell proliferation to a lesser degree in this analysis. It was further found that the 4D5 antibody sensitizes breast tumor cell lines overexpressing ErbB2 to the cytotoxic effects of TNF-a (US 567717). Anti-ErbB2 antibodies treated in Hudziak et al., Are further 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 Regulation 1: 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. Immunother., 37: 255-263; Pietras et al., (1994) Oncogene 9: 1829-1838; Vitetta et al., (1994) Cancer Research 54: 5301-5309; Sliwkowski et al., (1994) J. Biol. Chem., 269 (20): 14661-14665; Scott et al., (1991) J. Biol. Chem., 266: 14300-5; D'Souza et al., Proc. Nati Acad. Sci. , (1994) 91: 7202-7206; Lewis et al., (1996) Cancer Research 56: 1457-1465; and Schaefer et al., (1997) Oncogene 15: 1385-1394. The ErbB receptor will generally comprise an extracellular domain, which can bind to an ErbB ligand; a transmembrane lipophilic domain; a conserved intracellular tyrosine kinase domain; and a carboxyl-terminated signaling domain harboring various tyrosine residues that may be phosphorylated. The ErbB receiver can to be an ErbB receptor of "natural sequence" or an "amino acid sequence variant" thereof. Preferably, the ErbB receptor is a human ErbB receptor of natural sequence. Accordingly, a "member of the ErbB receptor family" is EGFR / ErbBl), ErbB2, ErbB4 or any other ErbB receptor currently known or to be identified in the future. The terms "ErbBl", "epidermal growth factor receptor", "EGFR" and "HERI" are used interchangeably herein and refer to EGFR as described for example in Carpenter et al., (1987) Ann. Rev. Biochem. , 56: 881-914, including their mutant forms of natural origin (eg, an EGFR mutant deletion as in Humphrey et al., (1990) Proc. Nati. Acad. Sci., (EUA) 87: 4207-4211) . The term erbBl refers to the gene that codes for the EGFR protein product. Antibodies against HERI are described, for example, in Murthy et al., (1987) Arch. Biochem. Biophys. , 252: 549-560 and in O 95/25167. The term "ERRP", "EGF receptor related protein", "EGFR-related protein" and "epidermal growth factor receptor related protein" are used interchangeably herein and refer to ERRP as described by example, in US 6399743 and US Publication No. 2003/0096373.

The terms "ErbB2" and "HER2" are used interchangeably herein and refer to the human HER2 protein described, for example, in Semba et al., (1985) Proc. Nati Acad. Sci. , (USA) 82: 6497-6501 and Yamamoto et al., (1986) Nature, 319: 230-234 (accession number Genebank X03363). The term "erbB2" refers to the gene encoding human ErbB2 and "neu" refers to the gene encoding rat pl85neu. The preferred ErbB2 is human ErbB2 of natural sequence. "ErbB3" and HER3"refers to the receptor polypeptide as described, for example, in U.S. Patent Nos. 5183884 and 5480968 as well as Kraus et al., (1989) Proc. Nati. Acad. Sci. (USA) 86: 9193-9197 Antibodies to ErbB3 are known in the art and are described, for example, in U.S. Patent Nos. 5183884, 5480968 and WO 97/35885. The terms "ErbB4 and" HER4"herein, are refer to the receptor polypeptide as described, for example, in EP Patent Application No. 599,274; Plowman et al., (1993) Proc. Nati Acad. Sci USA 90: 1746-1750; and Plowman et al., (1993) Nature 366: 473-475; including its isoforms, e.g., as described in WO 99/19488. Antibodies against HER4 are described, for example, in WO 02/18444. Antibodies to ErbB receptors are commercially available from a number of sources, including, for example, Santa Cruz Biotechnology, Inc., California, USA. The term "amino acid sequence variant" refers to polypeptides having amino acid sequences that differ to some degree from a natural sequence polypeptide. Commonly, amino acid sequence variants will possess less than about 70% sequence identity with at least one receptor binding domain of a natural ErbB ligand or with at least one ligand binding domain of a native ErbB receptor, and preferably , will be at least about 80%, more preferably at least about 90% homologs per sequence with such receptor or ligand binding domains. The amino acid sequence variants possess substitutions, deletions and / or insertions at certain positions within the amino acid sequence of the natural amino acid sequence. Amino acids are designated by the conventional names, one letter codes and three letters. "Sequence identity" is defined as the percentage of residues in the amino acid sequence variant, which is identical after aligning the sequences and entering spaces, if necessary, to achieve maximum percent sequence identity. The methods and computer programs for the alignment are very known in the art. Such a computer program is "Align 2", by Genentech, Inc., which was submitted with user documentation in the United States Copyright Office, Washington DC 20559, on December 10, 1991. "Antibody-dependent cell-mediated cytotoxicity "and" ADCC "refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing Fe receptors (FcRs). { e.g. , Natural Eliminator cells (NK), neutrophils and macrophages) recognize the bound antibody in a target cell and subsequently cause the lysis of the target cell. Primary cells to mediate ADCC, NK cells express only FcyRIII, while monocytes express FcyRI, FcyRII and FcyRIII. The expression FcR in hematopoietic cells is summarized in Table 3 on page 464 of Ravetech and Kinet, (1991) Annu. Rev. Immunol. 9: 457-92. To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in the U.S. Patent may be carried out. No. 5500362 and 5821337. Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and Natural Eliminator (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model such as that described by Clynes et al., (1998) PROC. NATL. ACAD. SCI (USA) 95: 652-656.

"Human effector cells" are leukocytes that express one or more constant region receptors (FcRs) and carry out effector functions. Preferably, the cells express at least FcyRIII and carry out the effector function of ADCC. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; PBMCs and NK cells being preferred. Effector cells can be isolated from a natural source thereof, e.g., from blood or PBMCs, as described herein. The terms "Fe receptor" or "FcR" are used to describe a receptor that binds to the Fe constant region of an antibody. The preferred FcR is a native human FcR sequence. In addition, a preferred FcR is one that binds to an IgG antibody (a gamma receptor) and includes receptors of the subclasses FcyRI, FcyRII and FcyRIII, including allelic variants and alternatively spliced forms of these receptors. FcyRII receptors include FcyRIIA (an "activation receptor") and FcyRIIB (an "inhibition receptor"), which have similar amino acid sequences that differ mainly in the cytoplasmic domains thereof. The activation receptor FcyRIIA contains an activation immunoreceptor motif based on tyrosine (ITAM) in its cytoplasmic domain. The inhibition receptor FcyRIIB contains an immunoreceptor motif of inhibition based on tyrosine (ITIM) in its cytoplasmic domain. (See review by M. in Daéron, Annu, Rev. Immunol., 15: 203-234 (1997)). The FcRs were summarized in Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991); Capel et al., Immunomethods (1994) 4: 25-34; and de Haas et al., (1995) J. Lab. Clin. Med. 126: 330-41. Other FcRs, including those to be identified in the future, are included by the term "FcR" herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs 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 pathway of complement activation is initiated by the binding of the first component of the complement system (Clq) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay can be carried out, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996). The "natural antibodies" are commonly heterotetrametric glycoproteins of approximately 150,000 daltons, composed of two identical light (L) chains and two identical heavy chains (H). Each light chain is linked to a heavy chain by a covalent bond of disulfide, while the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has intrachain chain disulfide bridges spaced regularly. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. It is believed that the particular amino acid residues form an interface between the variable domains of light chain and heavy chain. The term "variable" refers to the fact that certain portions of the variable domains differ widely in sequence among the antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed across the variable domains of antibodies. This is concentrated in three segments called hypervariable regions in both the variable domains of light chain and heavy chain.

The most highly conserved portions of the variable domains are called the structure regions (FRs). The variable domains of the heavy and light natural chains each comprise four FRs, largely adopting a β-sheet configuration, connected by three hypervariable regions, which form connection circuits and in some cases form part of the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and with the hypervariable regions of the other chain contributing to the formation of the antigen-binding site of the antibodies (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5a Ed. Public Health Service, National Institutes of Health, Bethesda, D). The constant domains do not directly involve the binding of an antibody to an antigen, but exhibit several effector functions, such as the participation of the antibody in cellular antibody-dependent cellular cytotoxicity (ADCC). The term "hypervariable region" when used herein refers to residues of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g., residues 24-34 (Ll), 50-56).

(L2) and 89-97 (L3) in the light chain variable domain and 31-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Supra) and / or those residues from a "hypervariable circuit" (eg, residues 26-32 (Ll), 50-52 (L2) and 91-96 (L3) in the variable domain of chain light and 96-101 (H3) in the heavy chain variable domain, Chothia and Lesk (1987) J. Mol. Biol., 196: 901-917). The "region of structure" or "FR" residues are those variable domain residues different from the hypervariable region residues as defined herein. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments each with a unique antigen binding site, and a residual "Fe" fragment, whose name reflects its ability to crystallize easily. The treatment of pepsin produces an F (ab ') 2 fragment that has two antigen-binding sites and is still capable of cross-linking the antigen. "Fv" is the minimum antibody fragment that contains a complete site of antigen recognition and antigen binding. This region consists of a dimer of a variable domain of a heavy chain and a light chain in close non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions confer the specificity of antigen binding to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind the antigen, albeit at a lower affinity than the entire binding site. The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab 'fragments differ from the Fab fragments by the addition of some residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody's articulation region. Fab '-SH is the designation herein for Fab' in which the cysteine residue (s) of the constant domains contain at least one free thiol group. The F (ab ') 2 antibody fragments were originally produced as pairs of Fab' fragments that have articulation cysteines between them. Other chemical couplings of antibody fragments are also known. The "light chains" of antibodies of any vertebrate species can be assigned to one or two clearly distinct types, called kappa (?) And lambda (?), Based on the amino acid sequences of their domains constants The "single chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of the antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide binding between the VH and VL domains that allows the scFv to form the desired structure for antigen binding. For a review of scFv, see Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. , Springer-Verlag, New York, pp. 269-315 (1994). The scFv fragments of the anti-ErbB2 antibody are described in WO 93/16185; U.S. Patent Nos. 5571894 and 5587458. The term "diabodies" refers to small fragments of antibody with two antigen-binding sites, the fragments of which comprise a variable heavy domain (VH) connected to a variable light domain (VL) in the same polypeptide chain (VH-VL). Using too short a union to allow pair formation between the two domains in the same chain, the domains are forced to pair with the complementary domains of another chain and to create two antigen-binding sites. The diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., (1993) Proc. Nati Acad. Sci. USA, 90: 6444-6448.

The "humanized" forms of non-human antibodies (e.g., rodents) are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. Humanization is a method for transferring murine antigen binding information to a non-immunogenic human antibody receptor, and has resulted in many therapeutically useful drugs. The humanization method is generally initiated by transferring all six murine complementarity determining regions (CDRs) to a human antibody structure (Jones et al., (1986) Nature, 321: 522-525). These CDR-grafted antibodies generally do not retain their original affinity for antigen binding, and in fact, affinity is often severely impaired. In addition to the CDRs, select residues of non-human antibody structure can also be incorporated to maintain an appropriate CDR conformation (Chothia et al., (1989) Nature 342: 877). The transfer of key residues of mouse structure to the human receptor in order to support the structural conformation of the grafted CDRs has been shown to restore antigen binding and affinity (Riechmann et al., (1992) J. Mol. Biol., 224 : 487-499; Foote and inter, (1992) J. Mol. Biol. 224: 487-499; Presta et al., (1993) J. Immunol., 151, 2623-2632; erther et al., (1996 J. Immunol, Methods 157: 4986-4995, and Presta et al (2001) Thromb. Haemost 85: 379-389). For the most part, antibodies humanized are human immunoglobulins (receptor antibody) whose residues from a hypervariable region of the receptor 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 specificity , affinity and capacity of the desired antibody. In some cases, the structure region (FR) residues of the human immunoglobulin are replaced by the corresponding non-human residues. In addition, the humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine the performance of the antibody. In general, the humanized antibody will comprise substantially all of at least one and typically two variable domains, in which all or substantially all of the hypervariable circuits correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a sequence of human immunoglobulin. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fe), typically that of a human immunoglobulin. For additional details, see US 6407213; Jones et al., (1986) Nature 321: 522-525; Riechman et al., (1988) Nature 332: 323-329; and Presta, (1992) Curr. Op. Struct. Biol. 2: 593-596. A "free cysteine amino acid" refers to an amino acid residue of cysteine that has been manufactured in an original antibody, has a thiol functional group (-SH), and is not paired as an intramolecular or intermolecular disulfide bridge. The term "thiol reactivity value" is a quantitative characterization of the free amino acid reactivity of cysteine. The thiol reactivity value is the percentage of a free cysteine amino acid in a designed cysteine antibody that reacts with a thiol reactive reagent and is converted to a maximum value of 1. For example, a free cysteine amino acid in an antibody designed cysteine that reacts in a 100% yield with a thiol-reactive reagent, such as a biotin-maleimide reagent, to form a biotin-labeled antibody, has a thiol reactivity value of 1.0. Another amino acid of cysteine made in the same or different original antibody that reacts in an 80% yield with a thiol reactive reagent, has a thiol reactivity value of 8.0. Another amino acid of cysteine made in the same or different original antibody that fails completely to react with a thiol reactive reagent has a thiol reactivity value of 0. The determination of the thiol reactivity value of a particular cysteine can conducted by ELISA analysis, mass spectroscopy, liquid chromatography, autoradiography or other quantitative analytical tests. An "original antibody" is an antibody comprising an amino acid sequence from which one or more amino acid residues are replaced by one or more cysteine residues. The original antibody may comprise a wild type or wild type sequence. The original antibody can have pre-existing amino acid sequence modifications (such as additions, deletions and / or substitutions) relative to other natural, wild-type or modified forms of an antibody. An original antibody can be targeted against a target antigen of interest, e.g., a biologically important polypeptide. Antibodies directed against non-polypeptide antigens (such as tumor-associated glycolipid antigens are also contemplated; see US 5091178. Exemplary antibodies of origin include antibodies that have affinity and selectivity for cell surface and transmembrane receptors and tumor-associated antigens ( TAA.) Other exemplary of origin antibodies include those selected from, and without limitation, anti-estrogen receptor antibody, anti-progesterone receptor antibody, anti-p53 antibody, anti-HER2 / neu antibody, anti-EGFR antibody, anti-cathepsin antibody D, anti-Bcl-2 antibody, anti-E-caderin antibody, anti-CA125 antibody, anti-CA15-3 antibody, anti-CA19-9 antibody, anti-c-erbB antibody -2, anti-P-glycoprotein antibody, anti-CEA antibody, anti-retinoblastoma protein antibody, anti-oncoprotein ras antibody, anti-Lewis X antibody, anti-Ki-67 antibody, anti-PCNA antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD9 / p24 antibody, anti-CD10 antibody, anti-CDIIc antibody, anti-CD13 antibody, anti-CD14 antibody, anti-CD14 antibody CD15, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD30 antibody, anti-CD31 antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD35 antibody, anti-CD35 antibody CD38, anti-CD41 antibody, anti-LCA / CD45 antibody, anti-CD45RO antibody, anti-CD45RA antibody, anti-CD39 antibody, anti-CD45 antibody -CD100, anti-CD95 / Fas antibody, anti-CD99 antibody, anti-CD106 antibody, anti-ubiquitin antibody, anti-CD71 antibody, anti-c-myc antibody, anti-cytokeratin antibody, anti-vimentin antibody, anti-anti-antibody HPV protein, anti-kappa light chain antibody, anti-lambda light chain antibody, anti-melanosome antibody, anti-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 separated and / or recovered from a component of its natural environment. The contaminating components of its natural environment are materials that can interfere with the diagnostic or therapeutic use for the antibody and may include enzymes, hormones and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method and more preferably more than 99% by weight (2) to a sufficient degree to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a rotary cup sequencer or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably, silver dye. The isolated antibody includes the antibody in itself within the recombinant cells since at least one component of the antibody's natural environment will not be present. However, commonly, the isolated antibody will be prepared by at least one purification step. An antibody "binding" to a molecular target or to an antigen of interest, e.g., ErbB2 antigen, is one capable of binding the antigen with sufficient affinity in order to that the antibody is useful for targeting a cell that expresses the antigen. When the antibody is one that binds to ErbB2, it preferentially binds ErbB2 to other ErbB receptors, and may be one that does not cross-react significantly with other proteins such as EGFR, ErbB3 or ErbB4. In such embodiments, the degree of antibody binding to these non-ErbB2 proteins (eg, cell surface binding to the endogenous receptor) will be less than 10% as determined by fluorescence-activated cell selection (FACS) or radioimmunoprecipitation ( RIA). Sometimes, the anti-ErbB2 antibody will not cross-react significantly with the rat neu protein, eg, as described in Schecter et al., (1984) Nature 312: 513 and Drebin et al., (1984) Nature 312_545 -548 Molecular targets for the antibodies encompassed by the present invention include CD proteins and their ligands, such as, but not limited to: (i) CD3, CD4, CD8, CD19, CD20, CD74, CD34, CD34, CD74, CD79a, CD79a , and CD79b (CD79b); (ii) members of the ErbB receptor family such as the EGF receptor, HER2 receptor, HER3 or HER4; (iii) cell adhesion molecules such as LFA-1, Macl, pl50.95, VLA-4, ICAM-1, VCAM and Vv / 53 integrin, including their subunits either alpha or beta (eg, anti-CDlla antibodies, anti-CD18 or anti-CDII); (iv) growth factors such as VEGF; IgE; blood group antigens; flk2 / flt3 receiver; Obesity receiver (OB); receiver mpl; CTLA-4; protein C, BR3m c-met, tissue factor, 37 etc .; and (v) cell surface and transmembrane tumor-associated antigens (TAA). Unless otherwise indicated, the term "monoclonal antibody 4D5" refers to an antibody having antigen-binding residues of, or derivatives of, the murine 4D5 antibody (ATCC CRL 10463). For example, the monoclonal antibody 4D5 can be the murine monoclonal antibody 4D5 or a variant thereof, such as a humanized 4D5. Exemplary humanized 4D5 antibodies include huMAb4D5-l, huMAb4D5-2, huMAb4D5-3, hu Ab4D5-4, huMAb4D5-6, huMAb4D5-7, and huMAb4D5-8 (trastuzumab, HERCEPTIN®) as in the US Patent. No. 5821337. The terms "treat" or "treatment" refer to both therapeutic treatment and prophylactic or preventive measures, wherein the object is to prevent or delay (decrease) an undesired physiological change or disorder, such as the development or extension of the cancer. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, decrease in the degree of disease, stability (ie, not worsening) of the disease state, delay or slowness. of the progress of the disease, improvement or palliation of the state of illness, and remission (either partial or total), whether detectable or undetectable. "Treatment" can also mean the prolongation of survival compared to the expected survival if they do not receive the treatment. Those in need of treatment include those who already have the condition or disorder as well as those prone to having the condition or disorder or those in whom the condition or disorder is to be avoided. The term "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a mammal. In the case of cancer, the therapeutically effective amount of the drug can reduce the number of cancer cells; reduce the size of the tumor; inhibit (i.e., retard to a certain degree and preferably stop) the infiltration of cancer cells into the peripheral organs; inhibit (i.e., delay to a certain degree and preferably stop) tumor metastasis; inhibit, to some degree, the growth of the tumor; and / or alleviating to some degree one or more of the symptoms associated with the cancer. To the extent that the drug can prevent the growth and / or destruction of existing cancer cells, it can be cytostatic and / or cytotoxic. For cancer therapy, efficacy can be calculated, for example, by setting the progress time of the disease (TTP) and / or determining the response rate (RR). The term "bioavailability" refers to the systemic availability (i.e., blood / plasma levels) of a given amount of drug administered to a patient. Bioavailability is an absolute term that indicates the measurement of both the time (proportion) and the total amount (degree) of the drug that reaches the general circulation of a dosage form administered. The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. A "tumor" comprises one or more cancer cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g., squamous cell epithelial cancer), lung cancer including small cell lung cancer, non-small cell lung cancer ("NSCLC"), adenocarcinoma of the lung and squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, cancer colon, cancer rectal, colorectal cancer, endometrial or uterine carcinoma, carcinoma of the salivary gland, kidney or kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, anal carcinoma, penile carcinoma, melanoma, as well as head cancer and neck. A "cancer expressing ErbB" is one that comprises cells that have ErbB protein present on its surface. A "cancer expressing ErbB2" is one that produces sufficient levels of ErbB2 on the surface of its cells so that an anti-ErbB2 antibody can bind to them and has a therapeutic effect with respect to cancer. A cancer that "overexpresses" an antigenic receptor is one that has significantly higher levels of the receptor, such as ErbB2, on the cell surface thereof as compared to a non-cancerous cell of the same type of tissue. Such overexpression can be caused by the amplification of the gene or by increased transcription or translation. Overexpression of the receptor can be determined in a diagnostic or prognostic assay by evaluating increased levels of the receptor protein present on the surface of a cell (e.g., through an immunohistochemical assay; IHC). Alternatively or additionally, the levels of nucleic acid encoding the receptor in the cell can be measured, e.g., through fluorescent in situ hybridization (FISH: see WO 98/45479), Southern blot techniques, or polymerase chain reaction (PCR), such as real-time quantitative PCR (RT-PCR). Tumors that overexpress ErbB2 (HER2) are assessed by immunohistochemical scores corresponding to the number of copies of HER2 molecules expressed per cell, and can be determined biochemically: 0 = 0-10,000 copies / cell, 1 + = at least approximately 200,000 copies / cell, 2 + = at least approximately 500,000 copies / cell, 3 + = approximately 1-2 x 106 copies / cell. Overexpression of HER2 at level 3+, which leads to the independent activation of the tyrosine kinase ligand (Hudziak et al., (1987) Proc. Nati. Acad. Sci., USA 84: 7159-7163), is presented in approximately 30% of breast cancers, and in these patients, relapse-free survival and total survival are decreased (Slamon et al., (1989) Science, 244: 707-712; Slamon et al., ( 1987) Science, 235: 177-182). The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of the cells and / or causes the destruction of the cells. The term is proposed to include radioactive isotopes (e.g., At211, I131, I125 Y90, Re186, Re188, Sm153, Bi212, p32 c6o and radioactive isotopes of Lu), chemotherapeutic agents and toxins such as molecule toxins. small or enzymatically active toxins of bacterial, fungal, plant or animal origin, including synthetic analogues and derivatives thereof. An "autoimmune disease" herein is a disease or disorder that arises from and is directed against the tissues or organs of the individual himself or a co-segregated or manifestation of the same or resulting condition therefrom. In many of these autoimmune and inflammatory disorders, there may be a number of clinical and laboratory markers, including, but not limited to, hypergammaglobulinemia, high levels of autoantibodies, antigen-antibody complex deposits in tissues, benefits of corticosteroid or immunosuppressive treatments. , and lymphoid cell aggregates in affected tissues. Without being limited to any theory concerning autoimmune disease mediated by B cell, it is believed that B cells demonstrate a pathogenic effect in human autoimmune diseases through a multitude of mechanical trajectories, including the production of autoantibodies, immune complex formation, dendritic activation and T cell, cytosine synthesis, direct release of chemosin, and provision of a nidus for ectopic neo-lymphogenesis. Each of these trajectories can participate to different degrees in the pathology of autoimmune diseases. An autoimmune disease can be a organ-specific disease (ie, the immune response is directed specifically against an organ system such as the endocrine system, the hematopoietic system, the skin, the cardiopulmonary system, the gastrointestinal and hepatic systems, the renal system, the thyroid, the ears , the neuromuscular system, the central nervous system, etc.) or a systemic disease that can affect multiple organ systems (for example, systemic lupus erythematosus (SLE), rheumatoid arthritis, polymyositis, etc.). The term "cytostatic" refers to the effect of limiting the function of cells, such as limiting cell growth or cell proliferation. A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include Arlotinib (TARCEVA®, Genentech / OSI Pharm.), Bortezomib (VELCADE®, Millenium Pharm.), Fulvestrant (FASLODEX®, Astra Zeneca), Sutent (SU11248, Pfizer), Letrozole (FEMARA®, Novartis). , Imatinib mesylate (GLEEVEC®, Novartis), PTK787 / ZK 222584 (Novartis), Oxaliplatin (Eloxatin®, Sanofi), 5-FU (5-fluoroacyl), Leucovorin, Rapamycin (Sirolimus, RAPAMUNE®, Wyeth), Lapatinib (GSK) 572016, GlaxoSmithKline), Lonafarnib (SCH 66336), Sorafenib (BAY 43-9006, Bayer Labs.), And Gefitinib (IRESSA®, Astra Zeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbocuone, meturedopa and uredopa; ethylene imines and methylamelamines including altretamine, triethylene-ammine, triethylene-phosphoramide, triethylene-thiophosphoramide and trimethyl-melamine; acetogenins (especially bulatacin and bulatacinone); a captothecin (including the synthetic analog topotecan); Bryostatin; Callistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin and bizelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmicin (including synthetic analogues, K -2189 and CB1-TM1); eleuterobin; pancratistatin; a sarcodicitin; spongistatin; nitrogen mustards such as chlorambucil, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembicin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediin antibiotics (eg, calicheamicin, especially gamma II calicheamicin and omegall calicheamicin, (Agnew, Chem Intl. Ed. Engl., (1994) 33: 183-186; dynemycin, including dynemycin A; bisphosphonates, such as clodronate A waitmycin, so as chromophore neocarzinostatin and chromophoric antibiotics of enediin related chromoprotein), aclacinomisins, actinomycin, autramycin, azaserin, bleomycins, cactinomycin, carabomycin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, Adriamycin® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydixorubicin), epirubicin, esububicin, idarubicin, marcelomycin, mitomycin such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, chelamicin, rodoubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluoroacyl (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, tiamiprin, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocythabin, floxuridine; androgens such as calusterone, dromostathionone propionate, epithiostanol, mepitiostane, testolactone, - anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid filler such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamin; demecolcine; diazicuone; alfornitin; eliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; Pentostatin; fenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rizoxin; sizofiran; spirogermanium; tenuazonic acid; triazicuone; 2, 2 ', 2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); uretan; vindesine; Dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacitosina; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids; eg, paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) CREAMofor-free ABRAXANETM, nanoparticle formulation manufactured from paclitaxel albumin (American Pharmaceutical Partners, Schaumberg, Illinois) and doxetaxel (TAXOTERE®, Rhone-Poulenc, Anthony , France); chlorambucil; gemicitabine (Gemzar "), 6-thioguanine, mercaptopurine, methotrexate, platinum analogs such as cisplatin and carboplatin, vinblastine, platinum, etoposide (VP-16), ifosfamide, mitoxantrone, vincristine, vinorebu (Navelbine); novantrone; teniposide; edatrexate; Daunomycin; aminopterin; xeloda; ibandronate; CPT-11; Topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Also included in this definition of "chemotherapeutic agent" are: (i) anti -hormone agents that act to regulate or inhibit the action of hormones in tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including Novaldex), raloxifene, droloxifene, 4-hydroxy tamoxifen, trioxifen, keoxifene, LY117018, onapristone and toremifene (Fareston); (ii) aromatase inhibitors that inhibit the aromatase enzyme, which regulates the production of estrogen in the adrenal glands, such as, for example, 4 (5) -imidazoles, aminoglutethimide, megestrol acetate Megace®, AROMASIN® exemestane, formestane, fadrozolo, Rivisor® vorozolo, Femara® letrozolo and Arimidex® anastrozolo; (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analogue; (iv) aromatase inhibitors; (v) protein kinase inhibitors; (vi) lipid kinase inhibitors; (vii) antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways involved in aberrant cell proliferation, such as for example, PKC-alpha, Ralf and H-Ras; (viii) ribozymes such as an inhibitor of VEGF expression (e.g., ribozyme A GIOZYME®) and an HER2 expression inhibitor; (ix) vaccines such as vaccines for gene therapy, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine and VAXID® vaccine; PROLEUKIN® rlL-2; Topoisomerase 1 inhibitor LURTOTECAN®; ABARELIX® rmRH; (x) anti-angiogenic agents such as bevacizuman (AVASTIN®, Genentech); and (xi) pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

As used herein, the term "EGFR-targeted drug" refers to a therapeutic agent that binds to EGFR and, optionally, inhibits EGFR activation. Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies that bind to EGFR include Mab 579 (ATCC CRL HB 8506), Mab 455 (ATCC CRL HB 8507), Mab 225 (ATCC CRL 8508), Mab 528 (ATCC CRL 8509) (see US 4943533, Mendelsohn et al. .) and variants thereof, such as 225 chimerized (C225 or Cetuximab; ERBITUX®) and reconfigured human 225 (H225) (see WO 96/40210, Imclone Systems Inc.); antibodies that bind mutant AGFR type II (U.S. Patent No. 5,212,290); humanized and chimeric antibodies that bind to EGFR as described in US 589 ° 996; and human antibodies that bind to EGFR such as ABX-EGF (see WO 98/50433, Abgenix). The antibody Anti-EGFR can be conjugated with a cytotoxic agent, thus generating an immunoconjugate (see e.g., EP 659,439a2, Merck Patent GmbH). Examples of small molecules that bind to EGFR include ZD1839 or Gefitinib (IRESSA ™, Astra Zeneca), Erlotinib HC1 (CP-358774, TARCEVA ™, Genentech / OSI) and AG1478, AG1571 (SU 5271; Sugen). Inhibitors of protein kinase include kinase inhibitors that inhibit to some degree the tyrosine kinase activity of a tyrosine kinase such as an ErbB receptor. Examples of such inhibitors include drugs directed to EGFR noted in the preceding paragraph as well as quinazolines such as PD 153035,4- (3-chloroanilino) quinazoline, pyridopyrimidines, pyrimidopyrimidines, pyrrolopyrimidines such as CGP 59326. CGP 60261 and CGP 62706, and pyrazolopyrimidines, 4- (phenylamino) -7H-pyrrolo [2, 3-d] pyrimidines, curcumin (diferuloylmethane, 4,5-bis (4-fluoroanilino) phthalimide), trifostins containing nitrothiophene residues; PD-0183805 (Warner-Lambert); antisense molecules (e.g., those that bind to the nucleic acid encoding ErbB); Quinoxalines (US 5804396); trifostins (US 5804396); ZD6474 (Astra Zeneca); PTK-787 (Novartis / Shering AG); pan-ErbB inhibitors such as CI-1033 (Pfizer); Affinitac (ISIS 3521; Isis / Lilly); Imatinib mesylate (Gleevec; Novartis); PKI 166 (Novartis); GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB- 569 (yeth); Semaxanib (Sugen); ZD6474 (Astra Zeneca); PTK-787 (Novartis / Shering AG); INC-ICII (Imclone); or as described in any of the following patent publications: WO 99/09016 (American Cyanamid); WO 98/43960 (American Cyanamid); WO 97/38983 (Warner Lambert); WO 99/06378 (Warner Lambert); WO 99/06396 (Warner Lambert); WO 96/30347 (Pfizer, Inc.); WO 96/33978 (Zeneca); WO 96/3397 (Zeneca); and WO 96/33980 (Zeneca). An "anti-angiogenic agent" refers to a compound that blocks, or interferes to some degree, the development of blood vessels. The anti-angiogenic factor, for example, can be a small molecule or antibody that binds to a growth factor or growth factor receptor involved in the promotion of angiogenesis. The preferred anti-angiogenic factor herein is an antibody that binds to vascular endothelial growth factor (VEGF). The term "cytosine" is a generic term for proteins released by a cell population that acts in another cell as cellular mediators. Examples of such cytosines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytosines are growth hormone such as human growth hormone, human growth hormone N-methionyl, and bovine growth hormone; hormone parathyroid; thyroxine; insulin; proinsulin; relaxin; Prorrelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), or luteinizing hormone (LH); liver growth factor; fibroblast growth factor; prolactin; placental lactogen; alpha-factor and -beta of tumor necrosis; Mullerian inhibition substance; peptide associated with mouse gonadotropin; inhibin; activin; Vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-b; platelet growth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; factor I and II of insulin-like growth; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-alpha, beta and gamma; colony stimulation factors (CFSs) such as CSF-macrophage (M-CSF); CSF-granulocyte-macrophage (GM-CSF); and CSF-granulocyte (G-CSF); interleukins (lys) such as IL-1, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; a tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors including LIF and ligand kit (KL). As used herein, the term "cytosine" includes proteins from natural or recombinant cell culture sources and biologically active equivalents of naturally occurring cytosines.

The term "prodrug" as used in this application refers to a precursor or derivative form of a pharmaceutically active substance that is less toxic to tumor cells compared to the original drug and is capable of being activated enzymatically or hydrolytically or become a more active form of origin. See, e.g., Wilman, "Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions, 14 pp. 375-382, 615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery, Borchardt et al., (Ed.) Pp. 247-267, Humana Press (1985). The prodrugs of this invention include, but are not limited to, prodrugs containing phosphate, prodrugs containing thiophosphate, prodrugs containing sulfate, prodrugs containing peptide, D-amino acid-modified prodrugs, glycosylated prodrugs, prodrugs containing b-lactam , prodrugs containing optionally substituted phenoxyacetamide or prodrugs containing optionally substituted phenylacetamide, 5-fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the most active cytotoxic free drug. Examples of cytotoxic drugs that can be derivatized into a prodrug form for use in this invention, include, but are not limited to, those chemotherapeutic agents described above. A "liposome" is a small vesicle composed of various types of lipids, phospholipids and / or surfactants which is useful for the delivery of a drug (such as the anti-ErbB antibodies described herein and, optionally, a chemotherapeutic agent) to a mammal. The components of the liposome are commonly ordered in a bilayer formation similar to the lipid ordering of the biological membranes. The term "package insert" is used to refer to instructions that are customarily included in commercial packages of therapeutic products, which contain information about indications, uses, doses, administration, contraindications and / or precautions concerning the use of such therapeutic products. "Phage display" is a technique by which variant polypeptides are deployed as fusion proteins to a coat protein on the surface of phage particles, e.g., filamentous phage. One utility of phage display lies in the fact that large libraries of randomized protein variants can be selected quickly and efficiently by those sequences that bind to a target molecule with high affinity. The deployment of peptide libraries and phage proteins has been used to visualize millions of polypeptides by ones with specific binding properties. Multipurpose phage display methods have been used to deploy small random peptides and small proteins, typically through fusions for filamentous phage pIII or pVIII (Wells and Lowman (1992) Curr Opin. Struct. Biol., 3: 355-362, and references cited therein). In the monovalent phage display, a protein or peptide library is fused to a phage coat protein or to a portion thereof, and is expressed at low levels in the presence of a wild-type protein. The effects of avidity are reduced in relation to the polyvalent phage so that selection is based on the intrinsic affinity of the ligand, and phagemid vectors are used that simplify DNA manipulations. Lowman and Wells, Methods: A companion to Methods in Enzymology, 3: 205-0216 (1991). Phage display includes techniques for producing antibody-like molecules (Janeway C, Travers, P., Walport M., Shlomchik (2001) Immunology, 5th edition, Garland Publishing, New York, p627-628, Lee et al). A "phagemid" is a plasmid vector that has a bacterial origin of replication, e.g., ColEl, and a copy of an intergenic region of a bacteriophage. The phagemid can be used in any known bacteriophage, including filamentous bacteriophage and lamdoid bacteriophage. The plasmid will also generally contain a selectable marker for resistance to the antibiotic. The DNA segments cloned within these vectors can propagate as plasmids. When the cells harboring these vectors are supplied with all the genes necessary for the production of phage particles, the mode of replication of the plasmid changes to rolling circle replication to generate copies of a strand of plasmid DNA and packaging phage particles. The phagemid can form infectious or non-infectious phage particles. This term includes phagemids that contain a phage coat protein gene or a fragment thereof linked to a heterologous polypeptide gene as a gene fusion such that the heterologous polypeptide is deployed on the surface of the phage particle. "Union", "binding unit" or "union" means a chemical residue comprising a covalent bond or a chain of atoms that covalently binds an antibody to a drug residue. In several embodiments, a linkage is specified as L. The linkages include a divalent radical such as an alkyldiyl, an arylene, a heteroarylene, residues such as: - (CR2) nO (CR2) n- / alkyloxy repeating units (eg , polyethyleneoxy, PEG, polymethyleneoxy) and alkylamino (eg, polyethyleneamino, Jeffamine ™); and diacid ester and amides including succinate, succinamide, diglycolate, malonate, and caproamide. The term "label" means any residue that can be covalently bound to an antibody and that it works to: (i) provide a detectable signal; (ii) interacting with a second tag to modify the detectable signal provided by the first or second tag, e.g., FRET (fluorescence resonance energy transfer); (iii) stabilize the interactions or increase the binding affinity, with antigen or ligand; (iv) affect mobility, eg, electrophoretic mobility, or cellular permeability, by charge, hydrophobicity, form or other physical parameters, or (v) provide a capture residue, to modulate ligand affinity, antibody binding / antigen or ionic complexation. The definitions and conventions used in the present generally follow S.P. Parker, Ed., MaGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel E., and ilen S., Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., New York. There are many organic compounds in optically active forms, i.e., they have the ability to rotate the flat-polarized plane of light. When describing an optically active compound, the prefixes D and L or R and S, are used to denote the absolute configuration of the molecule around its chiral center (s). The prefixes d and 1 or (+) and (-) are used to designate the sign of plane-polarized light rotation by the compound, meaning (-) or 1 that the compound is levorotatory. A compound with the prefix (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical, except that they are mirror images of each other. A specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often referred to as an enantiomeric mixture. A 50-50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which may occur when there has been no stereoselection or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, devoid of optical activity. The phrase "pharmaceutically acceptable salt", as used herein, refers to pharmaceutically acceptable organic or inorganic salts of an ADC. Exemplary salts include, but are not limited to, salts of sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (ie, 1, 1'-methylene-bis- (2-hydroxy-3-naphthoate)). A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, an ion of - - succinate or other counterion. The counterion can be any organic or inorganic residue that stabilizes the charge in the parent compound. In addition, a pharmaceutically acceptable salt can have more than one charged atom in its structure. Examples wherein the multiple charge atoms are part of the pharmaceutically acceptable salt may have multiple counterions. Thus, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counterions. "Pharmaceutically acceptable solvate" refers to an association of one or more solvent molecules and an ADC. Examples of solvents forming pharmaceutically acceptable solvates include, but are not limited to water, isopropanol, ethanol, methanol, DSO, ethyl acetate, acetic acid, and ethanolamine. The following abbreviations are used herein and have the stated definitions: BE is beta-mercaptoethanol, Boc is N- (t-butoxycarbonyl), cit is citrulline (2-amino-5-ureido pentanoic acid), dap is dolaproine, DCC is 1, 3-dicyclohexylcarbodiimide, DCM is diechloromethane, DEA is diethylamine, DEAD is diethylazodicarboxylate, DEPC is diethylphosphorylcyanate, DIAD is diisopropylazodicarboxylate, DIEA is?,? - diisopropylethylamine, dil is dolaisoleucine, DMA is dimethylacetamide, DMAP is 4-dimethylaminopyridine, DME is ethylene glycol dimethyl ether (or 1,2-dimethoxyethane), DMF is NN- dimethylformamide, DMSO is dimethylsulfoxide, doe is dolafenin, dov is NN-dimethylvaline, DTNB is 5,5'-dithiobis (2-dinitrobenzoic acid), DTPA is diethylenetriaminepentaacetic acid, DTT is dithiothreitol, EDCO is 1- (3-dimethylaminopropyl) - 3-ethylcarbodiimide hydrochloride, EEDQ is 2-ethoxy-l-ethoxycarbonyl-1,2-dihydroquinoline, ES-MS is mass spectrometry by electro-alloy, EtOAc is ethyl acetate, Fmoc is N- (9-fluorenolmethoxycarbonyl), gly is glycine, HATU is O- (7-azabenzotriazol-l-il) -?,?,? ' ,? ' - tetramethyluronium hexafluorophosphate, HOBt is 1- | hydroxybenzotriazolo, HPLC is high pressure liquid chromatography, ile is isoleucine, lys is lysine, MeCN (CH3CN) is acetonitrile, eOH is methanol, Mtr is 4-anisilyphenylmethyl (or 4-methoxytrityl), ñor is (1S, 2R) - (+) - norephedrine, PAB is p-aminobenzylcarbamoyl, PBS is phosphate-buffered saline (pH 7), PEG is polyethylene glycol, Ph is phenyl, Pnp is p-nitrophenyl, MC is 6 - maleimidacaproil, phe is L-phenylalanine, PyBrop is bromo tris-pyrrolidino phosphonium hexafluorophosphate, SEC is chromatography by size exclusion, Su is succinimide, TFA is trifluoroacetic acid, TLC is thin layer chromatography, OV is ultraviolet and val is valine. ANTIBODIES DESIGNED OF CYSTEINE The compounds of the invention include designed antibodies of cysteine wherein one or more amino acids of a original wild-type antibody are replaced with a cysteine amino acid. Any form of antibody can be designed in this way, i.e., to be mutated. For example, a Fab antibody fragment of origin can be designed to form a Fab made of cysteine, referred to herein as "ThioFab". Similarly, a monoclonal antibody of origin can be designed to form a "ThioMab". It should be noted that a single site mutation produces a single designed cysteine residue in a ThioFab, although a single single site mutation produces two designed cysteine residues in a ThioMab due to the dimeric nature of the IgG antibody. Mutants with replaced cysteine (Cys) residues ("manufactured") are evaluated by the reactivity of the newly introduced cysteine thiol designed groups. The thiol reactivity value is relative, numerical term in the range of 0 to 1.0 and can be calculated for any designed cysteine antibody. The thiol reactivity values of cysteine engineered antibodies of the invention are in the ranges of 0.6 to 1.0; 0.7 to 1.0; or 0.8 to 1.0. The methods of design, selection and preparation of the invention allow designed cysteine antibodies that are reactive with the electrophilic functionality. These methods also allow conjugated antibody compounds such as antibody-drug conjugates.

(ADC) with drug molecules in designated, designed selective sites. Reactive cysteine residues on an antibody surface allow a drug residue to be specifically conjugated through a reactive thiol group such as maleimide or haloacetyl. The nucleophilic reactivity of the thiol functionality of a Cys residue to a maleimide group is approximately 1000 times higher compared to any other amino acid functionality in a protein, such as the amino group of lysine residues or the amino group of N-terminus. Specific thiol functionality in iodoacetyl reagents and maleimide can react with amine groups, but a higher pH (> 9.0) and longer reaction times are required (Garman 1997, Non-Radioactive Labeling: A Practical Approach, Academic Press, London ). The designed cysteine antibodies of the invention preferably retain the antigen binding capacity of their original wild-type antibody counterparts. Thus, the designed cysteine antibodies are capable of binding, preferably specifically, to antigens. Such antigens include, for example, tumor associated antigens (TAA), cell surface receptor proteins and other cell surface molecules, transmembrane proteins, signaling proteins, cell survival regulatory factors, cell proliferation regulatory factors, molecules associated with (eg, that known or suspected to contribute to functionality a) tissue development or differentiation, lymphokines, cytokines, molecules involved in the regulation of the cell cycle, molecules involved in vasculogenesis and molecules associated with (eg, it is known or suspected that contribute to functionality a) angiogenesis. The tumor-associated antigen can be a cluster differentiation factor (i.e., a CD protein). An antigen to which a designed cysteine antibody is capable of binding can be a member of a subset of one of the aforementioned categories, wherein the other subset (s) of that category comprise other molecules / antigens. which have a distinct characteristic (with respect to the antigen of interest). The original antibody can also be a humanized antibody selected from huMAb4D5-l, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7, and huMAb4D5-8 (Trastuzumab, HERCEPTIN®), as described in Table 3 of US 5821337, expressly incorporated herein by reference; humanized 520C9 antibodies (WO 93/21319) and humanized 2C4 as described herein. The designed cysteine antibodies of the invention can be site-specifically and efficiently coupled with a thiol-reactive reagent. The thiol-reactive reagent can be a multifunctional linker reagent, a capture reagent, ie, affinity, labeling (eg, a biotin linker reagent), a detection label (eg, a fluorophore reagent), a solid phase immobilization reagent (eg, SEPHAROSE ™, polystyrene or glass), or a drug binding intermediary. An example of a thiol-reactive reagent is N-ethyl maleimide (NE). In an exemplary embodiment, the reaction of a ThioFab with a biotin linker reagent provides a biotinylated ThioFab by which the presence and reactivity of the manufactured cysteine residue can be detected and calculated. The reaction of a ThioFab with a multifunctional linker reagent provides a ThioFab with a functionalized linkage that can be reactivated further with a drug residue reagent or other label. The reaction of a ThioFab with a drug binding intermediate provides a ThioFab-drug conjugate. The exemplary methods described herein, can generally be applied to the identification and production of antibodies, and more generally, to other proteins through the application of the design and visualization steps described herein. Such a procedure can be applied to the conjugation of other thiol-reactive agents in which the reactive group is, for example, a maleimide, an iodoacetamide, a pyridyl disulfide, or another thiol-reactive conjugating partner (Haugland, 2003, Molecular Probes Handbook of - - Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.,; Brinkley 1992, Bioconjugate Chem., 3: 2; Garman, 1997, Non-Radioactive Labeling: A Practical Approach, Academic Press, London; Means (1990) Bioconjugate Chem., 1: 2; Hermanson, G., in Bioconjugate Techniques (1996) Academic Press, San Diego, pp. 40-55, 643-671). The partner can be a cytotoxic agent (eg, a toxin such as doxorubicin or pertussis toxin), a fluorophore such as a fluorescent dye such as fluorescein or rhodamine, a chelating agent for a visual or radiotherapeutic metal, a peptidyl tag or no peptidyl or detection mark, or a purity modifying agent such as various polyethylene glycol isomers, a peptide that binds to a third component or another carbohydrate or lipophilic agent. The sites identified in the exemplary antibody fragment, hu4D5Fabv8, are present mainly in the constant domain of an antibody that is highly conserved across all antibody species. These sites should be broadly applicable to other antibodies, without the need for additional structural design or knowledge of specific antibody structures, and without interference in the antigen-binding properties inherent to the variable domains of the antibody. The designed antibodies of cysteine that can - - being useful in the treatment of cancer include, but are not limited to, antibodies against cell surface receptors and tumor-associated antigens (TAA). Such antibodies can be used as naked antibodies (not conjugated to a drug or brand residue) or as antibody-drug conjugates of Formula I (ADC). Tumor-associated antigens are known in the art, and can be prepared for use in the generation of antibodies using methods and information well known in the art. In attempts to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify transmembrane or other tumor-associated polypeptides that are specifically expressed on the surface of one or more particular types of cancer cell compared to one or more noncancerous normal cancer cells. Frequently, such tumor-associated polypeptides are more abundantly expressed on the surface of cancer cells compared to the surface of non-cancerous cells. The identification of such tumor-associated cell surface antigen polypeptides has resulted in the ability to specifically target cancer cells for destruction through antibody-based therapies. Examples of TAA include, but are not limited to, TAA (1) - (36) listed below. For convenience, information related to these antigens, all of which are known in the art, is listed below and includes names, alternative names, Genbank access numbers and primary references, following protein and nucleic acid sequence identification conventions. National Center for Biotechnology Information (NCBI). The nucleic acid and protein sequences corresponding to TAA (1) - (36) are available in public databases such as GenBank. Antibodies associated to the tumor directed by antibodies include all the amino acid sequence variants and isoforms that possess at least about 70%, 80%, 85%, 90% or 95% sequence identity in relation to the sequences identified in the references cited, or that exhibit substantially the same properties or biological characteristics as TAA having the sequence found in the references cited. For example, a TAA having a variant sequence is generally capable of specifically binding to an antibody that binds specifically to TAA with the corresponding sequence listed. The sequences and description in the reference specifically cited herein are expressly incorporated by reference. ANTIGENS ASSOCIATED WITH TUMOR (l) - (36): (1) BMPR1B (bone morphogenic protein receptor type IB, Access to Genbank No. NM_001203) ten Dijke P., et al., Science 264 (5155): 101-104 (1994), Oncogene 14 (11): 1377-1382 (1997)); WO 200463362 (Claim 2); WO 2000342661 (Claim 12); US 2003134790-A1 (Page 38-39); WO 2002102235 (Claim 13, page 296); WO 200355443 (Page 91-92); WO 200299122 (Example 2; Page 528-530); WO 2003029421 (Claim 6); WO 2003024392 (Claim 2, Figure 112); WO 200298358 (Claim 1; Page 183); WO 200254940 (Page 100-101); WO200259377 (page 349-350); WO 200230268 (Claim 27; Page 376); WO200148204 (Example; Figure 4) NP_001194 bone morphogenic protein receptor type? / Pid = NP_001194.1- Cross references: M1: 603248; NP_001194.1; AY06599. (2) E16 (LATI, SLC7A5, Access to Genbank No. NM_003486) Biochem. Biophys. Res. Commun. 255 (2), 283-288 (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); WO200299074 (Claim 19; Page 127-129); WO200286443 (Claim 27; Pages 222, 393); WO2003003906 (Claim 10; Page 293); WO200264798 (Claim 33; Page 93-95); WO200014228 (Claim 5; Page 133-136); US2003224454 (Figure 3); WO2003025138 (Claim 12; Page 150); NP_003477 family 7 of solute vehicle 7 (cationic amino acid transporter, system y +), member 5 /pid=NP_003477.3 - Homo sapiens Cross-references: MIM: 600182; NP_003477.3; NM_015923; NM_003486_1 (3) STEAP1 (transmembrane six epithelial antigen of prostate, Genbank Access No. NM_012449) Cancer Res. 61 (15), 5857-5860 (2001), Hubert, R.S., et al (1999) Proc. Nati Acad. Sci. USES. 96 (25): 14523-14528); WO2004065577 (Claim 6); WO2004027049 (Figure 1L); EP1394274 (Example 11); WO2004016225 (Claim 2); O2003042661 (Claim 12); US2003157089 (Example 5); US2003185830 (Example 5); US2003064397 (Figure 2); WO200289747 (Example 5; Page 618-619); WO2003022995 (Example 9; Figure 13A, Example 53; Page 173, Example 2; Figure 2A); NP_036581 six transmembrane epithelial antigen of the prostate Cross-references: MIM: 604415; NP_036581.1; NM_012449_1 (4) 0772P (CA125, MUC16, Access to Genbank No. AF361486) J. Biol. Chem. 276 (29): 27371-27375 (2001)); WO2004045553 (Claim 14); O200292836 (Claim 6, Figure 12); WO200283866 (Claim 15; Page 116-121); US2003124140 (Example 16); Cross references: GI: 34501467; AAK74120.3; AF361486_1 (5) MPF (MPF, MSLN, S R, megakaryocyte enhancement factor, mesothelin, Access to Genbank No. NM_005823) Yamaguchi, N., et al Biol. Chem. 269 (2), 805-808 (1994), Proc. Nati Acad. Sci. U.S.A. 96 (20): 11531-11536 (1999), Proc. Nati Acad. Sci. U.S.A. 93 (1) .136-140 (1996), J. Biol. Chem. 270 (37): 21984-21990 (1995)); O2003101283 (Claim 14); (WO2002102235 (Claim 13; Page 287-288); O2002101075 (Claim 4; Page 308-309); O200271928 (Page 320-321); O9410312 (Page 52-57); Cross References: IM: 601051; NP_005814.2; NM_005823_1 (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, family 34 of solute vehicle (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, Access to Genbank No. NM_006424) J. Biol. Chem. 277 (22): 19665-19672 (2002), Genomics 62 (2): 281-284 (1999), Feild, JA , et al (1999) Biochem.

Biophys. Res. Commun. 258 (3): 578-582); WO2004022778 (Claim 2); EP1394274 (Example 11); WO2002102235 (Claim 13; Page 326); EP875569 (Claim 1; - - Page 17-19); WO200157188 (Claim 20; Page 329); O2004032842 (Example IV); WO200175177 (Claim 24; Page 139-140); Cross references: MIM: 604217; NP 006415.1; NM 006424 1 (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SE AG, Semaphorin 5b Hlog, sema domain, seven repeats of thrombospondin (type 1 and similar to type 1), transmembrane domain (TM) and short cytoplasmic domibnio , (semaphorin) 5B, Genbank Accession No. AB040878) Nagase T., et al (2000) DNA Res. 7 (2): 143-150); O2004000997 (Claim 1); WO2003003984 (Claim 1), - O200206339 (Claim 1; Page 50); WO200188133 (Claim 1; Page 41-43, 48-58); WO2003054152 (Claim 20); O2003101400 (Claim 11); Access: Q9P283; EMBL; AB040878; BAA95969.1. Genew; HGNC: 10737; (8) PSCA hlg (2700050C12Rik, C530008O16Rik, cDNA 2700050C12 RIKEN, cDNA gene 2700050C12 RIKEN, Access to Genbank No. AY358628); Ross et al (2002) Cancer Res. 62: 2546-2553; US2003129192 (Claim 2); US2004044180 (Claim 12); US2004044179 (Claim 11); US2003096961 (Claim 11); US2003232056 (Example 5); WO2003105758 (Claim 12); US2003206918 (Example 5); EP1347046 - - (Claim 1); WO2003025148 (Claim 20); Cross references: GI: 37182378; AAQ88991.1; AY358628_1 (9) ETBR (Endothelin type B recipient, Access to Genbank No. AY275463); Nakamuta M., et al Biochem. Biophys. Res. Commun. 177, 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., Yanagisawa M., et al Biochem. Biophys. Res. Commun. 178, 656-663, 1991; Elshourbagy N.A. , et al J. Biol. Chem. 268, 3873-3879, 1993; Haendler B., et al J. Cardiovasc. Pharmacol. 20, sl-S4, 1992; Tsutsumi M., et al Gene 228, 43-49, 1999; Strausberg R.L., et al Proc. Nati Acad. Sci. U.S.A. 99, 16899-16903, 2002; Bourgeois C, et al J. Clin. Endocrinol Metab. 82, 3116-3123, 1997; Okamoto Y., et al Biol. Chem. 272, 21589-21596, 1997; Verheij J.B., et al Am. J. Med. Genet. 108, 223-225, 2002; Hofstra R.M.W., et al. Eur. J. Hum. Genet 5, 180-185, 1997; Puffenberger E.G., et al Cell 79, 1257-1266, 1994; Attie T., et al, Hum. Mol. Genet 4, 2407-2409, 1995; Auricchio A., et al Hum. Mol. Genet 5: 351-354, 1996; Amiel J., et al Hum. Mol. Genet 5, 355-357, 1996; Hofstra R.M.W., et al Nat. Genet 12, 445-447, 1996; Svensson P.J., et al Hum. Genet 103, 145-148, 1998; Fuchs S., et al Mol. Med. 7, 115-124, 2001; Pingault V., et al (2002) Hum. Genet 111, 198-206; O2004045516 (Claim 1); O2004048938 (Example 2); O2004040000 (Claim 151); O2003087768 (Claim 1); O2003016475 (Claim 1); O2003016475 (Claim 1); O200261087 (Figure 1); WO2003016494 (Figure 6); O2003025138 (Claim 12; Page 144); WO200198351 (Claim 1; Page 124-125); EP522868 (Claim 8, Figure 2); O200177172 (Claim 1; Page 297-299); US2003109676; US6518404 (Figure 3); US5773223 (Claim la; Col 31-34); O2004001004; (10) SG783 (RNF124, hypothetical protein FLJ20315, Access to Genbank No. NM_017763); O2003104275 (Claim 1); WO2004046342 (Example 2); WO2003042661 (Claim 12); WO2003083074 (Claim 14; Page 61); WO2003018621 (Claim 1); O2003024392 (Claim 2, Figure 93); O200166689 (Example 6); Cross references: Locus ID: 54894; NP_060233.2; NM_017763_1 (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STA P1, STEAP2, STMP, gene 1 associated with prostate cancer, protein 1 associated with prostate cancer, transmembrane epithelial antigen 6 from prostate 2, transmembrane prostate protein six, Access to Genbank No. AF455138) Lab. Invest. 82 (11): 1573-1582 (2002)); WO2003087306; US2003064397 (Claim 1, Figure 1); WO200272596 (Claim 13; Page 54-55); WO200172962 (Claim 1, Figure 4B); WO2003104270 (Claim 11); WO2003104270 (Claim 16); US2004005598 (Claim 22); WO2003042661 (Claim 12); US2003060612 (Claim 12, Figure 10); WO200226822 (Claim 23, Figure 2); WO200216429 (Claim 12, Figure 10); Cross references: GI: 22655488; AAN04080.1; AF455138_1 (12) TrpM4 (BR22450, FLJ20041, TRP 4, TRPM4B, transient receptor potential cation channel, subfamily, member 4, Access to Genbank No. NM_017636) Xu, X.Z., et al Proc. Nati Acad. Sci. USES. 98 (19): 10692-10697 (2001), Cell 109 (3): 397-407 (2002), J. Biol. Chem. 278 (33): 30813-30820 (2003)); US2003143557 (Claim 4); WO200040614 (Claim 14; Page 100-103); O200210382 (Claim 1, Figure 9A); O2003042661 (Claim 12); O200230268 (Claim 27; Page 391); US2003219806 (Claim 4); O200162794 (Claim 14, Figure 1A-D); Cross references: MIM: 606936; NP_060106.2; N _017636_1 (13) CRYPT (CR, CR1, CRGF, CRYPT, TDGF1, factor of growth derived from teratocarcinoma, Access to Genbank No. NP_003203 or 40 NM_003212) Ciccodicola, A., et al EMBO J. 8 (7): 1987-1991 (1989), Am. J. Hum. Genet 49 (3): 555-565 (1991)); US2003224411 (Claim 1); O2003083041 (Example 1); O2003034984 (Claim 12); WO200288170 (Claim 2; Page 52-53); WO2003024392 (Claim 2, Figure 58); WO200216413 (Claim 1; Page 94-95, 105); WO200222808 (Claim 2, Figure 1); US5854399 (Example 2; Col 17-18); US5792616 (Figure 2); Cross references: MIM: 187395; NP_003203.1; NM_003212_1 (14) CD21 (CR2 (Complement Receptor 2) or C3DR (C3d / Epstein Barr Virus Receptor) or Hs.73792 Access to Genbank No. 26004) Fujisaku et al (1989) J. Biol. Chem. 264 (4): 2118-2125); Weis J.J., et al J. Exp. Med. 167, 1047-1066, 1988; Moore M., et al Proc. Nati Acad. Sci. USES. 84, 9194-9198, 1987; Barel. , et al Mol. Immunol. 35, 1025-1031, 1998; Weis J.J., et al Proc. Nati 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); WO2004045520 (Example 4); WO9102536 (Figure 9.1-9.9); WO2004020595 (Claim 1); Access: P20023; Q13866; Q14212; EMBL; M26004; AAA35786.1. (15) CD79b (CD79B, CD79p, IGb (beta associated with immunoglobulin), B29, Access to Genbank No. NM_000626 or 11038674) Proc. Nati Acad. Sci. USES. (2003) 100 (7) .4126-4131, Blood (2002) 100 (9): 3068-3076, Muller et al (1992) Eur. J. Immunol. 22 (6): 1621-1625); O2004016225 (claim 2, Figure 140); WO2003087768, US2004101874 (claim 1, page 102); WO2003062401 (claim 9); WO200278524 (Example 2); US2002150573 (claim 5, page 15); US5644033; O2003048202 (claim 1, pages 306 and 309); WO 99/558658, US6534482 (claim 13, Figure 17A / B); O200055351 (claim 11, pages 1145-1146); Cross references: MIM: 147245; NP_000617.1; NM_000626_1 (16) FcRH2 (IFGP4, IRTA4, SPAP1A (Protein the phosphatase anchor containing SH2 domain), SPAP1B, SPAP1C, Access to Genbank No. NM_030764, AY358130) Genome Res. 13 (10): 2265-2270 (2003) , Immunogenetics 54 (2): 87-95 (2002), Blood 99. (8): 2662-2669 (2002), Proc. Nati Acad. Sci. U.S.A. 98 (17): 9772-9777 (2001), Xu, M.J., et al. (2001) Biochem. Biophys. Res. Commun. 280 (3): 768-775; O2004016225 (Claim 2); O2003077836; O200138490 (Claim 5; Figure 18D-1-18D-2); WO2003097803 (Claim 12); WO2003089624 (Claim 25); Cross references: MIM: 606509; NP_110391.2; NM_030764_1 (17) HER2 (ErbB2, Access to Genbank No. M11730) Coussens L., et al Science (1985) 230 (4730): 1132-1139); Yamamoto T., et al Nature 319, 230-234, 1986; Semba K., et al Proc. Nati 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 (Figure II); WO2004009622; WO2003081210; O2003089904 (Claim 9); WO2003016475 (Claim 1); US2003118592; WO2003008537 (Claim 1); O2003055439 (Claim 29, Figure 1A-B); WO2003025228 (Claim 37; Figure 5C); WO200222636 (Example 13; Page 95-107); WO200212341 (Claim 68; Figure 7); WO200213847 (Page 71-74); WO200214503 (page 114-117); O200153463 (Claim 2; Page 41-46); O200141787 (Page 15); WO200044899 (Claim 52, Figure 7); WO200020579 (Claim 3, Figure 2); US5869445 (Claim 3; Col 31-38); O9630514 (Claim 2; Page 56-61); EP1439393 (Claim 7); WO2004043361 (Claim 7); WO2004022709; WO200100244 (Example 3, Figure 4); Access: P04626; EMBL; M11767; AAA35808.1. EMBL; M11761; AAA35808.1. - - (18) NCA (CEACAM6, Access to Genbank No. M18728), - Barnett T., et al Genomics 3, 59-66, 1988; Tawaragi Y., et al Biochem. Biophys. Res. Commun. 150, 89-96, 1988; Strausberg R.L., et al Proc. Nati Acad. Sci. U.S. A. 99: 16899-16903, 2002; O2004063709; EP1439393 (Claim 7); WO2004044178 (Example 4); WO2004031238; O2003042661 (Claim 12); WO200278524 (Example 2); O200286443 (Claim 27; Page 427); WO200260317 (Claim 2); Access: P40199; Q14920; EMBL; 29541; AAA59915.1. EMBL; 18728; (19) MDP (DPEP1, Access to Genbank No. BC017023) Proc. Nati Acad. Sci. U.S. A. 99 (26): 16899-16903 (2002)); WO2003016475 (Claim 1); WO200264798 (Claim 33; Page 85-87); JP05003790 (Figure 6-8); W09946284 (Figure 9); Cross references: MIM: 179780; AAH17023.1; BC017023_1 (20) IL20Ra (IL20Ra, ZCYTOR7, Access to Genbank No. AF184971); Clark H.F., et al Genome Res. 13, 2265-2270, 2003; ungall A.J., et al Nature 425, 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: 12617-12624; Sheikh F. , et al (2004) J. Immunol. 172, 2006-2010; EP1394274 (Example 11); US2004005320 (Example 5); WO2003029262 (Page 74-75); WO2003002717 (Claim 2; Page 63); WO200222153 (Page 45-47); US2002042366 (Page 20-21); WO200146261 (Page 57-59); WO200146232 (Page 63-65); W09837193 (Claim 1; Page 55-59); Access: Q9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971; AAF01320.1. (21) Brevican (BCAN, BEHAB, Access to Genbank No. AF229053) Gary S.C., et al Gene 256, 139-147, 2000; Clark H.F., et al Genome Res. 13, 2265-2270, 2003; Strausberg R.L., et al Proc. Nati Acad. Sci. U.S.A. 99, 16899-16903, 2002; US2003186372 (Claim 11); US2003186373 (Claim 11); US2003119131 (Claim 1, Figure 52); US2003119122 (Claim 1, Figure 52); US2003119126 (Claim 1); US2003119121 (Claim 1, Figure 52); US2003119129 (Claim 1); US2003119130 (Claim 1); US2003119128 (Claim 1, Figure 52); US2003119125 (Claim 1); WO2003016475 (Claim 1); WO200202634 (Claim 1); (22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Access to Genbank No. NM 004442) Chan, J and Watt, V.M. , Oncogene 6 (6), 1057-1061 (1991) Oncogene 10 (5): 897-905 (1995), Annu. Rev. Neurosci. 21: 309-345 (1998), Int. Rev. Cytol. 196: 177-244 (2000)); WO2003042661 (Claim 12); WO200053216 (Claim 1; Page 41); WO2004065576 (Claim 1); WO2004020583 (Claim 9); WO2003004529 (Page 128-132); WO200053216 (Claim 1; Page 42); Cross references: MIM: 600997; NP_004433.2; _004442_1 (23) ASLG659 (B7h, Access to Genbank No. AX092328) US20040101899 (Claim 2); WO2003104399 (Claim 11); WO2004000221 (Figure 3); US2003165504 (Claim 1); US2003124140 (Example 2); US2003065143 (Figure 60); WO2002102235 (Claim 13; Page 299); US2003091580 (Example 2); WO200210187 (Claim 6; Figure 10); WO200194641 (Claim 12, Figure 7b); WO200202624 (Claim 13; Figure 1A-1B); US2002034749 (Claim 54; Page 45-46); WO200206317 (Example 2; Page 320-321, Claim 34; Page 321-322); WO200271928 (Page 468-469); WO200202587 (Example 1; 1); WO200140269 (Example 3; Pages 190-192); WO200036107 (Example 2; Page 205-207); WO2004053079 (Claim 12); WO2003004989 (Claim 1); WO200271928 (Page 233-234, 452-453); WO 0116318; (24) PSCA (Prostate Germ Cell Antigen Precursor, Access to Genbank No. AJ297436) Reiter R.E., et al Proc. Nati 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); O2003008537 (Claim 1), -WO200281646 (Claim 1; Page 164); WO2003003906 (Claim 10; Page 288); WO200140309 (Example 1, Figure 17); US2001055751 (Example 1, Figure Ib); WO200032752 (Claim 18, Figure 1); O9851805 (Claim 17; Page 97); 09851824 (Claim 10; Page 94); WO9840403 (Claim 2, Figure IB); Access: 043653; EMBL; AF043498; AAC39607.1. (25) GEDA (Access to Genbank No. AY260763); AAP14954 protein similar to lipoma fusion partner HMGIC /pid=AAP14954.1 - Homo sapiens Species: Homo sapiens (human) WO2003054152 (Claim 20); WO2003000842 (Claim 1); WO2003023013 (Example 3, Claim 20); US2003194704 (Claim 45); Cross references: GI: 30102449; AAP14954.1; AY260763 1 (26) BAFF-R (B cell activation factor receptor, - - receiver 3 BLyS, BR3, Access to Genbank No. AF116456); BAFF receptor /pid=NP_443177.1 - Homo sapiens Thompson, J.S., et al Science 293 (5537), 2108-2111 (2001); WO2004058309; O2004011611; WO2003045422 (Example; Page 32-33); WO2003014294 (Claim 35; Figure 6B); WO2003035846 (Claim 70; Page 615-616); WO200294852 (Col 136-137); O200238766 (Claim 3; Page 133); O200224909 (Example 3, Figure 3); Cross references: 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, Access to Genbank No. AK026467); Wilson et al (1991) J. Exp. Med. 173: 137-146; WO2003072036 (Claim 1, Figure 1); Cross references: IM: 107266; NP 001762.1; NM 001771 1 (28) CD79a (CD79A, CD79a, alpha associated to immunoglogulin, a B-cell-specific protein that interacts covalently with Ig beta (CD79B) and forms a complex on the surface with IgM molecules, translates a signal involved in the B cell differentiation), pl: 4.84, M: 25028 TM: 2 [P] Gen Chromosome: 19ql3.2, Access to Genbank No. NP_001774.10) WO2003088808, US20030228319; WO2003062401 (claim 9); - - US2002150573 (claim 4, pages 13-14); W09958658 (claim 13, Figure 16); WO9207574 (Figure 1); US5644033; Ha et al (1992) J. Immunol. 148 (5): 1526-1531; Mueller et al (1992) Eur. J. Biochem. 22: 1621-1625; Hashimoto et al (1994) Immunogenetics 40 (4): 287-295; Preud'homme et al (1992) Clin. Exp. Immunol. 90 (1): 141-146; Yu et al (1992) J.

Immunol. 148 (2) 633-637; Sakaguchi et al (1988) EMBO J. 7 (11): 3457-3464; (29) CXCR5 (Burkitt lymphoma receptor 1, a G-protein coupled receptor that is activated by the chemokine CXCL13, functions in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and perhaps the development of AIDS, lymphoma, myeloma, and leukemia); aa 372, pl: 8.54: 41959 TM: 7 [P] Gen Chromosome: Llq23.3, Genbank Accession No. NP_001707.1) WO2004040000; WO2004015426; US2003105292 (Example 2); US6555339 (Example 2); WO200261087 (Figure 1); WO200157188 (Claim 20, page 269); WO200172830 (pages 12-13); WO200022129 (Example 1, pages 152-153, Example 2, pages 254-256); W09928468 (claim 1, page 38); US5440021 (Example 2, col 49-52); W09428931 (pages 56-58); W09217497 (claim 7, Figure 5); Dobner et al (1992) Eur. J. Immunol. 22: 2795-2799; Barella et al (1995) Biochem. J. 309: 773-779; (30) HLA-DOB (Beta subunit of MHC class II molecule II (la antigen) that binds the peptides and presents them to CD4 + T lymphocytes); aa 273, pl: 6.56 M: 30820 TM: 1 [P] Gen Chromosome: 6p21.3, Access to Genbank No. 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: 433-441; Strausberg et al (2002) Proc. Nati Acad. Sci USA 99: 16899-16903; Servenius et al (1987) J. Biol. Chem. 262: 8759-8766; Beck et al (1996) J. Mol. Biol. 255: 1-13; Naruse et al (2002) Tissue Antigens 59: 512-519; W09958658 (claim 13, Figure 15); US6153408 (Col 35-38); US5976551 (col 168-170); US6011146 (col 145-146); Kasahara et al (1989) Immunogenetics 30 (1): 66-68; Larhammar et al (1985) J. Biol. Chem. 260 (26): 14111-14119; (31) P2X5 (Channel 5 of the open ion of the P2X ligand of the purinergic receptor, an open ion channel by extracellular ATP, may be involved in synaptic transmission and neurogenesis, deficiency may contribute to the pathophysiology of idiopathic detrusor insetability); aa 422), pl: 7.63, MW: 47206 TM: 1 [P] Gen Chromosome: 17pl3.3, Access to Genbank No. 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); O2003093444 (claim 1); WO2003087768 (claim 1); O2003029277 (page 82); (32) CD72 (CD72 antigen of B cell differentiation, Lyb-2) PROTEIN SEQUENCE Complete maeaity ... tafrfpd (1.359; aa 359), pl: 8.66, MW: 40225 TM: 1 [P] Chromosome from Gen: 9pl3.3, Genbank Accession No. NP_001773.1) WO2004042346 (claim 65); WO2003026493 (pages 51-52, 57-58); WO200075655 (pages 105-106); Von Hoegen et al (1990) J. Immunol. 144 (12): 4870-4877; Strausberg et al (2002) Proc. Nati Acad. Sci USA 99: 16899-16903; (33) LY64 (Lymphocyte Antigen 64 (RP105), membrane type I family protein (LRR) repeat rich in leucine, regulates B cell activation and apoptosis, loss of function is associated with activity increased disease in patients with systemic lupus erythematosus); aa 661, pl: 6.20, MW: 74147 TM: 1 [P] Gen Chromosome: 5ql2, Access to Genbank No. NP_005573.1) US2002193567; WO9707198 (claim 11, pages 39-42); Miura et al (1996) Genomics 38 (3): 299-304; Miura et al (1998) Blood 92: 2815-2822; WO2003083047; W09744452 (claim 8, pages 57-61); WO200012130 (pages 24-26); (34) FcRH1 (protein 1 similar to the Fe receptor, a putative receptor for the immunoglobulin Fe domain containing Ig-like domains type C2 and ITA, may have a role in B lymphocyte differentiation); 429 aa, pl: 5.28, M: 46925 T: 1 [P] Gen Chromosome: Iq21-lq22, Access to Genbank No. NP_443170.1) WO2003077836; WO200138490 (claim 6, Figure 18E-1-18-E-2); Davis et al (2001) Proc. Nati Acad. Sci USA 98 (17): 9772-9777; WO2003089624 (claim 8); EP1347046 (claim 1); WO2003089624 (claim 7); (35) IRTA2 (Associated translocation 2 of the immunoglobulin superfamily receptor, a putative immunoreceptor with possible roles in B cell development and lymphomagenesis, - deregulation of the translocation gene occurs in the same B cell malignancies); aa 977, pl: 6.88 MW: 106468 TM: 1 [P] Gen Chromosome: lq21, Access to Genbank Human: AF343662, AF343663, AF343664, AF343665, AF369794, AF397453, AK090423, AK090475, AL834187, AY358085; Mouse: AK089756, AY158090, AY506558; NP_112571.1 WO2003024392 (claim 2, Figure 97); Nakayama et al (2000) Biochem. Biophys. Res. Commun. 277 (1): 124-127; WO2003077836; WO200138490 (claim 3, Figure 18B-1-18B-2); (36) TENB2 (T EFF2, tomoregulin, TPEF, HPP1, TR, putative transmembrane proteoglycan, related to EGF / heregulin family of growth factors and follistatin); aa 374, NCBI Access: AAD55776, AAF91397, AAG49451, NCBI RefSeq: NP_057276; NCBI Gene: 23671; OMIM: 605734; SwissProt Q9UIK5; Access to Genbank No. AF179274; AY358907, CAF85723, CQ782436 WO2004074320 (SEQ ID NO 810); JP2004113151 (SEQ ID NOS 2, 4, 8); O2003042661 (SEQ ID NO 580); O2003009814 (SEQ ID NO 411); EP1295944 (pages 69-70); WO200230268 (page 329); WO200190304 (SEQ ID NO 2706); US2004249130; US2004022727; O2004063355; US2004197325; US2003232350; US2004005563; US2003124579; Horie et al (2000) Genomics 67: 146-152; Uchida et al (1999) Biochem. Biophys. Res. Commun. 266: 593-602; Liang et al (2000) Cancer Res. 60: 4907-12; Glynne-Jones et al (2001) Int J Cancer. Oct 15; 94 (2): 178-84. The original antibody can also be a fusion protein comprising a sequence of albumin binding peptides (ABP) (Dennis et al., (2002) "Albumin Binding As a General Strategy for Improving the Pharmacokinetics of Proteins" J. Biol. Chem ., 277: 35035-35043; O 01/45746). Antibodies of the invention include fusion proteins with ABP sequences shown by: (i) Dennis et al., (2002) J. Biol. Chem., 277: 35035-35043 in Tables III and IV, page 35038; (ii) US 20040001827 to [0076] SEQ ID NOS: 9-22; and (iii) WO 01/45746 on pages 12-13, SEQ ID NOS: zl-zl4 all of which are incorporated herein by reference. MUTAGENESIS DNA that codes for an amino acid sequence variant of the starting polypeptide is prepared by a variety of methods known in the art. These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of a previously prepared DNA encoding the polypeptide. Variants of recombinant antibodies can also be constructed by restriction fragment manipulation or by extension PCR superimposed with synthetic oligonucleotides. Mutagenesis primers code for cysteine codon replacement (s). Standard mutagenesis techniques can be used to generate the DNA encoding such engineered mutant antibodies of cysteine. A general guide can be found in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience. , New York, NY, 1993. Site-directed mutagenesis is a method for the preparation of substitution variants, ie, proteins - - mutants. This technique is well known in the art (see for example, Cárter (1985) et al., Nucleic Acids Res., 13: 4431-4443, Ho et al., (1989) Gene (Amst) 77: 51-59; and Kunkel et al., (1987) Proc. Nati, Acad. Sci., USA 82: 488). Briefly, in carrying out site-directed mutagenesis of DNA, the starting DNA is altered by first hybridizing an oligonucleotide coding for the desired mutation to a single strand of such start DNA. After hybridization, a DNA polymerase is used to synthesize a second complete chain, using the hybridized oligonucleotide as the primer, and using the single strand of the start DNA as a template. In this way, the oligonucleotide coding for the desired mutation is incorporated into the resulting double-stranded DNA. Site-directed mutagenesis can be carried out within the gene expressing the protein to be mutagenized in an expression plasmid and the resulting plasmid can be sequenced to confirm the introduction of the desired cysteine replacement mutations (Liu et al., (1998) J. Biol. Chem., 273-20252-20260). Protocols and formats directed to the site including those commercially available, e.g., QuikChange® Multi Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). PCR mutagenesis is also suitable for producing amino acid sequence variants of the - - start polypeptide. See Higuchi, (1990) in PCR Protocols, pp. 177-183, Academic Press; Ito et al., (1991) Gene 102: 67-70; Bernhard et al., (1994) Bioconjugate Chem., 5: 126-132; and Vallette et al., (1989) Nuc. Acids Res., 17: 723-733. Briefly, when small amounts of template DNA are used as starting material in a PCR, primers that differ slightly in sequence from the corresponding region in a template DNA can be used to generate relatively large amounts of a specific DNA fragment that differs of the template sequence only in the positions where the initiators differ from the template. Another method for the preparation of variants, cassette mutagenesis, is based on the technique described by Wells et al., (1985) Gene 34: 315-323. The starting material is the plasmid (or other vector) comprising the DNA of the starting polypeptide to be mutated. The codon (s) in the home DNA to be mutated is identified. There must be a unique restriction endonuclease site on each side of the identified mutation site (s). If no such restriction sites exist, they can be generated using the oligonucleotide-mediated mutagenesis method described above to be introduced into appropriate locations in the DNA of the initiation polypeptide. The plasmid DNA is cut at these sites to linearize it A double-stranded oligonucleotide coding for the DNA sequence between the restriction sites, but containing the desired mutation (s) is synthesized using standard procedures, wherein the two oligonucleotide chains are synthesized separately and then they hybridize together using standard techniques. This double-stranded oligonucleotide is referred to as the cassette. This cassette is designed to have 5 'and 3' ends that are compatible with the ends of the linearized plasmid, so that they can be ligated directly to the plasmid. This plasmid now contains the mutated DNA sequence. The mutant DNA containing the encoded cysteine replacements can be confirmed by DNA sequencing. Unique mutations are also generated by oligonucleotide-directed mutagenesis using double-stranded plasmid DNA as a template by PCR-based mutagenesis (Sambrook and Russel, (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; Zoller et al., (1983) Methods Enzymol., 100: 468-500; Zoller MJ, and Smith M. (1982) Nucí.Aids Res., 10: 6487-6500). In the present invention, hu4D5Fabv8 displayed in M13 phage (Gerstner et al., (2000) "Sequence Plasticity in the Antigen Binding Site of a Therapeutic Anti-HER2 Antibody", J. Mol. Biol., 321: 851-62) used for experiments as a model system. Cysteine mutations were introduced in hu4D5Fabv8-phage; hu4D5Fabv8 and structures of ABP-hu4D5Fabv8. The preparations of hu4D5-ThioFab-Phage were carried out using the polyethylene glycol (PEG) precipitation method as described previously (Lowman, Henry B., (1998) Methods in Molecular Biology (Totowa, New Jersey) 87 (Combinatorial Peptide Library Protocols) 249-264). Oligonucleotides are prepared by the phosphoramide synthesis method (US 4415732; US 4458066; Beaucage S., and Iyler R., (1992) "Advances in the synthesis of oligonucleotides by the phosphoramidite approach", Tetrahedron 48: 2223-2311) . The phosphoramidite method involves the cyclic addition of nucleotide monomer units with a 3 'phosphoramidite residue reactive to an oligonucleotide chain that grows on a solid support comprised of controlled pore glass or highly crosslinked polystyrene, and most commonly in the 3-way direction. 'to 5' in which the 3 'terminating nucleoside is attached to the solid support at the start of the synthesis (US 5047524; US 5262530). The method is commonly practiced using coraercially available automatic synthesizers (Applied Biosystems, Foster City, CA). Oligonucleotides can be chemically labeled with non-isotopic residues for detection, capture, stabilization or other purposes (Andrus A., "Chemical methods for 5 'non-isotopic 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 H., and Anak M., in DNA Probes Second Edition (1993), Stockton Press, New York, pp. 121-23). PHESELECTOR ANALYSIS The PHESELECTOR analysis (phage ELISA for the selection of reactive thiols) allows the detection of reactive cysteine groups in antibodies in a phage ELISA format. The process of coating proteins (eg, antibody) of interest on well surfaces, followed by incubation with phage particles and then with secondary antibody labeled with HRP with absorbance detection is detailed in Example 2. Mutant proteins displayed in phage , they can be selected in a fast, robust and high performance way. The designed cysteine antibody libraries can be produced and subjected to binding selection using the same procedure to identify appropriate reactive sites of free Cys incorporation from random protein-phage libraries of antibodies or other proteins. This technique includes reacting cysteine mutant proteins deployed in phage with an affinity reagent or reporter group which is also thiol reactive. Figure 8 illustrates the PHESELECTOR analysis through a schematic representation illustrating the union - - from Fab or ThioFab to HER2 (upper part) and ThioFab biotinylated to streptavidin (lower part). EXPRESSION AND PURIFICATION OF PROTEINS DNA encoding designed antibodies to cysteine is readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells serve as a source of such DNA. Once isolated, the DNA can be placed in expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or other mammalian host cells, such as myeloma cells (US 5807715; US 2005/0048572; US 2004/0229310) that otherwise do not produce the antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The yields of hu4D5Fabv8 designed cysteine antibodies were similar to wild type hu4D5Fabv8. Review articles about the recombinant expression in bacteria of DNA encoding the antibody, include Skerra et al., (1993) Curr. Opinion in Immunol. , 5: 256-262 and Plückthun (1992) Immunol. Revs. , 130: 151-188. After design and selection, antibodies designed cysteine, eg, ThioFabs, with unreacted, highly reactive Cys residues, can be produced by: (i) expression in a bacterial system, eg, E. coli, or a mammalian cell culture system (WO 01/00245), eg, Chinese hamster ovary cells (CHO); and (ii) purification using common protein purification techniques (Lowman et al., (1991) J. Biol. Chem., 266 (17): 10982-10988). ThioFabs were expressed upon induction in 34B8, a non-suppressor E. coli species (Baca et al., (1997) Journal Biological Chemistry 272 (16): 10678-84). See Example 3a. The harvested cell pill was resuspended in PBS (phosphate buffered saline), the total cell lysis was carried out by passing it through a microfluidizer and the ThioFabs were purified by affinity chromatography with protein G SEPHAROSE ™ (Amersham). The ThioFabs were conjugated with biotin-PEO-maleimide as described above and the biotinylated ThioFabs were further purified by Superdex-200 ™ gel filtration chromatography (Amersham), which eliminated the free biotin-PEO-maleimide and the oligomeric fraction of ThioFabs. . ANALYSIS OF MASS SPECTROSCOPY The spectrometric analysis of ionization mass by electro-randomization by liquid chromatography (LC-ESI-MS) was used for the precise determination of the molecular weight of Fab conjugated by biotin (Colé R.B., Electro Spray Ionization ass Spectrometry: Fundamentals, Instrumentation and Applications, (1997), iley, New York). The amino acid sequence of the biotinylated hu4D5Fabv8 peptide (A121C) was determined by tryptic digestion followed by LC-ESI-Tandem MS analysis (Table 4, Example 3b). The Fab fragment of the hu4D5Fabv8 antibody contains approximately 445 amino acid residues, including 10 Cys residues (five in the light chain and five in the heavy chain). The high resolution structof the humanized variable fragment 4D5 (Fv4D5) has been evaluated, see: Eigenbrot et al., "X-Ray Struct of the Antigen Binding Domains from Three Variants of Humanized Anti-P185her2 Antibody 4D5 and Comparison with Molecular Modeling" (1993) J. Mol. Biol. , 229: 969-995). All Cys residues are present in the form of disulfide bonds, consequently these residues do not have any free thiol group to conjugate with drug-maleimide (unless treated with a reducing agent). In this way, the newly manufact Cys residue can remain unpaired and is capable of reacting with i.e., conjugated to, an electrophilic linker reagent or drug-binding intermediate, such as a drug-maleimide. Fig1A shows a three-dimensional representation of the hu4D5Fabv8 antibody fragment derived by X-ray crystal coordinates. The structpositions of the designed Cys residues of the heavy and light chains are They are numbered according to a sequential numbering system. This sequential numbering system is correlated with the Kabat numbering system (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda MD) for the trastuzumab 4d5v7fabH variant according to FigIB showing the sequential numbering scheme (upper row), starting at the N ending, which differs from the Kabat numbering scheme (lower row) by the insertions annotated by a, b, c. By using the Kabat numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or an insertion in, an FR or CDR of the variable domain. The designed heavy chain variant sites of cysteine are identified by sequential numbering and Kabat numbering schemes in the following scheme: The mutant Fabs of M13 phagemid-Cys (Fig 3A and 3B) can be visualized rapidly compared to Fab proteins. The binding of phagemid-ThioFab to antigen and streptavidin can be tested by coating HER2 and streptavidin, respectively on ELISA plates followed by a test with anti-Fab-HRP (Horse radish peroxidase) as described in Example 2 and illustrated in Fig8 This method allowed the simultaneous monitoring of the effect of antigen binding and of the reactivity of the thiol group by the molecule of Cys residue manufact / biotin conjugate. The method can also be applied to visualize reactive thiol groups by any protein displayed on M13 phage. The non-conjugated phagemid-ThioFabs are purified by simple precipitation of PEG. The antigen binding fragment of humanized 4D5 (hu4D5Fab) is expressed in E. coli and has been deployed in bacteriophages. (Garrard et al., (1993) Gene 128: 103-109). The Fab fragment of antibody hu4D5Fab was deployed in M13 phage as a model system in the ELISA-based assay to test the thiol reactivity. Fig8 is a graphical representation of the PHESELECTOR analysis, illustrating the binding of a biotinylated ThioFab phage and a anti-phage HRP antibody for HER2 (upper part) and streptavidin (lower part). Five amino acid residues (L-Ala43, H-Ala40, H-Serll9, H-Alal21 and H-Serl22) were initially selected from the crystal structinformation as remote from the antigen-binding surface (Eigenbrot et al., (1993) J. Mol. Biol. , 229: 969-995). The crystalline X-ray structure of the protein database was designated 1FVC. The Cys residues were manufactured in the positions by site-directed mutagenesis. The ThioFab-phage preparations were isolated and reactivated with the biotinylation reagent. The biotin-conjugated and unconjugated variants were tested for binding to HER2 and streptavidin using a PHESELECTOR analysis based on ELISA (Figure 8, Example 2) with an HRP (horseradish peroxidase) -anti-conjugated anti-phage antibody. The interaction of non-biotinylated phage-hu4D5Fabv8 (Figure 2a) and biotinylated phage-hu4D5Fabv8 (Figure 2B) with BSA (open cell), HER2 (gray cell) or streptavidin (solid cell) were monitored by anti-M13 peroxidase antibody -horseradish (HRP) developing a standard HRP reaction and measuring the absorbance at 450 nm. The absorbency produced by a change of a colorimetric substrate was measured at 450 nm. The reactivity of ThioFab with HER2 measures the antigen binding. The reactivity of ThioFab with streptavidin measures the degree of biotinylation. The - - ThioFab reactivity with BSA is a negative control for non-specific interaction. As seen in Figure 2a, all ThioFab-phage variants have a binding similar to HER2 compared to that of hu4D5Fabv8-wild-type phage. In addition, conjugation with biotin did not interfere in the binding of ThioFab to HER2 (Figure 2B). Surprisingly and unexpectedly, ThioFabs-phage samples showed varying levels of streptavidin-binding activity. Of all the phage-ThioFabs tested, the A121C antibody designed from cysteine exhibited the highest reactivity of thiol. Although the hu4D5Fabv8-wild type phage was incubated with the same amounts of biotin-maleimide, these phage had low binding to streptavidin indicating that the pre-existing cysteine residues (involved in disulfide bond formation) of the phage coat proteins hu4D5Fabv8 and 13 did not interfere with the specific conjugation of the biotin-maleimide site. These results demonstrated that phage ELISA analysis can be used successfully to visualize thiol groups on the Fab surface. The PHESELECTOR analysis allows the visualization of reactive thiol groups in antibodies. The identification of the variant A121C by this method is exemplary. The complete Fab molecule can be effectively investigated to identify more ThioFab variants with reactive thiol groups.

One parameter, fractional surface accessibility, was used to identify and quantify the accessibility of the solvent to the amino acid residues in a polypeptide. The surface accessibility can be expressed as the surface area (A2) that can be contacted by a solvent molecule, e.g., water. The occupied water space is approximated as a sphere of radio of 1.4 A. The software is freely available or licensed (Secretary for CCP4, Daresbury Laboratory, Warrington, WA4 4AD, United Kingdom, Fax: (+44) 1925 603825, or online: www.ccp4.ac.uk/dist/html/lNDEX.html) as the CCP4 Suite of crystallography programs that use algorithms to calculate the surface accessibility of each amino acid of a protein with coordinates derived from X-ray crystallography known ("The CCP4 Suite: Programs for Protein Crystallography" (1994) Acta Cryst., D50: 760-763). Two exemplary software modules that perform the surface accessibility calculations are "AREAIMOL" and "SURFACE", based on the algorithms of B. Lee and F.M. Richards (1971) J. Mol. Biol. , 55: 379-400. AREAIMOL defines the solvent-accessible surface of a protein as the site of the center of a probe sphere (representing a solvent molecule) as it travels over the Van der Waals surface of the protein. AREAIMOL calculates the surface area accessible to the solvent generating surface points in a sphere extended around each atom (at a distance from the center of the atom equal to the sum of the atom and the radius of the probe), and eliminating those that fall within the equivalent spheres associated with neighboring atoms. AREAIMOL finds the solvent accessible area of atoms in a PDB coordinate file, and summarizes the accessible area by residue, by chain and for the entire molecule. The accessible areas (or area differences) for individual atoms can be written to an output pseudo-PDB file. AREAIMOL assumes a single radio for each element, and only recognizes a limited number of different elements. They will be assigned to unknown types of atoms (i.e., those that are not found in the internal database of AREAIMOL), failure radius of 1.8 A. The list of atoms recognized is: Atom No. atomic radio Van der Waals (A) C 6 1.80 N 7 1.65 O 8 1.60 Mg 12 1.60 S 16 1.85 P 15 1.90 Cl 17 1.80 Co 27 1.80 AREAIMOL and SURFACE report absolute accessibilities, ie, the number of square Angstroms ( TO). the Fractional surface accessibility is calculated by reference to a standard state relevant to an amino acid within a polypeptide. The reference state is the tripeptide Gly-X-Gly, where X is the amino acid of interest, and the reference state must be an "extended" conformation, i.e., like that of the beta chains. The extended configuration maximizes the accessibility of X. A calculated accessible area is divided by the accessible area in a reference state of the tripeptide Gly-X-Gly and reports the quotient, which is fractional accessibility. Percent accessibility is fractional accessibility multiplied by 100. Another exemplary algorithm for calculating surface accessibility is based on the SOLV module of the xsae program (Broger, CF, Hoffma-LaRoche, Basel) that calculates the fractional accessibility of an amino acid residue to a water sphere based on the X-ray coordinates of the polypeptide. The fractional surface accessibility for each amino acid in hu4D5Fabv7 was calculated using the crystal structure information (Eigenbrot et al., (1993) J. Mol. Biol. 229: 969-995). The fractional surface accessibility values for the amino acids of the light chain and the heavy chain of hu4D5Fabv7 are shown in descending order in Table 1.

Table 1. hu4D5Fabv7-light chain BE A 202 frac acc = 101. 236 ASP A 151 frac acc = 41. 586 GLY A 41 frac acc = 90. 775 SER A 12 frac acc = 40. 633 GLY A 157 frac acc = 88. 186 ASN A 210 frac acc = 40. 158 ASP A 1 frac acc = 87. 743 SER A 63 frac acc = 39. 872 SER A 156 frac acc = 83. 742 ARG A 66 frac acc = 39. 669 GLY A 57 frac acc = 81. 611 PRO A 8 frac acc = 39. 297 BE A 168 frac acc = 79. 680 SER A 65 frac acc = 39. 219 BE A 56 frac acc = 79. 181 SER A 77 frac acc = 38. 820 LYS A 169 frac acc = 77. 591 THR A 180 frac acc = 38. 296 BE A 60 frac acc = 75. 291 ASP A 185 frac acc = 38. 234 THR A 109 frac acc = 74. 603 THR A 31 frac acc = 38. 106 CYS A 214 frac acc = 72. 021 THR A 94 frac acc = 37. 452 LYS A 126 frac acc = 71. 002 THR A 93 frac acc = 37. 213 BE A 67 frac acc = 66. .694 THR A 197 frac acc = 36. 709 ARG A 18 frac acc = 66. .126 SER A 182 frac acc = 36. 424 ASN A 152 frac acc = 65. .415 GLY A 128 frac acc = 35. 779 BE A 127 frac acc = 65., 345 LYS A 207 frac acc = 35., 638 LYS A 190 frac acc = 65., 189 ASP A 17 frac acc = 35., 413 LYS A 145 frac acc = 63. .342 GLY A 200 frac acc = 35., 274 GLN A 199 frac acc = 62. .470 GLU A 165 frac acc = 35., 067 GLU A 143 frac acc = 61. .681 WING A 112 frac acc = 34., 912 GLN A 3 frac acc = 59. .976 GLN A 79 frac acc = 34. .601 LYS A 188 frac acc = 59. .680 VAL A 191 frac acc = 33. .935 ARG A 24 frac acc = 59, .458 SER A 208 frac acc = 33. .525 PHE A 53 frac acc = 58, .705 LYS A 39 frac acc = 33. .446 BE A 9 frac acc = 58. .446 GLU A 123 frac acc = 32, .486 GLN A 27 frac acc = 57 .247 THR A 69 frac acc = 32, .276 WING A 153 frac acc = 56 .538 SER A 76 frac acc = 32, .108 BE A 203 frac acc = 55 .864 HIS A 189 frac acc = 31, .984 LYS A 42 frac acc = 54 .730 ARG A 108 frac acc = 31.915 GLY A 16 frac acc = 54 .612 ASN A 158 frac acc = 31 .447 LYS A 45 frac acc = 54 .464 VAL A 205 frac acc = 31 .305 PRO A 204 frac acc = 53 .172 SER A 14 frac acc = 31 .094 GLU A 213 frac acc = 53 .084 GLN A 155 frac acc = 30 .630 WING A 184 frac acc = 52 .556 GLU A 187 frac acc = 30 .328 VAL A 15 frac acc = 52 .460 ARG A 211 frac acc = 30 .027 BE A 7 frac acc = 51 .936 LYS A 183 frac acc = 29 .751 LEU A 154 frac acc = 51 .525 ASN A 138 frac acc = 29 .306 GLN A 100 frac acc = 51 .195 ASP A 170 frac acc = 29 .041 BE A 10 frac acc = 49 .907 SER A 159 frac acc = 27 .705 THR A 5 frac acc = 48 .879 GLN A 147 frac acc = 27 .485 THR A 206 frac acc = 48.853 THR A 22 frac acc = 27 .121 ASP A 28 frac acc = 48.758 ALA A 43 frac acc = 26 .801 GLY A 68 frac acc = 48 .690 ARG A 142 frac acc = 26 .447 THR A 20 frac acc = 48 .675 LEU A 54 frac acc = 25 .882 ASP A 122 frac acc = 47 .359 ASP A 167 frac acc = 25 .785 PRO A 80 frac acc = 46..984 THR A 129 frac acc = 23., 880 BE A 52 frac acc = 46. .917 ALA A 144 frac acc = 23. .652 BE A 26 frac acc = 46., 712 VAL A 163 frac acc = 22. .261 TYR A 92 frac acc = 46., 218 PRO A 95 frac acc = 20. .607 LYS A 107 frac acc = 45. .912 ALA A 111 frac acc = 19. .942 GLU A 161 frac acc = 45. .100 LYS A 103 frac acc = 18. .647 VAL A 110 frac acc = 44. .844 LEU A 181 frac acc = 18, .312 GLU A 81 frac acc = 44. .578 THR A 72 frac acc = 18, .226 PRO A 59 frac acc = 44. .290 GLU A 195 frac acc = 18 .006 ASN A 30 frac acc = 42, .721 THR A 178 frac acc = 17 .499 GLN A 160 frac acc = 42,692 THR A 85 frac acc = 17,343 BE A 114 frac acc = 42 .374 ASP A 70 frac acc = 17 .194 PRO A 40 frac acc = 41 .928 LEU A 11 frac acc = 16.568 PHE A 116 frac acc = 16. 406 LEU A 125 frac acc = 2. 398 THR A 97 frac acc = 16. 204 PRO A 96 frac acc = 2. 387 ARG A 61 frac acc = 16. 192 LEU A 47 frac acc = 2. 180 TYR A 49 frac acc = 16. 076 WING A 51 frac acc = 1. 837 BE A 50 frac acc = 15. 746 PHE A 118 frac acc = 1. 779 LYS A 149 frac acc = 15 510 PHE A 62 frac acc = 1. 581 GLU A 55 frac acc = 14. 927 WING A 25 frac acc = 1. 538 LEU A 201 frac acc = 14. 012 VAL A 133 frac acc = 1. .315 GLY A 64 frac acc = 13. .735 ASP A 82 frac acc = 1. .1 1 GLY A 212 frac acc = 13. 396 LEU A 179 frac acc = 0., 872 PHE A 98 frac acc = 12., 852 GLN A 124 frac acc = 0., 787 THR A 74 frac acc = 12., 169 ET A 4 frac acc = 0. .778 BE A 171 frac acc = 11., 536 SER A 177 frac acc = 0..693 PRO A 141 frac acc = 11. .073 SER A 131 frac acc = 0..693 PHE A 83 frac acc = 10. .871 LEU A 135 frac acc = 0. .654 THR A 164 frac acc = 10. .325 PHE A 71 frac acc = 0.593 ALA A 32 frac acc = 9. .971 TRP A 35 frac acc = 0. .448 HIS A 198 frac acc = 9 .958 PHE A 209 frac acc = 0, .395 VAL A 146 frac acc = 9.861 TYR A 186 frac acc = 0, .259 BE A 121 frac acc = 9, .833 LEU A 78 frac acc = 0.157 WING A 13 frac acc = 9, .615 VAL A 196 frac acc = 0, .000 GLU A 105 frac acc = 9, .416 VAL A 132 frac acc = 0, .000 BE A 162 frac acc = 9 .304 VAL A 104 frac acc = 0.000 ILE A 117 frac acc = 8 .780 VAL A 33 frac acc = 0 .000 HIS A 91 frac acc = 8 .557 VAL A 29 frac acc = 0 .000 WING A 193 frac acc = 8 .547 TYR A 192 frac acc = 0 .000 GLN A 37 frac acc = 8 .442 TYR A 86 frac acc = 0 .000 VAL A 58 frac acc = 8 .281 TYR A 36 frac acc = 0 .000 PRO A 120 frac acc = 8 .095 THR A 102 frac acc = 0 .000 GLN A 38 frac acc = 6 .643 SER A 174 frac acc = 0 .000 PRO A 113 frac acc = 6 .594 PHE A 139 frac acc = 0 .000 GLY A 101 frac acc = 6 .558 LEU A 136 frac acc = 0 .000 TYR A 140 frac acc = 5,894 LEU A 73 frac acc = 0,000 VAL A 115 frac acc = 5 .712 ILE A 75 frac acc = 0 .000 TYR A 87 frac acc = 4.539 ILE A 48 frac acc = 0., 000 BE A 176 frac acc =. , 106 ILE A 21 frac acc = 0., 000 ILE A 2 frac acc = 4., 080 GLN A 90 frac acc = 0., 000 ASN A 137 frac acc = 3., 906 GLN A 89 frac acc = 0.000 TRP A 148 frac acc = 3. .676 CYS A 194 frac acc = 0.000 GLY A 99 frac acc = 3..550 CYS A 134 frac acc = 0.000 PRO A 44 frac acc = 3..543 CYS A 88 frac acc = 0.000 LEU A 175 frac acc = 3.488 CYS A 23 frac acc = 0, .000 VAL A 19 frac acc = 3, .420 ALA A 130 frac acc = 0 .000 ILE A 106 frac acc = 3.337 ALA A 84 frac acc = 0 .000 PRO A 119 frac acc = 2. .953 ALA A 34 frac acc = 0 .000 LEU A 46 frac acc = 2 .887 GLN A 6 frac acc = 2 .860 TYR A 173 frac acc = 2 .825 VAL A 150 frac acc = 2 .525 GLN A 166 frac acc = 2 .525 THR A 172 frac acc = 2 .436 hu4D5Fabv7-heavy chain SER B 179 frac acc = 99. 479 PRO B 14 frac acc = 45. 729 GLY B 42 frac acc = 95. 850 THR B 54 frac acc = 45. 503 GLU B 1 frac acc = 87. 276 THR B 200 frac acc = 45. 369 GLY B 66 frac acc = 84. 541 LEU B 177 frac acc = 45. 337 ASP B 102 frac acc = 83. 794 GLY B 8 frac acc = 44. 898 BE B 75 frac acc = 80. 567 SER B 7 frac acc = 43., 530 GLY B 140 frac acc = 80. .344 THR B 69 frac acc = 43., 503 ASN B 211 frac acc = 79., 588 PRO B 220 frac acc = 43., 378 GLY B 197 frac acc = 78., 676 LYS B 208 frac acc = 43., 138 ASP B 62 frac acc = 77., 716 LYS B 30 frac acc = 42., 380 GLY B 103 frac acc = 77. .176 ALA B 23 frac acc = 41. .952 BE B 163 frac acc = 76. .664 GLU B 46 frac acc = 41. .430 BE B 139 frac acc = 74. .946 SER B 25 frac acc = 41. .323 LYS B 213 frac acc = 74. .442 ARG B 87 frac acc = 41. .282 ALA B 165 frac acc = 74. .339 LYS B 124 frac acc = 40. .888 THR B 167 frac acc = 73, .934 ASN B 28 frac acc = 40, .529 BE B 122 frac acc = 72. .870 GLN B 3 frac acc = 39, .824 SER B 194 frac acc = 71. .959 THR B 123 frac acc = 39 .306 PRO B 41 frac acc = 71. .540 SER B 63 frac acc = 38 .867 THR B 198 frac acc = 68,666 GLY B 56 frac acc = 38 .582 BE B 222 frac acc = 68 .128 GLY B 169 frac acc = 38 .469 LYS B 43 frac acc = 67 .782 THR B 172 frac acc = 38 .421 GLY B 26 frac acc = 67 .782 PRO B 209 frac acc = 38 .309 THR B 138 frac acc = 65 .826 GLY B 101 frac acc = 38 .040 ASP B 31 frac acc = 64 .222 TYR B 109 frac acc = 36 .829 GLY B 15 frac acc = 64 .172 LYS B 221 frac acc = 36 .520 BE B 168 frac acc = 62 .100 GLY B 44 frac acc = 35 .147 SER B 120 frac acc = 61 .332 GLY B 181 frac acc = 34 .735 LYS B 76 frac acc = 61 .092 THR B 58 ac ac acc = 34 .457 GLY B 141 frac acc = 59 .419 GLY B 9 frac acc = 34 .254 SER B 137 frac acc = 59 .179 VAL B 5 frac acc = 34 .198 TYR B 57 frac acc = 58.916 ALA B 121 frac acc = 33 .049 GLU B 89 frac acc = 58,483 SER B 127 frac acc = 32,390 BE B 180 frac acc = 56. 289 GLY B 10 frac acc = 32. 230 LYS B 65 frac acc = 55. 044 SER B 71 frac acc = 30. 659 ASP B 215 frac acc = 54. 656 ASP B 73 frac acc = 30. 245 GLN B 13 frac acc = 53. 719 LEU B 115 frac acc = 29., 867 GLN B 112 frac acc = 53., 215 LEU B 11 frac acc = 29., 825 TYR B 105 frac acc = 51., 940 ASN B 84 frac acc = 29.765 ALA B 88 frac acc = 51. .602 SER B 210 frac acc = 28. .656 GLY B 164 frac acc = 50. .259 GLU B 155 frac acc = 28. .162 PRO B 192 frac acc = 49. .826 SER B 160 frac acc = 26. .526 THR B 158 frac acc = 49. .694 CYS B 223 frac acc = 26. .270 THR B 142 frac acc = 48. .896 GLY B 16 frac acc = 26, .158 ASN B 55 frac acc = 48. .344 ILE B 202 frac acc = 26 .068 LYS B 136 frac acc = 48 .312 GLN B 82 frac acc = 25 .836 ARG B 19 frac acc = 48 .082 SER B 193 frac acc = 25 .550 PRO B 156 frac acc = 47 .366 ASN B 77 frac acc = 25 .418 PRO B 174 frac acc = 47 .157 ARG B 59 frac acc = 25 .301 LYS B 217 frac acc = 47 .102 VAL B 93 frac acc = 25 .254 GLN B 199 frac acc = 46 .650 THR B 74 frac acc = 24 .902 BE B 17 frac acc = 45 .980 GLU B 219 frac acc = 24 .778 SER B 85 frac acc = 45 .824 ASN B 206 frac acc = 24 .647 VAL B 170 frac acc = 24., 549 PRO B 154 frac acc = 6. 767 TYR B 52 frac acc = 2. , 298 PRO B 133 frac acc = 6., 767 ALA B 175 frac acc = 23. .804 TRP B 99 frac acc = 6., 502 LYS B 216 frac acc = 23., 277 THR B 32 frac acc = 6. .291 VAL B 214 frac acc = 23. .150 LEU B 45 frac acc = 4..649 GLY B 125 frac acc = 22.802 VAL B 128 frac acc = 4..515 ASN B 162 frac acc = 22. .245 ILE B 51 frac acc = 4. .307 ALA B 72 frac acc = 22. .166 SER B 186 frac acc = 4. .084 ALA B 40 frac acc = 21. .974 PHE B 173 frac acc = 3. .969 LEU B 18 frac acc = 20. .273 ARG B 38 frac acc = 3, .734 THR B 212 frac acc = 20, .170 TRP B 47 frac acc = 3, .561 LEU B 182 frac acc = 19, .619 VAL B 118 ac ac acc = 3, .409 TYR B 33 frac acc = 19, .398 ALA B 24 frac acc = 3 .376 THR B 190 frac acc = 19 .365 TYR B 95 frac acc = 3 .242 VAL B 176 frac acc = 18,941 GLU B 6 frac acc = 3 .216 BE B 21 frac acc = 18 .929 ALA B 144 frac acc = 3 .167 BE B 119 frac acc = 18 .877 ILE B 70 frac acc = 1 .958 THR B 91 frac acc = 18 .237 GLY B 111 frac acc = 1 .868 ASP B 151 frac acc = 17,849 LEU B 4 frac acc = 1,780 THR B 114 frac acc = 17. 601 TYR B 201 frac acc = 1. 758 SER B 134 frac acc = 17. 571 LEU B 148 frac acc = 1. 744 LEU B 196 frac acc = 17. 090 PHE B 68 frac acc = 1. 708 TYR B 60 frac acc = 16. 575 VAL B 188 frac acc = 1. 315 TYR B 183 frac acc = 15 968 CYS B 22 frac acc = 0. 935 VAL B 2 frac acc = 15. 901 TRP B 161 frac acc = 0. 876 PRO B 130 frac acc = 15. 342 LEU B 131 frac acc = 0.654 LEU B 166 frac acc = 15. 268 VAL B 205 frac acc = 0. 495 GLY B 100 frac acc = 15. 003 ALA B 92 frac acc = 0. 356 PHE B 27 frac acc = 14. 383 ALA B 79 frac acc = 0. 356 ASN B 204 frac acc = 13. 873 VAL B 64 frac acc = 0., 263 PHE B 104 frac acc = 13., 836 ILE B 29 frac acc = 0., 227 TYR B 80 frac acc = 13., 490 VAL B 218 frac acc = 0. .000 VAL B 159 frac acc = 12. .782 VAL B 189 frac acc = 0. .000 ARG B 67 frac acc = 12. .362 VAL B 149 frac acc = 0. .000 GLN B 178 frac acc = 12. .131 VAL B 116 frac acc = 0. .000 HIS B 171 frac acc = 11. .412 VAL B 48 frac acc = 0. .000 SER B 184 frac acc = 11. .255 VAL B 37 frac acc = 0. .000 ARG B 98 frac acc = 11. .115 TYR B 152 frac acc = 0. .000 PRO B 53 frac acc = 11. .071 TYR B 94 frac acc = 0.000 GLN B 39 frac acc = 11, .037 TRP B 36 frac acc = 0. .000 SER B 195 frac acc = 10. .909 SER B 187 frac acc = 0, .000 ASP B 108 frac acc = 10 .525 SER B 97 frac acc = 0 .000 LEU B 185 frac acc = 10 .464 MET B 107 frac acc = 0 .000 GLY B 113 frac acc = 10 .406 MET B 83 frac acc = 0 .000 THR B 78 frac acc = 10.213 LEU B 145 frac acc = 0 .000 THR B 117 frac acc = 9 .990 LEU B 86 frac acc = 0 .000 LYS B 150 frac acc = 9. 447 LEU B 81 frac acc = 0 .000 VAL B 157 frac acc = 9 .323 LEU B 20 frac acc = 0 .000 VAL B 12 frac acc = 9 .207 ILE B 34 frac acc = 0 .000 TRP B 110 frac acc = 9 .069 HIS B 207 frac acc = 0 .000 ALA B 143 frac acc = 8 .903 HIS B 35 frac acc = 0 .000 SER B 135 frac acc = 8 .897 GLY B 146 frac acc = 0 .000 PHE B 129 frac acc = 8 .895 CYS B 203 frac acc = 0 .000 ARG B 50 frac acc = 8 .639 CYS B 147 frac acc = 0 .000 ALA B 61 frac acc = 8 .547 CYS B 96 frac acc = 0 .000 ALA B 132 frac acc = 7,882 ASP B 90 frac acc = 0 .000 VAL B 191 frac acc = 7 .366 ALA B 106 frac acc = 0 .000 PRO B 126 frac acc = 7 .258 ALA B 49 frac acc = 0 .000 PHE B 153 frac acc = 6 .918 The following two criteria were applied to identify the hu4D5Fabv8 residues that can be designed to be replaced with Cys residues: 1. The amino acid residues that are completely buried are eliminated, i.e., less than 10% of the fractional surface accessibility. Table 1 shows that there are 134 residues (light chain) and 151 (heavy chain) of hu4D5Fabv8 that are more than 10% accessible (fractional surface accessibility). The ten most accessible residues Ser, Ala and Val were selected due to their close structural similarity to Cys over other amino acids, introducing only minimal structural restrictions in the antibody by means of newly manufactured Cys. Other cysteine replacement sites may also be visualized and may be useful for conjugation. 2. the residues are selected based on their role in the functional and structural interactions of Fab. We also selected residues that are not involved in antigen interactions and distant from the existing disulfide bonds. The newly designed Cys residues must be different from, and not interfere with, antigen binding, or form erroneous pairs with the cysteines involved in disulfide bond formation. The following residues of hu4D5Fabv8 possess the above criteria and were selected for replacement with Cys: L-V15, L-A43, L-V110, L-A144, L-S168, H-A88, H-A121, H-S122, H -A175 and G-S179 (shown in Figure 1). The thiol reactivity can be generalized to any antibody wherein the replacement of amino acids with reactive cysteine amino acids can be effected within the ranges in the light chain 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 within the ranges in the heavy chain selected from: H-35 to H-45; H-83 to H-93; H-114 to H-127; and H-170 to H-184, and in the Fe region within the selected ranges of H-268 to H-291; H-319 to H-344; H-370 to H-380; and H-395 to H-405. The thiol reactivity can also be generalized to certain domains of an antibody, such as the light chain constant domain (CL) and the heavy chain constant domains, CH1, CH2 and CH3. The cysteine replacements that result in values of thiol reactivity of 0.6 and greater can be made in the constant domains of heavy chain, d, e,?, And μ of intact antibodies: IgA, IgD, IgE, IgG and IgM, respectively, including subclasses of IgG: IgGl, IgG2, IgG3, IgG4 and IgA2. It is evident from the crystal structure data that the selected Cys mutants are very far from the antigen combining site, such as the HER2 interface in this case. These mutants can be experimentally tested for their indirect effects on functional interactions. The thiol reactivities of all Fab variants of Cys were measured and calculated as described in Examples 1 and 2, and are presented in Table 2. Residues L-V15C, L-V110C, H-A88C and H-A121C they have reactive and stable thiol groups (Figures 3A and 3B).

The mutants V15C, V110C, A144C, S168C are light chain Cys variants. The mutants A88C, A121C, A175C, S179C are Cys heavy chain variants. It was surprising and unexpected that sites with high fractional surface accessibility did not have the highest thiol reactivity calculated by the PHESELECTOR analysis (Table 2). In other words, the fractional surface accessibility (Tables 1, 2) does not correlate with the thiol reactivity (Table 2). In fact, Cys residues designed at sites with moderate surface accessibility of 20% to 80% (Figure 4A, Table 1), or at partially exposed sites, such as Ala or Val residues, exhibited better reactivity thiol, i.e., > 0.6 (Figure 3B, Table 2) that the Cys introduced in Ser residues, thus needing the use of PHESELECTOR analysis in the visualization of reactive thiol sites since the crystal structure information is not sufficient to select these sites (Figure 3B, and 4A). The thiol reactivity data are shown in Figures 3A and 3B for the amino acid residues of 4D5 ThioFab Cys mutants: (3A) non-biotinylated phago-ThioFabs (control) and (3B) biotinylated. the reactive thiol groups on the antibody / Fab surface were identified by PHESELECTOR analysis by the interaction of non-biotinylated phage-hu4D5Fabv8 (3A) and biotinylated phage-hu4D5Fabv8 (3B) with BSA (open cell), HER2 (gray box), or streptavidin (solid box). The analysis was carried out as described in Example 2. Light chain variants are found on the left side and heavy chain variants on the right side. The binding of 4D5 ThioFab Cys mutants is low as expected, but a strong binding to HER2 is retained. The ratio of streptavidin binding and HER2 of the 4D5 ThioFab Cys mutants gives the thiol reactivity values in Table 2. The background absorbance at 450 nm or the non-specific protein binding of the biotinylated ThioFab Cys 4D5 mutants to BSA is also evident in Figure 3B. The fractional surface accessibility values of the selected amino acid residues that were replaced with a Cys residue are shown in Figure 4A. The fractional surface accessibility was calculated from the structure of available hu4D5Fabv7 and is shown in Table 1 (Eigenbrot et al., (1993) J. Mol. Biol., 229: 969-995). The shaping parameters of the structures hu4D5Fabv7 and hu4D5Fabv8 are highly consistent and allow the determination of any correlation between the fractional surface accessibility calculations of the cysteine mutants hu4D5Fabv7 and the thiol reactivity of the hu4D5Fabv8. The thiol reactivity measured from ThioFab Cys phage residues introduced in partially exposed residues (Ala or Val) has better thiol reactivity compared to that introduced in Ser residues (Table 2). It can be observed at From the ThioFab Cys mutants of Table 2, there is 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 an antibody can generally be mutated (replaced) with free cysteine amino acids. Ranges within about 5 amino acid residues on each side of these positions can also be replaced with free cysteine acids, 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, as well as the ranges in the Fe region selected from H-268 to H-291; H-319 to H- 344; H-370 to H-380; and H-395 to H-405 to produce the designed cysteine antibodies of the invention.

Table 2. Thiol reactivity of phage-ThioFabs L = light chain, H = heavy chain, A = alanine, S = serine, V = valine, C = cysteine * Thiol reactivity is measured as the ratio of OD50 nm for binding to streptavidin at OD450 nm for binding to HER2 (antibody) (Example 2). The thiol reactivity value of 1 indicates the complete biotinylation of the cysteine thiol. Two Cys variants of the light chain (L-V15C and L-V110C) and two of the heavy chain (H-A88C and H-A121C) were selected for further analysis as the variants that showed the highest thiol reactivity (Table 2) . Unlike phage purification, the Fab preparation may require 2-3 days, depending on the scale of production. During this time, the thiol groups may lose the reactivity due to oxidation. To test the stability of the thiol groups in hu4D5Fabv8-phage, the stability of the thiol reactivity of phage-ThioFabs was calculated (Figure 4B). After purification of ThioFab- phage, on day 1, day 2 and day 4, all samples were - - conjugated with biotin-PEO-maleimide and were tested with a phage ELISA (PHESELECTOR) to test the binding to HER2 and streptavidin. L-V15C, L-V110C, H-A88C and H-A121C retain significant amounts of thiol reactivity compared to other ThioFab variants (Figure 4B). MARKED ANTIBODIES DESIGNED FROM CYSTEINE The designed cysteine antibodies of the invention can be conjugated to any labeled residue that can be covalently bound to the antibody through a reactive thiol cysteine group (Singh et al., (2002) Anal. Biochem., 304 : 147-15; Harlow E., and Lane D., (1999) Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Lundblad R.L., (1991) Chemical Reagents for Protein Modification, 2nd ed., CRC Press, Boca Raton, FL). The linked mark may function to: (i) provide a detectable signal; (ii) interacting with a second tag to modify the detectable signal provided by the first or second tag, e.g., to give FRET (resonance energy transfer by fluorescence); (iii) stabilize the interactions or increase the binding affinity, with antigen or ligand; (iv) affect mobility, e.g., electrophoretic mobility or cellular permeability, by charge, hydrophobicity, form, or other physical parameters, or (v) provide a capture residue, to modulate the affinity of the ligand, the binding of antibody / antigen or the ionic complex ion. The engineered labeled cysteine antibodies may be useful in diagnostic assays, e.g., to detect the expression of an antigen of interest in specific cells, tissues or serum. For diagnostic applications, the antibody will typically be labeled with a detectable residue. Numerous trademarks are available that can generally be grouped into the following categories: (a) Radioisotopes (radionuclides), such as 3H, "C, 14C 18F, 32P, 35S, 64Cu, S8Ga, 86Y, 99Tc, ^ In, 123I, 124I , 125I, 133Xe, 177Lu, 211At, or 213Bi Antibodies labeled with radioisotopes are useful in receptor-directed visualization experiments.The antibody can be labeled with ligand reagents that bind, chelate, or otherwise complex with a radioisotope metal wherein the reagent is reactive with the designed cysteine thiol of the antibody, using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed., Interscience, New York, NY, Pubs (1991) Chelate ligands that can complex a metal ion include DOTA, DOTP, DOTMA, DTPA, and TETA (Macrocyclics, Dallas, TX) .The radionuclides can be directed through complexation with the conjugates of antibody-d of the invention (Wu et al., (2005) Nature Biotechnology 23 (9) -.1137-1146.

- - Metal-chelate complexes suitable as antibody labels for visualization experiments are described: 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. Cancer 1990, Suppl. 10: 21-26; Izard et al., (1992) Bioconjugate Chem., 3: 346-350; Nikula et al., (1995) Nucí. Med. Biol. 22: 387-90; Camera et al., (1993) Nucí. Med. Biol., 20: 955-62; Kukis et al., (1998) J. Nucí. Med. 39: 2105-2110; Verel et al., (2003) J. Nucí. Med., 44: 1663-1670; Camera et al., (1994) J. Nucí. Med., 21: 640-646; Ruegg et al., (1990) Cancer Res. 50: 4221-4226; Verel et al., (2003) J. Nucí. Med., 44: 1663-1670; Lee et al., (2001) Cancer Res., 61: 4474-4482; Mitchell et al., (2003) J. Nucí. Med., 44: 1105-1112; Kobayashi et al., (1999) Bioconjugate Chem., 10: 103-111; Miederer et al., (2004) J. Nucí. Med., 45: 129-137; DeNardo et al., (1998) Clinical Cancer Research 4: 2483-90; Blend et al., (2003) Cancer Biotherapy & Radiopharmaceuticals 18: 355-363; Nikula et al., (1999) J. Nucí. Med., 40: 166-76; Kobayashi et al., (1998) J. Nucí. Med., 39: 829-36; Mardirossian et al., (1993) Nucí. Med. Biol. 20: 65-74; Roselli et al., (1999) Cancer Biotherapy & Radiopharmaceuticals 14: 209-20. (b) Fluorescent labels such as rare earth ground chelates (europium chelates), fluorescein types including FITC, 5-carboxyfluorescein, 6-carboxyfluorescein; rhodamine types including TAMRA; dansil; lysamine; cyanines; phycoerythrins; Red Texas; and its analogues. Fluorescent labels can be conjugated to antibodies using the techniques described in Current Protocols in Immunology, supra, for example. Fluorescent dyes and fluorescent labeling reagents include those commercially available from Invitrogen / Molecular Probes (Eugene, OR) and Pierce Biotechnology, Inc., (Rockford, IL). (c) Various enzyme-substrate labels are available or described (US 4275149). The enzyme generally catalyzes a chemical alteration of a chromogenic substrate that can be measured using various techniques. For example, the enzyme can catalyze a color change in a substrate, which can be measured spectrometrically. Alternatively, the enzyme can alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying a change in fluorescence were described above. the chemiluminescent substrate is excited electronically by a chemical reaction and can then emit light that can be measured (using, for example, a chemiluminometer) or donate energy to a fluorescent receptor. Examples of enzyme labels include luciferases (eg, firefly luciferase and bacterial luciferase; US 4737456), luciferin, 2,3-dihydrophthalazineadiones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, glucoamylase, lysozyme , saccharide oxidases (eg, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as 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 Immunoassay", in Methods in Enzym. , (ed J. Langone &H. Van Vunakis), Academic Press, New York, 73: 147-166. Examples of enzyme-substrate combinations include, for example: (i) Horseradish peroxidase (HRP) with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor (eg, orthophenylene diamine (OPD) or 3, 3 '. 5.5 '-tetramethylbenzidine hydrochloride (TB)); (ii) alkaline phosphatase (AP) with para-nitrophenyl phosphate as a chromogenic substrate; and (iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl-S-D-galactosidase) or a fluorogenic substrate 4 -methylumbeliferyl-β-D- galactosidase Numerous other enzyme-substrate combinations are available to those skilled in the art. For a general review, see US 4275149 and US 4318980. A label can be conjugated indirectly with a designed antibody of cysteine. For example, the antibody can be conjugated with biotin and any of the three broad categories of brands mentioned above can be conjugated with avidin or streptavidin or vice versa. Biotin binds selectively to streptavidin and therefore, the label can be conjugated to the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the tag with the polypeptide variant, the polypeptide variant is conjugated with a small hapten (eg, digoxin) and one of the different types of tag mentioned above is conjugated with an anti-polypeptide variant. hapten (eg, anti-digoxin antibody). In this way, indirect conjugation of the tag can be achieved with the polypeptide variant (Hermanson G., (1996) in Bioconjugate Techniques Academic Press, San Diego). The polypeptide variant of the present invention can be employed in any known assay method, such as ELISA, competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation analysis (Zola (1987) Monoclonal Antibodies: A Manual of Techniques, pp. 147-158, CRC Press, Inc.). A detection mark can be useful to locate, visualize, and quantify a union or recognition event. The labeled antibodies of the invention can detect cell surface receptors. Another use for detectably labeled antibodies is a pearl immunocapture method which comprises conjugating a bead with a fluorescently labeled antibody and detecting a fluorescence signal upon binding a ligand. Similar binding detection methodologies utilize the surface plasmon resonance (SPR) effect to measure and detect antibody-antigen interactions. Detection labels 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 a detectable signal and are generally applicable for labeling antibodies, preferably, with the following properties: (i) the labeled antibody must produce a very high signal with low background so that small amounts of antibodies can be detected in both free analysis of cells as cell-based; and (ii) the labeled antibody must be photostable so that the fluorescent signal can - - Observe, monitor and register without a photo significant whitening. For applications involving the binding of the cell surface of the labeled antibody to membranes or cell surfaces, especially living cells, the preferably (iii) labels have a good solubility in water to achieve an effective conjugate concentration and a detection sensitivity and (iv) They are non-toxic to living cells so as not to disrupt the normal metabolic processes of the cells or cause premature cell death. Direct quantification of cell fluorescence intensity and enumeration of fluorescently labeled events, eg, cell surface binding of peptide-dye conjugates can be conducted in a system (FMAT® 8100 HTS System, Applied Biosystems, Foster City, Calif. .) that automates non-radioactive analysis of mixing and reading with living cells or beads (Miraglia "Homogeneous cell and bead based assays for high throughput screening using fluorometric microvolume assay technology", (1999), J. of Biomolecular Screening 4: 193-204 ). Uses of labeled antibodies also include cell surface receptor binding assays, immunocapture assays, fluorescence-linked immunoabsorbent assays (ELISA), caspase split analysis (Zheng "Caspase 3 controls both cytoplasmic and nuclear events associated with Fas- - - mediated apoptosis in vivo ", (1998) Proc. Natl. Acad. Sci. USA 95: 618-23; US 6372907), of apoptosis (vermes" A novel assay for apoptosis. "Flow cytometric detection of phosphatidylserine expression in early apoptotic cells using fluorescein labelled Annexin V "(1995) J. Immunol. Methods, 184: 39-51) and cytotoxic The technology of microvolume fluorometric analysis can be used to identify the envelope or sub-regulation by a molecule directed to the cell surface ( Swartzman "A homogeneous and multiplexed immunoassay for high throughput screening using fluorometric microvolume assay technology" (1999) Anal. Biochem., 271: 143-51) The engineered designer cysteine antibodies of the invention are useful as display biomarkers and probes by the various methods and techniques of biomedical and molecular visualization such as: (i) MRI (magnetic resonance imaging), (ii) MicroCT (computed tomography), (iii) SPECT (tomography) computerized by single photon emission); (iv) PET (positron emission tomography) Chen et al., (2004) Bioconjugate Chem., 15: 41-49; (v) bioluminescence; (vi) fluorescence; and (vii) ultrasound. Immunoscintigraphy is a visualization procedure in which antibodies labeled with radioactive substances are administered to an animal or human patient and a virus is taken. photograph of the sites in the body where the antibody is located (US 6528624). Biomarkers of visualization can be measured and evaluated objectively as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. Biomarkers can be of several types: Type 0 are markers of the natural history of a disease and are correlated longitudinally with known clinical indexes, e.g., RI evaluation of synovial inflammation in rheumatoid arthritis; Type I markers capture the effect of an intervention according to a mechanism of action, although the mechanism may not be associated with clinical development; Type II markers function as surrogate endpoints where the change in, or signal from, the biomarker predicts a clinical benefit to "validate" the target response, such as measuring bone erosion in rheumatoid arthritis using CT. The display biomarkers can therefore provide therapeutic pharmacodynamic (PD) information about: (i) expression of an objective protein; (ii) the binding of a therapeutic to the target protein, i.e., selectivity, and (iii) cleaning data and pharmacokinetic half-life. The advantages of biomarkers of in vivo visualization in relation to biomarkers based on lab include: non-invasive treatment, quantifiable evaluation in the whole body, dosage and repetitive establishment, i.e., multiple time points, and potentially transferable effects from preclinical (small animal) to clinical (human) outcomes. For some applications, biovisualization supplants or minimizes the number of animal experiments in preclinical studies. Radionuclide display marks include radionuclides such as 3H, "C, 14C, 18F, 32P, 35S, 64Cu, 68Ga, 86Y," Te, ^ ln, 123I, 124I, 125I, 131I, 133Xe, 177Lu, 211At or 213Bi . The radionuclide metal ion can be complexed with a chelate linkage such as DOTA. Binding reagents such as DOTA-maleimide (4-maleimidabutyramidobenzyl-DOTA) can be prepared by the reaction of aminobenzyl-DOTA with 4 -maleimidabutyric acid (Fluka) activated with isopropylchloroformate (Aldrich), following the procedure of Axworthy et al., ( 2000 = Proc. Nati. Acad. Sci. USA 97 (4): 1802 - 1807). The DOTA-maleimide reagents react with the free cysteine amino acids of the antibodies designed with cysteine and provide a complex metal binding ligand in the antibody (Lewis et al., (1998) Bioconj. Chem., 9: 72-86 ). Chelate-binding labeling reagents such as DOTA-NHS acid (N-hydroxysuccinimide ester) (1,4,7,10-tetraazacyclododecane-1,4,7,7-tetraacetic are commercially available (Macrocyclics, Dallas, TX ) . The visualization of receptor target with radionuclide-labeled antibodies can provide a pathway activation marker by detecting and quantifying the progressive accumulation of antibodies in tumor tissue (Albert et al., (1998) Bioorg. Med. Chem. Lett., 8: 1207-1210). The conjugated radio metals can remain intracellular after lysosomal degradation. Peptide labeling methods are well known. See Haugland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc .; Brinkley 1992, Bioconjugate Chem., 3: 2; Garman (1997) Non-Radioactive Labeling: A Practical Approach, 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, Vols. I and II, CRC Press, New York; Pfleiderer G., (1985) "Chemical Modification of Proteins", Modern Methods in Protein Chemistry, H. Tschesche, Ed., Walter DeGryter, Berlin and New York; and Wong (1991) Chemistry of Protein Conjugation and cross linking, CRC Press, Boca Raton, Fia.); De Leon-Rodriguez et al., (2004) Chem. Eur. J. 10: 1149-1155; Lewis et al., (2001) Bioconjugate Chem. 12: 320-324; Li et al., (2002) Bioconjugate Chem., 13: 110-115; Mier et al., (2005) Bioconjugate Chem. 16: 240-237. Peptides and proteins labeled with two residues, a fluorescent reporter and sated in sufficient proximity, undergo fluorescence resonance energy transfer (FRET). Reporting groups are typically fluorescent dyes that are excited by light at a certain wavelength and transfer energy to a target or satiated group, with the appropriate Stokes shift for emission at maximum brightness. Fluorescent dyes include molecules with extended aromaticity, such as fluorescein and rhodamine, and their derivatives. The fluorescent reporter may be partially or significantly satiated by the satiated residue in an intact peptide. By dividing the peptide by a peptidase or protease, a detectable increase in fluorescence can be measured (Knight C, (1995) "Fluorometric Assays of Proteolytic Enzymes", Methods in Enzymology, Academic Press, 248: 18-34). The labeled antibodies of the invention can also be used as an affinity purifying agent. In this process, the labeled antibody is immobilized on a solid phase such as a Sephadex resin or filter paper, using methods well known in the art. the immobilized antibody is contacted with a sample containing the antigen to be purified, and then the The support is washed with a suitable solvent that will remove substantially all of the material in the sample except the antigen to be purified, which is bound to the immobilized polypeptide variant. Finally, the support is washed with another suitable solvent, such as glycine buffer, pH 5.0, which will release the antigen from the polypeptide variant. Labeling reagents typically contain reactive functionality that can react (i) directly with a thiol cysteine of an antibody designed with cysteine to form the labeled antibody, (ii) with a linker reagent to form a label-binding intermediate, or (iii) ) with a binding antibody to form the labeled antibody. The reactive functionality of labeling reagents includes: maleimide, haloacetyl, iodoacetamide succinimidyl ester (eg, NHS, N-hydroxysuccinimide), isothiocyanate, sulfonyl chloride, 2,6-dichlorotriazinyl, pentafluorophenyl ester and phosphoramidite, although other functional groups may also be used . An exemplary reactive functional group is N-hydroxysuccinimidyl ester (NHS) of a carboxyl group substituent of a detectable label, e.g., biotin or a fluorescent dye. The NHS ester of the brand can be preformed, isolated, purified and / or characterized or it can be formed in situ and reactivated with a nucleophilic group of an antibody. Typically, the carboxyl form of the label is activated by reacting with some combination of a carbodiimide reagent, eg, dicyclohexylcarbodiimide, diisopropylcarbodiimide or a uronium reagent, eg, TSTU (O- (N-succinimidyl) -?,?,? ' ,? '-tetramethyluronium tetrafluoroborate, HBTU (O-benzotriazol-1-yl) -?,?,',? '-tetramethyluronium hexafluorophosphate, or HATU (O- (7-azabenzotriazol-1-yl) -?,?,? ',?' -tetramethyluronium hexafluorophosphate), an activator, such as 1-hydroxybenzotriazolo (HOBt) and N-hydroxysuccinimide to give the NHS-branded ester.In some cases, the label and antibody can be coupled by in situ activation of the Mark and reaction with the antibody to form the antibody-label conjugate in one step Other activation and coupling reagents include TBTU (2- (1H-benzotriazo-l-yl) -1-1,3, 3-tetramethyluronium hexafluorophosphate ), TFFH (?.? ',?' ',?' '' -tetramethyluronium 2-fluoro-hexafluorophosphate), PyBOP (benzotriazolo-l-il-oxy-tr is-pyrrolidino-phosphonium hexafluorophosphate, EEDQ (2-ethoxy-l-ethoxycarbonyl-1,2-dihydro-quinoline), DCC (dicyclohexylcarbodiimide); DIPCDI (diisopropylcarbodiimide), MSNT (1- (mesitylene-2-sulfonyl) -3-nitro-lH-1, 2,4-triazolo, and aryl sulfonyl halides, e.g., triisopropylbenzenesulfonyl chloride CONJUGATION OF BIOTIN-MALEIMIDE TO THIOFABS - - The ThioFab properties described above were evaluated in the presence of phage because the fusion of Fab to the phage coating protein can potentially alter the accessibility or reactivity of Cys thiol. Accordingly, ThioFab structures were cloned into an expression vector under the alkaline phosphatase promoter (Chang et al., (1987) Gene 55: 189-196) and ThioFab expression was induced by culturing E. coli cells in the phosphate-free medium. The ThioFabs were purified on a SEPHAROSE ™ Protein G column and analyzed on SDS-PAGE reduction and non-reduction gels. These analyzes allow to evaluate if the ThioFab retain their reactive thiol group or become inactive forming intramolecular or intermolecular disulfide bonds. ThioFabs L-V15C, L-V110C, H-A88C, and H-A121C were expressed by SEPHAROSE ™ Protein G column chromatography (see methods sections for details). The purified proteins were analyzed in SDS-PAGE gel under conditions of reduction (with DTT) and non-reduction (without DTT). Other reducing agents such as BME (beta-mercaptoethanol) can be used in the gel to divide the interchain disulfide groups. It is evident from the analysis in SDS-PAGE gel that the main fraction (-90%) of ThioFab is in the monomeric form, while the wild-type hu4D5Fabv8 is essentially in the monomeric form (47 kDa).

The ThioFab (A121C) and the wild-type hu4D5Fabv8 were incubated with a 100-fold excess of biotin-maleimide for 3 hours at room temperature and the biotinylated Fabs were loaded onto a Superdex-200 ™ gel filtration column. This purification step was useful in the separation of the monomeric Fab from the oligomeric Fab and also from the excess of free biotin-maleimide (or free cytotoxic drug). Figure 5 shows the validation of the properties of the ThioFab variants in the absence of the phage context. Proteins without phage fusion, hu4D5Fabv8 and hu4D5Fabv8-A121C (ThioFab-A121C) were expressed and purified using G protein agarose beads followed by incubation with a 100-fold molar excess of biotin-maleimide. The binding to streptavidin and HER2 of a biotinylated ThioFab made of cys and a non-biotinylated wild-type Fab was compared. The degree of biotin conjugation (interaction with streptavidin) and its binding capacity to HER2 were monitored by ELISA analysis. Each Fab was tested at 2 ng and 20 ng. The biotinylated ThioFab A121C retained a binding to HER2 comparable to that of wild-type hu4D5Fabv8 (Figure 5). Wild-type Fab and ThioFab A121C were purified by column chromatography by gel filtration. The two samples were tested for binding to HER2 and - - Streptavidin by ELISA using goat anti-Fab-HRP as a secondary antibody. Both the wild type (open cell) and ThioFab · (dotted cell) have a similar union to HER2 but only ThioFab retained the binding to streptavidin. Only a background level of interaction with streptavidin was observed with non-biotinylated wild type hu4D5Fabv8 (Figure 5). Mass spectral analysis (LC-ESI-MS) of biotinylated ThioFab (A121C) resulted in a major peak with 48294.5 daltons compared to wild-type hu4D5Fabv8 (47737 daltons). The difference of 537.5 daltons between the two molecules corresponds exactly to a single biotin-maleimide conjugated to ThioFab. The results of the mass spectrum protein sequence (LC-ESI Tandem mass spec analysis) further confirmed that the conjugated biotin molecule was found in the newly manufactured Cys residue (Table 4, Example 3). SPECIFIC CONJUGATION OF THE BIOTIN SITE MALEIMIDA TO PEPTIDE OF UNION TO ALBUMIN (ABP) -THIOFABS The plasma-protein binding can be an effective means to improve the pharmacokinetic properties of short-lived molecules. Albumin is the most abundant protein in plasma. Serum albumin binding peptides (PBL) can alter the pharmacodynamics of fused active-domain proteins, including altered absorption, penetration and diffusion in tissue. These pharmacodynamic parameters can be modulated by specific selection of the appropriate serum albumin binding peptide sequence (US 20040001827). A series of albumin binding peptides were identified by phage display visualization (Dennis et al., (2002) "Albumin Binding as a General Strategy for Improving the Pharmacokinetics of Proteins", J. Biol. Chem., 277: 35035- 35043; WO 01/45746). The compounds of the invention include ABP sequences shown by: (i) Dennis et al., (2002) J. Biol. Chem., 277: 35035-35043 in Tables III and IV, page 35038; (ii) US 20040001827 in [0076] SEQ ID NOS: 9-22; and (iii) WO 01/45746 on pages 12-13, SEQ ID NOS: zl-zl4, all of which are incorporated herein by reference. The albumin binding (ABP) -Babs were manufactured by fusing an albumin-binding peptide to the X-terminus of the Fab heavy chain in a stoichiometric ratio of 1: 1 (1 ABP / l Fab). It was shown that the association of these ABP-Fabs with albumin increased its half-life by more than 25 times in rabbits and mice. The reactive Cys residues described above can, therefore, be introduced into these ABP-Fabs and used for site-specific conjugation with cytotoxic drugs followed by animal studies in vivo. Figure 9 shows a graph of a conjugate of fusion binding peptide-albumin-Fab binding drug (ABP-Fab). Exemplary albumin-binding peptide sequences include, but are not limited to, the amino acid sequences listed in SEQ ID NOS: 1-5: CDKTHTGGGSQRL EDICLPR GCLWEDDF SEQ ID NO: 1 QRLMEDICLPR GCL EDDF SEQ ID NO: 2 QRLIEDICLPR GCLWEDDF SEQ ID NO: 3 RLIEDICLPR GCLWEDD SEQ ID NO: 4 DICLPRWGCL SEQ ID NO: 5 Albumin-binding peptide (ABP) sequences bind to albumin of multiple species (mouse, rat, rabbit, bovine, rhesus, baboon and human) ) with Kd (rabbit) = 0.3 μ ?. The albumin binding peptide does not compete with the known ligands for binding to albumin and has a half-life (T ½) in rabbit of 2.3 hours. The ABP-ThioFab proteins were purified on BSA-SEPHAROSE ™ followed by biotin-maleimide conjugation and purification on Superdex-S200 column chromatography as described in the previous sections. The purified biotinylated proteins were homogeneous and devoid of any oligomeric form (Example 4). Figure 6 shows the properties of albumin-binding peptide (PBL) -thioFab variants. ELISA analyzes were carried out to test the binding capacity of ABP- hu4D5Fabv8-wt, ABP-hu4D5Fabv8 -VllOC and ABP-hu4D5Fabv8-A121C with rabbit albumin, streptavidin and HER2. Biotinylated ABP-ThioFabs are capable of binding to albumin and HER2 with affinity similar to that of wild-type ABP-hu4D5Fabv8 as confirmed by ELISA (Figure 6) and BIAcore binding kinetics (Table 3). An ELISA plate was coated with albumin, HER2 and SA as described. The binding of biotinylated ABP-ThioFabs to albumin, HER2 and SA with anti-Fab HRP was tested. The biotinylated ABP-ThioFabs were able to bind to streptavidin compared to the non-biotinylated ABP-hu4D5Fabv8-wt indicating that the ABP-ThioFabs were conjugated with biotin-maleimide as ThioFabs in a site-specific manner since the same mutants were used Cys for both variants (Figure 6).

Table 3. Analysis of BIAcore kinetics for binding of HER2 and rabbit albumin to wild-type biotinylated ABP-hu4D5Fabv8 and ThioFabs - - ABP = albumin binding peptide Alternatively, an albumin binding peptide can be bound to the antibody by covalent attachment through a linker residue. MANUFACTURE OF ABP-THIOFABS WITH TWO FREE TIOL GROUPS PER FAB The above results indicate that the four variants (L-V15C, L-V110C, H-A88C and H-A121C) of thiofab (Fab-designed cysteine antibodies) have reactive thiol groups which can be used for site-specific conjugation with a branded reagent, binding reagent, or drug-binding intermediate. L-V15C can be expressed and purified but with relatively low yields. However, the expression and purification yields of variants L-V110C, H-A88C and H-A121C were similar to those of hu4D5Fabv8. Accordingly, these mutants can be used for further analysis and recombined to obtain more than one thiol per Fab group. Towards this goal, a thiol group was constructed in the light chain and one in the heavy chain to obtain two thiol groups per Fab molecule (L-V110C / H-A88C and L-V110C / H-A121C). these two variants of double Cys were expressed in an E. coli expression system and they were purified. The homogeneity of purified biotinylated ABP-ThioFabs was found to be similar to that of unique variants of Cys. The effects of the manufacture of two Cys reagent residues per Fab were investigated (Figure 7). The presence of a second biotin was tested by testing the binding of biotinylated ABP-ThioFab to SA using streptavidin-HRP (Figure 7). For the HER2 / Fab analysis, an ELISA plate was coated with HER2 and tested with anti-Fab HRP. For the SA / Fab analysis, an ELISA plate was coated with SA and tested with anti-Fab HRP. For the SA / SA analysis, an ELISA plate was coated with SA and tested with SA-HRP. Figure 7. ELISA analyzes for the interaction of cys variants ABP-hu4D5Fabv8 with HER2, streptavidin (SA), HER2 / Fab, SA / Fab and SA / SA indicate that their interactions were monitored by HRP anti-Fab, SA-HRP, respectively. SA / Fab monitors the presence of biotin alone per Fab and more than one biotin per Fab is monitored by SA / SA analysis. The binding of HER2 with double mutants of cys is similar to that of the unique Cys variants (Figure 7). However, the degree of biotinylation in double mutants of Cys was higher compared to the unique Cys variants due to more than one free thiol group per Fab molecule (Figure 7). MANUFACTURE OF TIO VARIANTS IgG DE TRASTUZUMAB Cysteine was introduced into the full-length monoclonal antibody, trastuzumab (HERCEPTIN®, Genentech Inc.) in certain residues. The unique mutants of Cys H-A88C, H-A121C and L-V110C of trastuzumab, and the double mutants of cys V110C-A121C and V110C-A121C of trastuzumab were expressed in CHO (Chinese hamster ovarian) cells by transient fermentation in medium containing 1 mM of cysteine. The heavy chain sequence of mutant A88C (450 aa) is SEQ ID NO: 6. The heavy chain sequence of mutant A121C (450 aa) is SEQ ID NO: 7. The light chain sequence of mutant V110C (214 aa) is SEQ ID NO: 8.

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE VARIYPTNGYTRY ADSVKGRFTISADTSK TAYLQMNSLRCEDTAVYYCSRWGGDGFYAMDY GQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDK VEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF YVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE TK QVSLTCLVKGFYPSDIAVE ESNGQPENOTKTTPPVLDSDGSFFLYSIGJIVDKSRW QQG VFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 6 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRY ADSVKGRFTISADTSK TAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS CSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSWTVPSSSLGTQTYICNV HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQD LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN YKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID N0: 7 DIQMTQSPSSLSASVGDRVTITCRASQDV TAVAWYQQKPGKAPKLLIYSASFLYSGVPS RFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTCAAPSVFIFPP SDEQLKSGTASWCLLN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 8 According to one embodiment, the designed cysteine thio-trastuzumab antibodies comprise one or more of the following variable region heavy chain sequences with a free cysteine amino acid (SEQ ID NOS: 9-16).

Mutant Sequence SEQ ID NO: 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 designed cysteine thio-trastuzumab antibodies comprise one or more of the following variable region light chain sequences with an amino acid of free cysteine (SEQ ID NOS 17-27).

The resulting total length IgG thio-trastuzumab variants were analyzed by thiol reactivity and HER2 binding activity. Figure 13A shows an illustration of the binding of the biotinylated antibody to immobilized HER2 and secondary antibody labeled with HRP for absorbance detection. Figure 13B shows immobilized HER2 binding measurements with absorbance detection at 450 nm from (left to right): wild-type non-biotinylated trastuzumab (Wt), thio-trastuzumab variants of biotin-maleimide conjugate V110C (cys) single), A121C (single cys), V110C / A121C (double cys). Each thio IgG variant and trastuzumab was tested at l, 10 and 100 ng. The measurements show that the biotinylated anti-HER2 ThioMabs retain the HER2 binding activity. Figure 14A shows an illustration of the binding of the biotinylated antibody to immobilized HER2 with the binding of biotin to anti-IgG-HRP for absorbance detection. Figure 14B shows binding calculations with absorbance detection at 450 nm of thio trastuzumab variants of biotin-maleimide conjugate and wild type non-biotinylated trastuzumab at streptavidin binding. From left to right: V110C (single cys), A121C (single cys), V110C / A121C (double cys), and trastuzumab. Each variant of IgG trastuzumab and trastuzumab of origin was tested at 1, 10 and 100 ng. The measurements show that ThioMabs HER2 have high thiol reactivity. Cysteine was introduced into the full-length anti-EphB2R 2HP antibody in certain residues. The single mutant of cys H-A121C of 2H9 was expressed in CHO (Chinese hamster ovarian) cells by transient fermentation in medium containing 1 mM of cysteine. The heavy chain sequence of mutant A121C 2H9 (450 aa) is SEQ ID NO: 28.

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMH VRQAPGKGLEWVGFINPSTGYTDY NQKFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCTRRPKIPRHANVFWGQGTLVTVSS CSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNV HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY STYRWSVLTVLHQD LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVE ESNGQPEN YKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALH HYTQKSLSLSPGK SEQ ID NO: 28 The thio-2H9 antibodies designed of cysteine comprise the following heavy chain sequences of Fe constant region with a free cysteine amino acid (SEQ ID NOS: 29-38).

Mutant sequence SEQ ID NO: V273C HEDPECKFN YVDGVEVHNAKTKPR SEQ ID NO: 29 V279C HEDPEVKFNWYVDGVEVHNAKTKPR SEQ ID NO: 30 V28C HEDPEVKFN YVDGCEVHNAKTKPR 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 Figure 16 shows SDS-PAGE analysis (polyacrylamide gel electrophoresis) of denaturation of non-reduction (upper part) and reduction (lower part) of 2H9 ThioMab Fe variants (from left to right, fields 1-9); A339C; S337C; S324C; A287C; V284C; V282C; V279C; and V273C with wild-type 2H9 after purification on immobilized Protein A. The field on the right is a scale marker scale, indicating that intact proteins are approximately 150 kDa, heavy chain fragments approximately 50 kDa, and light chain fragments approximately 25 kDa. Figure 17A shows analysis of denatured polyacrylamide gel electrophoresis of nonreduction (left) and reduction (right) of 2H9 ThioMab variants (from left to right, fields 1-4): L-V15C; S179C; S375C; S400C, after purification on immobilized Protein A. Figure 17B shows denatured polyacrylamide gel electrophoresis analysis of nonreduction (left) and reduction (+ DTT) (right) of additional variants of 2H9 and 3A5 ThioMab after purification on immobilized Protein A. The 2H9 ThioMab variants (in the Fab as well as in the Fe region) were expressed and purified as described. As seen in Figures 16, 17A and 17B, all proteins are homogeneous in SDS-PAGE followed by the reduction and oxidation procedure of Example 11 to prepare ThioMabs reagents for conjugation (Example 12). Cysteine was introduced into the full-length anti-MUC16 antibody 3A5 in certain residues. The mutant The unique of cys H-A121C of 3A5 was expressed in CHO (Chinese hamster ovarian) cells by transient fermentation in medium containing 1 mM of cysteine. The heavy chain sequence of mutant A121C 3A5 (446 aa) comprises SEQ ID NO: 39.

DVQLQESGPGLVNPSQSLSLTCTVTGYSITNDYAWN IRQFPGNKLEWMGYINYSGYTTY NPSLKSRISITRDTSKNQFFLHLNSVTTEDTATYYCARWDGGLTYWGQGTLVTVSACSTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS NSGALTSGVHTFPAVLQSSGLYS LSSWTVPSSSLGTQTYICNVNHKPSNT VDKKVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFN YVDGVEVHNAKTKPREEQYNSTYR WSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVE ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK The cysteine-designed anti-MUC16 thio-3A5 antibodies comprise the following variable region heavy chain sequences with a free cysteine amino acid (SEQ ID NOS: 40-44).

Anti-MUC16 anti-MUC16 thio-3A5 antibodies designed from cysteine comprise the following variable region light chain sequences with a free cysteine amino acid (SEQ ID NOS: 45-49).

THIOMABS TIOL REACTIVITY The thiol reactivity of IgG antibodies designed for full-length cysteine (ThioMabs) was calculated by biotinylation and biotin binding. A western immunoassay was established to visualize the ThioMab that is specifically conjugated with biotin-maleimide. In this analysis, the antibodies are analyzed on SDS-REDUCTION PAGE and the presence of biotin is specifically tested by incubating with streptavidin-HRP. As seen in Figure 18, the streptavidin-HRP interaction is observed either in the heavy chain or in the light chain depending on which variant of manufactured cys is used, and no interaction with the wild type is observed, indicating that the ThioMab variants specifically conjugated biotin in the manufactured Cys residue. Figure 18 shows the denatured gel analysis of reduced biotinylated Thio-IgG variants after capture in immobilized anti-IgG-HRP (upper gel) and streptavidin-HRP (lower gel). Field 1: 3A5 H-A121C. Field 2: 3A5 L-V110C. Field 3: 2H9 H-A121C. Field 4: 2H9 L-V110C. Field 5: anti-EphB2R 2H9 of origin, wild type. Each mutant (fields 1-4) was captured by anti-IgG with detection of HRP (upper part) indicating that selectivity and affinity were retained. The capture by immobilized streptavidin with detection of HRP (lower part) confirmed the location of biotin in the heavy and light chains. The location of the cysteine mutation in the designed cysteine antibodies in fields 1 and 3 is the heavy chain. The location of the cysteine mutation in the designed cysteine antibodies in fields 2 and 4 is the light chain. The cysteine mutation site undergoes conjugation with the biotin-maleimide reagent. Analysis of the designed ThioMab antibodies of cysteine of Figure 18 and of the variant 2H9 V15C by LC / MS provided a quantitative indication of the thiol reactivity (Table 5).

Table 5 LC / MS quantification of ThioMabs biotinylation thiol reactivity Manufacturing with cysteine was conducted in the constant domain, i.e., Fe region, of the IgG antibodies. A variety of amino acid sites were converted to cysteine sites and the expressed mutants, i.e., designed cysteine antibodies, were evaluated for their thiol reactivity. The biotinylated variants of 2H9 ThioMab Fe were evaluated by thiol reactivity by HRP quantification by means of capture in immobilized streptavidin in an ELISA analysis (Figure 19). An ELISA analysis was established to quickly visualize Cys residues with reactive thiol groups. As illustrated in the schematic diagram of Figure 19, the streptavidin-biotin interaction is monitored by testing with an anti-IgG-HRP followed by absorbance measurement at 450 nm. These results confirmed that variants V282C, A287C, A339C, S375C and S400C had moderate to higher thiol reactivity. He Biotin conjugation degree of the 2H9 Thio ab Fe variants was quantified by LS / MS analysis as reported in Table 6. LS / MS analysis confirmed that variants A282C, S375C and S400C had 100% conjugation of biotin and V284C and A339C had 50% conjugation, indicating the presence of a reactive thiol group of cysteine. The other wild type, ThioFc and 2H9 variants had either little or no biotinylation.

Table 6 LC / MS quantification of ThioMabs Fe 2H9 biotinylation TIOL REACTIVITY OF TIO-4D5 VARIANTS FAB LIGHT CHAIN Visualization of a variety of light chain variant Fabs made of cysteine from the antiErbB2 antibody 4D5 yielded a number of variants with a thiol reactivity value of 0.6 and higher, (Table 7), calculated by means of the PHESELECTOR analysis of Figure 8. The thiol reactivity values of Table 7 are normalized to the heavy chain ThioFab 4D5 variant (HC-A121C) which is evaluated at 100%, assuming a complete biotinylation of the variant HC-A121C, and represented as percentage values. Table 7 Percent values of thiol reactivity of light chain ThioFab 4D5 variants CONJUGATES OF ANTIBODY-DRUG Antibodies designed invention can be conjugated with any therapeutic agent, i.e., drug residue, which can be covalently bound to the antibody through a reactive thiol group of cysteine. An exemplary embodiment of an antibody-drug conjugate (ADC) comprises a designed antibody of cysteine (Ab), and a drug residue (D), wherein the antibody has one or more free cysteine amino acids that have a reactivity value thiol in the range of 0.6 to 1.0, and the antibody is bound through one or more amino acids of free cysteine via a linker residue (L) to D; Formula I having the composition. Ab- (LD) p I where p is 1, 2, 3 or 4. The number of drug residues that can be conjugated through a thiol linker residue reactive to an antibody molecule is found limited by the number of cysteine residues that is introduced by the methods described herein. The exemplary ADC of Formula I thus comprises antibodies having 1, 2m3 or 4 amino acids designed of cysteine. Another exemplary embodiment of an antibody-drug conjugate compound (ADC) comprises a designed antibody of cysteine (Ab), an albumin binding peptide (ABP) and a drug residue (D) where the antibody is bound to the drug residue for a linker residue (L) and the antibody is bound to the albumin binding peptide by an amide linkage or a second linker residue; the composition having the formula: ABP-Ab- (L-D) p wherein p is 1, 2, 3 or 4. The ADC compounds of the invention include those with utility for anticancer activity. In particular, the compounds include an antibody conjugate designed from cysteine, i.e., covalently linked by a binding to a drug residue, i.e., toxin. When the drug is not conjugated to an antibody, the drug has a cytotoxic or cytostatic effect. The biological activity of the drug residue is then modulated by means of conjugation to an antibody. The antibody-drug conjugates (ADCs) of the invention selectively deliver an effective dose of a cytotoxic agent to a tumor tissue whereby higher selectivity, i.e., a lower effective dose can be achieved. In one embodiment, the bioavailability of the ADC of the invention, or an intracellular metabolite of the ADC, is improved in a mammal when compared to a drug compound comprising the ADC drug residue. Also, the bioavailability of ADC, or an intracellular metabolite of ADC is improved in a mammal compared to the analogue of the ADC that does not have the drug residue. DRUG RESIDUES The drug residue (D) of the antibody-drug conjugates (ADC) includes any compound, residue or group that has a cytotoxic or cytostatic effect. The drug residues include: (i) chemotherapeutic agents, which may function as microtubulin inhibitors, mitosis inhibitors, topoisomerase inhibitors, or DNA intercalators; (ii) protein toxins, which can function enzymatically; and (iii) radiosotopes. Exemplary drug residues include, but are not limited to, a maytansinoid, an auritantin, a dolastatin, a tricothecene, CC1065, a calicheamicin and other enediin antibiotics, a taxane, an anthracycline and stereoisomers, isosterers, analogs or derivatives thereof. same. Maytansine compounds suitable for use as maytansinoid drug residues are well known in the art and can be isolated from natural sources according to known methods, produced using genetic engineering techniques (see Yu et al., (2002) PROC. NATL ACAD, SCI (USA) 99: 7968-7973) or maytansinol or maytansinol analogues prepared synthetically according to known methods. The exempt maytansinoid drug residues - - include those having a modified aromatic ring, such as: C-19-dechloro (US 4256746) (prepared by reduction of lithium aluminum hydride of ansamitocin P2); C-20-hydroxy (or C-20-demethyl) + / C-19-dechloro (U.S. Patent Nos. 4361650 and 4307016) (prepared by demethylation using Streptomyces or Actinomyces or by dechlorination using LAH); and C-20-demethoxy, C-20-acyloxy (-OCOR), +/- dechloro (U.S. Patent No. 4,294,757) (prepared by acylation using acyl chlorides) and those having modifications in other positions. Exemplary maytansinoid drug residues also include those having modifications such as: C-9-SH (US 4424219) (prepared by the reaction of maytansinol with H2D or P2S5); C-14-alkoxymethyl (demethoxy / CH2OR) (US 4331598); C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2Oac) (US 4450254) (prepared by Nocardia); C-15-hydroxy / alkoxy (US 4364866) (prepared by the conversion of maytansinol by Streptomyces); C-15-methoxy (Patents of U.S. Nos. 4313946 and 4315929) (isolated from Trewia nudlflora); C-18-N-demethyl (U.S. Patent Nos. 436663 and 4322348) (prepared by the demethylation of maytansinol by Streptomyces); and 4,5-deoxy (US 4371533) (prepared by the reduction of titanium trichloride / LAH of maytansinol). The utility of many positions in the maytansin compounds as the binding position is known, depending on the type of Union. For example, for the formation of an ester linkage, the C-3 position having a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with a hydroxyl group and the C-20 position which is It has a hydroxyl group. The drug residue (D) of the antibody-drug conjugates (ADC) of Formula I include maytansinoids having the structure: wherein the wavy line indicates the covalent attachment of the sulfur atom of D to a linker (L) of an antibody-drug conjugate (ADC). R may independently be H or a C 1 -C 6 alkyl selected from methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl, 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2- hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2, 3 - dimethyl-2-butyl, and 3, 3-dimethyl-2-butyl. The alkylene chain linking the amide group to the sulfur atom may be methanil, ethanil, or propyl, ie, m is 1, 2 or 3. Maytansine compounds inhibit cell proliferation by inhibiting microtubule formation during mitosis through of the inhibition of the polymerization of the microtubulin protein, tubulin (Remillard et al., (1975) Science 189: 1002-1005). Maytansine and maytansinoids are highly cytotoxic but their clinical use in cancer therapy has been greatly limited by their severe systemic side effects mainly attributed to their low selectivity for tumors. Clinical trials with maytansine have been discontinued due to serious adverse effects on the central nervous system and the gastrointestinal system (Issel et al., (1978) Can. Treatment Rev., 5: 199-207). The maytansinoid drug residues are attractive drug residues in antibody-drug conjugates because they are: (i) relatively accessible in their preparation by fermentation or chemical modification, derivatization of fermentation products; (ii) docile for derivatization with functional groups suitable for conjugation through non-disulfide bonds to antibodies; (iii) stable in plasma, or (iv) effective against a variety of tumor cell lines (US 2005/0169933; 2005/037992; US 5208020). As with other drug residues, all stereoisomers of the maytansinoid drug residue are contemplated for the compounds of the invention, ie, any combination of the R and S configurations in the chiral carbons of D. In one embodiment, the maytansinoid drug residue (D) will have the following stereochemistry: Exemplary moieties of maytansinoid drug residues include: DM1, (CR2) m = CH2CH2; DM3, (CR2) m = CH2CH2CH (CH3); and DM4, (CR2) m = CH2CH2C (CH3) 2, which have the structures: - - The union can bind to the maytansinoid molecule in several positions, depending on the type of binding. For example, an ester linkage can be formed by reaction with a hydroxyl group using conventional coupling techniques. The reaction can take place at the C-3 position having a hydroxyl group, at the C-14 position modified with hydroxymethyl. In the C-15 position modified with a hydroxyl group, and in the C-20 position having a hydroxyl group. In a preferred embodiment, the linkage is formed at the C-3 position of maytansinol or a maytansinol analogue. The drug residue (D) of the antibody-drug conjugates (ADC) of Formula I also includes dolastatins and their analogues and peptide derivatives, auristatins (U.S. Patent Nos. 5635483; 5780588). Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al., (2001) Antimicrob Agents and Chemother., 45 (12): 3580-3584) and have anti-cancer activity (US 5663149) and anti-fungal activity (Pettit et al., (1988) Antimicrob Agents Chemother., 42: 2961-2965). Various forms of a drug residue of dolastatin or auristatin may be covalently linked to an antibody through the N (amino) terminus or the C (carboxyl) terminus of the peptide drug residue (O 02/088172; Doronina et al. , (2003) Nature Biotechnology 21 (7): 778-784; Francisco et al., (2003) Blood 102 (4): 1458-1465). The drug residues include dolastatins, auristatins (US 5635483; US 5780588; US 5767237; US 6124431), and analogs and derivatives thereof. Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Oyke et al., (2001) Antimicrob Agents and Chemother., 45 (12): 3580-3584) and have anti-cancer activity (US 5663149) and anti-fungal activity (Pettit et al., (1988) Antimicrob Agents Chemother., 42: 2961-2965). The drug residue of dolastatin or auristatin can be bound to the antibody via the N (amino) terminus or the C (carboxyl) terminus of the peptide drug residue (O 02/088172). Exemplary auristatin moieties include drug residues DE and DF of monomethylauristatin attached to the N terminus described in: WO 2005/081711; Senter et al., Proceedings of the American Association for Cancer Research, Volume 45, excerpt number 623, filed on March 28, 2004, the description of which is expressly incorporated by reference in its entirety. The drug residue (D) of the antibody-drug conjugates (ADC) of Formula 1 includes the drug residues of monomethylauristatin MMAE and MMAF linked through the N-terminus to the antibody, and have the structures: Typically, peptide-based drug residues can be prepared by forming a peptide linkage between two or more amino acid fragments and / or peptides. Such peptide linkages can be prepared, for example, according to the liquid phase synthesis method (see E. Schroder and K. Lübke, "The Peptides", volume 1, pp. 76-135, 1965, Academic Press) It is well known in the field of peptide chemistry. The drug residue includes calicheamicin and its analogs and derivatives. The calicheamicin family of antibiotics is capable of producing double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of conjugates of the calicheamicin family, see US 5712374; US 5714586; US 5739116; US 5767285; US 5770701; US 5770710; US 5773001; US 5877296. Calicheamicin structural analogs that may be used include, but are not limited to, 1 1, 21, I31, N-acetyl-1, PSAG. and T1! (Hinman et al., Cancer Research 53: 3336-3342 (1993), Lode et al., Cancer Research 58: 2925-2928 (1988) .The protein toxins include: diphtheria A chain, active fragments of non-union of Diphtheria toxin, A chain of exotoxin (from Pseudomonas aeruginosa), A chain of ricin (Vitteta et al., (1987) Science, 238: 1098), A chain of abrin, chain A of modecin, alpha-sarcin, proteins Aleurites fordii, diantine proteins, proteins of Phytolaca americana (PAPI, PAPII and PAP-S), inhibitor of momordica charantia, curcin, crotina, inhibitor of sapaonaria officinalis, gelonin, mitogeline, restrictocin, fenomycin, enomycin, and trichothecenes (OR 93 / 21232) Therapeutic radioisotopes include: 32P, 33P, 90Y (125I, 131ln, 153Sm, 186Re, 188Re, 211At, 212Bi, 212Pb, and the radioactive isotopes of Lu. The radioisotope or other brands can be incorporated into the conjugate in ways known (Fraker et al., (1987) Biochem. Biophys. Res. Commun., 80: 49-57; "Monoclone l Antibodies in Immunoscintigraphy "Chatal, CRC Press 1989). The l-isothiocyanatobenzyl-3-methyldiethylene triamine-penta-acetic acid labeled with carbon-14 (MX-DTPA) is an exemplary chelating agent for the conjugation of a radionuclide to the antibody (WO 94/11026). UNIONS A "binding" (L) is a bifunctional or mu? I-functional residue that can be used to join one or more drug residues (D) and one antibody unit (Ab) to form antibody-drug conjugates (ADCs) of Formula I. antibody-drug conjugates (ADCs) can conveniently be prepared using a linkage having reactive functionality to bind to the drug and the antibody. A cysteine thiol of a designed cysteine antibody (Ab) can form a binding with a functional group of a binding reagent, a drug residue or a drug-binding intermediate. In one aspect, a linkage has a reactive site having an electrophilic group that is reactive to a nucleophilic cysteine present in an antibody. The cysteine thiol of the antibody is reactive with an electrophilic group at a junction and forms a covalent bond to a junction. Useful electrophilic groups include, but are not limited to, maleimide and haloacetamide groups. The designed cysteine antibodies react with binding reagents or drug-binding intermediates, with electrophilic functional groups, such as maleimide or an α-halo carbonyl, according to the conjugation method on page 766 of Klussman, et al., (2004) Bioconjugate Chemistry 15 (4): 765-773, and in accordance with the protocol of Example 4. In one embodiment the L-junction of an ADC has the formula: -Aa-ww-Yy-where: -A- is an extensor unit covalently linked to a thiol of cysteine of the antibody (Ab); a is 0 or 1; each -W- is independently an amino acid unit; w is independently an integer ranging from 0 to 12; -Y. it is a separating unit covalently linked to the drug residue; and y is 0, 1 or 2. EXTENSIVE UNIT The extender unit (-A-) when present, is capable of binding an antibody unit to an amino acid unit (-W-). In this regard, an antibody (Ab) has a thiol group of free cysteine which can form a linkage with an electrophilic functional group of an extender unit. The stretching units representative of this embodiment are illustrated within the square brackets of the Formulas Illa and Illb. Where Ab-, -W-, -Y-, -D, w and y, are as defined above, and R17 is a divalent radical selected from (CH2) r / C3-C8 carbocyclyl, 0- (CH2) r, arylene , (CH2) r-arylene, -arylene- (CH2) r- (CH2) r- (carbocyclyl C3-C8), (C3-C8 carbocyclyl) - (CH2) r C3-C8 heterocyclyl, (CH2) r- (C3-C8 heterocyclyl), - (C3-C8 heterocyclyl) - (CH2) r-, - (CH2 ) rC (0) NRb (CH2) r-, - (CH2CH20) r-, - (CH2CH20) r -CH2-, (CH2) rC (0) NRb (CH2CH20) r-, - (CH2) rC (0) NRb (CH2CH20) r -CH2-, (CH2CH20) rC (0) NRb (CH2CH20) r-, - (CH2CH20) rC (O) NRb (CH2CH20) r-CH2- and - (CH2CH20) rC (0) NRb (CH2) r-; wherein Rb is H, d-C6 alkyl, phenyl, or benzyl; and r is independently an integer ranging from 1-10. Arylene includes divalent aromatic hydrocarbon radicals of 6-20 carbon atoms derived by the removal of two hydrogen atoms from an aromatic ring system of origin. Typical arylene groups include, but are not limited to, radicals derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, and the like. The heterocyclyl groups include a ring system in which one or more ring atoms is a heteroatom, e.g., nitrogen, oxygen and sulfur. The heterocycle radical comprises from 1 to 20 carbon atoms and from 1 to 3 heteroatoms selected from N, O, P and S. A heterocycle can be a monocycle having from 3 to 7 ring members (2 to 6 carbon atoms and from 1 to 3 heteroatoms selected from N, O, P and S) or a bicyclic having from 7 to 10 ring members (4 to 9 carbon atoms and from 1 to 3 heteroatoms selected from N, 0, P and S) , for example: a bicyclo system [4,5], [5,5], [5,6], or [6,6]. The heterocycles are described in Paquette, Leo A.: "Principies of Modern Heterocyclic Chemistry" (W.A. Benjamin, New York., 1968), particularly, chapters 1, 3, 4, 6, 7 and 9; "The Chemistry of Heterocyclic Compounds, A series of Monographs" (John iley &Sons, New York, 1950 to present), in particular volumes 13, 14, 16, 19 and 28; and J. Am. Chem. Soc. (1960) 82: 5566. Examples of heterocycles include, by way of example and not limitation, pyridyl, dihydropyridyl, tetrahydropyridyl, (piperidyl), thiazolyl, tetrahydrothiophenyl, tetrahydrothiophenyl oxidized with sulfur, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, tianaphthalenyl , indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, azocinyl, triazinyl, 6H -1, 2, 5-thiadiazinyl, 2H, 6H-1, 5, 2-dithiazinyl, thienyl, thiantrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxatinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl , 3H-indolyl, lH-indazolyl, purinyl, 4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinol nil, pteridinyl, 4Ah-carbazolyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, furazinyl, phenoxazinyl, isochromanil, chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl, oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazilinyl, and isatinoyl. The carbocyclyl groups include a saturated or unsaturated ring having from 3 to 7 carbon atoms as a monocycle or from 7 to 12 carbon atoms as a bicyclo. Monocyclic carbocycles have from 3 to 6 ring atoms, even more typically from 5 to 6 ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms, eg, arranged as a bicyclo system [4,5], [5,5], [5,6], or [6,6], or 9 or 10 carbon atoms. ring arranged as a bicycle system [5,6], or [6,6]. Examples of monocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl. 1-cyclohex-l-enyl, 1-cyclohex-2-enyl, l-cyclohex-3-enyl, cycloheptyl, and cyclooctyl. It should be understood from all exemplary embodiments of the ADC of Formula I, such as III-IV, that even where not expressly noted, from 1 to 4 drug residues are bound to an antibody (p. 1-4) ), depending on the number of cysteine residues manufactured.

An exemplary spreading unit is that of the Formula Illa, and is derived from maleimide-caproyl (MC) wherein R17 is - (CH2) 5-; An illustrative extension unit is that of Formula Illa, and is derived from maleimide-propanoyl (MP) wherein R17 is - (CH2) 2-; Another exemplary spreading unit is that of the Formula Illa, where R17 is - (CH2CH20) r-CH2- and r is 2: Another exemplary spreading unit is that of Formula Illa, where R17 is - (CH2) rC (0) NRb (CH2CH20) r-CH2- where Rb is H and each r is 2: Another exemplary spreading unit is that of Formula Illb, where R17 is - (CH2) 5: In another embodiment, the spreading unit is linked to the antibody unit through a disulfide bond between a sulfur atom of the antibody unit and a sulfur atom of the spreading unit. A spreading unit representative of this embodiment is illustrated within the square brackets of Formula IV, wherein R17, Ab-, - -, -Y-, -D, w and y, are as defined above.

In yet another embodiment, the reactive group of the spreading unit contains a reactive thiol functional group which can form a binding with a free cysteine thiol of an antibody. Examples of reactive thiol functional groups include, but are not limited to, maleimide, α-haloacetyl, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates and isothiocyanates. . The stretching units representative of this embodiment are illustrated within the brackets of the Formulas Va and Vb, where -R17, Ab-, -W-, -Y-, -D, w and y, are as defined above: In another embodiment, the linkage can be a dendritic junction for the covalent attachment of more than one drug residue through a branched, multifunctional linker residue to an antibody (Sun et al., (2002) Bioorganic &Medicinal Chemistry Letter 12: 2213-2215; Sun et al., (2003) Bioorganic &Medicinal Chemistry 11: 1761-1768; King (2002) Tetrahedron Letters 43: 1987-1990). Dendritic junctions can increase the molar ratio of drug to antibody, i.e., the charge, which is related to the potency of the ADC. Accordingly, when a designed cysteine antibody contains only one reactive cysteine thiol group, a multitude of drug residues can be linked through a dendritic junction. AMINO ACID UNIT The linkage can comprise amino acid residues. The amino acid unit (-Ww-), when present, binds the antibody (Ab) to the drug residue (D) of the cysteine-drug designed antibody conjugate (ADC) of the invention. -Ww- is a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, or dodecapeptide unit. The amino acid residues comprising the amino acid unit include those of natural origin, as well as minor amino acids and amino acid analogues not of natural origin, such as citrulline. Each unit -W-independently has the formula denoted below in the brackets, and w is an integer that varies from 0 to 12: wherein R19 is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, -CH2OH, CH (OH) CH3, -CH2CH2SCH3, -CH2CONH2, -CH2C00H, -CH2CH2CONH2, CH2CH2COOH, - (CH2) 3NHC (= NH) NH2, - (CH2) 3NH2, - (CH2) 3NHCOCH3, - (CH2) 3NHCHO, - (CH2) 4NHC (= NH) NH2, - (CH2) 4NH2, - (CH2) 4NHCOCH3, - (CH2 ) 4NHCHO, - (CH2) 3NHCONH2, - (CH2) 4NHCONH2, -CH2CH2CH (OH) CH2NH2, 2-pyridylmethyl, 3-pyridylmethyl, 4-pyridylmethyl, phenyl, cyclohexyl. enzymatically by one or more enzymes, including a tumor-associated protease, to release the drug residue (-D), which in one embodiment is protonated in vivo upon release to provide a drug (D). Useful units -Ww- can be designed and optimized in their selectivity for the enzymatic division by a particular enzyme, for example, a tumor-associated protease. In one modality, a unit -Ww- is one whose division is catalyzed by cathepsin B, C and D, or a plasmin protease. The amino acid units -Ww- exemplary include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptides include: valine-citrulline (ve or val-cit), alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include: glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-giy) · When R19 is different from hydrogen, the carbon atom to which R19 is attached is chiral . Each carbon atom to which R19 is attached is independently bound in the (S) or ® configuration, or a racemic mixture. The amino acid units can therefore be enantiomerically pure, racemic or diastereomeric. SEPARATING UNIT - - The separating unit (-Yy-) when present (y = 1 or 2), joins an amino acid unit (- w-) to the drug residue (D) when the amino acid unit is present (w = 1- 12). Alternatively, the separator unit links the extender unit to the drug residue when the amino acid unit is absent. The separator unit also binds the drug residue to the antibody unit when both the amino acid unit and the extender unit are absent (w, y = 0). The separation units are of two general types: self-immolative and not self-immolative. A non-self-immolative separating unit is one in which part or all of the separating unit remains attached to the drug residue after the division, particularly enzymatic, of an amino acid unit of the antibody-drug conjugate or drug-binding residue. . When an ADC containing a glycine-glycine separating unit or a glycine separating unit undergoes enzymatic cleavage through a tumor cell-associated protease, a cancer cell-associated protease or a lymphocyte-associated protease, a residue of glycine-glycine drug or a glycine-drug residue from Ab-Aa-Ww-. In one embodiment, an independent hydrolysis reaction takes place within the target cell, dividing the glycine-drug residue binding and releasing the drug.

In another embodiment, -Yy- is a p-aminobenzylcarbamoyl (PAB) unit (see Schemes 2 and 3) whose phenylene portion is substituted with Q, where Q is Ci-C8 alkyl, -0- (Ci-C8 alkyl), -halogen, -nitro or -ciano; and m is an integer that varies from 0.4. Exemplary modes of a non-self-immolative separating unit (-Y-) are: -Gly-Gly; -Gly-; -Ala-Phe- -Val-Cit-. In one embodiment, a drug-linker residue or an ADC is provided in which the spacer unit is absent (y = 0) or a pharmaceutically acceptable salt or solvate thereof. Alternatively, an ADC containing a self-immolative separating unit can release -D. In one embodiment, -Y- is a PAB group which is linked to -Ww-through the amino nitrogen atom of the PAB group, and directly connected to -D through a carbonate, carbamate or ether group, wherein the ADC has the following structure: wherein Q is Ci-C8 alkyl, -0- (Ci-C8 alkyl), -halogen, -nitro or -cyano; m is an integer that varies from 0.4; and p varies from 1 to 4. Other examples of self-immolative separators include, but are not limited to, aromatic compounds that are electronically similar to the PAB group such as 2-aminoimidazole-5-methanol derivatives (Hay et al., (1999) Bioorg. Med. Chem., Lett., 9: 2237) and another or for aminobenzylacetals. Separators that undergo cyclization to amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodriguez et al., (1995) Chemistry Biology 2; 223), bicyclic ring systems [2.2.1 ] and bicyclo [2.2.2] appropriately substituted (Storm et al., (1972) J. Amer. Chem. Soc., 94: 5815) and 2-aminophenylpropionic acid amides (Amsberry et al., (1990) J. Org. Chem., 55: 5867). The elimination of amine-containing drugs that are substituted in glycine (Kingsbury et al., (1984) J. Med. Chem., 27: 1447) are also examples of self-immolative separator useful in ADCs. In one embodiment, the separating unit is a branched bis (hydroxymethyl) styrene (BHMS), which can be used to incorporate and release multiple drugs, having the structure: comprising a 2- (4-aminobenzylidene) propane-1,3-diol dendrimer unit (O 2004/043493; de Groot et al., (2003) Angew., Chem. Int. Ed., 42: 4490-4494) , wherein Q is Ci-C8 alkyl, -O- (Ci-C8 alkyl), -halogen, -nitro or -cyano; m is an integer that varies from 0.4; n is 0 or 1; and p varies from 1 to 4. DENDRÍTICAS JOINTS In another modality, the union L can be a union dendritic type for the covalent union of more than a residue of drug through a branching multifunctional residue branched to an antibody (Sun et al., (2002) Bioorganic & Medicinal Chemistry Letter 12: 2213-2215; Sun et al., (2003) Bioorganic & Medicinal Chemistry 11: 1761-1768). Dendritic junctions can increase the molar ratio of drug to antibody, i.e., charge, which is related to the potency of the ADC. Accordingly, when a designed cysteine antibody contains only one reactive cysteine thiol group, a multitude of drug residues can be linked through a dendritic junction. The following exemplary embodiments of dendritic binding reagents allow to conjugate up to nine nucleophilic drug residue reagents by reaction with the functional groups of chloroethyl nitrogen mustard gold CH2OCH2CH2C IINHCH2CY3 In another embodiment of a separating unit, branched dendritic junctions can be employed with self-immolative 2,6-bis (hydroxymethyl) -p-cresol and 2,4,6-tris (hydroxymethyl) -phenol dendrimer units (WO 2004). / 01993 Szalai et al., (2003) J. Amer. Chem. Soc., 125: 15688-15689 Shamis et al., (2004) J.- Amer. Chem. Soc, 126: 1726-1731 Amir et al. (2003) Angew, Chem. Int. Ed., 42: 4494-4499) as bonds in the compounds of the invention. In another mode, the residuals D are the same. Even in another mode, the D residues are different. In one aspect, the separation units (-Yy-) are represented by the Formulas (X) - (XII): wherein Q is Ci-C8 alkyl, -O- (Ci-C8 alkyl), -halogen, -nitro or -cyano; and m is an integer that varies from 0.4; ^ - HN - CH2 - CO- ^ ?? Modes of antibody-drug conjugate compounds of Formula I include XlIIa (val-cit), XlIIb (MC-val-cit), XIIIc (MC-val-cit-PAB): Xlla Xlllb XIIIC Other exemplary embodiments of the antibody-drug conjugate compounds of the Formula include XlVa-e XIVc where X is OR And it is: and R is independently H or Ci-C6 alkyl; and n is 1 to 12. In another embodiment, a linkage has a reactive functional group that has a nucleophilic group that is reactive to an electrophilic group present in a antibody. Electrophilic groups useful in an antibody, include, but are not limited to, aldehyde and ketone carbonyl groups. The heteroatom of a nucleophilic group of a linkage can react with an electrophilic group on an antibody and form a covalent bond to an antibody unit. The nucleophilic groups in a binding include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate and aryl hydrazide. The electrophilic group in an antibody provides a convenient site for binding to a junction. Typically, peptide-type linkages can be prepared by forming a peptide linkage between two or more amino acids and / or peptide fragments. Such peptide linkages can be prepared, for example, according to the liquid phase synthesis method ((see E. Schroder and K. Lübke, "The Peptides", volume 1, pp. 76-135, 1965, Academic Press) which is well known in the field of peptide chemistry.The binding intermediates can be assembled with any combination or sequence of reactions including separation, stretch and amino acid units.The separation, stretch and amino acid units can employ reactive functional groups which are electrophilic, nucleophilic, or free radical in nature Reagent functional groups include, but are not limited to: wherein X is a leaving group, e.g., O-methyl, O-toxil, -Cl, -Br, -I; or maleimide. In another embodiment, the linkage can be substituted with groups that modulate solubility or reactivity. For example, a charged substituent such as sulfonate (-S03-) or ammonia, can increase the water solubility of the reagent and will facilitate the coupling reaction of the linker reagent with the antibody or drug residue, or will facilitate the coupling reaction of Ab-L (antibody-binding intermediate) with D, or DL (drug-binding intermediate) with Ab, depending on the synthetic route used to prepare the ADC. The compounds of the invention expressly contemplate, but are not limited to, the ADC prepared with binding reagents: B PEO, B PS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, - - sulfo-GMBS, sulfo-KMUS, sulfo-BS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB, and SVSB (succinimidyl- (4-vinyl sulphone) benzoate), and including bis-maleimide reagents: DTME, BMB , BMDB, DMOR, BM (PEO) 3 and BM (PEO) 4, which are commercially available from Pierce Biotechnology, Inc., Customer Service Department, P.O. Box 117, Rockford, IL. , 61105 U.S.A., U.S.A. 1-800-874-3723, International + 815 968-0747. See pages 467-498, 2003-2004 Applications Handbook and Catalog. The bis-maleimide reagents allow the binding of the thiol group of a designed cysteine antibody to a drug residue, label or thiol-containing binding intermediate, in a sequential or concurrent manner. Other functional groups besides maleimide, which are reactive with a thiol group of a designed antibody of cysteine, drug residue, label or binding intermediate, include iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyl disulfide, isocyanate and isothiocyanate.

BM (PEO) 3 BM (PEO) 4 Useful binding reagents can also be obtained through other commercial sources, such as Molecular Biosciences Inc., (Boulder, CO), or synthesized in accordance with the procedures described in Toki et al., (2002) J. Org. Chem. 67: 1866-1872; Walker M.A. , (1995) J. Org. Chem., 60: 5352-5355; Frisen et al., (1996) Bioconjugate Chem., 7: 180-186; US 6214345; WO 02/088172; US 2003130189; US 2003096743; WO 03/026577; WO 03/043583; and WO 04/032828. The stretching units of the Formula (Illa) can be introduced into a joint by reacting the following reagents with the N-terminus of an amino acid unit: wherein n is an integer ranging from 1-10 and T is -H or-S03Na; where n is an integer that varies from 0-3: Stretching units can be introduced into a junction by reacting the following bifunctional reagents with the N-terminus of an amino acid unit: where X is Br or I. Stretching units can also be introduced into a junction by reacting the following bifunctional reagents with the N-terminus of an amino acid unit: the stretching units of the formula (Va) can be introduced into a union by reacting the following intermediates with the N-terminus of an amino acid unit: the isothiocyanate stretching units of the formula shown below can be prepared from isothiocyanatocarboxylic acid chlorides as described in Angew Chem., (1975), 87 (14), 517. where -R17 is as described herein. An exemplary valine-citrulline dipeptide linker reagent having a maleimide extender unit and a self-immolating separator unit for parasite aminobenzylcarbamoyl (PAB) has the structure: wherein Q is Ci-C8 alkyl, -O- (Ci-C8 alkyl), -halogen, -nitro or -cyano; and m is an integer that varies from 0.4. An exemplary phe-lysylated dipeptide linker reagent (Mtr) having a maleimide extender unit and a p-aminobenzyl autoimmune immobilizing unit can be prepared according to Dubowchik et al., (1997) Tetrahedron Letters, 38: 5257 -60 and has the structure: wherein Mtr is mono-4-methoxytrityl, Q is Ci-C8 alkyl, -O- (Ci-C8 alkyl), -halogen, -nitro or -cyano; and m is an integer that varies from 0.4. The antibody-drug conjugated compounds of the invention include: Ab-MC-vc-PAB-MMAF Ab-MC-vc-PAB-MMAE Ab-MC-MMAE Ab-MC-MMAF where Val is valina; Cit is citrulline; p is 1, 2, 3 or 4; and Ab is a designed antibody of cysteine. Other exemplary antibody-drug conjugates wherein the DM1 residue of the maytansinoid drug is bound through a BMPEO binding to a thiol group of trastuzumab have the structure: wherein Ab is an antibody designed of cysteine; n is O, 1 or 2; and p is 1, 2, 3 or 4. PREPARATION OF ANTIBODY-DRUG CONJUGATES The ADC of Formula I can be prepared by several routes, employing reactions, conditions and reagents of organic chemistry, known to those skilled in the art, including: ( 1) reaction of a cysteine group of a designed cysteine antibody with a binding reagent, to form the antibody-linker Ab-L intermediate, through a covalent linkage, followed by the reaction with an activated drug D residue; and (2) reaction of a nucleophilic group of a drug residue with a binding reagent, to form the drug-linker intermediate DL through a covalent bond, followed by reaction with a cysteine group of a designed cysteine antibody . The conjugation methods (1) and (2) can be used with a variety of designed antibodies of cysteine, drug residues and junctions, to prepare the antibody-drug conjugates of Formula I. The thiol groups of cysteine antibody are nucleophilic and capable of reacting to form covalent linkages with electrophilic groups in binding reagents and drug-linker intermediates including: (i) active esters such as NHS esters, HOBt esters, haloformates and acid halides; (ii) alkyl and benzyl halides, such as haloacetamides; (iii) aldehydes, ketones, carboxyl groups and maleimide; and (iv) disulfides, including pyridyl disulfides through sulfide exchange. Nucleophilic groups in a drug residue include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups in binding residues and binding reagents. Maytansine, for example, can be converted to May-SSCH3, which can be reduced to the free thiol, May-SH, and reactivated with a modified antibody (Chari et al., (1992) Cancer Research 52: 127-131) to generate a Immunoconjugate of maytansinoid-antibody with a disulfide bond. Conjugates of antibody-maytansinoid with disulfide bonds have been reported (WO 04/016801, US 6884874, US 2004/039176 Al, WO 03/068144, US 2004/001838 Al; US Patents Nos. 6441163, 5208020, 5416064; WO 01/024763). The SPP disulfide bond is constructed with the linker reagent N-succinimidyl-4- (2-pyridylthio) entoate. Under certain conditions, designed cysteine antibodies can be prepared for conjugation with binding reagents by treatment with a reducing agent such as DTT (Cleland reagent, dithiothreitol) or TCEP (tris (2-carboxyethyl) phosphine hydrochloride; Gets et al. ., (1999) Anal. Biochem. Vol., 273: 73-80, Soltec Ventures, Beverly, MA). The designed full-length cysteine antibodies (ThioMabs) expressed in CHO cells were reduced with approximately a 50-fold excess of TCEP for 3 hours at 37 ° C to reduce the disulfide bonds that can be formed between the newly introduced cysteine residues and the cysteine present in the culture medium. The reduced ThioMab was diluted and loaded onto a HiTrap S column in 10 mM sodium acetate, pH 5, and eluted with PBS containing 0.3 M sodium chloride. The disulfide bonds were re-established between the cysteine residues present in the Mab of origin with dilute aqueous copper sulfate (CuS04) (200 nM) at room temperature overnight. Other oxidants i.e., oxidation agents, and oxidation conditions known in the art can be used. Oxidation to ambient air is also effective. This mild stage of partial reoxidation forms disulfides intrachain efficiently with high fidelity. An approximate 10-fold excess of drug-antibody intermediate, eg, BM (PEO) 4-DMI, was added, mixed and allowed to stand for about one hour at room temperature to effect conjugation and form the antibody-ThioMab conjugate. drug. The conjugation mixture was gel filtered and loaded and eluted through a HiTrap S column to remove excess drug-linker intermediate and other impurities. Figure 15 shows the general process for the preparation of a designed cysteine antibody expressed from cell culture for conjugation. The cysteine adducts, presumably in conjunction with several interchain disulfide bonds, divide reductively to give a reduced form of the antibody. The interchain disulfide bonds between paired cysteine residues are reformed under partial oxidation conditions, such as exposure to ambient oxygen. The newly introduced, designed and unpaired cysteine residues remain available for reaction with binding reagents or drug-linker intermediates to form the antibody conjugates of the invention. The ThioMabs expressed in mammalian cell lines result in an externally conjugated Cys adduct for a Cys manufactured through the formation of the S-S junction. of this Thus, purified ThioMabs have to be treated with reduction and oxidation procedures as described in Example 11 to produce reactive ThioMabs. These ThioMabs are used to conjugate with cytotoxic drugs containing maleimide, fluorophores and other brands. A variety of antibody-drug ThioFab and ThioMab conjugates was prepared (examples 4-8). The cysteine mutant hu4D5Fabv8 (V110C) was conjugated to the maytansinoid drug residue DM1 with a bis-maleimide linker reagent BMPEO to form hu4D5Fabv8 (V110C) -BMPEO-DM1 (Example 8). ANALYSIS OF IN VITRO CELLULAR PROLIFERATION Generally, the cytotoxic or cytostatic activity of an antibody-drug conjugate (ADC) is calculated: by exposing mammalian cells having receptor proteins, e.g., HER2, to the ADC antibody in a cell culture medium; culturing the cells for a period of about 6 hours to about 5 days; and calculating cell viability. Cell-based analyzes were used to calculate the viability (proliferation), and the induction of apoptosis (caspase activation) of the ADC of the invention. The in vivo potency of the antibody-drug conjugates was calculated by a cell proliferation assay (Figures 10 and 11, Example 9). He CellTiter-Glo® cell viability assay Luminescent is a commercially available homogeneous analysis method (Promega Corp., Madison, I) based on the recombinant expression of Coleoptera luciferase (U.S. Patent Nos. 5583024; 5674713 and 5700670). This analysis of cell proliferation determines the number of viable cells in culture based on the quantification of the ATP present, an indicator of metabolically active cells (Crouch et al., (1993) J. Immunol.eth 160: 81-88; 6602677). The CellTiter-Glo® analysis was conducted in a 96 well format, making it docile to high performance visualization (HTS) (Cree et al., (1995) AntiCancer Drugs 6: 398-404). The homogeneous analysis procedure involves adding the reagent alone (CellTiter-Glo® Reagent) directly to cells cultured in medium supplemented with serum. The steps of cell washing, media removal and multiple pipetting are not required. The system detects as few as 15 cells / well in a 384-well format in the 10 minutes after addition of the reagent and mixing. The cells can be treated continuously with ADC, or they can be treated and separated from the ADC. Generally, cells treated briefly, i.e., 3 hours, showed the same potency effects as continuously treated cells. The homogeneous "addition-mixing-measuring" format results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in the culture. The CellTiter-Glo® analysis generates a luminescent signal "brightness type" produced by the luciferase reaction, which has a half-life generally greater than five hours, depending on the cell type and the medium used. Viable cells are reflected in relative luminescence units (RLU). The substrate, Beetle Luciferin, is oxidatively decarboxylated by recombinant firefly luciferase with concomitant conversion of ATP to A P and generation of photons. The long half-life eliminates the need to use reagent injectors and provides flexibility for continuous or batch mode processing of multiple plates. This cell proliferation assay can be used with several multiple well formats, e.g., 96 or 384 well format. The data can be recorded by means of a luminometer or a CCD camera display device. The luminescence output is presented as relative light units (RLU), calculated by time. Alternatively, the luminescence photons can be counted in a scintillation counter in the presence of a scintillant. The light units can then be represented as CPS counts per second.

Luciferase ATP + Luciferin + 02 »Oxiluciferin + AMP + PP¡ + C02 + light M g + 2 The anti-proliferative effects of antibody-drug conjugates were calculated by analysis of cell proliferation, in vitro cell destruction against the cell line of breast tumor SK-BR-3 (Figures 10 and 11). IC50 values of the ADC were established against SK-BR-3 cells, which are known to overexpress the HER2 receptor protein. Figure 10 shows that trastuzumab SMCC-DMl (IC50 = 0.008-0.015 / μg / ml) was more potent than the mutant heavy chain cysteine conjugate hu4D5Fabv8 (A121C) -BMPEO-DM1 (IC50 = 0.04 / ug / ml) . Both conjugates were significantly more potent in cell destruction than naked trastuzumab (IC50 = 0.1 μg / ml). The loading of drug for trastuzumab SMCC-DMl was 2.8 DMl / Ab and for hu4D5Fabv8 (A121C) -BMPEO-DM1 was 0.6 DMl / Ab. Figure 11 shows that trastuzumab SMCC-DMl (IC50 = 0.008-0.015 μg / ml) was more potent than the light chain cysteine mutant conjugate hu4D5Fabv8 (V110C) -BMPEO-DM1 (IC50 = 0.07 μg / ml). Both conjugates were more potent in cell destruction than naked trastuzumab (IC50 = 0.1 μg / ml). The loading of drug for trastuzumab SMCC-DMl was 2.8 DMl / Ab and for hu4D5Fabv8 (V110-C) -BMPEO-DM1 was 0.9 DMl / Ab.

The full-length ThioMab IgG conjugates were tested for their cell proliferation efficiency in vitro and compared with the original antibodies. Figure 20 shows the results of an analysis of SK-BR-3 cells treated with: original antibody trastuzumab (HERCEPTIN®, Genentech, Inc.); trastuzumab SMCC-DM1 with a drug load of approximately 3.4 DMl / Ab; and thio-trastuzumab (A121C) BMPEO-DM1 with a drug loading of approximately 1.6 DMl / Ab. The conjugate of trastuzumab-SMCC-DM1 is bound to the antibody through the reagent reagent NHS ester SMCC amino reactive, while the conjugate of thio-trastuzumab (A121C) BMPEO-DM1 is bound through the reagent linker of maleimide BMPEO reactive thiol. Both conjugates' were potent against SK-BR-3 cells and showed comparable activity, whereas trastuzumab did not exert a cytotoxic effect. Figure 21A shows the results of an analysis of HT 1080EphB2 cells treated with: 2H9 anti-EphB2R of origin; and conjugate of thio 2H9 (A121C) BMPEO-DM1. Figure 21B shows the results of an analysis of BT 474 cells treated with 2H9 anti-EphB2R of origin; and conjugate of thio 2H9 (A121C) BMPEO-DM1. Against HT 1080EphB2 and BT 474 cells, the 2H9 ThioMab conjugate was more potent than the 2H9 antibody conjugate of origin. The conjugate Thio-2H9-BMPEO-DM1 showed a functional cell destruction activity in the specific cell line EphB2 (HT 1080EphB2) compared to a non-EphB2 cell line, BT 474 in which only marginal activity is observed. The drug antibody conjugates were compared when the antibody is an original antibody and when the antibody is a designed antibody of cysteine. Figure 22 shows the results of an analysis of PC3 / neo cells treated with: 3A5 anti-MUC16-SMCC-DM1; and uncle 3A5 (A121C) BMPE0-DM1. Figure 23 shows the results of an analysis of PC3 / MUC16 cells treated with: 3A5 anti-MUC16-S CC-DM1; and uncle 3A5 (A121C) BMPEO-DM1. Figure 24 shows the results of an analysis of OVCAR-3 cells treated with: 3A5 anti-MUC16-SMCC-DMl; and uncle 3A5 (A121C) BMPE0-DM1. The thio-3A5-BMPEO-DM1 did not show any significant cell destruction activity in the PC3 / neo control cell line, although it showed an activity comparable to 3A5-SMCC-DM1 in the cell line PC3 / MUC16. The 3A5-D 1 conjugate also showed activity in OVCAR-3 expressing the endogenous MUC16 antigen. IN VIVO EFFICACY The in vivo efficacy of two peptide-D l (maytansinoid) antibody-drug (ADC) albumin binding conjugates of the invention was calculated by a mouse model that highly expresses transgenic HER2 explant (Figure 12, Example 10 ). A transgenic mouse allograft was propagated Fo5 mmtv that does not respond to, or responds poorly to, therapy with HERCEPTIN®. Subjects were treated once with ABP-rhuFab4D5-cys (light chain) -DM1; ABP-rhuFab4D5- cys (heavy chain) -DM1; and placebo PBS control buffer (vehicle), and were monitored for 3 weeks to calculate tumor doubling time, cell trunk destruction, and tumor shrinkage.

The term Ti is the number of animals in the study group with tumor at T = 0 ÷ the total number of animals in the group. The term PR is the number of animals that achieve partial remission of the tumor ÷ animals with a tumor at T = 0 - in the group. The term TDV is the time of duplication of the tumor, i.e., the time in days to control the duplication of tumor volume. The seven mice treated with 25 mg per kg (1012 g / m2 of DM1) of ABP-rhuFab4D5-cys (light chain) -DM1 were all positive to the tumor and gave an animal with partial remission after 20 days. The seven mice treated with 37.5 mg per kg (1012 ^ g / m2 of DM1) of ABP-rhuFab4D5-cys (heavy chain) -DM1 were all positive to the tumor and gave four animals with partial remission after 20 days. The full-length ThioMab IgG antibody variant with the A121C cysteine mutation and conjugated to the BMPEO binding and the DM1 drug residue, it was tested against the conjugate of trastuzumab of origin-SMCC-DMl in MMTC-HER2 Fo5 tumor-bearing mice. The size of the tumor on day 0 of injection was approximately 100-200 mm in size. Figure 25 shows the mean change in tumor volume during 21 days in nude nude mice with breast tumor allografts MMTV-HER2 Fo5, after a single dose on day 0 with: vehicle (buffer); trastuzumab SMCC-DM1 10 mg / kg; thio trastuzumab (A121C) -SMCC-DM1 21 mg / kg and thio trastuzumab (A121C) -SMCC-DM1 10 mg / kg.

It can be seen from Figure 25 that each conjugate exerts a significant effect on the growth retardation of the tumor relative to the placebo (vehicle). Each of the ten mice in the four previous groups received a single injection on day 1. The conjugate of trastuzumab of origin-SMCC-DMl was loaded more than twice (3.4 DMl / Ab) the number of drug residues that the conjugate of thio-trastuzumab (A121C) -BMPEO-DM1 designed of cysteine (1.6 DMl / Ab). The effective amount of DM1 was then approximately equal between the trastuzumab of origin-SMCC-DMl and the highest dose (21 mg Ab) of thio-trastuzumab (A121C) - BMPEO-DM1. These two samples showed the greatest power. After 14 days post-injection, most of the animals that received these conjugates were found in partial or complete remission The lower efficacy of the lower dose of the thio-trastuzumab (A121C) -BMPE0-DM1 sample confirmed a dose-related response to DM1. The thio-trastuzumab DM1 was dosed either in an equivalent amount of antibody (10 mg / kg) or DM1 drug (21 mg / kg) to that of the control trastuzumab conjugate SMCC-DMl. As shown in Figure 25, the uncle BMPE0-DM1 (21 mg / kg) showed a slightly better response than that of the trastuzumab-SMCC-DM1 group since some of the animals showed a complete response with trastuzumab-DMl while only There was a partial response with trastuzumab-SMCC-DMl. ADMINISTRATION OF ANTIBODY-DRUG CONJUGATES The antibody-drug conjugates (ADC) of the invention can be administered by any route appropriate for the condition being treated. The ADC will typically be administered parenterally, i.e., infusion, subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and epidural. PHARMACEUTICAL FORMULATIONS Pharmaceutical formulations of antibody-drug therapeutic conjugates (ADCs) of the invention are typically prepared for parenteral administration, i.e., rapid injection, intravenous intratumor with a pharmaceutically acceptable parenteral vehicle and in a unit dose injectable form. An antibody conjugate- Drug (ADC) having the desired degree of purity is optionally mixed with pharmaceutically acceptable diluents, carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A., Ed.) in the form of a lyophilized formulation or a aqueous solution. Acceptable diluents, carriers, excipients and stabilizers are non-toxic to the receptors at the doses and concentrations employed, and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenyl, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight polypeptides (less than about 10 residues); 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); I nonionic surfactants such as TWEEN, PLURONICS or polyethylene glycol (PEG). For example, lyophilized formulations of anti-ErbB2 antibody are described in O 97/04801, expressly incorporated herein by reference. The active pharmaceutical ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization., for example, hydroxymethylcellulose or gelatin microcapsules and poly- (methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences 16th edition, Osol, A., Ed., (1980). Sustained release preparations can be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the ADC, 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-hydroxyethyl-methacrylate), or poly (vinyl alcohol), polylactides (US 3773919), L-acid copolymers glutamate and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, lactic acid-degradable glycolic acid copolymers such as LUPRON DEPOT ™ (injectable microspheres composed of lactic acid-glycolic acid and leuprolide acetate copolymer) and polyhydric acid D- (-) -3-Hydroxybutyric. Formulations that are used for in vivo administration must be sterile, which is easily achieved by filtration through sterile filtration membranes. The formulations include those suitable for the above administration routes. The formulations can conveniently be presented in unit dosage form and can be prepared by any of the methods known in the pharmaceutical art. The techniques and formulations are generally found in Remignton's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA). Such methods include the step of associating the active ingredient with the vehicle that constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately associating the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. The aqueous suspensions of the invention contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include a suspending agent, such as sodium carboxymethylcellulose, croscarmellose, povidone, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing agents or humectants such as phosphatide of natural origin (eg, lecithin ), a condensation product of an alkylene oxide with a fatty acid (eg, polyoxyethylene stearate), a condensation product of ethylene oxide with a long-chain aliphatic alcohol (eg, heptadecaethylene-ethanol), an oxide condensation product of ethylene with a partial ester derived from a fatty acid and a hexitol anhydride (eg, polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives such as 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. The ADC pharmaceutical compositions may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation can also be a sterile solution or suspension injectable in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butane diol or prepared as a lyophilized powder. Among the vehicles and acceptable solvents that can be used are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils may be conveniently employed as a solvent or suspension medium. For this purpose, any fixed soft oil including synthetic mono or diglycerides can be employed. Additionally, fatty acids such as oleic acid can also be used in the preparation of injectables. The amount of the active ingredient that can be combined with the carrier material to produce a single dose form will vary depending on the host treated and the particular mode of administration. For example, an aqueous solution intended for intravenous infusion may contain from about 3 to 500 μg of the active ingredient per milliliter of solution so that the infusion of a suitable volume can be presented at a rate of about 30 ml / hr. Formulations suitable for parenteral administration include sterile aqueous and non-aqueous injection solutions which may contain anti-oxidants, buffers, bacteriostats and solubles which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickeners. Although oral administration of protein therapeutics is disadvantaged due to hydrolysis and denaturation in the intestine, ADC formulations suitable for oral administration can be prepared as discrete units such as capsules, sachets, or tablets each containing a predetermined amount of the ADC. The formulations can be packaged in single dose or multiple dose containers, for example sealed vials and vials, and can be stored in a frozen-dry (lyophilized) condition requiring only the addition of the sterile liquid vehicle, for example water, for injection immediately. prior to its use. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the type previously described. Preferred unit dose formulations containing a daily dose or unit daily sub-dose, as recited hereinabove, or an appropriate fraction thereof, of the active ingredient. The invention further provides veterinary compositions comprising at least one active ingredient as defined above in conjunction with a veterinary vehicle therefor. Veterinary vehicles are useful materials for the purpose of administering composition and may be solid, liquid or gaseous materials that are otherwise inert or acceptable in the veterinary art and are compatible with the active ingredient. These veterinary compositions can be administered parenterally, orally or by any other desired route. ANTIBODY-DRUG CONJUGATE TREATMENTS It is contemplated that the antibody-drug conjugates of the present invention can be used to treat various diseases or disorders, e.g., characterized by overexpression of a tumor antigen. Exemplary conditions of hyperproliferative disorders include benign or malignant tumors; leukemia and lymphoid malignancies. Others include neuronal, glial, astrocytal, glandular, macrophagal, epithelial, stromal, blastocoelic, inflammatory, angiogenic and immunological disorders, including autoimmune. ADC compounds that are identified in animal models and in cell-based assays can also be tested in older primate tumor carriers and in clinical trials in humans. Clinical trials in humans can be designed similarly to clinical trials that test the efficacy of the anti-HER2 monoclonal antibody HERCEPTIN® in patients with metastatic breast cancers overexpressing HER2 who have received extensive prior therapy anti-cancer as reported by Baselga et al., (1996) J. Clin. Oncol. , 14: 737-744. The clinical test can be designed to evaluate the efficacy of an ADC in combinations with known therapeutic regimens, such as radiation and / or chemotherapy involving known chemotherapeutic and / or cytotoxic agents. Generally, the disease or disorder to be treated is a hyperproliferative disease such as cancer. Examples of cancer to be treated herein include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (eg, epithelial squamous cell cancer), lung cancer including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, carcinoma salivary gland, kidney or kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, and anal carcinoma, penile carcinoma, as well as cancer of the head and neck. Cancer can include cells that express HER2, whereby the ADC of the present invention is capable of binding to cancer cells. To determine ErbB2 expression in cancer, several diagnostic / prognostic analyzes are available. In one embodiment, overexpression of ErbB2 can be analyzed by IHC, e.g., using the HERCEPTEST (Dako). Sections of tissue embedded in paraffin from a tumor biopsy can be subjected to IHC analysis and according to a criterion of intensity of ErbB2 protein staining as follows: Score 0, no coloration is observed or membrane staining observed in less than 10 minutes. % of tumor cells; Score 1+, a weak / barely perceptible membrane coloration is detected in more than 10% of the tumor cells, the cells are colored only in part of its membrane; Score 2+, a weak to moderate complete membrane staining is observed in more than 10% of the tumor cells; Score 3+, a moderate to strong complete membrane staining is observed in more than 10% of the tumor cells. Those tumors with scores of 0 or 1+ to evaluate the overexpression of ErbB2 can be characterized as not overexpressing ErbB2, while those tumors with scores of 2+ or 3+ can be characterized overexpressing ErbB2. Alternatively or additionally, FISH analyzes such as the INFORM ™ (Ventana Co., Ariz.) Can be carried out. or PATHVISION (Vysys, III) in tumor tissue fixed in formalin or embedded in paraffin to determine the degree (if any) of overexpression of ErbB2 in the tumor. Autoimmune diseases for which the ADC compounds can be used in the treatment include rheumatological disorders (such as, for example, rheumatoid arthritis, Sjogren's syndrome, scleroderma, lupus such as SLE and lupus nephritis, polymyositis / dermatomyositis, cryoglobulinemia, anti-phospholipid antibody, and psoriatic arthritis), osteoarthritis, gastrointestinal and hepatic autoimmune disorders (such as, for example, inflammatory bowel diseases (eg, ulcerative colitis and Crohn's disease), autoimmune gastristis and pernicious anemia, autoimmune hepatitis, primary biliary cirrhosis , primary sclerosing cholangitis, and celiac disease), vasculitis (such as, for example, ANCA-associated vasculitis, including Churg-Strauss vasculitis, Wegener's granulomatosis, and polyarteritis), neurological autoimmune disorders (such as, for example, multiple sclerosis , opsoclonus myoclonus syndrome, myasthenia gravis, n optical euromyelitis, Parkinson's disease, Alzheimer's disease, and autoimmune polyneuropathies), kidney disorders (such as, for example, glomerulonephritis, Goodpasture's syndrome, and Berger's disease), autoimmune dermatological disorders (such as, for example, psoriasis, urticaria, hives, pemphigus vulgaris, bullous pemphigoid and cutaneous lupus erythematosus), hematological disorders (such as, for example, thrombocytopenic purpura, thrombotic thrombocytopenic purpura, post-transfusion purpura, and autoimmune hemolytic anemia), atherosclerosis, uveitis, autoimmune auditory diseases ( such as, for example, inner ear disease and hearing loss), Behcet's disease, Raynaud's syndrome, organ transplantation, and autoimmune endocrine disorders (such as, for example, autoimmune diseases related to diabetes such as diabetes mellitus dependent of insulin (IDDM), Addison's disease, and autoimmune thyroid disease (eg, Graves' disease and thyroiditis)). More preferred diseases include, for example, rheumatoid arthritis, ulcerative colitis, ANCA-associated vasculitis, lupus, multiple sclerosis, Sjogren's syndrome, Graves' disease, IDDM, pernicious anemia, thyroiditis, and glomerulonephritis. For the prevention or treatment of the disease, the appropriate dose of an ADC will depend on the type of disease to be treated, as defined above, of the severity and course of the disease, whether the molecule is administered for preventive purposes or therapeutic, previous therapy, patient's clinical history and response to the antibody, and at the discretion of the attending physician. The The molecule is properly administered to the patient at one time or in a series of treatments. Depending on the type and severity of the disease, approximately 1 / kg / kg to 15 mg / kg (eg, 1-20 mg / kg) of the molecule is an initial candidate dose for administration to the patient, either for example, by one or more separate administrations, or by continuous infusion. A typical daily dose may vary from about 1 μg / kg to 100 mg / kg or more, depending on the aforementioned factors. An exemplary dose of ADC for administration to a patient is in the range of about 0.1 to about 10 mg / kg of the patient's weight. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of the symptoms of the disease occurs. An exemplary dose regimen comprises administration of an initial loading dose of about 4 mg / kg, followed by a weekly maintenance dose of about 2 mg / kg of an anti-ErbB2 antibody. Other dose regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and analysis. COMBINATION THERAPY An antibody-drug conjugate (ADC) of the invention can be combined in a combination formulation pharmaceutical, or a dosage regimen as combination therapy, with a second compound having anti-cancer properties. The second compound of the pharmaceutical combination formulation or dose regimen preferably has complementary activities to the ADC of the combination such that they do not adversely affect each other. The second compound may be a chemotherapeutic agent, cytotoxic agent, cytosine, growth inhibitory agent, anti-hormonal agent, and / or cardioprotective agent. Such molecules are suitably present in combination in effective amounts for the purpose proposed. A pharmaceutical composition containing an ADC of the invention can also have a therapeutically effective amount of a chemotherapeutic agent such as a tubulin-forming inhibitor, a topoisomerase inhibitor or a DNA-binding. Other therapeutic regimens may be combined with the administration of an anticancer agent identified in accordance with this invention. The combination therapy can be administered as a simultaneous or sequential regimen. When administered sequentially, the combination can be administered in two or more administrations. The combined administration includes co-administration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in any order, where preferably there is a period of time while both (or all) active agents simultaneously exercise their biological activities. In one embodiment, treatment with an ADC involves the combined administration of an anticancer agent identified herein, and one or more chemotherapeutic agents or growth inhibitory agents, including co-administration of cocktails of different chemotherapeutic agents. Chemotherapeutic agents include taxanes (such as paclitaxel and docetaxel) and / or anthracycline antibiotics. The preparation and dosage schedules for such chemotherapeutic agents may be used according to the manufacturer's instructions or determined empirically by the physician. The preparation and dose schemes for such chemotherapy are also described in "Chemotherapy Service" (1992) Ed., M.C. Perry, Williams / Wilkins, Baltimore, Md. ADC can be combined with an anti-hormonal compound, e.g., an anti-estrogen compound such as tamoxifen; an anti-progesterone such as onapristone (EP 616812); or an anti-androgen such as flutamide, in known doses for such molecules. When the cancer to be treated is a hormone-dependent cancer, the patient may have previously undergone anti-hormone therapy and, after the cancer becomes hormone-independent, the ADC (and optionally other agents described herein) can be administered to the patient. It may be beneficial to also co-administer a cardioprotective (to avoid or reduce the myocardial dysfunction associated with the therapy) or one or more cytokines to the patient. In addition to the above therapeutic regimens, the patient may undergo surgical removal of the cancer cells and / or radiation therapy. Suitable doses for any of the above coadministered agents are those currently used and may be decreased due to the combined action (synergy) of the newly identified agent and other chemotherapeutic agents or treatments. The combination therapy can provide "synergy" and prove to be "synergistic", i.e., the effect achieved when the active ingredients used together is greater than the sum of the effects resulting from the use of the compound separately. A synergistic effect can be achieved when the active ingredients: (1) are co-formulated and administered or delivered simultaneously in a combined unit dose formulation; (2) are supplied alternately or in parallel as separate formulations; or (3) through some other regime. When given in alternating therapy, a synergistic effect can be achieved when the compounds are administered or they are delivered sequentially, e.g., by different injections into separate syringes. In general, during the alternating therapy, an effective dose of each active ingredient is administered sequentially, i.e., serially, while in the combination therapy, effective doses of two or more active ingredients are administered together. METABOLITES OF ANTIBODY-DRUG CONJUGATES Also within the scope of this invention are the in vivo metabolic products of the ADC compounds described herein, since such products are new and not obvious in the prior art. Such products may result, for example, from the oxidation, reduction, hydrolysis, amidation, esterification, enzymatic cleavage, and the like, of the compound administered. Accordingly, the invention includes novel and non-obvious compounds produced by a process comprising contacting a compound of this invention with a mammal for a period of time sufficient to produce a metabolic product thereof. The metabolite products are typically identified by preparing a radiolabelled ADC (eg, 14C or 3H), by administering them parenterally at a detectable dose (eg, greater than about 0.5 mg / kg) to an animal such as rat, mouse, guinea pig, monkey, or man, allowing sufficient time for the metabolism to present itself (typically approximately 30 seconds to 30 hours) and isolating its conversion products from urine, blood or other biological samples. These products are easily isolated since they are marked (others are isolated by the use of antibodies capable of binding to epitopes that survive in the metabolite). The metabolite structures are determined in conventional manner, e.g., by MS, LC / MS or NMR analysis. In general, metabolite analyzes are carried out in the same manner as conventional drug metabolism studies well known to those skilled in the art. the conversion products, while not otherwise found in vivo, are useful in diagnostic assays for therapeutic dosing of the ADC compounds of the invention. METHODS OF VISUALIZATION OF MARKING ANTIBODY In another embodiment of the invention, designed cysteine antibodies can be labeled through the cysteine thiol with radionuclides, fluorescent dyes, bioluminescent activating substrate residues, chemoluminescence activating substrate residues, enzymes and other labels. of detection for visualization experiments with diagnostic, pharmacodynamic and therapeutic applications. Generally, the designed antibody of labeled cysteine, i.e., "biomarker" or "probe", is administered by injection, perfusion or oral ingestion to a living organism, e.g., human, rodent or other small animal, a perfused organ, or tissue sample. The distribution of the probe is detected during a time course and is represented by an image. MANUFACTURING ARTICLES In another embodiment of the invention, there is provided an article of manufacture, or "equipment", containing materials useful for the treatment of the disorders described above. The article of manufacture comprises a package and a packaging label or insert in or associated with the package. Suitable containers include, for example, bottles, vials, syringes, ampoules, etc. The containers can be formed from a variety of materials such as glass and plastic. The package contains an antibody-drug conjugate composition (ADC) which is effective to treat the condition and may have a sterile access port (eg, the package may be an intravenous solution bag or a vial having a pierceable plug. using a hypodermic injection needle). At least one active agent in the composition is an ADC. The label or packaging insert indicates that the composition is used to treat the selected condition, such as cancer. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container that it comprises a pharmaceutically acceptable buffer such as bacteriostatic water for injection (B FI), phosphate buffered saline, Ringer's solution and dextrose solution. It may also include other desirable materials from the commercial and user's point of view, including other shock absorbers, diluents, filters, needles and syringes. EXAMPLES Example 1: Preparation of ThioFab Biotinylated Phage ThioFab-phage (5 x 1012 phage particles) was reactivated with a 150-fold excess of biotin-PEO-maleimide ((+ = - biotinyl-3-maleimidepropiomamidyl-3,6-dioxaoctanediamine, Oda et al., (2001) Nature Biotechnology 19: 379-382, Pierce Biotechnology Inc.) for 3 hours at room temperature Excess biotin-PEO-maleimide was removed from the biotin-conjugated phage by repeated PEG precipitations (3). -4 times) Other commercially available biotinylation reagents can be used with electrophilic groups that are reactive with cysteine thiol groups including biotin-BMCC, PEO-iodoacetyl biotin, iodoacetyl-LC-biotin and biotin-HPDP (Pierce Biotechnology, Inc. ) and Na- (3-maleimidylpropionyl) biocytin (MPB, Molecular Probes, Eugene, OR) Other commercial sources for bifunctional and multifunctional biotinylation binding reagents include Molecular Probes, Eugene, OR, and Sigma, St. Louis, MO.

Biotin-PEO-maleimide Example 2 PHESELECTOR analysis Bovine serum albumin (BSA), extracellular domain erbB2 (HER2) and streptavidin (100 μ? 2 they were coated separately on Maxisorp 96-well plates. After blocking with 0.5% Tween-20 (in PBS), biotinylated and non-biotinylated hu4D5Fabv8-ThioFab-Fago (2 x 1010 phage particles) was incubated for 1 hour at room temperature followed by incubation with horseradish peroxidase-labeled secondary antibody ( HRP) (anti-M13 phage coat protein, pVIII protein antibody). Figure 8 illustrates the PHESELECTOR analysis by a schematic representation illustrating the binding of Fab or ThioFab to HER2 (upper part) and biotinylated ThioFab to streptavidin (lower part). The standard HRP reaction was carried out and the absorbance at 450 nm was calculated. The thiol reactivity was measured by calculating the ratio between OD450 for streptavidin / OD450 for HER2. A thiol reactivity value of 1 indicates the complete biotinylation of the cysteine thiol.

In the case of measurement of Fab protein binding, hu4D5Fabv8 (2-20 ng) was used followed by incubation with goat polyclonal anti-Fab antibodies labeled with HRP.

Example 3a Expression and Purification of ThioFabs ThioFabs were expressed at induction in 34B8, a non-suppressor species of E. coli (Baca et al., (1997) Journal Biological Chemistry 272 (16): 10678-84). The harvested cell pill was resuspended in PBS (phosphate buffered saline), the total cell lysis was carried out by passing it through a microfluidizer and the ThioFabs were purified by affinity chromatography with protein G SEPHAROSE ™ (Amersham). The ThioFabs L-V15C, L-V110C, H-A88C and H-A121C were expressed and purified by G protein chromatography SEPHAROSE ™. Oligomeric Fab was present in fractions 26 to 30, and most of the monomeric forms were found in fractions 31-34. The consistent fractions of the monomeric form were deposited and analyzed by SDS-PAGE in conjunction with the wild-type hu4D5Fabv8 and analyzed on SDS-PAGE gel under conditions of reduction (with DTT or BME) and non-reduction (without DTT or BME) ). The gel filtration fractions of A121C-ThioFab were analyzed in SDS-PAGE of no reduction. The ThioFabs were conjugated with biotin-PEO- maleimide as described above and the biotinylated ThioFabs were further purified by Superdex-200 ™ gel filtration chromatography (Amersham), which removed the free biotin-PEO-maleimide and the oligomeric fraction of the ThioFabs. The wild-type hu4D5Fabv8 and hu4D5Fabv8 A121C-ThioFab (0.5 mg in quantity) were each incubated separately with a 100-fold molar excess of biotin-PEO-maleimide for 3 hours at room temperature and loaded onto a Superdex-200 gel to separate the free biotin as well as the oligomeric Fabs from the monomeric form. Example 3b Analysis of ThioFabs Enzymatic digestion fragments of biotinylated hu4D5Fabv8 (A121C) ThioFab and wild type hu4D5Fabv8 were analyzed by mass ionization spectroscopy by liquid chromatography electrospray (LS-ESI-MS). The difference between the primary mass of 48294.4 of biotinylated hu4D5Fabv8 (A121C) and the primary mass of 47737.0 of wild type hu4D5Fabv8 was 557.5 mass units. This fragment indicates the presence of a single residue of biotin-PEO-maleimide (C23H36 507S2). Table 4 shows the assignment of the fragmentation values confirming the sequence.

Table 4. LC-ESI-mass spectral analysis of ThioFab A121C biotinylated hu4D5Fabv8 after tryptic digestion Before and after the Superdex-200 gel filtration, SDS-PAGE gel analyzes were conducted with and without reduction by DTT or BME, of ABP-hu4D5Fabv8-A121C biotinylated, biotinylated ABP-hu4D5Fabv8-biotinylated VV, biotinylated Cys ABP-hu4D5Fabv8- (V110C-A88C), and double biotinylated Cys ABP-hu4D5Fabv8- (V110C-A121C). Mass spectroscopy analysis (MS / MS) of hu4D5Fabv8- (V110C) -BMPE0-DM1 (after purification of Superdex-200 gel filtration): Fab + 1 51607.5, Fab 50515.5. These data show a conjugation of 91.2%. MS / MS analysis of hu4D5Fabv8- (V110C) -BMPEO-DM1 (reduced): LC 23447.2, LC + 1 24537.3, HC (Fab) 27072.5. These data show that all conjugation of DM1 is found in the light chain of Fab. Example 4 Preparation of ABP-hu4D5Fabv8 - (V110C) -MC-MMAE by conjugation of ABP-hu4D5Fabv8 - (V110C) and MC-MMAE The drug-linker reagent, maleimidacaproyl-monomethyl auristatin E (MMAE), ie, MC-MMAE, dissolved in DMSO, diluted in acetonitrile and water to a known concentration, and added to ice-cold ThioFab ABP-hu4D5Fabv8 - (V110C) in phosphate buffered saline (PBS). After about one hour, an excess of maleimide is added to quench the reaction and cap all thiol group of unreactivated antibody. The reaction mixture is concentrated by centrifugal ultrafiltration and the ABP-hu4D5Fabv8- (V110C) -MC-MMAE is purified and desalted by elution through G25 resin in PBS, filtered through 0.2 μt filters. under sterile conditions and freezes for storage.

Example 5 Preparation of ABP-hu4D5Fabv8- (V110C) -MC-MMAF by conjugation of ABP-hu4D5Fabv8 - (V110C) and MC-MMAF The ABP-hu4D5Fabv8- (V110C) -MC-MMAF is prepared by conjugation of ThioFab ABP-hu4D5Fabv8 - (V110C) and MC-MMAF following the procedure of Example 4. Example 6 Preparation of ABP-A121C-ThioFab-MC-val-cit-PAB-MMAE by conjugation of ABP-A121C-ThioFab and MC-val-cit-PAB -MMAE The ABP-hu4D5Fabv8- (A121C) -MC-val-cit-PAB-MMAE is prepared by conjugation of ABP-hu4D5Fabv8 - (A121C) and MC-val-cit-PAB-MMAE following the procedure of Example 4. Example 7 Preparation of ABP-A121C-ThioFab-MC-val-cit-PAB-MMAF by conjugation of ABP-A121C-ThioFab and MC-val -cit-PAB-MMAF The ABP-hu4D5Fabv8 - (A121C) -MC-val -cit -PAB-MMAF is prepared by conjugation of ABP-hu4D5Fabv8 - (A121C) and MC-val -cit-PAB-MMAF following the procedure of Example 4.

MC-val-ct-PAB-MMAF Example 8 Preparation of hu4D5Fabv8- (V110C) ThioFab-BMPEO-DM1 Hu4D5Fabv8- (V110C) ThioFab was modified by the bis-maleimide reagent BM (PE0) 4 (Pierce Chemical) leaving a maleimide group without reacting on the surface of the antibody. This was achieved by dissolving BM (PE0) 4 in a 50% ethanol / water mixture at a concentration of 10 mM and adding a ten-fold molar excess of BM (PEO) 4 to a solution containing hu4D5Fabv8- (V110C) ThioFab in saline buffered with phosphate at a concentration of approximately 1.6 mg / ml (10 micromolar) and allowing it to react for 1 hour. The excess BM (PE0) 4 was removed by gel filtration (HiTrap column, Pharmacia) in 30 mM citrate, pH 6, with 150 mM NaCl buffer. A molar excess of about ten times of DM1 dissolved in dimethyl acetamide (DMA) was added to the intermediate of hu4D5Fabv8- (V110C) ThioFab-BMPEO. Dimethylformamide (DMF) can also be used to dissolve the drug residue reagent. The reaction mixture was allowed to react overnight before gel filtration or dialysis in PBS to remove the unreacted drug. Gel filtration in S200 columns in PBS was used to remove the high molecular weight aggregates and form the purified hu4D5Fabv8- (V110C) ThioFab-BMPEO-DM1. Through the same protocol, hu4D5Fabv8- (A121C) ThioFab-BMPEO-DM1 was prepared. Example 9 In vitro cell proliferation analysis The efficacy of ADC was calculated by a cell proliferation analysis using the following protocol (CellTiter Luminiscent Cell Viability Assay, Promega Corp., Technical Bulletin TB288, Mendoza et al., (2002) Cancer Res., 62: 5485-5488): 1. An aliquot of 100 μ? Cell culture medium containing approximately 104 cells (SKBR-3, BT474, MCF7 or MDA-MB-468) in medium was deposited in each well of a 96-well opaque-walled plate. 2. The control wells were prepared containing medium and without cells. 3. ADC was added to the experienced wells and incubated for 3-5 days. 4. The plates were equilibrated at room temperature for approximately 30 minutes. 5. One volume of CellTiter-Glo reagent was added equal to the volume of the cell culture medium present in each well. 6. The contents were mixed for 2 minutes in an orbital shaker to induce cell lysis. 7. The plate was incubated at room temperature for 10 minutes to stabilize the luminescence signal. 8. The luminescence was recorded and reported in graphs as RLU = units of relative luminescence.

Certain cells are seeded at 1000-2000 / well (PC3 lines) or at 2000-3000 / well (OVCAR-3) in a 96 well plate, 50 μ? / ????. After one (PC3) to two (OVCAR-3) days, ADC is added in 50 μ? volumes up to a final concentration of 9000, 3000, 1000, 333, 111, 37, 12.4, 4.1 or 1.4 ng / ml, with control wells of "no ADC" receiving the medium alone. The conditions are in duplicate or triplicate. After 3 (PC3) or 4-5 (OVCAR-3) days, add 100 μ? / ???? of CellTiter-Glo II (luciferase-based assay, proliferation measured by ATP levels) and cell counts are determined using a luminometer. The data is illustrated as the luminescence average for each set of replicas, with error bars of standard deviation. The protocol is a modification of the CellTiter Glo Luminiscent Cell Viability analysis (Promega). 1. Plate 1000 cells / well of PC3 / Mucl6, PC3 / neo (in 50 μ? / ????) of medium. OVCAR-3 cells should be plated at 2000 cells / well (in 50 μ?) Of their medium, (formulas below). Let the cells come together at night. 2. ADC is serially diluted 1: 3 in medium starting at a working concentration of 18 μg / ml (this results in a final concentration of 9 μg / ml). 50 μ? of diluted ADC is added to the 50 μ? of cells and to the medium already in the well. 3. Incubate 72-96 hours (the standard is 72 hours, but the concentration / ml is monitored to stop the analysis when the cells are 85-95% confluent). 4. Add 100 μ? / ???? of Promega Cell Titer Glo reagent, shake 3 minutes, and read in the luminometer.

Medium: PC3 / neo and PC3 / MUC16 grown in 50/50/10% / FBS / glutamine / 250 μg / l G-418 OVCAR-3 grown in RPMl / 20% / FBS / glutamine. Example 10 Inhibition of tumor growth, in vivo efficacy in transgenic explant mice with high HER2 expression. Animals suitable for transgenic experiments can be obtained from standard commercial sources such as Taconic (Germantown, N.Y.). Many species are suitable, but female FVB mice are preferred because of their high susceptibility to tumor formation. FVB males were used to compare and CD.l reinforcements were used to stimulate pseudo pregnancy. Vasectomized mice can be obtained from any commercial provider. The founders are raised either with FVB mice or with heterozygous 129 / BL6 x FVB p53 mice. Mice with heterozygosity in the p53 allele were used to potentially increase tumor formation. However, this has proven to be unnecessary. Consequently, the Fl tumors are of mixed species. The tumors founders are only FVB. Six founders were found with some tumors in development without having offspring. Animals with tumors (allografts propagated from Fo5 mmtv transgenic mice) were treated with a single or multiple dose by IV injection of ADC. The volume of the tumor was evaluated at several time points after the injection. Tumors arise easily in transgenic mice that express a mutationally activated form of neu, the HER2 rat homologue, but HER2 that is overexpressed in human breast cancers is not mutated and tumor formation is less robust in transgenic mice overexpressing non-mutated HER2 (Webster et al., (1994) Semin. Cancer Biol., 5: 69-76). To enhance tumor formation with unmutated HER2, transgenic mice were produced using a plasmid of HER2 cDNA in which an upstream ATG was suppressed in order to avoid initiation of translation in such upstream ATG codons, which would otherwise would reduce the frequency of translation initiation of the authentic start codon downstream of HER2 (e.g., see Child et al., (1999) J. Biol. Chem., 274: 24335-24341).

Additionally, a chimeric intron was added to the 5 'end, which must also improve the level of expression as previously reported (Neuberger and Williams (1988) Nucleic Acids Res., 16: 6713; Buchman and Berg (1988) Mol. Cell. Biol. 8: 4395; Brinster et al. , (1988) Proc. Natl. Acad. Sci. , USA 85: 836). The chimeric intron was derived from a Promega vector, mammalian expression vector Pci-neo (bp 890-1022). The 3 'end of DNA is surrounded by human growth hormone exons 4 and 5, and polyadenylation sequences. In addition, FBV mice were used because this species is more susceptible to tumor development. The MMTV-LTR promoter was used to ensure the expression of tumor-specific HER2 in the mammary gland. The animals were fed the AIN diet in order to increase the susceptibility to tumor formation (Rao et al., (1997) Breast Cancer Res. And Treatment 45: 149-158). EXAMPLE 11 Reduction / Oxidation of ThioMabs for Conjugation The designed monoclonal antibodies of cysteine (ThioMabs) expressed in CHO cells were reduced with an approximate 50-fold excess of TCEP (tris (2-carboxyethyl) phosphine hydrochloride; Getz et al., (1999 ) Anal. Biochem. Vol. 273: 73-80; Soltec Ventures, Beverly, MA) for 3 hours at 37 ° C. The reduced ThioMab (Figure 15) was diluted and loaded onto a HiTrap S column in 10 mM sodium acetate, pH 5, and eluted with PBS containing 0.3 M sodium chloride. ThioMab reduced eluate was treated with 200 nM aqueous copper sulfate (CuS04) at temperature environment, during the night. Oxidation to ambient air was also effective. Example 12 Conjugation of ThioMabs The re oxidized ThioMabs of Example 11, including thio-trastuzumab (A121C), thio-2H9 (A121C) and thio-3A5 (A121C), were combined with a 10-fold excess of drug-binding intermediate, BM (PEO) 4-DMl, mixed and allowed to stand for about one hour at room temperature to effect conjugation and form the ThioMab-drug antibody conjugates, including thio-trastuzumab (A121C) -BMPEO-DM1, thio-2H9 (A121C) -BMPEO-DM1 and thio-3A5 (A121C) -BMPEO-DM1. The conjugation mixture was gel filtered or charged and eluted through a HiTrap S column to remove excess drug-linker intermediate and other impurities. The present invention should not be limited in scope by the specific embodiments described in the examples which are intended as illustrations of some aspects of the invention, and all modalities that are functionally equivalent are within the scope of this invention. Indeed, various embodiments 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.

Claims (77)

1. CLAIMS 1. A designed cysteine antibody comprising one or more free cysteine amino acids having a thiol reactivity value in the range of 0.6 to 1.0, wherein the designed cysteine antibody is prepared by a process comprising the replacement of one or more amino acid residues of an original antibody by cysteine. The designed cysteine antibody of claim 1, wherein the designed cysteine antibody is more reactive than the original antibody with a thiol-reactive reagent. The designed cysteine antibody of claim 1, wherein the process further comprises determining the thiol reactivity of the designed cysteine antibody by reacting the designed cysteine antibody with a thiol-reactive reagent; wherein the designed cysteine antibody is more reactive than the original antibody with the thiol reactive reagent. The designed cysteine antibody of claim 1, wherein the one or more free cysteine amino acid residues are located in a light chain. 5. The designed cysteine antibody of the claim 4, wherein the one or more free cysteine amino acid residues are located in the light chain in the selected ranges of: L-10 to L-20; L-38 to L-48; L-105 to L-115; L-139 to L-149; and L-163 to L-173. The designed cysteine antibody of claim 1 comprising one or more sequences selected from: (i) SLSASCGDRVT (SEQ ID NO: 17) (ii) QKPGKCPKLLI (SEQ ID NO: 18) (iii) EIKRTCAAPSV (SEQ ID NO : 19) (iv) TCAAPCVFIFPP (SEQ ID NO: 20) (v) FIFPPCDEQLK (SEQ ID NO: 21) (vi) DEQLKCGTASV (SEQ ID NO: 22) (vii) FYPRECKVQWK (SEQ ID NO: 23) (viii) WKVDNCLQSGN (SEQ ID NO: 24) (ix) ALQSGCSQESV (SEQ ID NO: 25) (x) VTEQDCKDSTY (SEQ ID NO: 26) and (xi) GLSSPCTKSFN (SEQ ID NO: 27) 7. The designed antibody of cysteine of claim 1 comprising one or more sequences selected from: (i) NWIRQCPGNK (SEQ ID NO: 40) (ii) LNSCTTEDTAT (SEQ ID NO: 41) (iii) GQGTLVTVSACSTKGPSVFPL (SEQ ID NO: 42) (iv) HTFPCVLQSSGLYS (SEQ ID NO: 43) and (v) HTFPACLQSSGLYS (SEQ ID NO: 44) 8. The designed cysteine antibody of claim 1 comprising one or more sequences selected from: (i) FLSVSCGGRVT (SEQ ID NO: 45) (ii) QKPGNCPRLLI (SEQ ID NO: 46) (iii) EIKRTCAAPSV (SEQ ID NO: 47) (iv) FYPRECKVQWK (SEQ ID NO: 48) and (v) VTEQDCKDSTY (SEQ ID NO: 49) 9 The designed cysteine antibody of claim 1 wherein the one or more free cysteine amino acid residues are located in a heavy chain. The designed cysteine antibody of claim 9 wherein the one or more free cysteine amino acid residues are located in the heavy chain in the selected ranges of: H-35 to H-45; H-83 to H-93; H-114 to H-127; and H-170 to H-184. The designed cysteine antibody of claim 1 comprising one or more sequences selected from: (i) WVRQCPGKGL (SEQ ID NO: 9) (Ü) NSLRCEDTAV (SEQ ID NO: 10) (i) LVTVCSASTKGPS (SEQ ID NO: 11) (iv) LVTVSCASTKGPS (SEQ ID NO: 12) (v) LVTVSSCSTKGPS (SEQ ID NO: 13) (vi) LVTVSSACTKGPS (SEQ ID NO: 14) (ii) HTFPCVLQSSGLYS (SEQ ID NO: 15) and (viii) HTFPAVLQCSGLYS (SEQ ID NO: 16) 12. The designed cysteine antibody of claim 9 wherein the one or more free cysteine amino acid residues are located in the Fe region of the heavy chain in the selected ranges from H-268 to H-291; H-319 to H-344; H-370 to H-380; and H-395 to H-405. The designed cysteine antibody of claim 1 comprising one or more of the sequences selected from: (i) HEDPECKFNWYVDGVEVHNAKTKPR (SEQ ID NO: 29) (ii) HEDPEVKFNWYCDGVEVHNAKTKPR (SEQ ID NO: 30) (iü) HEDPEVKFNWYVDGCEVHNAKTKPR (SEQ ID NO: 31) (iv) HEDPEVKFNWYVDGVECHNAKTKPR (SEQ ID NO: 32) (v) HEDPEVKFNWYVDGVEVHNCKTKPR (SEQ ID NO: 33) (vi) YKCKVCNKALP (SEQ ID NO: 34) (vii) IEKTICKAKGQPR (SEQ ID NO: 35) (viii) IEKTISKCKGQPR (SEQ ID NO: 36) (ix) KGFYPCDIAVE (SEQ ID NO: 37) (x) PPVLDCDGSFF (SEQ ID NO: 38) 14. The designed cysteine antibody of claim 1 wherein the one or more free cysteine amino acid residues are selected from the positions on the heavy chain or the light chain of the variable region. The designed cysteine antibody of claim 1 wherein the one or more free cysteine amino acid residues are selected from the positions in the constant region. 16. The antibody of claim 1 wherein the thiol reactivity value is in the range of 0.7 to 1.0. 17. The designed cysteine antibody of claim 1 wherein the thiol reactivity value is in the range of 0.8 to 1.0. 18. The designed cysteine antibody of claim 1 prepared by a process comprising: (i) mutagenesis of a nucleic acid sequence encoding the designed cysteine antibody; (ii) the expression of the designed cysteine antibody; and (iii) the isolation and purification of the designed cysteine antibody. 19. The designed cysteine antibody of the claim 18 wherein the mutagenesis comprises site-directed mutagenesis. The designed cysteine antibody of claim 18 wherein the designed cysteine antibody is expressed in a viral particle selected from a phage or phagemid particle. The designed cysteine antibody of claim 18 further comprising: (i) reacting the designed cysteine antibody with a thiol-reactive affinity reagent to generate an affinity-labeled cysteine-labeled antibody; and (ii) measuring the binding of the designed affinity tagged cysteine antibody to a capture medium. 22. The designed cysteine antibody of claim 21 wherein the thiol reactive affinity reagent comprises a biotin residue. 23. The designed cysteine antibody of claim 22 wherein the thiol reactive reagent comprises a maleimide residue. 24. The designed cysteine antibody of claim 21 wherein the capture medium comprises streptavidin. 25. The designed cysteine antibody of the claim 1 wherein the original antibody is a fusion protein comprising the albumin binding peptide (ABP). 26. The designed cysteine antibody of claim 25 wherein the ABP comprises a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 27. The designed cysteine antibody of claim 1 wherein the original antibody is selected from a monoclonal antibody, a bispecific antibody, a chimeric antibody, a human antibody and a humanized antibody. 28. The designed cysteine antibody of claim 27 wherein the original antibody is huMAb4D5-8 (trastuzumab). 29. The designed cysteine antibody of claim 27 wherein the original antibody is an anti-EphB2R antibody. 30. The designed cysteine antibody of claim 27 wherein the original antibody is an anti-MUC16 antibody. 31. The designed cysteine antibody of claim 1 comprising an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 28 and SEQ ID NO: 39. 32. The designed cysteine antibody of claim 1 wherein the original antibody is an intact antibody selected from IgA, IgD, IgE, IgG and IgM. 33. The designed cysteine antibody of claim 32 wherein the IgG is selected from the subclasses IgG1, IgG2, IgG3, IgG4 and IgA2. 34. The designed cysteine antibody of claim 1 wherein the original antibody is an antibody fragment. 35. The designed cysteine antibody of claim 34 wherein the antibody fragment is a Fab fragment. 36. The designed cysteine antibody of claim 35 wherein the Fab fragment is hu4D5Fabv8. 37. The designed cysteine antibody of claim 36 wherein the one or more amino acid residues of hu4D5Fabv8 replaced by cysteine are selected from L-V15, L-A43, L-V110, L-A144, L-S168, H-A40, H-A88, H-S119, H-S120, H-A121, H-S122, H-A175 and H-S179. 38. The designed cysteine antibody of claim 1 wherein the designed cysteine antibody or the original antibody binds to one or more of the receptors (1) - (36): (1) BMPRIB (bone morphogenic protein receptor type IB Access to Genbank No. NM_001203); (2) E16 (LATI, SLC7A5, Access to Genbank No. NM_003486); (3) STEAPI (transmembrane epithelial antigen six of prostate, access to Genbank No. NM_012449); (4) 0772P (CA125, MUC16 Access to Genbank No. AF361486); (5) MPF (PF, MSLN, SMR, megakaryocyte enhancing factor, mesothelin, access to Genbank No. NM_005823). (6) Napi3b (NAPI-3B, NPTllb, SLC34A2, solute vehicle family 34 (sodium phosphate), member 2, sodium-dependent phosphate transporter 3b type II Access to Genbank No. N _006424); (7) Sema 5b (FLJ10372, KIAA1445, Mm 42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven repeats of thrombospondin (type 1 and similar to type 1), transmembrane domain (T) and short cytoplasmic domain, ( semaphorin) 5B Access to Genbank No. AB040878; (8) PSCA gene hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 Access to Genbank No. AY358628; (9) ETBR (endothelin type B receptor. Genbank No. AY275463); (10) MSG783 (RNF124, hypothetical protein FLK20315, Access to Genbank No. NM_017763); (11) STEAP2 (HGNC 8639, IPCA-1, PCANAPI, STAMP1, STEAP2, STMP, gene 1 associated with prostate cancer, protein 1 associated with prostate cancer, transmembrane six epithelial antigen of prostate 2, six transmembrane prostate protein. Access to Genbank No. AF455138); (12) Trp 4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient channel of potential receptor cation, subfamily M, member 4. Access to Genbank No. NM_017636); (13) CRYPT (CR, CR1, CRGF, CRYPT, TDGF1, growth factor derived from teratocarcinoma Access to Genbank No. NP_003203 or N _003212); (14) CD21 (CR2 (complement 2 receptor) or C3DR (C3d / Epstein Barr virus receptor) or Hs.73792. Access to Genbank No. M26004); (15) CD79b (CD79B, 0α79β, Igb (beta associated with immunoglobulin), B29 Access to Genbank No. NM_000626); (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing anchored phosphatase protein la), SPAP1B, SPAP1C Access to Genbank No. NM_030764); (17) HER2 (Access to Genbank No. M11730); (18) NCA (Access to Genbank No. 18728); (19) MDP (Access to Genbank No. BC017023); (20) IL20Ra (Access to Genbank No. AF184971); (21) Brevican (Access to Genbank No. AF229053); (22) EphB2R (Access to Genbank No. NM_004442); (23) ASLG659 (Access to Genbank No. AX092328); (24) PSCA (Access to Genbank No. AJ297436); (25) GEDA (Access to Genbank No. AY260763); (26) BAFF-R (B cell activation factor receptor, BLyS 3 receptor, BR3, NP_443177.1); (27) CD22 (CD22-B isoform B cell receptor, NP_001762.1); (28) CD79a (CD79A, CD79a, immunoglobulin-associated alpha, a B cell-specific protein that interacts covalently with beta Ig (CD79B) and forms a complex on the surface with IgM molecules, transduces a signal involved in the differentiation of cell B. Access to Genbank No. NP_001774.1); (29) CXCR5 (Burkitt's lymphoma receptor 1, a G-protein coupled receptor that is activated by chemokine CXCL13, works in lymphocyte migration and human defense, plays a role in HIV-2 infection and perhaps in the development of AIDS, lymphoma, myeloma, and leukemia Access to Genbank No. NP_001707.1), - (30) HLA-DOB (beta subunit of MHC class II molecule (antigen) that binds to peptides and presents them to CD4 + T lymphocytes. Access to Genbank No. NP_002111.1); (31) P2X5 (ion channel 5 entry to ligand of the P2X purinergic receptor, an ion channel with entry by extracellular ATP, may be involved in synaptic transmission and neurogenesis, the deficiency may contribute to the pathophysiology of idiopathic detrusor instability. Access to Genbank No. NP_002552.2); (32) CD72 (CD72 antigen of B-cell differentiation, Lyb-2 Access to Genbank No. NP_001773.1); (33) LY64 (lymphocyte antigen 64 (RP105), membrane protein type 1 of the leucine-rich repeat family (LRR), regulates B cell activation and apoptosis, loss of function is associated with increased activity of the disease in patients with systemic lupus erythematosus Access to Genbank No. NP_005573.1); (34) FcRHl (protein 1 similar to the Fe receptor, a putative receptor for the immunoglobulin Fe domain containing Ig-like C2-like domains and ITAM, may have a role in B lymphocyte differentiation. Access to Genbank No. NP_443170. 1); (35) IRTA2 (receptor of the immunoglobulin superfamily associated with translocation 2, a putative immunoreceptor with possible roles in the development and B cell lymphomagenesis, the deregulation of the gene by translocation occurs in some B cell malignancies. Access to Genbank No NP_112571.1); and (36) TENB2 (putative transmembrane proteoglycan, related to the EGF / heregulin family of growth factors and follistatin. Access to Genbank No. AF179274. 39. The designed cysteine antibody of claim 1 wherein the antibody is covalently bound to a drug residue. 40. The designed cysteine antibody of claim 1 wherein the antibody is covalently linked to a capture tag, a detection tag or a solid support. 41. The designed cysteine antibody of claim 40 wherein the antibody is covalently bound to a biotin capture tag. 42. The designed cysteine antibody of claim 40 wherein the antibody is covalently bound to a fluorescent dye detection label. 43. The designed cysteine antibody of claim 42 wherein the fluorescent dye is selected from a type of fluorescence, a type of rhodamine, dansyl, Lissamine, a cyanine, a phycoerythrin, Texas Red, and an analogue thereof. 44. The designed cysteine antibody of claim 40 wherein the antibody is covalently bound to a radionuclide detection tag selected from 3H, "c, 14C, 18F, 32P, 35S, 6Cu, 68Ga, 86Y, 99Tc, ^ ln, 123I, 124I, 125I, 131I, 133Xe, 177Lu, 211At and 213Bi. 45. The designed cysteine antibody of claim 40 wherein the antibody is covalently bound to a detection tag by a chelating ligand. 46. The designed cysteine antibody of claim 45 wherein the chelating ligand is selected from DOTA, DOTP, DOTMA, DTPA and TETA. 47. A method for selecting designed cysteine antibodies for their thiol reactivity, comprising: (a) introducing one or more amino acids of cysteine into an original antibody in order to generate a designed cistern antibody; (b) reacting the designed cysteine antibody with a thiol-reactive affinity reagent to generate an affinity-labeled cysteine-labeled antibody; and (c) measuring the binding of the designed affinity td cysteine antibody to a capture medium. 48. The screening method of claim 47 further comprising determining the thiol reactivity of the designed cysteine antibody with the thiol reactive reagent. 49. The selection method of claim 47 wherein step (a) of the process comprises: (i) site-directed mutagenesis of a gene encoding the designed cysteine antibody in a double-stranded plasmid (ds); and (ii) the expression of the designed cysteine antibody. 50. The method of selection of claim 49 further comprising the isolation and purification of the engineered antibody of expressed cysteine. 51. The selection method of claim 47 wherein the thiol-reactive affinity reagent comprises a biotin residue. 52. The selection method of claim 47 wherein the thiol-reactive reagent comprises a maleimide residue. 53. The method of selection of claim 47 wherein the capture means comprises streptavidin. 54. The method of selection of claim 47 wherein the original antibody is a fusion protein comprising an albumin binding peptide (ABP). 55. The method of selection of claim 54 wherein the ABP comprises a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. 56. The method of selection of claim 47 wherein the original antibody is an antibody fragment Fab. 57. An antibody-drug conjugate compound comprising a designed antibody of cysteine (Ab), and a drug residue (D) wherein the designed antibody of cysteine is linked through one or more amino acids of free cysteine by a linker residue (L) to D; the compound having Formula I: Ab- (L-D) p I wherein p is 1, 2, 3 or 4; and where the designed cysteine antibody is prepared by a process comprising the replacement of one or more amino acid residues of an original antibody by one or more free cysteine amino acids. 58. The antibody-drug conjugate compound of claim 57 wherein the designed cysteine antibody is more reactive than the original antibody with a thiol-reactive reagent. 59. The antibody-drug conjugate compound of claim 57 wherein the designed cysteine antibody is prepared by a process comprising: (a) the replacement of one or more amino acid residues of an original antibody by cysteine; and (b) determining the thiol reactivity of the designed cysteine antibody by reacting theantibody designed from cysteine with a thiol-reactive reagent. wherein the designed cysteine antibody is more reactive than the original antibody with the thiol reactive reagent. 60. The antibody-drug conjugate compound of claim 57 further comprising a sequence of albumin binding peptides (ABP). 61. The antibody-drug conjugate compound of claim 60 wherein the ABP comprises a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO. : 5. 62. The antibody-drug conjugate compound of claim 57 wherein the designed cysteine antibody binds to an ErbB receptor selected from EGFR, HER2 and HER4. 63. The antibody-drug conjugate compound of claim 57 wherein the designed antibody of cysteine or the original antibody binds to one or more of the receptors (1) - (36): (1) BMPRIB (morphogenic protein receptor Bone Type IB Access to Genbank No. NM_001203); (2) E16 (LATI, SLC7A5, Access to Genbank No. NM_003486); (3) STEAPI (transmembrane epithelial antigen) six of prostate. Access to Genbank No. NM_012449; (4) 0772P (CA125, MUC16 Access to Genbank No. AF361486); (5) MPF (PF, MSLN, SMR, megakaryocyte enhancing factor, mesothelin, access to Genbank No. NM_005823). (6) Napi3b (API-3B, NPTllb, SLC34A2, solute vehicle family 34 (sodium phosphate), member 2, sodium-dependent phosphate transporter 3b type II Access to Genbank No. NM_006424); (7) Sema 5b (FLJ10372, KIAA1445, Mm 42015, SEMA5B, SE AG, Semaphorin 5b Hlog, sema domain, seven repeats of thrombospondin (type 1 and similar to type 1), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B. Access to Genbank No. AB040878; (8) PSCA gene hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12. Access to Genbank No. AY358628; (9) ETBR (endothelin receptor type B. Access to Genbank No. AY275463); (10) MSG783 (RNF124, hypothetical protein FLK20315. Access to Genbank No. NM_017763); (11) STEAP2 (HGNC_8639, IPCA-1, PCANAPI, STAMPI, STEAP2, STMP, gene 1 associated with prostate cancer, protein 1 associated with prostate cancer, transmembrane epithelial antigen 6 from prostate 2, protein 6 from prostate transmembrane Access to Genbank No. AF455138); (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient channel of potential receptor cation, subfamily M, member 4. Access to Genbank No. NM_017636); (13) CRYPT (CR, CR1, CRGF, CRYPT, TDGFI, growth factor derived from teratocarcinoma Access to Genbank No. NP_003203 or NM_003212); (14) CD21 (CR2 (complement 2 receptor) or C3DR (C3d / Epstein Barr virus receptor) or Hs.73792. Access to Genbank No. M26004); (15) CD79b (CD79B, CD79b, Igb (immunoglobulin-associated beta), B29 Access to Genbank No. NM_000626); (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing anchored phosphatase protein la), SPAP1B, SPAP1C. Access to Genbank No. NM_030764); (17) HER2 (Access to Genbank No. MI1730); (18) NCA (Access to Genbank No. M18728); (19) MDP (Access to Genbank No. BC017023); (20) IL20Ra (Access to Genbank No. AF184971); (21) Brevican (Access to Genbank No. AF229053); (22) EphB2R (Access to Genbank No. NM_004442); (23) ASLG659 (Access to Genbank No. AX092328); (24) PSCA (Access to Genbank No. AJ297436); (25) GEDA (Access to Genbank No. AY260763); (26) BAFF-R (B cell activation factor receptor, BLyS 3 receptor, BR3, NP_443177.1); (27) CD22 (CD22-B isoform cell receptor B, NP_001762.1); (28) CD79a (CD79A, CD79a, alpha-associated immunoglobulin, a B cell-specific protein that covalently interacts with beta Ig (CD79B) and forms a complex on the surface with IgM molecules, transduces a signal involved in cell differentiation B. Access to Genbank No. NP_001774.1); (29) CXCR5 (Burkitt's lymphoma receptor 1, a G-protein coupled receptor that is activated by chemokine CXCL13, works in lymphocyte migration and human defense, plays a role in HIV-2 infection and perhaps in the development of AIDS, lymphoma, myeloma, and leukemia Access to Genbank No. NP_001707.1); (30) HLA-DOB (beta subunit of MHC class II molecule (la antigen) that binds to peptides and presents them to CD4 + T lymphocytes. Access to Genbank No. NP_002111.1); (31) P2X5 (P2X purinergic receptor ligand ion 5 input channel, an ion channel with extracellular ATP input), may be involved in synaptic transmission and neurogenesis, deficiency may contribute to the pathophysiology of cell instability. idiopathic detrusor Access to Genbank No. NP_002552.2); (32) CD72 (CD72 antigen of B-cell differentiation, Lyb-2 Access to Genbank No. NP_001773.1); (33) LY64 (lymphocyte antigen 64 (RP105), membrane protein type 1 of the leucine-rich repeat family (LRR), regulates B cell activation and apoptosis, loss of function is associated with increased activity of the disease in patients with systemic lupus erythematosus Access to Genbank No. NP_005573.1); (34) FcRHl (protein 1 similar to the Fe receptor, a putative receptor for the immunoglobulin Fe domain containing Ig-like C2-like domains and ITAM, may have a role in B lymphocyte differentiation. Access to Genbank No. NP_443170. 1); (35) IRTA2 (receptor of the immunoglobulin superfamily associated with translocation 2, a putative immunoreceptor with possible roles in the development and B cell lymphomagenesis, the deregulation of the gene by translocation occurs in some B cell malignancies. Access to Genbank No NP_112571.1); and (36) TENB2 (putative transmembrane proteoglycan, related to the EGF / heregulin family of growth factors and follistatin.) Access to Genbank No. AF179274 64. The antibody-drug conjugate compound of claim 57 wherein p is 1. 65. The antibody-drug conjugate compound of claim 57 wherein p is 2. 66. The antibody-drug conjugate compound of claim 57 wherein L has the Formula: -Aa -Ww-Yy- where: A is an extensor unit covalently linked to a cysteine thiol of the designed cysteine antibody (Ab); a is 0 or 1; each W is independently an amino acid unit; w is an integer that varies from 0 to 12; Y. is a separating unit covalently linked to the drug residue; and y is 0, 1 or 2. 67. The antibody-drug conjugate compound of claim 66 having the formula: wherein PAB is para-aminobenzylcarbamoyl, and R17 is a divalent radical selected from (CH2) r, C3-C8 carbocyclyl, 0- (CH2) r » arylene, (CH2) r-arylene, -arylene- (CH2) r- (CH2) r- (C3-C8 carbocyclyl), (C3-C8 carbocyclyl) - (CH2) r, C3-C8 heterocyclyl, (CH2) r - (C3-C8 heterocyclyl), - (C3-C8 heterocyclyl) - (CH2) r-, - (CH2) rC (0) NRb (CH2) r-, - (CH2CH20) r-, - (CH2CH20) r- CH2 -, (CH2) rC (0) NRb (CH2CH20) r-, - (CH2) rC (O) NRb (CH2CH20) r -CH2-, (CH2CH20) rC (0) NRb (CH2CH20) r-, - (CH2CH20) rC (0) NRb (CH2CH20) r -CH2- and - (CH2CH20) rC (0) NRb (CH2) r-; wherein Rb is H, Cx-C6 alkyl, phenyl, or benzyl; and r is independently an integer ranging from 1-10. 68. The antibody-drug conjugate compound of claim 67 wherein Ww is valine-citrulline. 69. The antibody-drug conjugate compound of claim 67 wherein R17 is (CH2) 5 or (CH2) 2. 70. The antibody-drug conjugate compound of claim 66 having the formula: 71. The antibody-drug conjugate compound of claim 70 wherein R17 is (CH2) 5 or (CH2) 2. 72. The antibody-drug conjugate compound of claim 66 having the formula: 73. The antibody-drug conjugate compound of claim 57 wherein L is SMCC. 74. The antibody-drug conjugate compound of claim 57 wherein L is BMPEO. 75. The antibody-drug conjugate compound of claim 57 wherein the drug residue D is selected from a microtubulin inhibitor, a mitotic inhibitor, a topoisomerase inhibitor, and a DNA intercalator. 76. The antibody-drug conjugate compound of claim 57 wherein the drug residue D is selected from a maytansinoid, an auristatin, a dolastatin and a calicheamicin. 77. The antibody-drug conjugate compound of claim 57 wherein D is MMAE, having the structure: albumin (ABP). 81. The antibody-drug conjugate compound of claim 57 wherein the original antibody is selected from a monoclonal antibody, a bispecific antibody, a chimeric antibody, a human antibody and a humanized antibody. 82. The antibody-drug conjugate compound of claim 57 wherein the original antibody is huMAb4D5.8 (trastuzumab). 83. The antibody-drug conjugate compound of claim 57 wherein the original antibody is an anti-ErbB2 antibody. 84. The antibody-drug conjugate compound of claim 57 wherein the original antibody is an anti-EphB2R antibody. 85. The antibody-drug conjugate compound of claim 57 wherein the original antibody is an anti-CD22 antibody. 86. The antibody-drug conjugate compound of claim 57 wherein the original antibody is an anti-MUC16 antibody. 87. The antibody-drug conjugate compound of claim 57 wherein the original antibody is an intact antibody selected from IgA, IgD, IgE, IgG and IgM. 88. The antibody-drug conjugate compound of claim 87 wherein the IgG is selected from the subclasses IgG1, IgG2, IgG3, IgG4, IgA and IgA2. 89. The antibody-drug conjugate compound of claim 57 wherein the original antibody is an antibody fragment. 90. The antibody-drug conjugate compound of claim 89 wherein the antibody fragment is a Fab fragment. 91. The antibody-drug conjugate compound of claim 90 wherein the Fab fragment is hu4D5Fabv8. 92. The antibody-drug conjugate compound of claim 91 wherein the one or more amino acid residues of hu4D5Fabv8 replaced by cysteine are selected from L-V15, L-A43, L-V110, L-A144, L-S168, H-A40, H-A88, H-S119, H-A121, H-S122, H-A175 and H-S179. 93. An antibody-drug conjugate compound selected from the structures: Ab-MC-MMAE and Ab-MC-MMAF where Val is valina; Cit is citrulline; p is 1, 2, 3 or 4; and Ab is a designed cysteine antibody prepared by a process comprising the replacement of one or more amino acid residues of an original antibody with one or more free cysteine amino acids. 94. An antibody-drug conjugate compound having the structure: where p is 1, 2, 3 or 4; n is 0, 1 or 2; and Ab is a designed cysteine antibody prepared by a process comprising the replacement of one or more amino acid residues of an original antibody with one or more free cysteine amino acids. 95. A pharmaceutical composition comprising the antibody-drug conjugate compound of claim 57 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, carrier or excipient. 96. The pharmaceutical composition of claim 95 which further comprises a therapeutically effective amount of an additional chemotherapeutic agent. 97. A method for destroying or inhibiting the proliferation of tumor cells or cancer cells comprising the treatment of tumor cells or cancer cells with an amount of the conjugated compound of The antibody-drug of claim 57, or a pharmaceutically acceptable salt or solvate thereof, being effective to destroy or inhibit the proliferation of tumor cells or cancer cells. 98. A method for inhibiting cell proliferation, comprising: (a) exposing mammalian cells in a cell culture medium to the antibody-drug conjugate compound of claim 57; and (b) measuring the cytotoxic or cytostatic activity of the antibody-drug conjugate compound, whereby the proliferation of the cells is inhibited. 99. A method for inhibiting the growth of tumor cells overexpressing a growth factor receptor selected from an HER receptor and an EGF receptor, comprising administering to a patient an antibody-drug conjugate compound of claim 57 which binds specifically to said growth factor receptor and a chemotherapeutic agent, wherein said antibody-drug conjugate and said chemotherapeutic agent are each administered in effective amounts to inhibit the growth of tumor cells in the patient. 100. The method of claim 99 wherein said antibody-drug conjugate compound sensitizes the tumor cells to said chemotherapeutic agent. 101. A manufacturing article comprising: an antibody-drug conjugate compound of claim 57, a package, and a package insert or label indicating that the compound can be used for the treatment of cancer. 102. A method for preparing an antibody-drug conjugate compound comprising a designed antibody to cysteine (Ab), and a drug residue (D), wherein the designed cysteine antibody is linked through one or more amino acids designed from cysteine by a linker residue (L) to D, the compound having Formula I: Ab- (LD) p I wherein p is 1, 2, 3 or 4; the method comprising replacing one or more amino acid residues of an original antibody with cysteine to prepare the designed cysteine antibody. 103. The method of claim 102 further comprising the steps of: (a) reacting a designed cysteine group of the designed cysteine antibody with a linker reagent to form the antibody-linker Ab-L intermediate; Y (b) reacting Ab-L with an activated drug D residue; with which the antibody-drug conjugate is formed. 104. The method of claim 102 further comprising the steps of: (a) reacting a nucleophilic group of a drug residue with a linker reagent to form the drug-linker intermediate D-L; and (b) reacting the D-L with a designed cysteine group of the designed cysteine antibody; whereby the antibody-drug conjugate is formed. 105. The method of claim 102 further comprising the step of expressing the designed cysteine antibody in Chinese hamster ovarian (CHO) cells. 106. The method of claim 102 wherein the designed cysteine engineered antibody is an IgG antibody. 107. The method of claim 102 further comprising the step of treating the designed cysteine antibody expressed with a reducing agent. 108. The method of claim 107 wherein the reducing agent is selected from TCEP and DTT. 109. The method of claim 107 further comprising the step of treating the antibody designed cysteine expressed with an oxidizing agent after treatment with the reducing agent. 110. The method of claim 109 wherein the oxidizing agent is copper sulfate.