Cysteine engineered antibodies and conjugates

Abstract

The invention relates to cysteine engineered antibodies and conjugates. Preparing a cysteine engineered antibody comprising a free cysteine amino acid in the heavy chain or light chain by mutagenizing the nucleic acid sequence of the parent antibody and replacing one or more amino acid residues with cysteine in order to encode the cysteine engineered antibody; expressing the cysteine engineered antibody; and isolating the cysteine engineered antibody. Certain highly reactive cysteine engineered antibodies were identified by a PHESELECTOR assay method. The isolated cysteine engineered antibody may be covalently attached to a capture label, a detection label, a drug moiety, or a solid support.

Description

[0001] This application is a re-divisional application of a divisional application with an application date of June 7, 2011, Chinese application number 202111072839.2, and invention name “Cysteine-modified antibodies and conjugates” (the original application number is 201180039258.7).

[0002] Citation to Related Applications

[0003] This application is a nonprovisional application filed under 37 CFR §1.53(b) and claims the benefit of U.S. Provisional Application Serial No. 61 / 352,728, filed on June 8, 2010, pursuant to 35 USC §119(e), which is incorporated herein by reference. Technical Field

[0004] The present invention generally relates to antibodies engineered with reactive cysteine ​​residues, and more specifically, the present invention relates to antibodies having therapeutic or diagnostic applications. Cysteine ​​engineered antibodies can be conjugated to chemotherapeutic drugs; toxins; affinity ligands such as biotin and detection labels such as fluorophores. The present invention also relates to methods of using antibodies and antibody-drug conjugate compounds to diagnose or treat mammalian cells or related pathological conditions in vitro, in situ and in vivo. Background Art

[0005] Antibody drug conjugates (ADCs) are attractive targeted chemotherapy molecules because they combine the desirable properties of both antibodies and cytotoxic drugs, namely by targeting potent cytotoxic drugs to tumor cells expressing antigens, thereby enhancing their anti-tumor activity. Successful ADC development for a given target antigen depends on optimization of antibody selection, linker stability, cytotoxic drug potency, and the linker-drug conjugation antibody approach.

[0006] Conventional attachment, i.e., by covalent bonding, of drug moieties to antibodies generally produces a heterogeneous mixture of molecules in which the drug moieties are bound to a number of sites on the antibody. For example, cytotoxic drugs are generally conjugated to antibodies through the generally large number of lysine residues of the antibody, thereby producing a heterogeneous mixture of antibody-drug conjugates. Depending on the reaction conditions, the heterogeneous mixture generally contains a distribution of antibodies and 0 to about 8 or more attached drug moieties. In addition, within each subgroup of conjugates having a specific integer ratio of drug moieties to antibodies there may be a heterogeneous mixture in which the drug moieties are bound to different sites on the antibody. Analytical and preparative methods are not adequate to separate and characterize antibody-drug conjugate molecules in a heterogeneous mixture produced by a conjugation reaction. Antibodies are large, complex, and structurally diverse biomolecules, typically with many reactive functional groups. Their reactivity with linker reagents and drug-linker intermediates depends on factors such as pH, concentration, salt concentration, and cosolvents. In addition, multi-step conjugation processes may not be reproducible due to difficulties in controlling reaction conditions and characterizing reactants and intermediates.

[0007] Cysteine ​​thiols are reactive at neutral pH, unlike most amines that are protonated and nucleophilic when approaching pH 7. Since free thiols (RSH, sulfhydryl) are relatively reactive, proteins with cysteine ​​residues are usually present in their oxidized form as disulfide-linked oligomers or have internally bridged disulfide groups. Antibody cysteine ​​thiols are generally more reactive, i.e. more nucleophilic, to electrophilic coupling reactants than antibody amines or hydroxyls. Designs on cysteine ​​thiols by mutating different amino acid residues of proteins to cysteine ​​amino acids may be problematic, particularly with respect to unpaired (free Cys) residues or those residues that are relatively easy to react or oxidize. In concentrated solutions of proteins, whether in the periplasm of E. coli, or in culture supernatants or partially or completely purified proteins, unpaired Cys residues on the surface of proteins can be paired and oxidized to intramolecular disulfides and thus protein dimers or polymers. The formation of disulfide dimers renders the new Cys unavailable for coupling reactions with drugs, ligands, or other labels. In addition, if the protein forms an intramolecular disulfide bond between the newly modified Cys and an existing Cys residue in an oxidative manner, both Cys groups are unavailable for participation and interaction in the active site. In addition, proteins can be inactivated or rendered nonspecific by misfolding or loss of tertiary structure (Zhang et al. (2002) Anal. Biochem. 311: 1-9).

[0008] Antibodies (ThioMabs) with cysteine ​​substitutions at sites where the engineered cysteine ​​is available for conjugation but does not perturb immunoglobulin folding and assembly or alter antigen binding and effector function (Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al (2009) Blood 114(13):2721-2729; US7521541; US ​​7723485; WO2009 / 052249). These ThioMabs can then be coupled to cytotoxic drugs via the engineered cysteine ​​thiol groups to obtain ThioMab drug conjugates (TDCs) with uniform stoichiometry (approximately 2 drugs per antibody). Studies of multiple antibodies against different antigens have shown that TDCs are as effective as conventional ADCs in xenograft models and are tolerated at higher doses in relevant preclinical models. ThioMab drug conjugates are engineered to attach drugs at different parts of the antibody (light chain-Fab, heavy chain-Fab and heavy chain-Fc). Due to their homogeneity and site-specific conjugation of cytotoxic drugs, the in vitro and in vivo stability, efficacy and PK properties of TDCs provide unique advantages over conventional ADCs. Summary of the invention

[0009] The invention includes isolated cysteine ​​engineered antibodies comprising a free cysteine ​​amino acid in either the heavy or light chain.

[0010] One aspect of the invention is a method for preparing an isolated cysteine ​​engineered antibody by mutagenizing the nucleic acid sequence of a parent antibody by replacing one or more amino acid residues with cysteine ​​so as to encode the cysteine ​​engineered antibody; expressing the cysteine ​​engineered antibody; and isolating the cysteine ​​engineered antibody.

[0011] Another aspect of the invention is a conjugate of an isolated cysteine ​​engineered antibody, wherein the antibody is covalently attached to a capture label, a detection label, a drug moiety, or a solid support.

[0012] The present disclosure relates to the following embodiments.

[0013] 1. An isolated cysteine ​​engineered antibody comprising a free cysteine ​​amino acid and a sequence selected from SEQ ID NOs: 50-98 in the heavy chain or a sequence selected from SEQ ID NOs: 99-147 in the light chain, wherein the cysteine ​​in the sequence is a free cysteine ​​amino acid.

[0014] 2. The isolated cysteine ​​engineered antibody of embodiment 1, prepared by a method comprising the following steps:

[0015] (i) mutagenizing the nucleic acid sequence of a parent antibody by replacing one or more amino acid residues with cysteine ​​so as to encode a cysteine ​​engineered antibody;

[0016] (ii) expressing a cysteine ​​engineered antibody; and

[0017] (iii) Isolation of cysteine ​​engineered antibodies.

[0018] 3. The cysteine ​​engineered antibody of embodiment 2, wherein the mutagenesis comprises site-directed mutagenesis.

[0019] 4. The cysteine ​​engineered antibody of embodiment 2, wherein the cysteine ​​engineered antibody is expressed on a viral particle selected from a phage or a phagemid particle.

[0020] 5. The cysteine ​​engineered antibody of embodiment 2, further comprising:

[0021] (i) reacting the cysteine-modified antibody with a thiol-reactive affinity reagent to generate an affinity-tagged cysteine-modified antibody; and

[0022] (ii) Measuring binding of the affinity-tagged cysteine ​​engineered antibody to the capture matrix.

[0023] 6. The cysteine ​​engineered antibody of embodiment 5, wherein the thiol-reactive affinity reagent comprises a biotin moiety.

[0024] 7. The cysteine ​​engineered antibody of embodiment 5, wherein the thiol-reactive reagent comprises a maleimide moiety.

[0025] 8. The cysteine ​​engineered antibody of embodiment 5, wherein the capture medium comprises streptavidin.

[0026] 9. The cysteine ​​engineered antibody of embodiment 1, wherein the cysteine ​​engineered antibody is a fusion protein comprising an albumin binding peptide (ABP).

[0027] 10. The cysteine ​​engineered antibody of embodiment 9, wherein the ABP comprises a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

[0028] 11. The cysteine ​​engineered antibody of embodiment 1, wherein the cysteine ​​engineered antibody is selected from the group consisting of a monoclonal antibody, an antibody fragment, a bispecific antibody, a chimeric antibody, a human antibody, and a humanized antibody.

[0029] 12. The cysteine ​​engineered antibody of embodiment 11, wherein the antibody fragment is a Fab fragment.

[0030] 13. The cysteine-modified antibody of embodiment 11, wherein the cysteine-modified antibody is an anti-HER2 antibody.

[0031] 14. The cysteine ​​engineered antibody of embodiment 1, wherein the cysteine ​​engineered antibody binds to one or more of the receptors (1)-(36):

[0032] (1) BMPR1B (bone morphogenetic protein receptor type IB);

[0033] (2) E16 (LAT1, SLC7A5);

[0034] (3) STEAP1 (six-transmembrane epithelial cell antigen of the prostate);

[0035] (4) 0772P (CA125, MUC16);

[0036] (5) MPF (MPF, MSLN, SMR, megakaryocyte enhancer factor, mesothelin);

[0037] (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b);

[0038] (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B);

[0039] (8) PSCAhlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA2700050C12, RIKENcDNA2700050C12 gene);

[0040] (9) ETBR (endothelin type B receptor);

[0041] (10) MSG783 (RNF124, putative protein FLJ20315);

[0042] (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-associated gene 1, prostate cancer-associated protein 1, six-transmembrane epithelial cell antigen of the prostate 2, six-transmembrane prostate protein);

[0043] (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4);

[0044] (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratoma-derived growth factor);

[0045] (14) CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein-Barr virus receptor) or Hs.73792);

[0046] (15) CD79b (CD79B, CD79β, IGb (immunoglobulin-associated β), B29);

[0047] (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchoring protein 1a), SPAP1B, SPAP1C);

[0048] (17) HER2;

[0049] (18) NCA;

[0050] (19) MDP;

[0051] (20)IL20Rα;

[0052] (21) Brevican;

[0053] (22) EphB2R;

[0054] (23)ASLG659;

[0055] (24) PSCA;

[0056] (25)GEDA;

[0057] (26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3);

[0058] (27) CD22 (B-cell receptor CD22-B isoform);

[0059] (28) CD79a (CD79A, CD79α, immunoglobulin-related α, B cell-specific protein);

[0060] (29) CXCR5 (Burkitt lymphoma receptor 1, G protein-coupled receptor);

[0061] (30) HLA-DOB (MHC class II molecule β subunit (Ia antigen));

[0062] (31) P2X5 (purinergic receptor P2X ligand-gated ion channel 5);

[0063] (32) CD72 (B-cell differentiation antigen CD72, Lyb-2);

[0064] (33) LY64 (lymphocyte antigen 64 (RP105), a type I membrane protein of the leucine-rich repeat (LRR) family);

[0065] (34) FcRH1 (Fc receptor-like protein 1);

[0066] (35) IRTA2 (immunoglobulin superfamily receptor translocation-associated protein 2); and

[0067] (36) TENB2 (putative transmembrane proteoglycan).

[0068] 15. The cysteine ​​engineered antibody of embodiment 1, wherein the antibody is covalently attached to a capture label, a detection label, a drug moiety, or a solid support.

[0069] 16. The cysteine ​​engineered antibody of embodiment 15, wherein the antibody is covalently attached to a biotin capture label.

[0070] 17. The cysteine ​​engineered antibody of embodiment 15, wherein the antibody is covalently attached to a fluorescent dye detection label.

[0071] 18. The cysteine ​​engineered antibody of embodiment 17, wherein the fluorescent dye is selected from fluoresceins, rhodamines, dansyl, lissamine, cyanine, phycoerythrin, Texas Red and analogs thereof.

[0072] 19. The cysteine ​​engineered antibody of embodiment 15, wherein the antibody is covalently attached to a 3 H. 11 C. 14 C. 18 F. 32 P. 35 S. 64 Cu, 68 Ga, 86 Y. 89 Zr, 99 Tc, 111 In, 123 I. 124 I. 125 I. 131 I. 133 Xe, 177 Lu, 211 At, and 213 Radionuclide detection labeling of Bi.

[0073] 20. The cysteine ​​engineered antibody of embodiment 15, wherein the antibody is covalently attached to a detection label via a chelating ligand.

[0074] 21. The cysteine ​​engineered antibody of embodiment 20, wherein the chelating ligand is selected from DOTA, DOTP, DOTMA, DTPA and TETA.

[0075] 22. The cysteine ​​engineered antibody of embodiment 15, wherein the antibody is covalently attached to a drug moiety selected from the group consisting of a maytansinoid, an auristatin, a dolastatin, a trichothecene, CC1065, a calicheamicin, an enediyne antibiotic, a taxane, and an anthracycline antibiotic to form an antibody-drug conjugate having Formula I:

[0076] Ab-(LD) p I

[0077] wherein Ab is an antibody, L is a linker, D is a drug moiety, and p is 1, 2, 3, or 4.

[0078] 23. The cysteine ​​engineered antibody of embodiment 22, wherein the antibody-drug conjugate has the following structure:

[0079]

[0080] 24. The cysteine ​​engineered antibody of embodiment 22, wherein D is a maytansinoid having the following structure:

[0081]

[0082] The wavy line indicates the covalent attachment of the sulfur atom of D to the linker;

[0083] R is independently selected from H, 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; and

[0084] m is 1, 2, or 3;

[0085] 25. The cysteine ​​engineered antibody of embodiment 24, wherein D is selected from the following structures:

[0086]

[0087]

[0088] 26. The cysteine ​​engineered antibody of embodiment 25, which has the following structure:

[0089]

[0090] Where n is 0, 1, or 2.

[0091] 27. The cysteine ​​engineered antibody of embodiment 22, wherein D is a monomethyl auristatin drug moiety MMAE or MMAF having the following structure:

[0092]

[0093] 28. The cysteine ​​engineered antibody of embodiment 27, wherein the antibody-drug conjugate is selected from the following structures:

[0094]

[0095] wherein Val is valine; Cit is citrulline; and p is 1, 2, 3 or 4. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1A A three-dimensional representation of the hu4D5Fabv7 antibody fragment derived by X-ray crystal coordinates is shown. The structural positions of exemplary engineered Cys residues of the heavy and light chains are numbered (according to the sequential numbering system).

[0097] Figure 1B The Kabat numbering scheme (lower row) of 4D5v7fabH is shown compared to the sequence numbering scheme starting at the N-terminus (upper row). The Kabat numbering inserts are marked by a, b, c.

[0098] Figure 2A and Figure 2B Represents the binding measurements of hu4D5Fabv8 and hu4D5Fabv8 Cys mutant (ThioFab) phage variants by PHESELECTOR assay interacting with BSA (open bars), HER2 (striped bars) or streptavidin (solid bars) using absorbance detection at 450 nm: (A) non-biotinylated phage-hu4D5Fabv8 and (B) biotinylated phage-hu4D5Fabv8 (B).

[0099] Figure 3A and Figure 3BRepresents the binding measurements of hu4D5Fabv8 (left) and hu4D5Fabv8 Cys mutants (ThioFab) by the PHESELECTOR assay using absorbance detection at 450 nm for interaction with BSA (open bars), HER2 (striped bars), and streptavidin (solid bars): (A) non-biotinylated phage-hu4D5Fabv8 and (B) biotinylated phage-hu4D5Fabv8. Light chain variants are on the left and heavy chain variants are on the right. Thiol reactivity = OD of streptavidin binding 450nm ÷HER2(antibody) binding OD 450nm .

[0100] Figure 4A Represents the Fractional Surface Accessibility Value of the residues on wild-type hu4D5Fabv8. The light chain sites are on the left, while the heavy chain sites are on the right.

[0101] Figure 4B Binding measurements of biotinylated hu4D5Fabv8 and hu4D5Fabv8Cys variants (ThioFab) for interaction with HER2 (Day 2), streptavidin (SA) (Day 2), HER2 (Day 4) and SA (Day 4) measured by monitoring absorbance at 450 nm. Phage-hu4D5Fabv8 Cys variants were isolated and stored at 4°C. Biotin coupling was performed at Day 2 or Day 4 followed by PHESELECTOR analysis to monitor interaction with Her2 and streptavidin as described in Example 2 and to probe the stability of reactive thiols on engineered ThioFab variants.

[0102] Figure 5 Represents the binding measurements of biotin-maleimide-coupled-hu4D5Fabv8 (A121C) and non-biotinylated wild-type hu4D5Fabv8 in binding streptavidin and HER2 measured by monitoring absorbance at 450 nm. Each Fab was tested at 2 ng and 20 ng.

[0103] Figure 6 Represents ELISA analysis of biotinylated ABP-hu4D5Fabv8 wild type (wt) and ABP-hu4D5Fabv8 cysteine ​​mutants V110C and A121C in binding to rabbit albumin, streptavidin (SA) and HER2 by detecting the absorbance at 450 nm.

[0104] Figure 7Represents ELISA analysis of biotinylated ABP-hu4D5Fabv8 cysteine ​​mutants (ThioFab variants): (left to right) single Cys variant ABP-V110C, ABP-A121C and double Cys variants ABP-V110C-A88C and ABP-V110C-A121C in combination with rabbit albumin, HER2 and streptavidin (SA) and probed with Fab-HRP or SA-HRP by detecting absorbance at 450 nm.

[0105] Figure 8 Represents the binding of biotinylated ThioFab phage and anti-phage HRP antibody to HER2 (top) and streptavidin (bottom).

[0106] Fig.9A Cartoon depiction showing biotinylated antibody bound to immobilized HER2 in conjunction with HRP-labeled secondary antibody for absorbance detection.

[0107] Fig. 9B Represents a binding assay of biotin-maleimide-coupled thio-trastuzumab variants and non-biotinylated wild-type trastuzumab binding to immobilized HER2 measured by absorbance detection at 450 nm. From left to right: V110C (single cys), A121C (single cys), V110C / A121C (double cys) and trastuzumab. Each thio IgG variant and trastuzumab were tested at 1, 10 and 100 ng.

[0108] Fig. 10A Cartoon depiction showing biotinylated antibody binding to immobilized HER2 and biotin conjugated to anti-IgG-HRP for absorbance detection.

[0109] Fig. 10B Represents the binding assay values ​​of biotin-maleimide coupled-thio trastuzumab variants and non-biotinylated wild-type trastuzumab in binding to immobilized streptavidin with absorbance detected at 450 nm. From left to right: V110C (single cys), A121C (single cys), V110C / A121C (double cys) and trastuzumab. Each thio IgG variant and trastuzumab were tested at 1, 10 and 100 ng.

[0110] Fig.11 A general method for preparing cysteine ​​engineered antibodies (ThioMab) expressed by cell culture for conjugation is presented. DETAILED DESCRIPTION

[0111] The details are with reference to certain embodiments of the present invention, examples of which are illustrated in the accompanying structures and formulas. Although the present invention has been described in conjunction with the enumerated embodiments, it should be understood that they are not intended to limit the present invention to those embodiments. On the contrary, the present invention covers all alternatives, modifications and equivalent technical solutions, which are all included within the scope of the present invention as defined in the claims.

[0112] Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used to practice the present invention. The present invention is in no way limited to the methods and materials described.

[0113] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and are consistent with those described in the following documents: Singleton et al. (1994) Dictionary of Microbiology and Molecular Biology, 2nd Ed., J. Wiley & Sons, New York, NY; and Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immonobiology, 5th Ed., Garland Publishing, New York.

[0114] definition

[0115] Unless otherwise stated, the following terms and expressions used herein have the following meanings:

[0116] When a trade name is used herein, Applicants intend to independently include the trade name product formulation of the product, the generic drug, and the active pharmaceutical ingredient of the trade name product.

[0117] The term "antibody" herein is used in its broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired biological activity (Miller et al. (2003) Jour. of Immunology 170: 4854-4861). Antibodies can be murine, human, humanized, chimeric, or derived from other species. Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). Target antigens generally have a large number of binding sites, also called epitopes, recognized by the CDRs of multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Therefore, an antigen can have more than one corresponding antibody. Antibodies include full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules containing antigens or portions thereof that specifically bind to targets of interest, such targets including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein can have any type (e.g., IgG, IgE, IgM, IgD, and IgA), category (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. Immunoglobulins can be derived from any species. However, in one aspect, immunoglobulins are derived from humans, mice, or rabbits.

[0118] "Antibody fragments" comprise a portion of a full-length antibody, generally its antigen binding or variable region. Examples of antibody fragments include: Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; minibodies (Olafsen et al. (2004) Protein Eng. Design & Sel. 17(4): 315-323); fragments prepared from Fab expression libraries; anti-idiotypic (anti-Id) antibodies; CDRs (complementarity determining regions); and epitope-binding fragments of any of the above that immunospecifically bind to cancer cell antigens, viral antigens, or microbial antigens; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0119] The term "monoclonal antibody" herein refers to an antibody obtained from a substantially homogeneous antibody group, i.e., except for possible mutations of natural occurrence that may be present in small amounts, each antibody contained in the group is identical. A monoclonal antibody is an antibody that is highly specific and targets a single antigenic site. Moreover, in contrast to polyclonal antibody preparations that typically include different antibodies targeting different determinants (epitopes), each monoclonal antibody only targets a single determinant on an antigen. In addition to its specificity, the advantage of monoclonal antibodies is that they can be synthesized in a manner that is not contaminated by other antibodies. The modifier "monoclonal" represents the antibody properties obtained from a substantially homogeneous antibody group, and is not interpreted as requiring antibodies to be produced by any ad hoc method. For example, the monoclonal antibody for 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 a recombinant DNA method (e.g., see: US 4816567; US 5807715). For example, monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature, 352: 624-628; Marks et al. (1991) J. Mol. Biol., 222: 581-597.

[0120] The monoclonal antibodies herein particularly include "chimeric" antibodies, in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody type or subtype, and the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody type or subtype, and fragments of chimeric antibodies are also included herein, as long as they exhibit the desired biological activity (US 4816567; and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA, 81: 6851-6855). The chimeric antibodies of interest herein include "primatized" antibodies, which comprise variable region antigen-binding sequences derived from non-human primates (e.g., Old World Monkey, Ape, etc.) and human constant region sequences.

[0121] "Complete antibodies" herein are antibodies comprising VL and VH domains and light chain constant domains (CL) and heavy chain constant domains CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. A complete antibody may have one or more "effector functions," meaning those biological activities attributed to the Fc constant region (native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); endocytosis; and downregulation of cell surface receptors, such as B cell receptors and BCRs.

[0122] Depending on the amino acid sequence of its heavy chain constant domain, intact antibodies can be assigned to different "classes". There are 5 major classes of intact immunoglobulin antibodies: IgA, IgD, IgE, IgG and IgM, and several of them can be further divided into "subclasses" (subtypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different antibody classes are called α, δ, ε, γ and μ, respectively. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known. Ig types include hinge-modified or hingeless types (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).

[0123] "ErbB receptors" are protein tyrosine kinases that belong to the ErbB receptor family of receptors, whose members are important mediators of cell growth, differentiation, and survival. The ErbB receptor family includes four different members, including epidermal growth factor receptor (EGFR, ErbB1, HER1), HER2 (ErbB2 or p185neu), HER3 (ErbB3), and HER4 (ErbB4 or tyro2). A panel of anti-ErbB2 antibodies has been characterized using the human breast tumor cell line SKBR3 (Hudziak et al. (1989) Mol. Cell. Biol. 9(3):1165-1172). Maximum inhibition was obtained using an antibody called 4D5 that inhibited cell proliferation by 56%. Other antibodies in the panel of antibodies tested reduced cell proliferation to a lesser extent. Antibody 4D5 was further found to sensitize breast tumor cell lines overexpressing ErbB2 to the cytotoxic effects of TNF-α (US 5677171). The anti-ErbB2 antibodies discussed by Hudziak et al. have been 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. (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. Natl. Acad. Sci. (1994) 91:7202-7206; Lewis et al. (1996) Cancer Research 56:1457-1465; and Schaefer et al. (1997) Oncogene 15:1385-1394.

[0124] ErbB receptors generally comprise an extracellular domain that can bind ErbB ligands; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a carboxyl-terminal signaling domain comprising several tyrosine residues that can be phosphorylated. The ErbB receptor can be a "native sequence" ErbB receptor or an "amino acid sequence variant" thereof. Preferably, the ErbB receptor is a native sequence human ErbB receptor. Thus, "members of the ErbB receptor family" include EGFR (ErbB1), ErbB2, ErbB3, ErbB4.

[0125] The term "amino acid sequence variant" means a polypeptide having an amino acid sequence that differs to some extent from a native sequence polypeptide. Amino acid sequence variants generally have at least about 70% sequence identity with at least one receptor binding domain of a native ErbB ligand or with at least one ligand binding domain of a native ErbB receptor, and preferably they are at least about 80%, more preferably at least about 90% homologous to such receptor or ligand binding domains. Amino acid sequence variants have substitutions, deletions and / or insertions at certain positions within the amino acid sequence of the native amino acid sequence. Amino acids are named according to conventional names, i.e., one-letter codes and three-letter codes.

[0126] "Sequence identity" is defined as the percentage of identical residues in amino acid sequence variants after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Methods and computer programs for performing sequence alignments are well known in the art. One such computer program is "Align2" created by Genentech, Inc., which was filed as a user file on December 10, 1991 in the United States Copyright Office, Washington, DC 20559.

[0127] "Native antibodies" are usually heterotetrameric glycoproteins of about 150,000 daltons composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced interchain disulfide bonds. Each heavy chain carries a variable domain (V H ), followed by a number of constant domains. Each light chain carries a variable (V L ), and a constant domain on the other end. The constant domain of the light chain is compared with the first constant domain of the heavy chain and the variable domain of the light chain is compared with the variable domain of the heavy chain. It is believed that specific amino acid residues form an interface between the light chain domain and the heavy chain variable domain.

[0128] The term "variable" refers to the fact that some parts of the variable region vary widely in the antibody sequence and are used for the binding and specificity of each specific antibody for its specific antigen. However, variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments called hypervariable regions in the light chain and heavy chain variable regions. The more highly conserved parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FRs, most of which adopt a β-folded conformation, connected by three hypervariable regions that form a loop connection and in some cases form a part of the β-folded structure. The hypervariable regions in each chain are kept together very closely by the FRs, and together with the hypervariable regions of the other chain, contribute to the formation of the antigen binding site of the antibody (see Kabat et al., 1991, "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD). The constant region is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions, such as the participation of antibodies in antibody-dependent cellular cytotoxicity (ADCC).

[0129] The term "hypervariable region" as used herein refers to the amino acid residues in an antibody that are responsible for antigen binding. A hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable region and residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable region; Kabat et al., supra) and / or those residues from a "hypervariable loop" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable region and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable region; Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917). "Framework Region" or "FR" residues are those variable region residues other than the hypervariable region residues as herein defined.

[0130] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with one antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0131] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and antigen binding site. This region consists of a dimer of one heavy chain variable region and one light chain variable region in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable region interact to form a single variable region in V H -V L An antigen binding site is defined on the surface of the dimer. The six hypervariable regions together give the antibody antigen binding specificity. However, even a single variable region (or half of an Fv containing only three hypervariable regions specific for an antigen) has the ability to recognize and bind to an antigen, although the affinity is lower than that of the complete binding site.

[0132] The Fab fragment also contains the constant region of the light chain and the first constant region of the heavy chain (C H 1) Fab' fragment is due to the heavy chain C H The Fab fragment differs from the Fab fragment by the addition of a few residues at the carboxyl terminus of the 1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains carry at least one free thiol group. F(ab')2 antibody fragments were originally generated as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0133] The "light chains" of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0134] "Single-chain Fv" or "scFv" antibody fragments comprise the V H and V L domains, wherein these domains are present on one polypeptide chain. Preferably, the Fv polypeptide is present in V H and V L A polypeptide linker is also included between the domains to enable the scFv to form the desired structure for antigen binding. For a review of scFv, see Plückthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore, ed., Springer-Verlag, New York, pp. 269-315, 1994. Anti-ErbB2 antibody scFv fragments are described in WO 93 / 16185; U.S. Pat. Nos. 5,571,894; and 5,587,458.

[0135] The "humanized" form of non-human (e.g., rodent) antibodies refers to chimeric antibodies that contain sequences derived from non-human immunoglobulins at a minimum. Humanization is a method in which mouse antigen binding information is transferred to non-immunogenic human antibody receptors, and many therapeutically useful drugs have been produced. Humanization methods generally begin by transferring all 6 mouse complementary determining regions (CDRs) to human antibody frameworks (Jones et al., (1986) Nature 321:522-525). The antibodies transplanted with these CDRs generally do not retain their initial affinity for antigen binding, and in fact, affinity is usually severely impaired. In addition to CDR, selected non-human antibody framework residues must also be introduced to maintain correct CDR conformation (Chothia et al. (1989) Nature 342:877). It has been demonstrated that transferring key mouse framework residues to the human acceptor to support the structural conformation of the grafted CDRs restores antigen binding and affinity (Riechmann et al. (1992) J. Mol. Biol. 224, 487-499; Foote and Winter, (1992) J. Mol. Biol. 224:487-499; Presta et al. (1993) J. Immunol. 151, 2623-2632; Werther et al. (1996) J. Immunol. Methods 157:4986-4995; and Presta et al. (2001) Thromb. Haemost. 85:379-389). To a great extent, humanized antibodies refer to immunoglobulins in which the hypervariable region residues in human immunoglobulins (recipient antibodies) are replaced with hypervariable region residues of non-human species (donor antibodies) such as mice, rats, rabbits or non-human primates with desired specificity, affinity and ability. In some cases, the framework region (FR) residues of human immunoglobulins are replaced with corresponding non-human residues. In addition, humanized antibodies may include residues not found in the receptor antibody or the donor antibody. These modifications are made in order to further improve the performance of the antibody. Generally, humanized antibodies include at least one, usually two, substantially the entire variable region, wherein the entire or substantially the entire hypervariable loop corresponds to the hypervariable loop of a non-human immunoglobulin, and the entire or substantially the entire FR is the FR of a human immunoglobulin sequence. Humanized antibodies optionally also include at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin. For more details, see US 6,407,213; Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-329; Presta (1992) Curr. Op. Struct. Biol. 2:593-596.

[0136] "Free cysteine ​​amino acid" refers to a cysteine ​​amino acid residue that has been engineered into a parent antibody, has a thiol functional group (-SH), and is not paired as an intramolecular or intermolecular disulfide bond.

[0137] The term "thiol reactivity value" is a quantitative characterization of the reactivity of free cysteine ​​amino acids. The thiol reactivity value is the percentage of free cysteine ​​amino acids in a cysteine ​​engineered antibody that reacts with a thiol-reactive reagent, and is converted to a maximum value of 1. For example, a free cysteine ​​amino acid on a cysteine ​​engineered antibody that reacts with a thiol-reactive reagent such as a biotin-maleimide reagent at a 100% yield to form a biotin-labeled antibody has a thiol reactivity value of 1.0. Another cysteine ​​amino acid engineered into the same or different parent antibody that reacts with a thiol-reactive reagent at an 80% yield has a thiol reactivity value of about 0.8. Another cysteine ​​amino acid engineered into the same or different parent antibody that is completely unable to react with a thiol-reactive reagent has a thiol reactivity value of 0. The thiol reactivity value of a specific cysteine ​​can be determined by ELISA assay, mass spectrometry, liquid chromatography, autoradiography or other quantitative analysis tests.

[0138] "Parent antibody" is an antibody in which one or more amino acid residues in the contained amino acid sequence are replaced with one or more cysteine ​​residues. The parent antibody may comprise a native or wild-type sequence. The parent antibody may have pre-existing amino acid sequence modifications (such as additions, deletions and / or substitutions) relative to other native, wild-type or modified forms of antibodies. The parent antibody may be directed against a target antigen of interest, such as a biologically important polypeptide. Antibodies directed against non-polypeptide antigens (such as tumor-associated glycolipid antigens; see US 5,091,178) are also of interest.

[0139] Exemplary parent antibodies include antibodies having affinity and selectivity for cell surface and transmembrane receptors and tumor associated antigens (TAA).

[0140] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminating components of its natural environment are substances that interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody is purified to (1) greater than 95% by weight, most preferably greater than 99% by weight, as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequencer, or (3) to homogeneity according to SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Since at least one component of the antibody's natural environment will not be present, an isolated antibody includes the antibody in situ within a recombinant cell. However, an isolated antibody is typically prepared by at least one purification step.

[0141] An antibody that "binds" a molecular target or antigen of interest, such as an ErbB2 antigen, is one that binds the antigen with sufficient affinity so that the antibody can be used to target cells expressing the antigen. If the antibody is an antibody that binds ErbB2, it will typically bind ErbB2 preferentially over other ErbB receptors and may be one that does not significantly cross-react with other proteins, such as EGFR, ErbB3, or ErbB4. In such embodiments, the extent of binding of the antibody to these non-ErbB2 proteins (e.g., cell surface binding to endogenous receptors) will be less than 10%, as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA). Sometimes, anti-ErbB2 antibodies do not significantly cross-react with rat neu protein, such as described in Schecter et al. (1984) Nature 312:513 and Drebin et al. (1984) Nature 312:545-548.

[0142] The molecular targets of 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, CD22, CD34, CD40, CD79α (CD79a) and CD79β (CD79b); (ii) members of the ErbB receptor family, such as EGF receptor, HER2, HER3 or HER4 receptor; (iii) cell adhesion molecules, such as LFA-1, Mac1, p150, 95, VLA-4, ICAM-1, VCAM and αv / β3 integrins, including their α or β subunits (e.g., anti-CD11a, anti-CD18 or anti-CD11b antibodies); (iv) growth factors, such as VEGF; IgE; blood group antigens; flk2 / flt3 receptors; obesity (OB) receptor; mpl receptor; CTLA-4; protein C, BR3, c-met, tissue factor, β7, etc.; and (v) cell surface and transmembrane tumor-associated antigens (TAA).

[0143] Unless otherwise stated, the term "monoclonal antibody 4D5" refers to an antibody having or derived from the antigen-binding residues of the murine 4D5 antibody (ATCC CRL10463). For example, monoclonal antibody 4D5 can be murine monoclonal antibody 4D5 or a variant thereof, such as humanized 4D5. Exemplary humanized 4D5 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7, and huMAb4D5-8 (trastuzumab, ).

[0144] The terms "treat" and "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the goal is to prevent or slow (mitigate) an unwanted physiological change or disorder, such as the formation or spread of cancer. For purposes of the present invention, favorable or desired clinical results include, but are not limited to, relief of symptoms, attenuation of the extent of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and recovery (whether partial or complete), whether detectable or undetectable. "Treatment" or "treatment" may also refer to prolonging survival compared to expected survival if not receiving treatment. Subjects in need of treatment include subjects already suffering from a condition or disorder as well as subjects predisposed to developing a condition or disorder or subjects in whom a condition or disorder is to be prevented.

[0145] The term "therapeutically effective amount" refers to an amount of a drug that is effective in treating a disease or disorder in a mammal. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce the size of a tumor; inhibit (i.e., slow down and preferably prevent to some extent) the infiltration of cancer cells into surrounding organs; inhibit (i.e., slow down and preferably prevent to some extent) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate to some extent one or more symptoms associated with cancer. To the extent that a drug can prevent growth and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic. For cancer therapy, efficacy can be measured, for example, by assessing the time to disease progression (TTP) and / or determining the response rate (RR).

[0146] 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 cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer including small cell lung cancer, non-small cell lung cancer ("NSCLC"), adenocarcinoma of the lung and squamous carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer.

[0147] "ErbB-expressing cancer" refers to cancers that contain cells with ErbB proteins on their cell surfaces. "ErbB2-expressing cancer" refers to cancers that produce sufficient levels of ErbB2 on their cell surfaces to allow anti-ErbB2 antibodies to bind to and have a therapeutic effect on the cancer.

[0148] Cancers that "overexpress" antigenic receptors refer to cancers that have significantly higher levels of receptors such as ErbB2 on their cell surfaces compared to non-cancerous cells of the same tissue type. Such overexpression can be caused by gene amplification or by increased transcription or translation. Receptor overexpression can be determined in diagnostic or prognostic assays by assessing elevated levels of receptor protein present on the cell surface (e.g., by immunohistochemical assays; IHC). Alternatively or in addition, the level of receptor encoding nucleic acid in the cell can be measured, for example, by fluorescent in situ hybridization (FISH; see WO 98 / 45479), Southern blotting, or polymerase chain reaction (PCR) techniques, such as real-time quantitative PCR (RT-PCR).

[0149] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes cell destruction. The term is intended to include: radioactive isotopes, such as At 211 ,I 131 ,I 125 , Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 , P 32 , C 60 and radioactive isotopes of Lu; chemotherapeutic agents; and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including synthetic analogs and derivatives thereof.

[0150] "Phage display" is a technique in which variant polypeptides are displayed on the surface of phage, such as filamentous phage particles, as fusion proteins with coat proteins. A kind of utility of phage display is that large libraries of randomized protein variants can be quickly and effectively sorted for the fact that those sequences that bind to target molecules with high affinity. Displaying peptide and protein libraries on phage has been used to screen millions of polypeptides for polypeptides with specific binding properties. Multivalent phage display methods have been used to display small libraries of peptides and small proteins, usually by merging with pill or pVIII of filamentous phage (Wells and Lowman, (1992) Curr.Opin.Struct.Biol.3:355-362 and references cited therein). In monovalent phage display, protein or peptide libraries are fused with phage coat proteins or parts thereof, and expressed at low levels when there is wild-type protein. The avidity effect is reduced compared with multivalent phage, so that sorting is based on intrinsic ligand affinity, and phagemid vectors that simplify DNA operations are used. Lowman and Wells, (1991) Methods: A companion to Methods in Enzymology 3: 205-0216. Phage display includes technologies for generating antibody-like molecules (Janeway, C., Travers, P., Walport, M., Shlomchik, (2001) Immunobiology, 5th Ed., Garland Publishing, New York, p627-628; Lee et al.).

[0151] "Phagemid" is a phage vector having a bacterial replication origin such as Co1E1 and a copy of the phage gene interval. Phagemids can be used for any known phage, including filamentous phages and lambda phages. Plasmids will also generally include a selection marker for antibiotic resistance. The DNA segments cloned into these vectors can be propagated like plasmids. When cells containing these vectors are equipped with all the genes necessary for generating phage particles, the replication mode of the plasmid is changed to rolling circle replication to generate a copy of one chain of the plasmid DNA and package the phage particles. Phagemids can form infectious or non-infectious phage particles. The term includes phage coat protein genes or fragments thereof and connected to heterologous polypeptide genes to form gene fusions so that the heterologous polypeptide is displayed on the surface of the phage particle.

[0152] "Linker", "Linker unit" or "connector" refers to a chemical moiety comprising a covalent bond or a chain of atoms that covalently links an antibody to a drug moiety. In various embodiments, the linker is designated as L. Linkers include: divalent groups, such as alkyldiyl, arylene, heteroarylene, such as -(CR2) n O(CR2) n -, alkoxy repeating units (e.g., polyethyleneoxy, PEG, polymethyleneoxy) and alkamino (e.g., polyethyleneamino, Jeffamine TM ) and dioic acid esters and amides, including succinate, succinamide, diglycolate, malonate and caproamide.

[0153] The term "label" refers to any moiety that can be covalently attached to an antibody and performs the following functions: (i) providing a detectable signal; (ii) interacting with a second label to change the detectable signal provided by the first or second label, such as FRET (fluorescence resonance energy transfer); (iii) stabilizing the interaction with an antigen or ligand or providing affinity for binding thereto; (iv) affecting mobility such as electrophoretic mobility or cell permeability through charge, hydrophobicity, shape or other physical parameters; or (v) providing a capture module to modulate ligand affinity, antibody / antigen binding, or ion complexation.

[0154] The definitions and rules of stereochemistry used herein generally follow those of SP Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., New York. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane polarized light. In describing optically active compounds, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule about its chiral center. The prefixes d and 1 or (+) and (-) are used to indicate the sign of the rotation of a compound with respect to plane polarized light, where (-) or 1 means that the compound is left-handed. Compounds prefixed with (+) or d are right-handed. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. Specific stereoisomers may also be referred to as enantiomers, and mixtures of such isomers are often referred to as enantiomeric mixtures. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can occur in a chemical reaction or process without stereoselectivity or stereospecificity. The terms "racemic mixture" and "racemate" refer to a mixture in which two enantiomers are mixed in equimolar amounts, resulting in the loss of optical activity.

[0155] The phrase "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of an ADC when used herein. Exemplary salts include, but are not limited to, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbates, succinates, maleates, gentisates, fumarates, gluconates, glucuronates, sugarates, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, and pamoates (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)). Pharmaceutically acceptable salts may involve the inclusion of another molecule, such as acetate ions, succinate ions, or other counterions. Counterions may be any organic or inorganic module that stabilizes the charge of the parent compound. In addition, pharmaceutically acceptable salts can have more than one charged atom in their structure. In the case where multiple charged atoms are components of pharmaceutically acceptable salts, multiple counterions can be present. Therefore, pharmaceutically acceptable salts can have one or more charged atoms and / or one or more counterions.

[0156] "Pharmaceutically acceptable solvate" refers to the association of one or more solvent molecules with the ADC. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

[0157] Cysteine-modified antibodies

[0158] The compounds of the present invention include cysteine ​​engineered antibodies, wherein one or more amino acids in the wild-type or parent antibody are replaced by cysteine ​​amino acids. Any form of antibody can be transformed, even if it is mutated. For example, the parent Fab antibody fragment can be transformed into a cysteine ​​engineered Fab, referred to herein as "ThioFab". Similarly, the parent monoclonal antibody can be transformed into a "ThioMab". It should be noted that a single site mutation produces a single engineered cysteine ​​residue in ThioFab, while a single site mutation produces two engineered cysteine ​​residues in ThioMab, due to the dimerization properties of IgG antibodies. The newly introduced engineered cysteine ​​thiol reactivity of the mutant replaced by a cysteine ​​(Cys) residue ("engineered") is evaluated. The thiol reactivity value is a relative numerical range in the range of 0-1.0 and can be determined for any cysteine ​​engineered antibody. The thiol reaction value of the cysteine ​​modified antibody of the present invention is in the range of 0.6-1.0; 0.7-1.0; or 0.8-1.0.

[0159] The design, selection and preparation methods of the present invention enable the antibody of cysteine ​​modification to react with electrophilic functional groups (functionality). These methods further enable antibody conjugate compounds, such as antibody-drug conjugate (ADC) compounds, to react with drug molecules at designated, designed, selected sites. Reactive cysteine ​​residues on the antibody surface can be specifically coupled to drug moieties through thiol reactive groups, such as maleimide or haloacetyl groups. The nucleophilic reactivity of the thiol functional group of Cys residues and maleimide groups is approximately higher than any other amino acid functional group in protein, such as the amino group of lysine residues or the N-terminal amino group is about 1000 times. The thiol-specific functional groups in iodoacetyl reagents and maleimide reagents can react with amine groups, but require higher pH (> 9.0) and longer reaction times (Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London).

[0160] The cysteine-modified antibodies of the present invention preferably retain the antigen binding ability of their wild-type parent antibody counterparts. Therefore, the cysteine-modified antibodies are capable of binding, preferably specifically binding, to antigens. Such antigens include, for example, tumor-associated antigens (TAA), cell surface receptor proteins and other cell surface molecules, transmembrane proteins, signal transduction proteins, cell survival regulators, cell proliferation regulators, molecules related to tissue development or differentiation (e.g., known or suspected to be functionally related), lymphokines, cytokines, molecules related to cell cycle regulation, molecules related to angiogenesis, and molecules related to angiogenesis (e.g., known or suspected to be functionally related). Tumor-associated antigens can be differentiation cluster factors (i.e., CD proteins). The antigen that can bind to the cysteine-modified antibodies can be a member of one of the above-mentioned subgroups, wherein another subgroup in the type contains other molecules / antigens (with respect to the antigen of interest) having different properties.

[0161] The parent antibody may also be selected from huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (trastuzumab, ), which is specifically incorporated herein by reference; humanized 520C9 (WO 93 / 21319) and the humanized 2C4 antibody as described herein.

[0162] The cysteine-modified antibodies of the present invention can be site-specifically and efficiently coupled to thiol-reactive reagents. The thiol-reactive reagents can be multifunctional linker reagents; capture substances, i.e., affinity, labeling reagents (e.g., biotin-linker reagents); detection reagents (e.g., fluorophore reagents); solid phase immobilization reagents (e.g., SEPHAROSE TM , polystyrene or glass) or a drug-linker intermediate. An example of a thiol-reactive reagent is N-ethylmaleimide (NEM). In a typical embodiment, ThioFab is reacted with a biotin-linker reagent to obtain a biotinylated ThioFab, by which the presence and reactivity of the engineered cysteine ​​residues can be detected and measured. ThioFab is reacted with a multifunctional linker reagent to obtain a ThioFab with a functionalized linker that can be further reacted with a drug moiety reagent or other label. ThioFab is reacted with a drug-linker intermediate to obtain a ThioFab drug conjugate.

[0163] The exemplary methods described herein can be applied generally to the identification and production of antibodies, and more generally to other proteins by using the design and screening procedures described herein.

[0164] This type of approach can be applied to coupling other thiol-reactive reagents, where the reactive group is, for example, maleimide, iodoacetamide, pyridyl disulfide or other thiol-reactive coupling partners (Haugland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London; Means (1990) Bioconjugate Chem. 1:2; Hermanson, G. in Bioconjugate Techniques (1996) Academic Press, San Diego, pp. 40-55, 643-671). The partner can be a cytotoxic agent (e.g., a toxin such as doxorubicin or pertussis toxin); a fluorophore such as the fluorescent dyes fluorescein or rhodamine; a chelator or radiotherapeutic metal for imaging; a peptidyl or non-peptidyl label or detection marker; or a clearance-modifying agent such as different isomers of polyethylene glycol; a peptide or another carbohydrate or lipophilic agent conjugated to a third component.

[0165] The sites identified on the representative antibody fragment hu4D5Fabv8 herein are mainly located in the constant domain of the antibody, which is well conserved between all antibody classes. These sites are widely applicable to other antibodies without further structural design or knowledge of specific antibody structure, and will not interfere with the antigen binding properties inherent to the antibody variable domain.

[0166] Cysteine-modified antibodies that can be used to treat cancer include, but are not limited to antibodies for cell surface receptors and tumor-associated antigens (TAA). Such antibodies can be used as naked antibodies (not coupled to a drug or labeling moiety) or as formula I antibody-drug conjugates (ADC). Tumor-associated antigens are well known in the art and can be prepared so that they can be used to produce antibodies using methods and information well known in the art. In the attempt to find effective cell targets for cancer diagnosis and therapy, researchers seek to identify transmembrane polypeptides that are specifically expressed on the surface of one or more specific types of cancer cells compared to one or more normal non-cancerous cells, or tumor-associated polypeptides. Compared with non-cancerous cells, usually such tumor-associated polypeptides are expressed on the surface of cancer cells in greater quantities. The identification of such tumor-associated cell surface antigen polypeptides has enabled people to specifically target cancer cells and destroy them by antibody-based therapy.

[0167] Examples of TAAs include, but are not limited to, the following TAAs (1)-(36). For convenience, information about these antigens, all of which are well known in the art, is listed below and is in accordance with the National Center for Biotechnology Information (NCBI) nucleic acid and protein sequence identification guidelines, including names, alternative names, Genbank accession numbers and primary references. Nucleic acid and protein sequences equivalent to TAAs (1)-(36) can be obtained in public databases such as GenBank. Tumor-associated antigens targeted by antibodies include all amino acid sequence variants and isoforms that have at least about 70%, 80%, 85%, 90% or 95% sequence identity compared to the sequences identified in the cited references, or exhibit substantially the same biological properties or characteristics as the TAA having the sequence found in the cited references. For example, a TAA having a variant sequence is generally capable of specifically binding to an antibody that specifically binds to the corresponding sequence TAA as shown in the literature. The sequences and disclosures in the references specifically cited herein are specifically incorporated by reference.

[0168] Tumor-associated antigens (1)-(36):

[0169] (1) BMPR1B (bone morphogenetic receptor-type IB, Genbank accession number NM_001203) ten Dijke, P., et al. Science 264(5155):101-104 (1994), Oncogene 14(11):1377-1382(1997)); WO2004063362(claim 2); WO2003042661(claim 12); US2003134790-A1(pp. 38-39); WO2002102235(claim 13; page 296); WO2003055443(pp. 91-92); WO200299122(Example 2; pages 528-530); WO2003029421(claim 6); WO2003024392(claim 2; FIG. 112); WO200298358 (claim 1; page 183); WO200254940 (pages 100-101); WO200259377 (pages 349-350); WO200230268 (claim 27; page 376); WO200148204 (Example; FIG. 4); NP_001194 Bone morphogenetic protein receptor, type IB / pid=NP_001194.1. Cross reference: MIM:603248; NP_001194.1; AY065994

[0170] (2) E16 (LAT1, SLC7A5, Genbank accession number NM_003486) Biochem. Biophys. Res. Commun. 255 (2), 283-288 (1999), Nature 395 (6699): 288-291 (1998), Gaugitsch, HW, 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 IV); 19; pages 127-129); WO200286443 (claim 27; 222, 393 pages); WO2003003906 (claim 10; 293 pages); WO200264798 (claim 33; pages 93-95); WO200014228 (claim 5; pages 133-136); US2003224454 (Figure 3); WO2003025138 (claim 12; page 150); NP_003477 solute carrier family 7 (solute Carrier family 7) (cationic amino acid transporter, y+ system), member 5 / pid = NP_003477.3 – human; Cross Ref: MIM:600182; NP_003477.3; NM_015923; NM_003486_1

[0171] (3) STEAP1 (six transmembrane epithelial antigen of prostate, Genbank accession number NM_012449); Cancer Res. 61(15), 5857-5860 (2001), Hubert, RS, et al. (1999) Proc. Natl. Acad. Sci. USA 96(25): 14523-14528); WO2004065577 (claim 6); WO2004027049 (Figure 1L); EP1394274 (Example 11); WO20040162 25 (claim 2); WO2003042661 (claim 12); US2003157089 (Example 5); US2003185830 (Example 5); US2003064397 (Fig. 2); WO200289747 (Example 5; pages 618-619); WO2003022995 (Example 9; Fig. 13A, Example 53; page 173, Example 2; Figure 2A );NP_036581 Six-transmembrane epithelial antigen of the prostate;Cross reference:MIM:604415;NP_036581.1;NM_012449_1

[0172] (4) 0772P (CA125, MUC16, Genbank accession number AF361486); J. Biol. Chem. 276 (29): 27371-27375 (2001); WO2004045553 (claim 14); WO200292836 (claim 6; FIG. 12); WO200283866 (claim 15; pages 116-121); US2003124140 (Example 16); Cross-reference P: GI: 34501467; AAK74120.3; AF361486_1

[0173] (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession number NM_005823) Yamaguchi, N., et al. Biol. Chem. 269 (2), 805-808 (1994), Proc. Natl. Acad. Sci. USA 96 (20): 11531-11536 (1999), Proc. Natl. Acad. Sci. USA 93 (1): 136-140 (1996), J. Biol. Chem. 270(37):21984-21990(1995)); WO2003101283(claim 14); (WO2002102235(claim 13; pages 287-288); WO2002101075(claim 4; pages 308-309); WO200271928(pages 320-321); WO9410312(pages 52-57); cross reference: MIM:601051; NP_005814.2; NM_005823_1

[0174] (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession number 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); WO2004022 778 (claim 2); EP1394274 (Example 11); WO2002102235 (claim 13; page 326); EP875569 (claim 1; pages 17-19); WO200157188 ​​(claim 20; pages 329); WO2004032842 (Example IV); WO200175177 (claim 24; pages 139-140); Cross reference: MIM:604217; NP_006415.1; NM_006424_1

[0175] (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (Semaphorin 5B, Genbank accession number AB040878); Nagase T., et al. (2000) DNA Res.7(2):143-150); WO2004000997 (claim 1); WO2003003984 (claim 1); WO200206339 (claim 1; 50 pages); WO200188133 (claim 1; pages 41-43, 48-58); WO2003054152 (claim 20); WO2003101400 (claim 11); accession number: Q9P283; EMBL; AB040878; BAA95969.1.Genew; HGNC:10737

[0176] (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession number 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 reference: GI:37182378; AAQ88991.1; AY358628_1

[0177] (9) ETBR (Endothelin type B receptor, Genbank accession number 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 NA, et al. J. Biol. Chem. 268, 3873-3879, 1993; Haendler B., et al. J. Cardiovasc. Pharmacol. 20, s1-S4, 1992; Tsutsumi M., et al. Gene 228, 43-49, 1999; Strausberg RL, et al. Proc. Natl. Acad. Sci. USA 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 JB, et al. Am. J. Med. Genet. 108, 223-225, 2002; Hofstra RMW, et al. Eur. J. Hum. Genet. 5, 180-185, 1997; Puffenberger EG, 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 RMW, et al. Nat. Genet. 12, 445-447, 1996; Svensson PJ, 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; WO2004045516 (claim 1); WO2004048938 (Example 2); WO2004040000 (claim 151); WO2003087768 (claim 1); WO2003016475 (claim 1); WO2003016475 (claim 1); WO200261087 (Figure 1); WO2003016494 (. Figure 6 ); WO2003025138 (claim 12; page 144); WO200198351 (claim 1; pages 124-125); EP522868 (claim 8; FIG2); WO200177172 (claim 1; pages 297-299); US2003109676; US6518404 (FIG3); US5773223 (claim 1a; Col31-34); WO2004001004

[0178] (10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession number NM_017763); WO2003104275 (claim 1); WO2004046342 (Example 2); WO2003042661 (claim 12); WO2003083074 (claim 14; page 61); WO2003018621 (claim 1); WO2003024392 (claim 2; Figure 93); WO200166689 (Example 6); Cross reference: Locus ID: 54894; NP_060233.2; NM_017763_1

[0179] (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-associated gene 1, prostate cancer-associated protein 1, six-transmembrane epithelial cell antigen of the prostate 2, six-transmembrane prostate protein, Genbank accession number AF455138); Lab. Invest. 82(11):1573-1582 (2002)); WO2003087306; US2003064397 (claim 1; Figure 1); WO200272596 (claim 13; pages 54-55); WO200172962 (claim 1; Figure 4B); WO2003104270 (claim 11); WO2003104270 (claim 16); US2004005598 (claim 22); WO2003042661 (claim 12); US2003060612 (claim 12; FIG. 10); WO200226822 (claim 23; FIG. 2); WO200216429 (claim 12; FIG. 10); Cross-references: GI:22655488; AAN04080.1; AF455138_1

[0180] (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession number NM_017636); Xu, XZ, et al. Proc. Natl. Acad. Sci. USA 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; pages 100-103); WO200210382 (claim 1; pages 101-103); Fig.9A ); WO2003042661 (claim 12); WO200230268 (claim 27; 391 pages); US2003219806 (claim 4); WO200162794 (claim 14; Figure 1A -D); Cross reference: MIM:606936; NP_060106.2; NM_017636_1

[0181] (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, Genbank accession number NP_003203 or 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); WO2003083041(Example 1); WO2003034984(claim 12); WO200288170(claim 2; pages 52-53); WO2003024392(claim 2; Figure 58); WO200216413(claim 1; pages 94-95, 105); WO200222808(claim 2; Figure 1); US5854399(Example 2; Col 17-18); US5792616 (Figure 2); Cross reference: MIM:187395; NP_003203.1; NM_003212_1

[0182] (14) CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein Barr virus receptor) or Hs.73792 Genbank accession number M26004); Fujisaku et al. (1989) J. Biol. Chem. 264 (4): 2118-2125); Weis JJ, et al. J. Exp. Med. 167, 1047-1066, 1988; Moore M., et al. Proc. Natl. Acad. Sci. USA 84, 9194-9198, 1987; Barel M., et al. Mol. Immunol. 35, 1025-1031, 1998; Weis JJ, et al. Proc. Natl. Acad. Sci. USA 83, 5639-5643, 1986; Sinha SK, et al. (1993) J. Immunol. 150, 5311-5320; WO2004045520 (Example 4); US2004005538 (Example 1); WO2003062401 (Claim 9); WO2004045520 (Example 4); WO9102536 (Fig. 9.1-9.9); WO2004020595 (Claim 1); Registration No.: P20023; Q13866; Q14212; EMBL; M26004; AAA35786.1

[0183] (15) CD79b (CD79B, CD79β, IGb (immunoglobulin-associated beta), B29, Genbank accession number NM_000626 or 11038674); Proc. Natl. Acad. Sci. USA (2003) 100(7): 4126-4131, Blood (2002) 100(9): 3068-3076, Muller et al. (1992) Eur. J. .Immunol.22(6):1621-1625); WO2004016225 (claim 2, FIG. 140); WO2003087768, US2004101874 (claim 1, page 102); WO2003062401 (claim 9); WO200278524 (Example 2); US2002150573 (claim 5, page 15); US5644033; WO2003048202 (claim 1, pages 306 and 309); WO 99 / 558658, US6534482 (claim 13, FIG. 17A / B); WO200055351 (claim 11, pages 1145-1146); cross reference: MIM:147245; NP_000617.1; NM_000626_1

[0184] (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2domain containing phosphatase anchor protein 1a), SPAP1B, SPAP1C, Genbank accession number NM_030764, AY358130); Genome Res. 13(10): 2265-2270 (2003), Immunogenetics 54(2):87-95(2002), Blood99(8):2662-2669(2002), Proc.Natl.Acad.Sci.USA98(17):9772-9777(2001), Xu, MJ, et al.(2001) Biochem.Biophys.Res.Commun.280(3):768-775; WO2004016225(claim 2); WO2003077836; WO200138490(claim 5; Figures 18D-1-18D-2); WO2003097803(claim 12); WO2003089624(claim 25); Cross reference: MIM:606509; NP_110391.2; NM_030764_1

[0185] (17) HER2 (ErbB2, Genbank accession number 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. Natl. Acad. Sci. USA 82, 6497-6501, 1985; Swiercz JM, et al. J. Cell Biol. 165, 869-880, 2004; Kuhns JJ, 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 1I); WO2004009622; WO2003081210; WO2003089904 (claim 9); WO2003016475 (claim 1); US2003118592; WO2003008537 (claim 1); WO2003055439 (claim 29; Figure 1A -B); WO2003025228 (claim 37; Figure 5 C); WO200222636 (Example 13; pages 95-107); WO200212341 (Claim 68; Figure 7 ); WO200213847 (pages 71-74); WO200214503 (pages 114-117); WO200153463 (claim 2; pages 41-46); WO200141787 (page 15); WO200044899 (claim 52; Figure 7 ); WO200020579 (claim 3; Figure 2); US5869445 (claim 3; Col 31-38); WO9630514 (claim 2; pages 56-61); EP1439393 (claim 7); WO2004043361 (claim 7); WO2004022709; WO200100244 (Example 3; Figure 4); Registration No.: P04626; EMBL; M11767; AAA35808.1.EMBL; M11761; AAA35808.1

[0186] (18) NCA (CEACAM6, Genbank accession number M18728); Barnett T., et al. Genomics 3, 59-66, 1988; Tawaragi Y., et al. Biochem. Biophys. Res. Commun. 150, 89-96, 1988; Strausberg RL, et al. Proc. Natl. Acad. Sci. USA 99:16899-16903, 2002; WO2004063709; EP1439393 (claim 7); WO2004044178 (Example 4); WO2004031238; WO2003042661 (claim 12); WO200278524 (Example 2); WO200286443 (claim 27; page 427); WO200260317 (claim 2); Accession No.: P40199; Q14920; EMBL; M29541; AAA59915.1.EMBL; M18728

[0187] (19) MDP (DPEP1, Genbank accession number BC017023); Proc. Natl. Acad. Sci. USA 99 (26): 16899-16903 (2002)); WO2003016475 (claim 1); WO200264798 (claim 33; pages 85-87); JP05003790 ( Figure 6-8 );WO9946284 (Figure 9); Cross reference: MIM:179780;AAH17023.1;BC017023_1

[0188] (20) IL20Rα (IL20Ra, ZCYTOR7, Genbank accession number AF184971); Clark HF, et al. Genome Res. 13, 2265-2270, 2003; Mungall AJ, 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 (74-75 pages); WO2003002717 (claim 2; 63 pages); WO200222153 (45-47 pages); US2002042366 (20-21 pages); WO200146261 (57-59 pages); WO200146232 (63-65 pages); WO9837193 (claim 1; 55-59 pages); registration numbers: Q9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971; AAF01320.1

[0189] (21) Brevican (BCAN, BEHAB, Genbank accession number AF229053); Gary SC, et al. Gene 256, 139-147, 2000; Clark HF, et al. Genome Res. 13, 2265-2270, 2003; Strausberg RL, et al. Proc. Natl. Acad. Sci. USA 99, 16899-16903, 2002; US2003186372 (claim 11); US2003186373 (claim 11); US2003119131 (claim 1; Figure 52); US2003119122 (claim 1; Figure 52); US2003119126 (claim 1; Figure 52); Claim 1); US2003119121 (claim 1; FIG. 52); US2003119129 (claim 1); US2003119130 (claim 1); US2003119128 (claim 1; FIG. 52); US2003119125 (claim 1); WO2003016475 (claim 1); WO200202634 (claim 1)

[0190] (22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession number NM_004442); Chan, J. and Watt, VM, Oncogene 6(6), 1057-1061 (1991) 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(pages 128-132); WO200053216(claim 1; page 42); cross reference: MIM:600997; NP_004433.2; NM_004442_1

[0191] (23) ASLG659 (B7h, Genbank accession number 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 7 b); WO200202624 (claim 13; Figure 1A-1B ); US2002034749 (claim 54; pages 45-46); WO200206317 (Example 2; pages 320-321, claim 34; pages 321-322); WO200271928 (pages 468-469); WO200202587 (Example 1; Figure 1); WO200140269 (Example 3; pages 190-192); WO200036107 (Example 2; pages 205-207); WO2004053079 (claim 12); WO2003004989 (claim 1); WO200271928 (pages 233-234, 452-453); WO 0116318

[0192] (24) PSCA (prostate stem cell precursor, Genbank accession number AJ297436); Reiter RE, et al. Proc. Natl. Acad. Sci. USA 95, 1735-1740, 1998; Gu Z., et al. Oncogene 19, 1288-1296, 2000; Biochem. Biophys. Res. Commun. (2000) 275(3): 783-788; WO2004022709; EP1394274 (Example 11); US2004018553 (Claim 17); WO2003008537 (Claim 1); WO200281646 (Claim 1; Page 1 64); WO2003003906 (claim 10; page 288); WO200140309 (Example 1; Figure 17); US2001055751 (Example 1; Figure 1b); WO200032752 (claim 18; Figure 1); WO9851805 (claim 17; page 97); WO9851824 (claim 10; page 94); WO9840403 (claim 2; Figure 1B ); Accession No.: O43653; EMBL; AF043498; AAC39607.1

[0193] (25) GEDA (Genbank accession number AY260763); AAP14954 lipoma HMGIC fusion-partner-like protein / pid=AAP14954.1-human (human); WO2003054152 (claim 20); WO2003000842 (claim 1); WO2003023013 (Example 3, claim 20); US2003194704 (claim 45); Cross reference: GI:30102449; AAP14954.1; AY260763_1

[0194] (26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3, Genbank accession number AF116456); BAFF receptor / pid=NP_443177.1-human: Thompson, JS, et al. Science 293(5537), 2108-2111 (2001); WO2004058309; WO2004011611; WO2003045422 (Example; pages 32-33); WO2003014294 (claim 35; Figure 6 B); WO2003035846 (claim 70; pages 615-616); WO200294852 (Col 136-137); WO200238766 (claim 3; page 133); WO200224909 (Example 3; Figure 3); Cross reference: MIM: 606269; NP_443177.1; NM_052945_1; AF132600

[0195] (27) CD22 (B-cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814, Genbank accession number AK026467); Wilson et al. (1991) J. Exp. Med. 173:137-146; WO2003072036 (claim 1; FIG1 ); cross references: MIM:107266; ​​NP_001762.1; NM_001771_1

[0196] (28) CD79a (CD79A, CD79α, immunoglobulin-related α, a B cell-specific protein that covalently interacts with Igβ (CD79B) and forms a complex with Ig M molecules on the surface, transducing signals involved in B-cell differentiation), pI: 4.84, MW: 25028TM: 2 [P] Gene Chromosome: 19q13.2, Genbank accession number NP_001774.10); WO2003088808, US20030228319; WO2003062401 (claim 9); US2002150573 (claim 4, pages 13-14); WO9958658 (claim 13, 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

[0197] (29) CXCR5 (Burkitt's lymphoma receptor 1), a G protein-coupled receptor activated by the CXCL13 chemokine, plays a role in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and may play a role in AIDS, lymphoma, melanoma and leukemia development); 372aa, pI: 8.54MW: 41959TM: 7[P]Gene Chromosome:11q23.3, Genbank accession number 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); WO9928468 (claim 1, page 38); US5440021 (Example 2, col 49-52); WO9428931 (pages 56-58); WO9217497 (claim 7, Figure 5 ); Dobner et al. (1992) Eur. J. Immunol. 22: 2795-2799; Barella et al. (1995) Biochem. J. 309: 773-779

[0198] (30) HLA-DOB (β subunit of MHC class II molecule (Ia antigen), which binds to peptides and presents them to CD4+ T lymphocytes); 273aa, pI: 6.56, MW: 30820. TM: 1 [P] Gene Chromosome: 6p21.3, Genbank accession number NP_002111.1); Tonnelle et al. (1985) EMBO J. 4(11): 2839-2847; Jonsson et al. (1989) Immunogenetics 29(6): 411-413; Beck et al. (1992) J. Mol. Biol. 228: 433-441; Strausberg et al. (2002) Proc. Natl. 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; WO9958658 (claim 13, FIG. 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

[0199] (31) P2X5 (purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, and its defects may contribute to the pathophysiology of idiopathic detrusor instability); 422aa), pI: 7.63, MW: 47206TM: 1 [P] Gene Chromosome: 17p13.3, Genbank accession number NP_002552.2); Le et al. (1997) FEBS Lett. 418 (1-2): 195-199; WO2004047749; WO2003072035 (claim 10); Touchman et al. (2000) Genome Res.10:165-173; WO200222660 (claim 20); WO2003093444 (claim 1); WO2003087768 (claim 1); WO2003029277 (82 pages);

[0200] (32) CD72 (B-cell differentiation antigen CD72, Lyb-2; 359aa), pI: 8.66, MW: 40225TM: 1 [P] GeneChromosome: 9p13.3, Genbank accession number NP_001773.1); WO2004042346 (claim 65); WO2003026493 (pp. 51-52, 57-58); WO200075655 (pp. 105-106); Von Hoegen et al. (1990) J. Immunol. 144(12): 4870-4877; Strausberg et al. (2002) Proc. Natl. Acad. Sci USA 99: 16899-16903

[0201] (33) LY64 (lymphocyte antigen 64 (RP105), a type I memberane protein of the leucine rich repeat (LRR) family, regulates B-cell activation and programmed cell death, and its functional loss is associated with increased disease activity in patients with systemic lupus erythematosus); 661aa, pI: 6.20, MW: 74147TM: 1 [P] Gene Chromosome: 5q12, Genbank accession number 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; WO9744452 (claim 8, pages 57-61); WO200012130 (pages 24-26)

[0202] (34) FcRH1 (Fc receptor-like protein 1, a putative receptor for the Fc domain of immunoglobulins containing a C2-type Ig-like domain and an ITAM domain, which may play a role in B-lymphocyte differentiation); 429aa, pI: 5.28, MW: 46925TM: 1 [P] Gene Chromosome: 1q21-1q22, Genbank accession number NP_443170.1); WO2003077836; WO200138490 (claim 6, Figures 18E-1-18-E-2); Davis et al. (2001) Proc. Natl. Acad. Sci USA 98(17):9772-9777; WO2003089624 (claim 8); EP1347046 (claim 1); WO2003089624 (claim 7)

[0203] (35) IRTA2 (immunoglobulin superfamily receptor translocation-associated 2, a putative immune receptor with a possible role in B-cell development and lymphomagenesis; gene dysregulation due to translocation occurs in certain B-cell malignancies); 977aa, pI: 6.88, MW: 106468, TM: 1 [P] Gene Chromosome: 1q21, Genbank accession number 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, Figures 18B-1-18B-2)

[0204] (36) TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembrane proteoglycan related to EGF / heregulin family growth factors and follistatin); 374aa, NCBI accession number: AAD55776, AAF91397, AAG49451, NCBI RefSeq: NP_057276; NCBI gene: 23671; OMIM: 605734; SwissProt Q9UIK5; Genbank accession number AF179274; AY358907, CAF85723, CQ782436; WO2004074320; JP2004113151; WO2003042661; WO2003009814; EP1295944 (pages 69-70); WO200230268 (page 329); WO200190304; US2004249130; US2004 022727; WO2004063355; US2004197325; US2003232350; US2004005563; US2003124579; Horie et al. (2000) Ge nomics67:146-152; 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.

[0205] The parent antibody may also be a fusion protein comprising an albumin-binding peptide (ABP) sequence (Dennis et al. (2002) "Albumin Binding As A General Strategy For Improving The Pharmacokinetics Of Proteins" J Biol Chem. 277:35035-35043; WO 01 / 45746). The antibodies of the present invention include fusion proteins having the ABP sequences taught in the following documents: (i) Dennis et al. (2002) J Biol Chem. 277:35035-35043, Tables III and IV, page 35038; (ii) US20040001827, at

[0076] ; and (iii) WO 01 / 45746, at pages 12-13, and all of these documents are incorporated herein by reference.

[0206] Mutagenesis

[0207] DNA encoding amino acid sequence variants of the starting polypeptide can be 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 the earlier prepared DNA encoding the polypeptide. Variants of recombinant antibodies can also be constructed by restriction fragment manipulation or by overlap extension PCR using synthetic oligonucleotides. Mutagenic primers encode cysteine ​​codon substitutions. Standard mutagenesis techniques can be used to generate DNA encoding antibodies with cysteine ​​modifications of such mutations. General guidelines can be found in the following literature: 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.

[0208] Site-directed mutagenesis is a method for preparing substitution variants, i.e., mutant proteins. This technology is well known in the art (e.g., see Carter (1985) Nucleic Acids Res. 13: 4431-4443; Ho et al. (1989) Gene (Amst.) 77: 51-59; and Kunkel et al. (1987) Proc. Natl. Acad. Sci. USA 82: 488). In short, in the process of performing DNA site-directed mutagenesis, the starting DNA is changed by first hybridizing an oligonucleotide encoding the desired mutation with a single strand of such starting DNA. After hybridization, a DNA polymerase is used to synthesize a complete second strand using the hybridized oligonucleotide as a primer and the starting DNA single strand as a template. Therefore, the oligonucleotide encoding the desired mutation is incorporated into the resulting double-stranded DNA. Site-directed mutagenesis can be performed in a gene expressing a pre-mutagenized protein in an expression plasmid, and the resulting plasmid can be sequenced to confirm that the desired cysteine ​​substitution mutation has been introduced (Liu et al. (1998) J. Biol. Chem. 273: 20252-20260). Site-directed protocols and methods, including those commercially available, such as Multi Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA).

[0209] PCR mutagenesis is also suitable for preparing amino acid sequence variants of the starting polypeptide. See Higuchi, (1990): 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. In short, when a small amount of template DNA is used as a starting material in PCR, primers that are slightly different in sequence from the corresponding region in the template DNA can be used to generate relatively large amounts of specific DNA fragments that differ from the template sequence only at the position where the primer differs from the template.

[0210] Another method for preparing variants, i.e., cassette mutagenesis, is based on the technology described in Wells et al. (1985) Gene 34:315-323. The starting material is a plasmid (or other vector) containing a premutated starting polypeptide DNA. The codons in the premutated starting DNA are identified. Unique restriction endonuclease sites must be present on each side of the identified mutation site. If such restriction sites do not exist, the above-mentioned oligonucleotide-mediated mutagenesis method can be used to generate them so that they are introduced into the appropriate position of the starting polypeptide DNA. The plasmid DNA is cut at these sites to linearize it. Double-stranded oligonucleotides encoding the DNA sequence between the restriction sites but containing the desired mutation are synthesized using standard operating procedures. The two chains of the oligonucleotides synthesized separately are then hybridized to each other using standard techniques. Oligonucleotides are prepared by phosphoramidite synthesis method (US 4415732; US 4458066; Beaucage, S. and Iyer, R. (1992) "Advances in the synthesis of oligonucleotides by the phosphoramidite approach", Tetrahedron 48: 2223-2311). This double-stranded oligonucleotide is called a cassette. This cassette is designed to have 5' and 3' ends compatible with the ends of the linearized plasmid so that it can be directly connected to the plasmid. This plasmid currently contains a mutated DNA sequence. The mutant DNA containing the encoded cysteine ​​substitute can be confirmed by DNA sequencing.

[0211] Single mutations were also generated by oligonucleotide-directed mutagenesis using double-stranded plasmid DNA mutagenized by PCR as a template (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) Nucl. Acids Res. 10:6487-6500).

[0212] In the present invention, hu4D5Fabv8 displayed on M13 phage (Gerstner et al. (2002) "Sequence Plasticity In The Antigen-Binding Site Of A Therapeutic Anti-HER2 Antibody", J Mol Biol. 321: 851-62) was used as a model system for experiments. Cysteine ​​mutations were introduced into hu4D5Fabv8-phage, hu4D5Fabv8 and ABP-hu4D5Fabv8 constructs. hu4D5-ThioFab-phage preparation was performed using polyethylene glycol (PEG) precipitation as described above (Lowman, Henry B. (1998) Methods in Molecular Biology (Totowa, New Jersey) 87 (Combinatorial peptide Library Protocols) 249-264).

[0213] PHESELECTOR assay

[0214] The PHESELECTOR (Phage ELISA for Selection of Reactive Thiols) assay enables detection of reactive cysteine ​​groups in antibodies in an ELISA phage format. The process of coating the protein of interest (e.g., antibody) on the well surface, followed by incubation with phagemid and then detection of HRP-labeled secondary antibodies using absorbance is described in detail in Example 2. Mutant proteins displayed on phage can be screened in a rapid, powerful, and high-throughput manner. Libraries of cysteine-engineered antibodies can be produced and screened for binding using the same means used to identify appropriate reactive sites for free Cys incorporation from random protein-phage libraries of antibodies or other proteins. This technique involves reacting cysteine ​​mutants displayed on phage with an affinity reagent or reporter group that is also thiol-reactive. Figure 8The PHESELECTOR assay is illustrated by schematics depicting Fab or ThioFab binding to HER2 (top) and biotinylated ThioFab to streptavidin (bottom).

[0215] Protein expression and purification

[0216] DNA encoding cysteine ​​engineered antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding murine antibody heavy and light chains). Hybridoma cells are used as a source of such DNA. Once isolated, the DNA can be placed into 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 that do not additionally produce antibody protein, such as myeloma cells (US 5807715; US 2005 / 0048572; US 2004 / 0229310), to obtain monoclonal antibodies synthesized in recombinant hosts. The yield of hu4D5Fabv8 cysteine ​​engineered antibodies is similar to that of wild-type hu4D5Fabv8. Review articles on recombinant expression of antibody-encoding DNA in bacteria include Skerra et al. (1993) Curr. Opinion in Immunol. 5:256-262 and Plückthun (1992) Immunol. Revs. 130:151-188.

[0217] After design and selection, cysteine-engineered antibodies, such as ThioFabs, with highly reactive unpaired Cys residues can be produced by: (i) expression in bacteria, such as E. coli systems or mammalian cell culture systems (WO 01 / 00245), such as Chinese hamster ovary cells (CHO); and (ii) purification using commonly used protein purification techniques (Lowman et al. (1991) J. Biol. Chem. 266(17): 10982-10988).

[0218] ThioFab is expressed upon induction in 34B8, a non-repressive E. coli strain (Baca et al. (1997) Journal Biological Chemistry 272(16):10678-84). See Example 3a. The collected cell pellet was resuspended in PBS (phosphate buffered saline), total cell lysis was performed by passing through a microfluidizer and the protein GSEPHAROSE was used. TMThioFab was purified by affinity chromatography using Amersham. ThioFab was coupled to biotin-PEO-maleimide as described above and purified by Superdex-200. TM Biotinylated-ThioFab was further purified by gel filtration chromatography (Amersham) to eliminate free biotin-PEO-maleimide and oligomerized fractions of ThioFab.

[0219] Mass spectrometry analysis

[0220] The accurate molecular weight of the biotin-coupled Fab was determined using liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) analysis (Cole, RB Electro Spray Ionization Mass Spectrometry: Fundamentals, Instrumentation and Applications. (1997) Wiley, New York). The amino acid sequence of biotinylated hu4D5Fabv8 (A121C) was determined by trypsin digestion followed by LC-ESI-tandem MS analysis (Table 4, Example 3b).

[0221] The antibody Fab fragment hu4D5Fabv8 contains about 445 amino acid residues, including 10 Cys residues (5 on the light chain and 5 on the heavy chain). The high-resolution structure of the humanized 4D5 variable fragment (Fv4D5) has been established, see: Eigenbrot et al. "X-Ray Structures Of The Antigen-Binding Domains From Three Variant Of Humanized Anti-P185her2Antibody 4D5 and Comparison With Molecular Modeling" (1993) J Mol Biol. 229: 969-995). All Cys residues exist in the form of disulfide bonds, so these residues do not have any reactive thiol groups (unless treated with a reducing agent) for drug-maleimide coupling. Thus, the newly engineered Cys residue can remain unpaired and can react with, ie, be conjugated to, an electrophilic linker reagent or a drug-linker intermediate, such as a drug-maleimide. Figure 1AA three-dimensional schematic diagram of the hu4D5Fabv8 antibody fragment derived from X-ray crystal coordinates. The structural positions of the modified Cys residues of the heavy and light chains are numbered according to a sequential numbering system. This sequential numbering system is related to the Kabat numbering system (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD), which is used in Figure 1B 4d5v7fabH variant of trastuzumab, the figure shows the sequential numbering scheme starting from the N-terminus (upper row), which is different from the Kabat numbering scheme (lower row) in that a, b, c are marked. Using the Kabat numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the shortening or insertion of the FR or CDR of the variable domain. The heavy chain variant sites of cysteine ​​engineering are identified by the sequential numbering and Kabat numbering scheme in the following table:

[0222] 4D5Fab heavy chain variant Sequential Number Kabat Number A40C Ala-40 Ala-40 A88C Ala-88 Ala-84 S119C Ser-119 Ser-112 S120C Ser-120 Ser-113 A121C Ala-121 Ala-114 S122C Ser-122 Ser-115 A175C Ala-175 Ala-168

[0223] Compared with Fab protein, M13 phagemid-Cys mutant Fab ( Figure 3A and Figure 3B ). HER2 and streptavidin can be coated on ELISA plates, respectively, and then as described in Example 2 and Figure 8 Phagemid-ThioFabs bound to antigen and streptavidin were tested by probing with anti-Fab-HRP (horseradish peroxidase) as depicted in Figure 2. This method enables simultaneous monitoring of the effects on antigen binding and thiol reactivity with engineered Cys residues / conjugated biotin molecules. Furthermore, this method can be applied to screen for reactive thiols of any protein displayed on M13 phage. Conjugated or unconjugated phagemid-ThioFabs were purified by simple PEG precipitation.

[0224] The antigen-binding fragment of humanized 4D5 (hu4D5Fab) is fully expressed in E. coli and has been displayed on phage (Garrard et al. (1993) Gene 128: 103-109). Antibody Fab fragment hu4D5Fabv8 was displayed on M13 phage as a model system in an ELISA-based assay to probe thiol reactivity. Figure 8Schematic diagram of PHESELECTOR assay, which depicts the combination of biotinylated ThioFab phage and anti-phage HRP antibody to HER2 (upper) and streptavidin (lower). Five amino acid residues (L-Ala43, H-Ala40, H-Ser119, H-Ala121 and H-Ser122) were initially selected from the crystal structure information as being farther from the antigen binding surface (Eigenbrot et al. (1993) J Mol Biol. 229: 969-995). The protein database X-ray crystal structure was named 1FVC. Cys residues were transformed at these positions by site-directed mutagenesis. ThioFab-phage preparations were separated and reacted with biotinylation reagents.

[0225] Using the ELISA-based PHESELECTOR assay ( Figure 8 , Example 2) HER2 and streptavidin binding of biotin-conjugated and unconjugated variants were tested with HRP (horseradish peroxidase)-conjugated anti-phage antibodies. The un-biotinylated phage-hu4D5Fabv8 ( Figure 2A ) and biotinylated phage-hu4D5Fabv8 ( Figure 2B ) with BSA (open bars), HER2 (grey bars) or streptavidin (solid bars). The absorbance due to turnover of the chromogenic substrate was measured at 450 nm. The reactivity of ThioFab with HER2 determined antigen binding. The reactivity of ThioFab with streptavidin determined the degree of biotinylation. The reactivity of ThioFab with BSA was a negative control for nonspecific interactions. As in Figure 2A As observed in Figure 2, all ThioFab-phage variants had similar binding to HER2 as wild-type hu4D5Fabv8-phage. In addition, conjugation to biotin did not interfere with ThioFab binding to HER2 ( Figure 2B ).

[0226] Surprisingly and unexpectedly, the ThioFab-phage samples exhibited variable levels of streptavidin binding activity. Of all the phage-ThioFabs tested, the A121C cysteine ​​engineered antibody exhibited the greatest thiol reactivity. Although wild-type hu4D5Fabv8-phage were incubated with the same amount of biotin-maleimide, these phages showed little streptavidin binding, indicating that pre-existing cysteine ​​residues from hu4D5Fabv8 and M13 phage envelope proteins (involved in disulfide bond formation) do not interfere with site-specific coupling of biotin-maleimide. These results indicate that the phage ELISA assay can be successfully used to screen for reactive thiols on the surface of Fabs.

[0227] The PHESELECTOR assay is able to screen for reactive thiols in antibodies. The identification of the A121C variant by this method is typical. The intact Fab molecule can be effectively studied to identify more ThioFab variants with reactive thiols. The parameter, fractional surface accessibility, is used to identify and quantify the accessibility of the solvent to the amino acid residues in the polypeptide. Surface accessibility is expressed as the surface area that can be contacted by solvent molecules, such as water. The space occupied by water is approximately The software is freely available or licensed (Secretary to CCP4, Daresbury Laboratory, Warrington, WA4 4AD, United Kingdom, Fax: (+44) 1925 603825, or via the Internet: www.ccp4.ac.uk / dist / html / INDEX.html), such as the crystallographic program CCP4 Suite ("The CCP4 Suite: Programs for Protein Crystallography" (1994) Acta. Cryst. D50:760-763) which uses an algorithm to calculate the surface accessibility of the amino acids of each protein with known X-ray crystallography-derived coordinates. Two typical software modules that perform surface accessibility calculations are "AREAIMOL" and "SURFACE", which are based on the algorithm 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 position of the center of a probe sphere (representing a solvent molecule) when it is flipped on the Van der Waals surface of the protein. AREAIMOL calculates the solvent accessible surface area by creating surface points on an extended sphere for each atom (at a distance from the center of the atom equal to the sum of the atom and probe radii) and eliminating those points that belong to equivalent spheres bound to neighboring atoms. AREAIMOL determines the solvent accessible areas of atoms in the PDB coordinate files and summarizes the accessible areas for residues, chains and complete molecules. The accessible areas (or area differences) for each atom can be stored as a hypothetical-PDB output file. AREAIMOL infers a single radius for each component and recognizes only a limited number of different components. Unknown atom types (i.e. those not in the AREAIMOL internal database) are assigned as The default radius of . The list of recognized atoms is:

[0228]

[0229] AREAIMOL and SURFACE report the absolute accessibility, i.e., square angstroms. The surface accessible fraction is calculated by comparing the standard state of the relevant amino acids in the reference polypeptide. The reference state is the tripeptide Gly-X-Gly, where X is the amino acid of interest, and the reference state should be an 'extended' conformation, i.e., such as those in β-strands. The extended conformation maximizes the accessibility X. The calculated accessible area is divided by the accessible area in the reference state of the Gly-X-Gly tripeptide and the quotient is reported, which is the accessible fraction. The accessibility percentage is the accessible fraction multiplied by 100.

[0230] Another typical algorithm for calculating surface accessibility is based on the SOLV module of the program xsae (Broger, C., F. Hoffman-LaRoche, Basel), which calculates the accessible scores of amino acid residues to the water sphere based on the X-ray coordinates of the polypeptide.

[0231] The surface accessibility score for each amino acid in hu4D5Fabv7 was calculated using crystal structure information (Eigenbrot et al. (1993) J Mol Biol. 229:969-995; US 7521541). The following two criteria were applied to identify residues of hu4D5Fabv8 that could be engineered to be replaced with Cys residues:

[0232] 1. Eliminate completely masked amino acid residues, i.e., less than 10% surface accessible fraction. There are 134 (light chain) and 151 (heavy chain) residues in hu4D5Fabv8 with greater than 10% accessibility (surface accessible fraction). The top 10 most accessible Ser, Ala, and Val residues were selected because the other amino acids are more similar in structure to Cys, thus introducing only minimal structural constraints into the antibody by the newly engineered Cys. Other cysteine ​​substitution sites can also be screened and used for conjugation.

[0233] 2. Sorting residues based on their role in the functional and structural interactions of the Fab. Further selection of residues that are not involved in antigen interactions and are far from existing disulfide bonds. The newly engineered Cys residues should not be involved (distinct from) and interfere with antigen binding, nor mispair with cysteines involved in disulfide bond formation.

[0234] Thiol reactivity can be generalized to any antibody in which amino acid replacement with a reactive cysteine ​​amino acid is performed within a light chain selected from the group consisting of: L-10 - L-20; L-38 - L-48; L-105 - L-115; L-139 - L-149; L-163 - L-173; and within a heavy chain selected from the group consisting of: H-35 - H-45; H-83 - H-93; H-114 - H-127; and H-170 - H-184; and within an Fc region selected from the group consisting of: H-268 - H-291; H-319 - H-344; H-370 - H-380; and H-395 - H-405.

[0235] Thiol reactivity can be generalized to certain domains of the antibody, such as the light chain constant domain (CL) and the heavy chain constant domains CH1, CH2 and CH3. Cysteine ​​substitutions resulting in thiol reactivity values ​​of about 0.8 and above 0.8 can be made in the heavy chain constant domains α, δ, ε, γ and μ of the following intact antibodies: IgA, IgD, IgE, IgG and IgM, respectively, including the IgG subclasses: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

[0236] It is clear from the crystal structure data that the 10 selected Cys mutants are away from the antigen-binding site, such as the interface with HER2 in this case. The indirect effects of these mutants on functional interactions can be tested experimentally. The thiol reactivity of all Cys Fab variants was determined and calculated as described in Examples 1 and 2 and listed in Table 1. Residues L-V15C, L-V110C, H-A88C and H-A121C have reactive and stable thiols ( Figure 3A and Figure 3B ). Mutants V15C, V110C, A144C, S168C are light chain Cys variants. Mutants A88C, A121C, A175C, S179C are heavy chain Cys variants. Surprisingly and unexpectedly, sites with high surface accessible scores do not have the highest thiol reactivity calculated by the PHESELECTOR assay (Table 1). In other words, the surface accessible score ( Figure 1A ) is not correlated with thiol reactivity (Table 1). In fact, Cys residues engineered at sites with moderate surface accessibility of 20% to 80% ( Figure 4A ) or partially exposed sites, such as Ala or Val residues, showed better thiol reactivity than Cys introduced on Ser residues, i.e., >0.6 ( Figure 3B , Table 1), thus necessitating the use of the PHESELECTOR assay in the screening of thiol-reactive sites, as crystal structure information alone is insufficient to select these sites ( Figure 3B and 4A ).

[0237] Thiol reactivity data such as Figure 3A and Figure 3B The amino acid residues of 4D5 ThioFab Cys mutants: (3A) non-biotinylated (control group) and (3B) biotinylated phage-ThioFab are shown. Reactive thiols on antibodies / Fabs were identified by PHESELECTOR assay analysis of the interaction of non-biotinylated phage-hu4D5Fabv8 (3A) and biotinylated phage-hu4D5Fabv8 (3B) with BSA (open bars), HER2 (grey bars) or streptavidin (solid bars). The assay was performed as described in Example 2. The light chain variants are on the left and the heavy chain variants are on the right. The binding of the non-biotinylated 4D5 ThioFab Cys mutants was low as expected, but strong binding to HER2 was maintained. The HER2 binding ratios with streptavidin and with biotinylated 4D5 ThioFab Cys mutants gave the thiol reactivity values ​​in Table 1. Background absorbance at 450 nm or a small amount of nonspecific protein binding of biotinylated 4D5 ThioFab Cys mutant to BSA was also Figure 3BThe surface accessibility score of the selected amino acid residues replaced by Cys residues is as follows: Figure 4A As shown in . The surface accessible score was calculated based on the available hu4D5Fabv7 structure (Eigenbrot et al. (1993) J Mol Biol. 229: 969-995). The conformational parameters of the hu4D5Fabv7 and hu4D5Fabv8 structures are highly consistent and any correlation between the calculated surface accessible score of hu4D5Fabv7 and the thiol reactivity of the hu4D5Fabv8 cysteine ​​mutants can be determined. The measured thiol reactivity of the phage ThioFab Cys residues introduced on partially exposed residues (Ala or Val) has better thiol reactivity than the Cys residue thiol reactivity introduced on the Ser residue (Table 1). It can be observed from the ThioFabCys mutants from Table 1 that there is little or no correlation between the thiol reactivity values ​​and the surface accessible score.

[0238] The amino acids at positions L-15, L-43, L-110, L-144, L-168, H-40, H-88, H-119, H-121, H-122, H-175 and H-179 of the antibody can generally be mutated (replaced) with free cysteine ​​amino acids. The range of about 5 amino acid residues on each side of these positions can also be replaced by free cysteines, i.e., L-10-L-20; L-38-L-48; L-105-L-115; L-139-L-149; L-163-L-173; H-35-H-45; H-83-H-93; H-114-H-127; and H-170-H-184, as well as within the range of the Fc region selected from the following: H-268-H-291; H-319-H-344; H-370-H-380; and H-395-H-405, thereby obtaining the cysteine-modified antibodies of the present invention.

[0239] Table 1 Thiol reactivity of phage-ThioFab

[0240] Phage-ThioFab constructs <![CDATA[Thiol reactivity * > Surface accessibility fraction (%) hu4D5Fabv8-wt 0.125 - L-V15C 0.934 52.46 L-A43C 0.385 26.80 L-V110C 0.850 44.84 L-A144C 0.373 23.65 L-S168C 0.514 79.68 H-A40C 0.450 21.97 H-A88C 0.914 51.60 H-S119C 0.680 18.88 H-A121C 0.925 33.05 H-S122C 0.720 72.87 H-A175C 0.19 23.80 H-S179C 0.446 99.48

[0241] L = light chain, H = heavy chain, A = alanine, S = serine, V = valine, C = cysteine * Thiol reactivity was measured as the OD of streptavidin binding. 450nm OD bound to HER2 (antibody) 450nm A thiol reactivity value of 1 indicates complete biotinylation of cysteine ​​thiols.

[0242] Two Cys variants from the light chain (L-V15C and L-V110C) and two Cys variants from the heavy chain (H-A88C and H-A121C) were selected for further analysis because these variants showed the highest thiol reactivity (Table 1).

[0243] Unlike phage purification, Fab preparation may take 2-3 days, depending on the scale of production. During this period, thiol reactivity is lost due to oxidation. To probe the stability of thiols on hu4D5Fabv8-phage, the stability of thiol reactivity of phage-thioFab was determined ( Figure 4B ). After ThioFab-phage purification on days 1, 2, and 4, all samples were coupled with biotin-PEO-maleimide and probed with a phage ELISA assay (PHESELECTOR) to test HER2 and streptavidin binding. L-V15C, L-V110C, H-A88C, and H-A121C retained significant thiol reactivity compared to other ThioFab variants ( Figure 4B ).

[0244] Cysteine-modified antibody

[0245] The cysteine-modified antibodies of the present invention can be coupled to any label moiety, which can be covalently bound to the antibody through a reactive cysteine ​​thiol (Singh et al. (2002) Anal. Biochem. 304: 147-15; Harlow E. and Lane, D. (1999) Using Antibody: A Laboratory Manual, Cold Springs Harbor Laboratory Press, Cold Spring Harbor, NY; Lundblad R.L. (1991) Chemical reagents for Protein Modification, 2nd ed. CRC Press, Boca Raton, FL). The bound label can serve the following functions: (i) to provide a detectable label signal; (ii) to interact with a second label to change the detectable signal provided by the first or second label, for example to obtain FRET (fluorescence resonance energy transfer); (iii) to stabilize the interaction or increase the binding affinity to the antigen or ligand; (iv) to affect mobility through charge, hydrophilicity, shape or other physical parameters, such as electrophoretic mobility or cell permeability; or (v) to provide a capture moiety to modulate ligand affinity, antibody / antigen binding or ion complexation.

[0246] Labeled cysteine ​​engineered antibodies can be used in diagnostic tests, for example, to detect the expression of an antigen of interest in specific cells, tissues or serum. For diagnostic applications, the antibody is generally labeled with a detectable moiety. A large number of labels are available, which can generally be divided into the following categories:

[0247] (a) Radioactive isotopes (radionuclides), such as 3 H. 11 C. 14 C. 18 F. 32 P. 35 S. 64 Cu, 68 Ga, 86 Y. 89 Zr, 99 Tc, 111 In, 123 I. 124 I. 125 I. 131 I. 133 Xe, 177 Lu, 211 At, or 213Bi. Radioisotope-labeled antibodies are used for receptor-targeted imaging experiments. Antibodies can be labeled with ligand reagents using techniques described in Current Protocols in Immunology, (1991) Volumes 1 and 2, Coligen et al., Ed. Wiley-Interscience, New York, NY, Pubs., which bind, chelate, and even compound radioisotope metals, wherein the reagent reacts with the cysteine ​​thiol of the modified antibody. Chelated ligands that can compound metal ions include DOTA, DOTP, DOTMA, DTPA, and TETA (Macrocyclics, Dallas, TX). Radionuclides can be compounded with the antibody-drug conjugates of the present invention (Wu et al. (2005) Nature Biotechnology 23 (9): 1137-1146). DOTA-maleimide reagent reacts with free cysteine ​​amino acids of cysteine ​​engineered antibodies and yields metal complexed ligands on the antibody (Lewis et al. (1998) Bioconj. Chem. 9: 72-86). Chelating linker labeling reagents such as DOTA-NHS (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono(N-hydroxysuccinimide ester) are commercially available (Macrocyclics, Dallas, TX). Receptor targets for imaging with radionuclide-labeled antibodies can provide activation pathway markers by detecting and quantifying the progressive accumulation of antibodies in tumor tissues (Albert et al. (1998) Bioorg. Med. Chem. Lett. 8: 1207-1210).

[0248] Metal-chelate complexes suitable as antibody labels for use in imaging experiments (US 2010 / 0111856; 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) Nucl. Med. Biol. 22:387-90; Camera et al. (1993) Nucl. Med. Biol. 20:955-62; Kukis et al. (1998) J. Nucl. Med. 39:2105-2110; Verel et al. (2003) J. Nucl. Med. 44:1663-1670; Camera et al. (1994) J. Nucl. Med. 21:640-646; Ruegg et al. (1990) Cancer Res.50:4221-4226; Verel et al. (2003) J.Nucl.Med.44:1663-1670; Lee et al. (2001) Cancer Res.61:4474-4482; Mitchell et al. (2003) J.Nucl.Med.44:1105-1112; Kobayashi et al. (1999) Bioconjugate Chem.10:103-111; Miederer et al. (2004) J.Nucl.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. Nucl. Med. 40: 166-76; Kobayashi et al. (1998) J. Nucl. Med. 39: 829-36; Mardirossian et al. (1993) Nucl. Med. Biol.20:65-74; Roselli et al. (1999) Cancer Biotherapy & Radiopharmaceuticals, 14:209-20). .

[0249] (b) Fluorescent labels, such as rare earth chelates (europium chelates); fluoresceins, including FITC, 5-carboxyfluorescein, 6-carboxyfluorescein; rhodamines, including TAMRA; dansyl; Lissamine; cyanines; phycoerythrin; Texas Red; and analogs thereof. For example, fluorescent labels can be coupled to antibodies using the techniques disclosed in Current Protocols in Immunology, supra. Fluorescent dyes and fluorescent labeling agents include those commercially available from Invitrogen / Molecular Probes (Eugene, OR) and Pierce Biotechnology, Inc. (Rockford, IL).

[0250] Detection labels, such as fluorescent dyes and chemiluminescent dyes (Briggs et al. (1997) "Synthesis of Functionalised Fluorescent Dyes and Their Coupling to Amines and Amino acids," J. Chem. Soc., Perkin-Trans. 1: 1051-1058) provide detectable signals and are generally used to label antibodies, which preferably have the following properties: (i) the labeled antibody should produce a very high signal with low background, so that small amounts of antibody can be sensitively detected in cell-free and cell-based assays; and (ii) the labeled antibody should be photostable so that the fluorescent signal can be observed, monitored and recorded without significant photobleaching. For applications involving the binding of the labeled antibody to membranes or cell surfaces, especially the cell surface of living cells, the label preferably (iii) has good water solubility to obtain effective conjugate concentrations and detection sensitivity and (iv) is nontoxic to living cells so as not to disrupt the normal metabolic processes of the cells or cause premature cell death.

[0251] (c) Various enzyme-substrate labels are available or disclosed (US 4275149). Enzymes generally catalyze chemical changes in chromogenic substrates that can be measured using various techniques. For example, enzymes can catalyze a color change in a substrate, and this change can be measured by spectrophotometry. Alternatively, the enzyme can change the fluorescence or chemiluminescence of the substrate. Techniques for quantifying fluorescence changes are described above. Chemiluminescent substrates become electrically excited by a chemical reaction and can then emit light that can be measured (e.g., using a chemiluminometer) or provide energy to a fluorescent receptor. Examples of enzymatic labels include: luciferases (e.g., firefly luciferase and bacterial luciferase; US 4737456); luciferin; 2,3-diphthalazinediones; malate dehydrogenase; urease; peroxidases, such as horseradish peroxidase (HRP); alkaline phosphatase (AP); β-galactosidase; glucoamylase; lysozyme; sugar oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase); heterocyclic oxidases (such as uricase and xanthine oxidase); lactoperoxidase; microperoxidase, etc. Techniques for coupling enzymes to antibodies are described in O'Sullivan et al. (1981) "Methods for the Preparation of Enzyme-Antibody Cojugate for use in Enzyme Immunoassay": Methods in Enzym. (ed J. Langone & H. Van Vunakis), Academic Press, New York, 73: 147-166.

[0252] Examples of enzyme-substrate combinations (US 4275149; US 4318980) include, for example:

[0253] (i) horseradish peroxidase (HRP) with catalase as substrate, wherein the catalase oxidizes a dye precursor (e.g. o-phenylenediamine (OPD) or 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB));

[0254] (ii) alkaline phosphatase (AP) with p-nitrophenyl phosphate as a chromogenic substrate; and

[0255] (iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g. p-nitrophenyl-β-D-galactosidase) or a fluorogenic substrate 4-methylumbelliferyl-β-D-galactosidase.

[0256] The label can be indirectly coupled to the cysteine ​​engineered antibody. For example, the antibody can be coupled to biotin, and any of the three major types of labels described above can be coupled to avidin or streptavidin, or vice versa. Biotin selectively binds streptavidin, and thus the label can be coupled to the antibody in this indirect manner. Alternatively, to achieve indirect coupling of the label to the polypeptide variant, the polypeptide variant is coupled to a small hapten (e.g., digoxigenin) and one of the above different types of labels is coupled to an anti-hapten polypeptide variant (e.g., an anti-digoxigenin antibody). Thus, indirect coupling of the label to the polypeptide variant can be achieved (Hermanson, G. (1996) in Biocojugate Techniques Academic Press, San Diego).

[0257] The polypeptide variants of the present invention can be used in any known assay method, such as ELISA, competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Zola, (1987) Monoclonal Antibodies: A Manual of Techniques, pp. 147-158, CRC Press, Inc.).

[0258] Detection labels can be used to locate, develop and quantify binding or recognition results. Antibodies labeled in the present invention can detect cell-surface receptors. Another application for detection labeled antibodies is bead-based immunocapture, which includes coupling beads to fluorescently labeled antibodies and detecting fluorescent signals when ligands are bound. Similar binding detection methods use surface plasmon resonance (SPR) effects to measure and detect antibody-antigen interactions.

[0259] The labeled cysteine ​​engineered antibodies of the present invention are used as imaging biomarkers and probes for various methods and techniques of biomedical and molecular imaging, such as: (i) MRI (magnetic resonance imaging); (ii) MicroCT (computerized tomography); (iii) SPECT (single photon emission computed tomography); (iv) PET (positron emission tomography) Tinianow, J. et al (2010) Nuclear Medicine and Biology, 37 (3): 289-297; Chen et al (2004) Bioconjugate Chem. 15: 41-49; US2010 / 0111856; (v) bioluminescence; (vi) fluorescence; and (vii) ultrasound. Immunoscintigraphy is an imaging method in which antibodies labeled with radioactive substances are administered to animals or human patients and images of the site where the antibodies are localized are taken (US 6528624). Imaging biomarkers can be measured objectively and evaluated as indicators of normal physiological processes, pathological processes, or pharmacological responses to therapeutic interventions. Biomarkers can be of several types: Type 0 are natural history markers of disease and are longitudinally correlated with known clinical indicators, such as MRI evaluation of synovial inflammation in rheumatoid arthritis; Type I markers capture the effects of an intervention according to a mechanism of action, even if the mechanism is not related to clinical outcome; Type II markers act as surrogate endpoints, where changes or signals in the biomarker predict clinical benefit in order to "validate" a targeted response, such as bone erosion measured by CT in rheumatoid arthritis. Imaging biomarkers can thus provide pharmacodynamic (PD) treatment information regarding: (i) expression of the target protein; (ii) binding of the therapeutic agent to the target protein, i.e., selectivity; and (iii) clearance and half-life pharmacokinetic data. Advantages of in vivo imaging biomarkers relative to laboratory-based biomarkers include: non-invasive treatment; quantifiable; global evaluation; repeated dosing and evaluation, i.e., multiple time points; and potentially transferable effects from preclinical (small animal) to clinical (human) results. For some applications, bioimaging has replaced or minimized the use of animal testing in preclinical studies.

[0260] 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 Labelling: A Practical Approach, Academic Press, London; Means (1990) Biogconjugate 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, Fla.); 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.

[0261] Peptides and proteins labeled with two parts, i.e., fluorescent reporter groups and quenchers, can fully approximate fluorescence resonance energy transfer (FRET). The reporter group is generally a fluorescent dye excited by light of a certain wavelength and can transfer energy to an acceptor or quencher group, wherein there is an 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 molecule can be partially or significantly quenched by the quencher portion of the intact peptide. When the peptide is cleaved by a peptidase or protease, the detectable increase in fluorescence can be determined (Knight, C. (1995) "Fluorimetric Assays of Proteolytic Enzymes", Methods in Enzymology, Academic Press, 248: 18-34).

[0262] The antibody of the present invention labeling can also be used as affinity purification reagent. In this method, the antibody of labeling is fixed on a solid phase, such as a Sephadex resin or a filter paper, using methods well known in the art. The immobilized antibody is contacted with a sample containing a pre-purified antigen, and thereafter the support is washed with a suitable solvent that can substantially remove all materials in the sample except the pre-purified antigen, and the antigen is combined with the fixed polypeptide variant. Finally, another suitable solvent is used, such as a glycine buffer solution at pH 5.0 to wash the support, which can release the antigen from the polypeptide variant.

[0263] The labeling reagent generally has a reactive functional group that can react with: (i) the cysteine ​​thiol of the cysteine ​​modified antibody directly to form a labeled antibody; (ii) react with a linker reagent to form a linker-labeled intermediate; or (iii) react with a linker antibody to form a labeled antibody. Reactive functional groups of labeling reagents include: maleimide, haloacetyl, iodoacetamide succinimidyl ester (e.g., NHS, N-hydroxysuccinimide), isothiocyanate, sulfonyl chloride, 2,6-dichlorotriazine, pentafluorophenyl ester and phosphoramidite, however, other functional groups may also be used.

[0264] Conjugation of biotin-maleimide to ThioFab

[0265] The above ThioFab properties are established in the presence of phage, because fusions of Fab to phage envelope proteins can alter Cys thiol accessibility or reactivity. Therefore, the ThioFab construct was cloned into an expression vector under the control of an alkaline phosphatase promoter (Chang et al. (1987) Gene 55:189-196) and ThioFab expression was induced by growing E. coli cells in phosphate-free medium. Protein G SEPHAROSE was used.TM ThioFab was purified by column and analyzed using reducing and non-reducing SDS-PAGE gels. These analyses allowed the evaluation of whether ThioFab could retain its reactive thiol or be inactivated by forming intra- or intermolecular disulfide bonds. TM ThioFab L-V15C, L-V110C, H-A88C and H-A121C were expressed and purified by column chromatography (see Detailed Description of Methods). The purified proteins were analyzed using SDS-PAGE gels under reducing (with DTT) and non-reducing (without DTT) conditions. Other reducing agents, such as BME (β-thiolethanol) can be used on the gel to cleave interchain disulfide bonds. From the SDS-PAGE gel analysis it was apparent that the major (~90%) fraction of ThioFab was in monomeric form, whereas wild-type hu4D5Fabv8 was essentially in monomeric form (47 kDa).

[0266] ThioFab (A121C) and wild-type hu4D5Fabv8 were incubated with 100-fold excess of biotin-maleimide at room temperature for 3 hours and the biotinylated Fab was loaded onto Superdex-200 TM Gel filtration column. This purification step is used to separate monomeric Fab from oligomeric Fab and also from excess free biotin-maleimide (or free cytotoxic drug).

[0267] Figure 5 : The validation of ThioFab variant properties is shown in the absence of phage background. Proteins without phage fusions, hu4D5Fabv8 and hu4D5Fabv8-A121C (ThioFab-A121C) were expressed and purified using a protein-G agarose column, followed by incubation with a 100-fold molar excess of biotin-maleimide. The streptavidin and HER2 binding of biotinylated cys-engineered ThioFab and non-biotinylated wild-type Fab were compared. The degree of biotin coupling (interaction with streptavidin) and its binding ability to HER2 were monitored by ELISA analysis. Each Fab was tested at 2ng and 20ng.

[0268] Biotinylated A121C ThioFab maintained HER2 binding similar to wild-type hu4D5Fabv8 ( Figure 5). Wild-type Fab and A121C-ThioFab were purified by gel filtration column chromatography. Both samples were tested for HER2 and streptavidin binding by ELISA using goat anti-Fab-HRP as a secondary antibody. Both wild-type (open bars) and ThioFab (dashed bars) had similar binding to HER2, but only ThioFab retained streptavidin binding. Only background levels of interaction with streptavidin were observed using non-biotinylated wild-type hu4D5Fabv8 ( Figure 5 ). Mass spectrometry (LC-ESI-MS) analysis of biotinylated-ThioFab (A121C) produced a major peak with 48294.5 Daltons compared to wild-type hu4D5Fabv8 (47737 Daltons). The 537.5 Dalton difference between the two molecules corresponds to a single biotin-maleimide coupled to ThioFab. Mass spectrometry protein sequencing (LC-ESI-Tandem mass spec analysis) results further confirmed that the coupled biotin molecule was on the newly engineered Cys residue (Table 8, Example 3b).

[0269] Site-specific conjugation of biotin-maleimide to albumin binding peptide (ABP)-ThioFab

[0270] Plasma protein binding can be an effective way to improve the pharmacokinetic properties of short-lived molecules. Albumin is the most abundant protein in plasma. Serum albumin binding peptides (ABPs) can alter the pharmacodynamic properties of fused active domain proteins, including changes in tissue uptake, penetration and diffusion. These pharmacodynamic parameters can be adjusted by specifically selecting appropriate serum albumin binding peptide sequences (US 20040001827). A series of albumin binding peptides were identified by phage display screening (Dennis et al. (2002) "Albumin Binding As A General Strategy For Improving The Pharmacokinetics Of Proteins" J Biol Chem. 277: 35035-35043; WO 01 / 45746). The compounds of the present invention include ABP sequences taught by: (i) Dennis et al. (2002) J Biol Chem. 277:35035-35043, Tables III and IV, page 35038; (ii) US20040001827,

[0076] ; and (iii) WO 01 / 45746, pages 12-13, and all of these documents are incorporated herein by reference.

[0271] Albumin binding (ABP)-Fabs were engineered by fusing an albumin binding peptide to the C-terminus of the Fab heavy chain in a 1:1 (1 ABP / 1 Fab) stoichiometric ratio. Binding of these ABP-Fabs to albumin was shown to increase their half-life by more than 25-fold in rabbits and mice. The above-mentioned reactive Cys residues can thus be introduced into these ABP-Fabs and used for site-specific conjugation with cytotoxic drugs followed by in vivo animal studies.

[0272] Typical albumin binding peptide sequences include, but are not limited to, the amino acid sequences listed in SEQ ID NOs: 1-5:

[0273]

[0274] The albumin binding peptide (ABP) sequence binds to albumin from multiple species (mouse, rat, rabbit, cow, macaque, baboon and human) with a Kd (rabbit) = 0.3 μM. The albumin binding peptide does not compete with known ligands for binding to albumin and has a half-life (T1 / 2) of 2.3 hours in rabbit. BSA-SEPHAROSE was used as described in the above section TM ABP-ThioFab protein was purified, followed by biotin-maleimide coupling and purification using Superdex-S200 column chromatography. The purified biotinylated protein was homogeneous and free of any oligomeric forms (Example 4).

[0275] Figure 6 Figure 5 shows the properties of albumin binding peptide (ABP)-ThioFab variants. ELISA analysis was performed to test the binding ability of ABP-hu4D5Fabv8-wt, ABP-hu4D5Fabv8-V110C and ABP-hu4D5Fabv8-A121C to rabbit albumin, streptavidin and HER2. Biotinylated ABP-ThioFab was able to bind to albumin and HER2 with similar affinity as wild-type ABP-hu4D5Fabv8, as shown by ELISA ( Figure 6 ) and BIAcore binding kinetic analysis (Table 2). ELISA plates were coated with albumin, HER2 and SA as described. Anti-Fab HRP was used to probe the binding of biotinylated ABP-ThioFab to albumin, HER2 and SA. Compared with the non-biotinylated control ABP-hu4D5Fabv8-wt, the biotinylated ABP-ThioFab was able to bind streptavidin, indicating that ABP-ThioFab is coupled to biotin maleimide ThioFab in a site-specific manner, because the same Cys mutant is used for both variants ( Figure 6 ).

[0276] Table 2 BIAcore kinetic analysis of biotinylated ABP-hu4D5Fabv8 wild-type and ThioFab binding to HER2 and rabbit albumin

[0277]

[0278]

[0279] ABP = albumin binding peptide

[0280] Alternatively, the albumin-binding peptide may be linked to the antibody by covalent bonding via a linker moiety.

[0281] Each FAB uses two free thiol groups to transform ABP-ThioFab

[0282] The above results show that all four (L-V15C, L-V110C, H-A88C and H-A121C) thioFab (Fab antibody with cysteine ​​modification) variants have reactive thiols that can be used for site-specific coupling with labeling reagents, linker reagents or drug-linker intermediates. L-V15C can be expressed and purified, but the yield is relatively low. However, the expression and purification yields of L-V110C, H-A88C and H-A121C variants are similar to those of hu4D5Fabv8. Therefore, these mutants can be used for further analysis and recombined to have more than one thiol group in each Fab. For this purpose, a thiol on the light chain and a thiol on the heavy chain are constructed to obtain two thiols (L-V110C / H-A88C and L-V110C / H-A121C) in each Fab molecule. These two double Cys variants are expressed and purified in an E. coli expression system. The homogeneity of purified biotinylated ABP-ThioFab was found to be similar to that of the single Cys variants.

[0283] To investigate the effect of engineering two reactive Cys residues in each Fab ( Figure 7 The presence of the second biotin was tested by probing the binding of biotinylated ABP-ThioFab to SA using streptavidin-HRP ( Figure 7 ). For HER2 / Fab analysis, ELISA plates were coated with HER2 and probed with anti-Fab HRP. For SA / Fab analysis, ELISA plates were coated with SA and probed with anti-Fab HRP. For SA / SA analysis, ELISA plates were coated with SA and probed with SA-HRP. Figure 7. The interaction of biotinylated ABP-hu4D5Fabv8 cys variants with HER2 and streptavidin (SA) was analyzed by ELISA. HER2 / Fab, SA / Fab and SA / SA indicate that the interaction was monitored by anti-Fab-HRP and SA-HRP, respectively. SA / Fab monitors the presence of a single biotin in each Fab and the SA / SA assay monitors more than one biotin per Fab. The binding of HER2 to the double cys mutant was similar to that of HER2 to the single Cys variant ( Figure 7 ). However, the degree of biotinylation on the double-Cys mutant was higher than that on the single-Cys variant, due to the presence of more than one free thiol group per Fab molecule ( Figure 7 ).

[0284] Engineering of thioIgG variants of trastuzumab

[0285] Introduction of cysteine ​​into the full-length monoclonal antibody trastuzumab ( The single cys mutants H-A88C, H-A121C and L-V110C of trastuzumab and the double cys mutants V110C-A121C and V110C-A121C of trastuzumab were expressed in CHO (Chinese Hamster Ovary) cells by transient fermentation in medium containing 1 mM cysteine. The heavy chain sequence (450 aa) of the A88C mutant is SEQ ID NO: 6. The heavy chain sequence (450 aa) of the A121C mutant is SEQ ID NO: 7. The light chain sequence (214 aa) of the V110C mutant is SEQ ID NO: 8.

[0286]

[0287]

[0288] According to one embodiment, the cysteine ​​engineered thio-trastuzumab antibody comprises one or more of the following variable region heavy chain sequences (SEQ ID NOs: 9-16) having a free cysteine ​​amino acid.

[0289] Mutants 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

[0290] According to another embodiment, the cysteine ​​engineered Sulfo-Trastuzumab antibody comprises one or more of the following variable region light chain sequences (SEQ ID NOs: 17-27) having a free cysteine ​​amino acid.

[0291] Mutants sequence SEQ ID NO: V15C SLSASCGDRVT SEQ ID NO:17 A43C QKPGKCPKLLI SEQ ID NO:18 V110C EIKRTCAAPSV SEQ ID NO:19 S114C TCAAPCVFIFPP SEQ ID NO:20 S121C FIFPPCDEQLK SEQ ID NO:21 S127C DEQLKCGTASV SEQ ID NO:22 A144C FYPRECKVQWK SEQ ID NO:23 A153C WKVDNCLQSGN SEQ ID NO:24 N158C ALQSGCSQESV SEQ ID NO:25 S168C VTEQDCKDSTY SEQ ID NO:26 V205C GLSSPCTKSFN SEQ ID NO:27

[0292] The resulting full-length thio-trastuzumab IgG variants were tested for thiol reactivity and HER2 binding activity. Fig. 9A Cartoon depiction showing biotinylated antibody binding to immobilized HER2 and HRP-labeled secondary antibody for absorbance detection. Fig. 9B Binding measurements to immobilized HER2 are shown for the following (left to right) substances detected by absorbance at 450 nm: non-biotinylated wild-type trastuzumab (Wt), biotin-maleimide-coupled thio-trastuzumab variants V110C (single cys), A121C (single cys), and V110C-A121C (double cys). Each thioIgG variant and trastuzumab were tested at 1, 10, and 100 ng. The measurements indicate that the biotinylated anti-HER2 ThioMab retains HER2 binding activity.

[0293] Fig. 10A Cartoon depiction showing biotinylated antibody binding to immobilized HER2 with biotin conjugated to anti-IgG-HRP for absorbance detection. Fig. 10B Figure 2 shows the binding measurements of biotin-maleimide coupled-thio trastuzumab variants and non-biotinylated wild-type trastuzumab in bound streptavidin with absorbance detected at 450 nm. From left to right: trastuzumab, V110C (single cys), A121C (single cys), V110C / A121C (double cys). Each thioIgG trastuzumab variant and parent trastuzumab were tested at 1, 10 and 100 ng. The measurements show that the HER2 ThioMab has high thiol reactivity.

[0294] Cysteine ​​was introduced at certain residues of the full-length 2H9 anti-EphB2R antibody. The single cys mutant H-A121C of 2H9 was expressed in CHO (Chinese Hamster Ovary) cells by transient fermentation in medium containing 1 mM cysteine. The heavy chain sequence (450 aa) of the A121C2H9 mutant is SEQ ID NO: 28.

[0295]

[0296] The cysteine ​​engineered thio-2H9 antibody comprises the following Fc constant region heavy chain sequences with free cysteine ​​amino acids (SEQ ID NOs: 29-38).

[0297]

[0298]

[0299] Cysteine ​​was introduced at certain residues of the full-length 3A5 anti-MUC16 antibody. The single cys mutant H-A121C of 3A5 was expressed in CHO (Chinese Hamster Ovary) cells by transient fermentation in medium containing 1 mM cysteine. The A121C 3A5 mutant heavy chain sequence (446 aa) comprises SEQ ID NO:39.

[0300]

[0301] The cysteine ​​engineered thio-3A5 anti-MUC16 antibodies comprise the following variable region heavy chain sequences with free cysteine ​​amino acids (SEQ ID NOs: 40-44).

[0302] Mutants sequence SEQ ID NO: F45C NWIRQCPGNK SEQ ID NO:40 A90C LNSCTTEDTAT SEQ ID NO:41 A121C GQGTLVTVSACSTKGPSVFPL SEQ ID NO:42 A175C HTFPCVLQSSGLYS SEQ ID NO:43 V176C HTFPACLQSSGLYS SEQ ID NO:44

[0303] The cysteine ​​engineered thio-3A5 anti-MUC16 antibodies comprise the following variable region light chain sequences with free cysteine ​​amino acids (SEQ ID NOs: 45-49).

[0304] Mutants sequence SEQ ID NO: L15C FLSVSCGGRVT SEQ ID NO:45 A43C QKPGNCPRLLI SEQ ID NO:46 V110C EIKRTCAAPSV SEQ ID NO:47 A144C FYPRECKVQWK SEQ ID NO:48 S168C VTEQDCKDSTY SEQ ID NO:49

[0305] Modification and thiol reactivity of 4D5 anti-HER2 ThioFab

[0306] Cysteine ​​was introduced into every position of the heavy and light chains of the anti-HER2 hu4D5Fabv8 Fab fragment antibody. All 440 heavy chain mutants and light chain mutants were prepared according to the methods described herein. Thiol reactivity was measured according to the PHESELECTOR assay. The heavy chain sequences are numbered according to the sequential numbering system. The light chain sequences follow the Kabat numbering system. In the light chain, both Kabat and sequential numbers represent the same number.

[0307] The heavy chain hu4D5Fabv8 mutant was selected for its effective binding to the HER2 receptor protein ( Figure 2A , Figure 2B and Figure 3A , Figure 3B) and thiol reactivity with biotinylation reagent Biotin-PEO-maleimide (Examples 1 and 2). Certain heavy chain mutants have limited or impaired binding to HER2 ECD because residues located in the variable region CDR of antibody-Fab are important for antigen binding (HER2). Some residues located in the constant domain of Fab also produce poor HER2 binding because these residues can promote the structure and folding of Fab, resulting in poor display of 4D5-Fab on M13 phage (Junutula, JR et al. (2008) J. Immunol Methods, 332: 41-52). Heavy chain hu4D5Fabv8 mutants with poor HER2ECD binding include cysteine ​​mutations at positions 1, 21, 31, 33-36, 38, 48-50, 59, 87, 95, 101, 104, 129, 131, 132, 136, 153, 155, 159, 166, 169, 170, 172, 197, 198, 202, 215, 219. Wild-type cysteine ​​variants 22, 96, 147, 203, 223 were measured. Other heavy chain mutants have limited thiol reactivity with biotinylation reagents. The free cysteine ​​amino acid residue is located in the center and the flanking residues are in the sequence of the middle column of Table 3. The left column indicates the alternative amino acid and position in the heavy chain. Table 3 SEQ ID NO: 50-98 Heavy chain hu4D5Fabv8 mutants retain HER2 binding and thiol reactivity values ​​of about 0.8 or above, excluding wild-type cysteine ​​variants. Antibodies with SEQ ID NO: 50-98 (Table 3) exhibit thiol reactivity and can be used to form covalent attachments to capture labels, detection labels, drug moieties, or solid supports. The heavy chain mutants of Table 3 can be coupled to a ThioFab or ThioMab, such as an antibody-drug conjugate.

[0308] Table 3 Efficient binding, thiol-reactive heavy chain hu4D5Fabv8 mutant

[0309]

[0310]

[0311] The light chain hu4D5Fabv8 mutant was selected for its effective binding to the HER2 receptor protein ( Figure 2A , Figure 2B and Figure 3A , Figure 3B) and thiol reactivity with biotinylation reagent Biotin-PEO-maleimide (Examples 1 and 2). Some light chain mutants have limited or impaired binding to HER2 because residues located in the antibody-Fab variable region CDR are important for antigen binding (HER2). Some residues located in the Fab constant domain also produce poor HER2 binding because these residues can promote the structure and folding of Fab, resulting in poor display of 4D5-Fab on M13 phage (Junutula, JR et al. (2008) J. Immunol Methods, 332: 41-52). Light chain hu4D5Fabv8 mutants with poor HER2 binding include cysteine ​​mutants at positions 4, 29-32, 35, 36, 50, 82, 86, 89-91, 113, 115, 117, 120, 126, 128, 139, 141, 146, 148, 179, 186, 192, 202. Wild-type cysteine ​​variants 23, 134, 194, 214 were measured. Other light chain mutants have limited thiol reactivity with biotinylation reagents. The free cysteine ​​amino acid residue is located in the center and the flanking residues are in the sequence of the middle column of Table 4. The left column indicates the alternative amino acid and position in the light chain. Table 4 SEQ ID NO: 99-147 Light chain hu4D5Fabv8 mutants retain HER2 binding and thiol reactivity values ​​of about 0.8 or above, excluding wild-type cysteine ​​variants. Antibodies having SEQ ID NOs: 99-147 (Table 4) exhibit thiol reactivity and can be used to form covalent attachments to capture labels, detection labels, drug moieties, or solid supports. The light chain mutants of Table 4 can be coupled to a ThioFab or ThioMab, such as an antibody-drug conjugate.

[0312] Table 4 Efficient binding, thiol-reactive light chain hu4D5Fabv8 mutant

[0313]

[0314]

[0315] Thiol reactivity of ThioMab

[0316] Thiol reactivity of antibodies (ThioMab) with full-length IgG cysteine ​​modifications was determined by biotinylation and streptavidin binding (US 7521541). A Western blot assay was set up to screen for ThioMab specifically coupled to biotin-maleimide. In this assay, antibodies were analyzed on reduced SDS-PAGE and the presence of biotin was specifically detected by incubation with streptavidin-HRP. Depending on which modified cys variant was used and the wild type was not observed to interact, streptavidin-HRP interactions were observed in the heavy chain or light chain, indicating that the ThioMab variant specifically coupled biotin on the modified Cys residue. Denaturing gel analysis of the biotinylated Thio-IgG variant reduced after capture on immobilized anti-IgG-HRP and streptavidin-HRP was implemented. Lane 1: 3A5 H-A121C. Lane 2: 3A5 L-V110C. Lane 3: 2H9 H-A121C. Lane 4: 2H9 L-V110C. Lane 5: anti-EphB2R 2H9 parent wild type. Each mutant (lanes 1-4) was captured using anti-IgG using HRP detection, indicating that selectivity and affinity were maintained. The location of biotin on the heavy and light chains was confirmed by capturing immobilized streptavidin using HRP detection. The cysteine ​​mutation position on the cysteine ​​engineered antibody in lanes 1 and 3 is the heavy chain. The cysteine ​​mutation position on the cysteine ​​engineered antibody in lanes 2 and 4 is the light chain. The cysteine ​​mutation sites were subjected to coupling with biotin-maleimide reagent.

[0317] LC / MS analysis of the ThioMab cysteine ​​engineered antibodies and the 2H9 V15C variant gave a quantitative indication of thiol reactivity (Table 5).

[0318] Table 5 LC / MS Quantification of Biotinylation of ThioMab - Thiol Reactivity

[0319] ThioMab variants The amount of biotin in each ThioMab 2H9 wt 0.0 2H9L-V15C 0.6 2H9 L-V110C 0.5 2H9 H-A121C 2.0 3A5 L-V110C 1.0 3A5 H-A121C 2.0

[0320] Cysteine ​​modification is carried out in the constant domain of IgG antibody, i.e., Fc region. Multiple amino acid sites are converted into cysteine ​​sites and the mutants expressed, i.e., the thiol reactivity of the cysteine ​​modified antibodies are evaluated. In the ELISA assay, the thiol reactivity of the biotinylated 2H9 ThioMab Fc variants is evaluated by quantification by HRP, and the thiol reactivity of the biotinylated 2H9 ThioMab Fc variants is captured and evaluated in immobilized streptavidin. An ELISA assay for rapid screening of Cys residues with reactive thiols is established. By detecting with anti-IgG-HRP, the absorbance is subsequently measured at 450nm to monitor the streptavidin-biotin interaction. These results confirm that 2H9-ThioFc variants V282C, A287C, A339C, S375C, and S400C have moderate to highest thiol reactivity. The biotin coupling degree of 2H9 ThioMab Fc variants is quantitatively analyzed by LS / MS as reported in Table 6. LS / MS analysis confirmed that the A282C, S375C and S400C variants had 100% biotin coupling and V284C and A339C had 50% coupling, indicating the presence of reactive cysteine ​​thiols. The other ThioFc variants and the parental wild-type 2H9 had very weak or no biotinylation.

[0321] Table 6 LC / MS Quantification of Biotinylated 2H9 Fc ThioMab

[0322] 2H9 ThioMab Fc variant Biotinylation% V273C 0 V279C 31 V282C 100 V284C 50 A287C 0 S324C 71 S337C 0 A339C 54 S375C 100 S400C 100 (Wild-type 2H9) 0

[0323] Thiol reactivity of Thio-4D5 FAB light chain variants

[0324] As through Figure 8 Screening various cysteine ​​engineered light chain variant Fabs of anti-ErbB2 antibody 4D5, as determined by the PHESELECTOR assay of , yielded a number of variants with thiol reactivity values ​​of 0.6 and above (Table 7). The thiol reactivity values ​​of Table 7 were calibrated to the heavy chain 4D5 ThioFab variant (HC-A121C) set to 100%, and the HC-A121C variant was presumed to be fully biotinylated and expressed as a percentage value.

[0325] Table 7 Thiol reactivity percentage values ​​of 4D5 ThioFab light chain variants

[0326]

[0327]

[0328] Antibody-drug conjugates

[0329] The cysteine ​​engineered antibodies of the present invention can be coupled to any therapeutic agent, ie, a drug moiety, which can be covalently bound to the antibody via the reactive cysteine ​​thiol.

[0330] Typical embodiments of antibody-drug conjugate (ADC) compounds comprise a cysteine ​​engineered antibody (Ab) and a drug moiety (D), wherein the antibody has one or more free cysteine ​​amino acids, and the antibody is attached (bound) to the one or more free cysteine ​​amino acids via a linker moiety (L) connected to D; the composition has Formula I:

[0331] Ab-(LD) p I

[0332] wherein p is 1, 2, 3 or 4. The number of drug moieties that can be coupled to the antibody molecule via a thiol-reactive linker moiety is limited by the number of cysteine ​​residues introduced by the methods described herein. Typical ADCs of Formula I thus comprise antibodies with 1, 2, 3 or 4 engineered cysteine ​​amino acids.

[0333] Another typical embodiment of an antibody-drug conjugate compound (ADC) comprises a cysteine ​​engineered antibody (Ab), an albumin-binding peptide (ABP), and a drug moiety (D), wherein the antibody is conjugated to the drug moiety via a linker moiety (L) and the antibody is conjugated to the albumin-binding peptide via an amide bond or a second linker moiety; the composition has Formula Ia:

[0334] ABP-Ab-(LD) p Ia

[0335] Where p is 1, 2, 3 or 4.

[0336] The ADC compounds of the present invention include those compounds for anticancer activity. In particular, the compounds include a cysteine-modified antibody to which a drug moiety, i.e., a toxin, is coupled via a linker, i.e., covalently bound thereto. When the drug is not coupled to the antibody, the drug has a cytotoxic or cell-inhibiting effect. Thus, the biological activity of the drug moiety is modulated by coupling to the antibody. The antibody-drug conjugate (ADC) of the present invention selectively delivers an effective dose of a cytotoxic agent to tumor tissue, thereby achieving greater selectivity, i.e., a lower efficacious dose.

[0337] Drug section

[0338] The drug portion (D) of an antibody-drug conjugate (ADC) includes any compound, moiety or group that has a cytotoxic or cytostatic effect. Drug portions include: (i) chemotherapeutic agents that can act as tubulin inhibitors, mitotic inhibitors, topoisomerase inhibitors or DNA intercalators; (ii) protein toxins that can act enzymatically; and (iii) radioactive isotopes.

[0339] Typical drug moieties include, but are not limited to, maytansinoids, auristatins, dolastatins, trichothecenes, CC1065, calicheamicin and other enediyne antibiotics, taxanes, anthracyclines, and stereoisomers, isosteres, analogs or derivatives thereof.

[0340] Maytansine compounds suitable for use as maytansine alkaloid drug moieties are well known in the art and maytansine and maytansine analogs can be isolated from natural sources according to known methods, produced using genetic engineering techniques (see Yu et al. (2002) PROC. NAT. ACAD. SCI. (USA) 99:7968-7973) or prepared synthetically according to known methods.

[0341] Typical maytansinoid drug moieties include those with modified aromatic rings, such as: C-19-dechloro (US 4256746) (prepared by lithium aluminum hydride reduction of ansamytocin P2); C-20-hydroxy (or C-20-demethyl) + / - C-19-dechloro (US 4361650 and 4307016) (prepared by demethylation using Streptomyces or Actinomyces or dechlorination using LAH); and C-20-demethoxy, C-20-acyloxy (-OCOR), + / -dechloro (US 4,294,757) (prepared by acylation using acyl chlorides) and those with modifications at other positions.

[0342] Typical maytansinoid drug moieties also include those with modifications such as: C-9-SH (US 4424219) (prepared by reacting maytansinol with H2S or P2S5); C-14-alkoxymethyl (demethoxy / CH2 OR) (US 4331598); C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (US 4450254) (prepared by Nocardia); C-15-hydroxy / acyloxy (US 4364866) (prepared by converting maytansinol by Streptomyces); C-15-methoxy (US 4313946 and 4315929) (isolated from Trewia nudlflora); C-18-N-demethyl (US 4362663 and 4322348) (prepared by demethylating maytansinol with Streptomyces); and 4,5-deoxy (US 4362663 and 4322348) (prepared by demethylating maytansinol with Streptomyces); 4371533) (prepared by phthalide trichloride / LAH reduction of maytansine). Many positions on known maytansine compounds are used as attachment positions, depending on the type of attachment. For example, in order to form an ester bond, the C-3 position with a hydroxyl group, the C-14 position modified with a hydroxymethyl group, the C-15 position modified with a hydroxyl group, and the C-20 position with a hydroxyl group are all suitable.

[0343] The drug portion (D) of the antibody-drug conjugate (ADC) of Formula I comprises a maytansinoid having the following structure:

[0344]

[0345] Wherein the wavy line represents that the sulfur atom of D is covalently bound to the linker (L) of the antibody-drug conjugate (ADC). R can be independently H or C1-C6 alkyl, and the C1-C6 alkyl is 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 that binds the amide group to the sulfur atom may be methanyl, ethanyl or propyl, ie, m is 1, 2 or 3.

[0346] Maytansine compounds inhibit cell proliferation by inhibiting tubulin formation during mitosis, and the inhibition of tubulin formation during mitosis is carried out by inhibiting tubulin, i.e., the polymerization of tubulin (Remillard et al. (1975) Science 189: 1002-1005). Maytansine and maytansine alkaloids are highly toxic, but their clinical application in cancer therapy is very limited due to the serious systemic side effects caused by poor selectivity for tumors. Clinical trials using maytansine have been interrupted due to serious adverse effects on the central nervous system and gastrointestinal system (Issel et al. (1978) Can. Treatment. Rev. 5: 199-207).

[0347] Maytansinoid drug moieties are attractive drug moieties in antibody-drug conjugates because they are: (i) relatively accessible for preparation by fermentation or chemical modification, derivatization of fermentation products; (ii) easily derivatized with functional groups suitable for conjugation to antibodies via non-disulfide linkers; (iii) stable in plasma; and (iv) active against a variety of tumor cell lines (US 2005 / 0169933; WO 2005 / 037992; US 5208020).

[0348] As additional drug moieties, all stereoisomers of the maytansinoid drug moiety are contemplated for the compounds of the invention, i.e., any combination of R and S configurations at the chiral carbon of D. In one embodiment, the maytansinoid drug moiety (D) has the following stereochemistry:

[0349]

[0350] Typical embodiments of the maytansinoid drug moiety include: DM1, (CR2) m =CH2CH2; DM3, (CR2) m =CH2CH2CH(CH3); and DM4,(CR2) m =CH2CH2C(CH3)2, they have the following structure:

[0351]

[0352]

[0353] According to the difference of connection type, joint can be combined with maytansinoid molecule in different positions.For example, ester bond can be formed by using conventional coupling technology and hydroxyl reaction.This reaction can be carried out on the C-3 position with hydroxyl.This reaction can be carried out on the C-14 position with hydroxyl, the C-15 position with hydroxyl modification and the C-20 with hydroxyl at the C-3 position with hydroxyl.In a preferred embodiment, the bond is formed on the C-3 position of maytansinol or maytansinol analog.

[0354] The drug portion (D) of the antibody-drug conjugate (ADC) of formula I also includes dolastatin and its peptide analogs and derivatives auristatins (U.S. Pat. Nos. 5,635,483 and 5,780,588). Dolastatin 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 anticancer (U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42: 2961-2965). Different forms of the dolastatin or auristatin drug moiety can be covalently attached to the antibody via the N (amino) terminus or the C (carboxyl) terminus of the peptide drug moiety (WO 02 / 088172; Doronina et al. (2003) Nature Biotechnology 21(7):778-784; Francisco et al. (2003) Blood 102(4):1458-1465).

[0355] Drug moieties 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 (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45 (12): 3580-3584) and have anticancer (US 5663149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42: 2961-2965). Different forms of dolastatins or auristatins drug moieties can be conjugated to antibodies via the N (amino) terminus or the C (carboxyl) terminus of the peptide drug moiety (WO 02 / 088172).

[0356] Typical auristatin embodiments include N-terminally linked monomethylauristatin drug moieties DE and DF, which are disclosed in US Pat. No. 7,498,298 and US Pat. No. 7,659,241, the entire disclosures of each of which are specifically incorporated herein by reference.

[0357] The drug moiety (D) of the antibody-drug conjugate (ADC) of Formula I comprises monomethylauristatin drug moieties MMAE and MMAF linked to the antibody via the N-terminus and has the following structure:

[0358]

[0359] In general, peptide-based drug moieties can be prepared by forming peptide bonds between two or more amino acids and / or peptide fragments. For example, such peptide bonds can be prepared by liquid phase synthesis methods well known in the art of peptide chemistry (see E. and K. Lübke, "The Peptides", volume 1, pp76-136, 1965, Academic Press).

[0360] The drug moiety includes calicheamicin and its analogs and derivatives. The calicheamicin antibiotics are capable of producing sub-picomolar double-strand DNA breaks. For the preparation of conjugates of the calicheamicin antibiotics, see US 5712374; US 5714586; US 5739116; US 5767285; US 5770701, US 5770710; US 5773001; US ​​5877296. Structural analogs of calicheamicin that can be used include, but are not limited to γ1 I , α2 I , α3 I , N-acetyl-γ1 I , PSAG and θ I 1 (Hinman et al. Cancer Research 53:3336-3342 (1993), Lode et al. Cancer Research 58:2925-2928 (1998).

[0361] Protein toxins include: diphtheria toxin A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain (Vitetta et al. (1987) Science, 238:1098), abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), momordica charantia proteins (PAP-S), and schizonepeta tenuifolia proteins (Science 238:1098). charantia inhibitors, curcin, crotin, sapaonaria officinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin and tricothecenes (WO 93 / 21232).

[0362] Therapeutic radioisotopes include: 32 P. 33 P. 90 Y. 125 I. 131 I. 131 In, 153 Sm, 186 Re, 188 Re, 211 At 212 Bi, 212 Radioactive isotopes of Pb and Lu.

[0363] Radioactive isotopes or other labels can be incorporated into the conjugate in a known manner (Fraker et al. (1978) Biochem. Biophys. Res. Commun. 80: 49-57; "Monoclonal Antibody in Immunoscintigraphy" Chatal, CRC Press 1989). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is a typical chelating agent for conjugating radionuclides to antibodies (WO 94 / 11026).

[0364] Connectors

[0365] "Linker" (L) is a bifunctional or multifunctional moiety having a function of connecting one or more drug moieties (D) and antibody units (Ab) to form an antibody-drug conjugate (ADC) of Formula I. Antibody-drug conjugates (ADC) can be conveniently prepared using linkers having reactive functional groups that bind to drugs and antibodies. The cysteine ​​thiol of the cysteine ​​engineered antibody (Ab) can form a bond with a functional group of a linker reagent, a drug moiety, or a drug-linker intermediate.

[0366] In one aspect, the linker has a reactive position with an electrophilic group that reacts with a nucleophilic cysteine ​​present on the antibody. The cysteine ​​thiol of the antibody reacts with the electrophilic group on the linker and forms a covalent bond with the linker. Useful electrophilic groups include, but are not limited to maleimide and haloacetamide groups.

[0367] The cysteine ​​engineered antibody is reacted with a linker reagent or drug-linker intermediate with an electrophilic functional group such as maleimide or α-halogenated carbonyl according to the method of Klussman et al. (2004) at Bioconjugate Chemistry 15(4):765-773, 766 and the protocol of Example 4.

[0368] In yet another embodiment, the reactive group of the linker reagent or drug-linker intermediate contains a thiol-reactive functional group that can form a bond with a free cysteine ​​thiol of the antibody. Examples of thiol-reactive functional groups include, but are not limited to: maleimide; α-haloacetyl; activated esters, such as succinimidyl esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters; anhydrides; acyl chlorides; sulfonyl chlorides; isocyanates and isothiocyanates.

[0369] In another embodiment, the linker L can be a dendritic type linker, which is used to covalently bind more than one drug moiety to an antibody through a branched multifunctional linker moiety (Sun et al. (2002) Bioorganic & Medicinal Chemistry Letters 12: 2213-2215; Sun et al. (2003) Bioorganic & Medicinal Chemistry 11: 1761-1768;). Dendritic linkers can increase the molar ratio of drug to antibody, i.e., loading, which is related to the potency of the ADC. Thus, if a cysteine ​​engineered antibody carries only one reactive cysteine ​​thiol, then numerous drug moieties can be bound through a dendritic linker.

[0370] The linker may comprise amino acid residues that connect the antibody (Ab) to the drug portion (D) of the cysteine ​​engineered antibody-drug conjugate (ADC) of the present invention. The amino acid residues may form dipeptides, tripeptides, tetrapeptides, pentapeptides, hexapeptides, heptapeptides, octapeptides, nonapeptides, decapeptides, undecapeptides or dodecapeptides units. The amino acid residues include those naturally occurring as well as minimal amino acids and non-naturally occurring amino acid analogs, such as citrulline.

[0371] Useful amino acid residue units can be designed and optimized for selectivity in enzymatic cleavage by specific enzymes, such as tumor-associated proteases, to release active drug moieties. In one embodiment, amino acid residue units such as valine-citrulline (vc or val-cit) are units whose cleavage is catalyzed by cathepsins B, C and D or plasmin proteases.

[0372] The linker unit may be of the self-immolative type, such as a p-aminobenzylcarbamoyl (PAB) unit, wherein the ADC has the following typical structure:

[0373]

[0374] wherein Q is -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro or -cyano; m is an integer of 0-4; and p is 1-4.

[0375] Other examples of self-immolative spacers include, but are not limited to, aromatic compounds that are similar in charge pattern to the PAB group, such as 2-aminoimidazole-5-methanol derivatives (US 7375078; Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho- or para-aminobenzyl acetals. Spacers that undergo cyclization upon amide bond hydrolysis may be used, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al. (1995) Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm et al. (1972) J. Amer. Chem. Soc. 94:5815) and 2-aminophenylpropionic acid amides (Amsberry, et al. (1990) J. Org. Chem. 55:5867). Elimination of amine-containing drugs substituted on glycine (Kingsbury et al. (1984) J. Med. Chem. 27:1447) is also an example of a self-immolative spacer for ADCs.

[0376] In another embodiment, the linker L can be a dendritic type linker (Sun et al. (2002) Bioorganic & Medicinal Chemistry Letters 12: 2213-2215; Sun et al. (2003) Bioorganic & Medicinal Chemistry 11: 1761-1768) for covalently binding more than one drug moiety to an antibody through a branched multifunctional linker moiety. Dendritic linkers can increase the molar ratio of drug to antibody, i.e., loading, which is related to the potency of the ADC. Thus, if a cysteine ​​engineered antibody carries only one reactive cysteine ​​thiol, a wide variety of drug moieties can be conjugated via a dendritic linker (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).

[0377] Embodiments of Formula Ia antibody-drug conjugate compounds include (val-cit), (MC-val-cit), and (MC-val-cit-PAB):

[0378]

[0379] Other typical embodiments of Formula Ia antibody-drug conjugate compounds include the following structures:

[0380]

[0381] Where X is:

[0382]

[0383] Y is:

[0384]

[0385] R is independently H or C1-C6 alkyl; and n is 1-12.

[0386] In another embodiment, the joint carries a reactive functional group having a nucleophilic group that reacts with an electrophilic group present on the antibody. Useful electrophilic groups on the antibody include, but are not limited to aldehydes and ketone carbonyls. The heteroatom of the nucleophilic group of the joint can react with the electrophilic group on the antibody and form a covalent bond with the antibody unit. Useful nucleophilic groups on the joint include, but are not limited to hydrazides, oximes, aminos, hydrazines, thiosemicarbazones, hydrazine carboxylates, and aryl hydrazides. The electrophilic group on the antibody provides a convenient position for binding (attaching) joints.

[0387] In general, peptide-type linkers can be prepared by forming a peptide bond between two or more amino acids and / or peptide fragments. For example, the peptide-type linkers can be prepared by the well-known liquid phase synthesis method (E. and K. Lübke (1965) "The Peptides", volume 1, pp 76-136, Academic Press) for the preparation of such peptide bonds.

[0388] In another embodiment, the linker can be substituted with groups that modulate solubility or reactivity. For example, a charged substituent such as a sulfonic acid group (-SO3 - ) or ammonium can increase the water solubility of the reagent and facilitate the coupling reaction of the linker reagent with the antibody or drug portion, or facilitate the coupling reaction of Ab-L (antibody-linker intermediate) with D or DL ​​(drug-linker intermediate) with Ab, depending on the synthetic route used to prepare the ADC.

[0389] Of particular interest are compounds of the invention, but not limited to: ADCs prepared with linker reagents: BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB and SVSB (succinimidyl-(4-vinylsulfone)benzoate); and including bis-maleimide reagents: DTME, BMB, BMDB, BMH, BMOE, BM(PEO)3 and BM(PEO)4, which are commercially available from Pierce Biotechnology, Inc., Customer Service Department, PO Box 117, Rockford, IL. 61105 U.S.A., 1-800-874-3723, International +815-968-0747. See pp. 467-498, 2003-2004 Applications Handbook and Catalog, bis-maleimide reagents enable the thiol of a cysteine ​​engineered antibody to be conjugated to a thiol-containing drug moiety, label or linker intermediate in a sequential or simultaneous manner. Other functional groups other than maleimide that react with the thiol of a cysteine ​​engineered antibody, drug moiety, label or linker intermediate include iodoacetamide, bromoacetamide, vinylpyridine, disulfide, pyridyl disulfide, isocyanate (salt) and isothiocyanate (salt).

[0390]

[0391] Useful linker reagents can also be obtained through other commercial sources, such as Molecular Biosciences Inc. (Boulder, CO) or synthesized according to the procedures described in the following literature: Toki et al. (2002) J. Org. Chem. 67: 1866-1872; Walker, MA (1995) J. Org. Chem. 60: 5352-5355; Frisch et al. (1996) Bioconjugate Chem. 7: 180-186; US 6214345; WO 02 / 088172; US2003130189; US2003096743; WO 03 / 026577; WO 03 / 043583; and WO 04 / 032828.

[0392] A typical valine-citrulline (val-cit or vc) dipeptide linker reagent with a maleimide stretcher and a p-aminobenzylcarbamoyl (PAB) self-immolative spacer has the following structure:

[0393]

[0394] wherein Q is -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro or -cyano; and m is an integer of 0-4.

[0395] A typical phe-lys (Mtr) dipeptide linker reagent having a maleimide stretcher unit and a p-aminobenzyl self-immolative spacer can be prepared as described by Dubowchik et al. (1997) Tetrahedron Letters, 38: 5257-60 and has the following structure:

[0396]

[0397] Wherein Mtr ​​is a 4-methoxytrityl group, Q is a -C1-C8 alkyl group, -O-(C1-C8 alkyl group), -halogen, -nitro or -cyano group; and m is an integer of 0-4.

[0398] Typical antibody-drug conjugate compounds of the present invention include:

[0399]

[0400]

[0401] Wherein Val is valine; Cit is citrulline; p is 1, 2, 3 or 4; and Ab is a cysteine-modified antibody. Other typical antibody-drug conjugates have the following structure, in which the maytansinoid drug moiety DM1 is linked to the thiol of trastuzumab via a BMPEO linker:

[0402]

[0403] wherein Ab is a cysteine ​​engineered antibody; n is 0, 1 or 2; and p is 1, 2, 3 or 4.

[0404] Preparation of Antibody-Drug Conjugates

[0405] ADCs of Formula I can be prepared by several routes using organic chemical reactions, conditions and reagents known to those skilled in the art, including: (1) reacting the cysteine ​​group of a cysteine ​​engineered antibody with a linker reagent to form an antibody-linker intermediate Ab-L via a covalent bond, followed by reaction with an activated drug moiety D; and (2) reacting the nucleophilic group of the drug moiety with a linker reagent to form a drug-linker intermediate DL via a covalent bond, followed by reaction with the cysteine ​​group of the cysteine ​​engineered antibody. Coupling methods (1) and (2) can be used with a variety of cysteine ​​engineered antibodies, drug moieties and linkers to prepare antibody-drug conjugates of Formula I.

[0406] Antibody cysteine ​​thiols are nucleophilic and can react with electrophilic groups on linker reagents and drug-linker intermediates to form covalent bonds, including: (i) active esters, such as NHS esters, HOBt esters, haloformates, and acyl halides; (ii) alkyl and benzyl halides, such as haloacetamides; (iii) aldehydes, ketones, carboxyls, and maleimido groups; and (iv) disulfides via sulfide exchange, including pyridyl disulfides. Nucleophilic groups on drug moieties include, but are not limited to, amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and aryl hydrazide groups, which can react with electrophilic groups on linker moieties and linker reagents to form covalent bonds.

[0407] For example, maytansine can be converted to May-SSCH3, which can be reduced to the free thiol May-SH and reacted with a modified antibody (Chari et al. (1992) Cancer Research 52: 127-131) to generate a maytansine alkaloid-antibody immunoconjugate with a disulfide linker. Antibody-maytansine alkaloid conjugates with disulfide linkers have been reported (WO 04 / 016801; US ​​6884874; US 2004 / 039176 A1; WO 03 / 068144; US 2004 / 001838 A1; U.S. Pat. Nos. 6441163, 5208020, 5416064; WO 01 / 024763). The disulfide linker SPP was constructed using the linker reagent N-succinimidyl 4-(2-pyridylthio) pentanoate.

[0408] Under certain conditions, cysteine ​​engineered antibodies can be reacted to couple linker reagents by treatment with a reducing agent, such as DTT (Cleland's reagent, dithiothreitol) or TCEP (tris(2-carboxyethyl)phosphine hydrochloride; Getz et al. (1999) Anal. Biochem. Vol 273:73-80; Soltec Ventures, Beverly, MA). Full-length cysteine ​​engineered monoclonal antibodies (ThioMab) expressed in CHO cells were reduced with approximately 50-fold excess TCEP for 3 hours at 37°C to reduce disulfide bonds that can be formed between newly introduced cysteine ​​residues and cysteines present in the culture medium. The reduced ThioMab was diluted with 10 mM sodium acetate, pH 5 and loaded onto a HiTrap S column in 10 mM sodium acetate, pH 5 and eluted with PBS containing 0.3 M sodium chloride. Spend the night under the condition of dilute (200nM) copper sulfate aqueous solution (CuSO4) at room temperature, make the disulfide bond between the cysteine ​​residues in the parental Mab be rebuilt.Other oxidants known in the art, i.e. oxidants and oxidation conditions can be used.Ambient air oxidation is also effective.This moderate partial reoxidation step effectively forms an interchain disulfide bond with high fidelity.Add about 10 times of excess drug-joint intermediates, such as BM (PEO) 4-DM1, mix and keep about 1 hour at room temperature, so as to couple and form ThioMab antibody-drug conjugate.The coupling mixture is subjected to gel filtration and upper HiTrap S column and eluted to remove excess drug-joint intermediates and other impurities.

[0409] Fig.11 A general method for preparing cysteine ​​engineered antibodies expressed by cell culture for conjugation is presented. Cysteine ​​adducts presumably bearing various interchain disulfide bonds are reductively cleaved to the reduced form of the antibody. Under partial oxidation conditions, such as exposure to ambient oxygen, the interchain disulfide bonds between paired cysteine ​​residues are reformed. Newly introduced, engineered and unpaired cysteine ​​residues are still available for reaction with linker reagents or drug-linker intermediates to form antibody conjugates of the present invention. ThioMabs expressed in mammalian cell lines produce Cys adducts for external conjugation with engineered Cys through –SS-bond formation, therefore, purified ThioMabs must be treated by reduction and oxidation procedures as described in Example 11 in order to produce reactive ThioMabs. These ThioMabs are used for conjugation of maleimide-containing cytotoxic drugs, fluorophores and other labels.

[0410] Various ThioFab and ThioMab antibody-drug conjugates were prepared (Examples 4-8). The cysteine ​​mutant hu4D5Fabv8(V110C) was conjugated to the maytansinoid drug moiety DM1 via the bis-maleimido linker reagent BMPEO to form hu4D5Fabv8(V110C)-BMPEO-DM1 (Example 8).

[0411] In vitro cell proliferation assay

[0412] In general, the cytotoxic or cell growth inhibitory activity of an antibody-drug conjugate (ADC) is determined by the following steps: contacting mammalian cells with a receptor protein, such as HER2, with the antibody of the ADC in a cell culture medium; culturing the cells for a period of about 6 hours to about 5 days; and determining cell viability. Cell-based in vitro assays are used to determine viability (proliferation), cytotoxicity, and programmed cell death induction (caspase activation) of the ADC of the present invention.

[0413] In vitro efficacy of antibody-drug conjugates was measured by cell proliferation assay ( Fig. 10A , Fig. 10B and 11 , Example 9). CellTiter- The Luminescent Cell Viability Assay is a commercially available (Promega Corp., Madison, WI) homogeneous assay method based on recombinant expression of Coleoptera luciferase (U.S. Pat. Nos. 5,583,024, 5,674,713, and 5,700,670). This cell proliferation assay is based on quantifying the number of viable cells in culture, where the presence of ATP is an indicator of metabolically active cells (Crouch et al. (1993) J. Immunol. Meth. 160:81-88; U.S. Pat. No. 6,602,677). CellTiter- Assay, making it easy to perform automated high throughput screening (HTS) (Cree et al. (1995) AntiCancer Drugs 6:398-404). The homogeneous assay procedure involves mixing a single reagent (CellTiter- Reagent) is added directly to cells cultured in serum-supplemented medium. No cell washing, medium removal and multiple pipetting steps are required. The system can detect as low as 15 cells / well in a 384-well grid within 10 minutes after adding the reagent and mixing. Cells can be treated with ADC continuously, or they can be treated and separated from the ADC. In general, cells treated briefly, i.e., for 3 hours, show the same efficacy as cells treated continuously.

[0414] The homogenous "add-mix-measure" format results in cell lysis and the 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 assay generates a "glow-type" luminescent signal produced by a luciferase reaction with a half-life generally greater than 5 hours, depending on the cell type and culture medium used. Viable cells are reflected in relative luminescence units (RLU). The substrate beetle luciferin is oxidatively decarboxylated by recombinant firefly luciferase, while ATP is converted to AMP and photons are generated.

[0415] In vivo effects

[0416] The in vivo efficacy of two albumin-binding peptide-DM1 (maytansinoid)-antibody-drug conjugates (ADCs) of the present invention was determined in a transgenic explant mouse model that highly expresses HER2 (Example 10). Fo5 mmtv transgenic mice that respond or respond poorly to therapy proliferate. Subjects were treated with ABP-rhuFab4D5-cys(light chain)-DM1; ABP-rhuFab4D5-cys(heavy chain)-DM1; and placebo PBS buffer control (vehicle) and monitored over 3 weeks to determine time to tumor volume doubling, log cell kill, and tumor shrinkage.

[0417] Administration of Antibody-Drug Conjugates

[0418] The antibody-drug conjugate (ADC) of the present invention can be administered by any route suitable for the disease to be treated.ADC is generally administered by parenteral routes, i.e., infusion, subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural.

[0419] Pharmaceutical preparations

[0420] The therapeutic antibody-drug conjugate (ADC) of the present invention is usually formulated into a unit dose injectable pharmaceutical formulation for parenteral administration, i.e., bolus, intravenous injection, intratumoral injection, together with a pharmaceutically acceptable parenteral vehicle. Optionally, the antibody-drug conjugate (ADC) having the desired purity is mixed with a pharmaceutically acceptable diluent, carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.) into a lyophilized dosage form or an aqueous solution.

[0421] Antibody-drug conjugate therapy

[0422] It is contemplated that the antibody-drug conjugates (ADCs) of the invention may be used to treat a variety of diseases or conditions, such as those characterized by overexpression of tumor antigens. Typical diseases or hyperproliferative conditions include benign or malignant tumors; leukemias and lymphoid malignancies. Other diseases include neuronal, glial, astrocyte, hypothalamic, glandular, macrophage, epithelial, mesenchymal, blastocoel, inflammatory, angiogenic, and immune diseases, including autoimmune diseases.

[0423] In general, the disease or condition treated is a hyperproliferative disease, such as cancer. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specifically, such cancers include: squamous cell carcinoma (e.g., epithelial squamous cell carcinoma); lung cancer, including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma; peritoneal cancer; hepatocellular carcinoma; gastric cancer, including gastrointestinal cancer; pancreatic cancer, glioblastoma; cervical cancer; ovarian cancer; liver cancer; bladder cancer; hepatocellular carcinoma (hepatoma); breast cancer; colon cancer; rectal cancer; colorectal cancer; endometrial or uterine cancer; salivary gland cancer; kidney cancer; prostate cancer, vulvar cancer; thyroid cancer; hepatic carcinoma; anal cancer; penile cancer, and head and neck cancer.

[0424] Autoimmune diseases that ADC compounds can be used to treat include rheumatic diseases (such as, for example, rheumatoid arthritis; Sjögren's syndrome; scleroderma; lupus, such as SLE and lupus nephritis; polymyositis / dermatomyositis; cryoglobulinemia; anti-phospholipid antibody syndrome; and psoriatic arthritis), osteoarthritis, autoimmune gastrointestinal and liver diseases (such as, for example, inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), autoimmune gastritis 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), autoimmune neurological diseases (such as, for example, multiple sclerosis, opsoclonus myoclonus syndrome, syndrome), myasthenia gravis, oculoencephalomyelopathy, Parkinson's disease, Alzheimer's disease, and autoimmune polyneuropathy), kidney diseases (such as, for example, glomerulonephritis, Goodpasture's syndrome, and Berger's disease), autoimmune skin diseases (such as, for example, psoriasis, urticaria, hives, pemphigus vulgaris, bullous pemphigoid, and cutaneous lupus erythematosus), blood diseases (such as, for example, thrombocytopenic Purpura, thrombotic thrombocytopenic purpura, post-transfusion purpura and autoimmune hemolytic anemia), atherosclerosis, uveitis, autoimmune hearing diseases (such as, for example, inner ear diseases and hearing loss), Behcet's disease, Raynaud's syndrome, organ transplantation and autoimmune endocrine disorders (such as, for example, diabetes-related autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM), Addison's disease and autoimmune thyroid diseases (such as Graves' disease and thyroiditis). More preferred such diseases include, for example, rheumatoid arthritis, ulcerative colitis, ANCA-associated vasculitis, lupus, multiple sclerosis, Sjögren's syndrome, Graves' disease, IDDM, pernicious anemia, thyroiditis and glomerulonephritis.

[0425] For the prevention or treatment of disease, the appropriate dose of the ADC depends on the type of disease being treated, the severity and duration of the disease, whether the molecule is administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The molecule is appropriately administered to the patient once or over a series of treatments. Depending on the type and severity of the disease, the initial candidate dose of the molecule administered to the patient is about 1 μg / kg-15 mg / kg (e.g., 0.1-20 mg / kg), for example, whether by one or more separate administrations or by continuous infusion. A typical daily dose may be in the range of about 1 μg / kg-100 mg / kg or more, depending on the above factors. The typical dose of ADC administered to a patient is in the range of about 0.1-about 10 mg / kg of the patient's body weight.

[0426] For repeated administration over a period of several days or more, depending on the condition, the treatment is continued until the desired disease symptoms are suppressed. A typical dosing regimen comprises administering an initial loading dose of about 4 mg / kg, followed by a weekly maintenance dose of about 2 mg / kg of anti-ErbB2 antibody. Other dosage regimens are also useful. The progress of the therapy is easily monitored by conventional techniques and tests.

[0427] Labeled Antibody Imaging Methods

[0428] In another embodiment of the invention, cysteine ​​engineered antibodies can be labeled with radionuclides, fluorescent dyes, substrate moieties that stimulate bioluminescence, moieties that stimulate chemiluminescence, enzymes and other detection labels through cysteine ​​thiols for use in imaging experiments with diagnostic, pharmacodynamic and therapeutic applications. Generally, labeled cysteine ​​engineered antibodies, i.e., "biomarkers" or "probes," are administered to living organisms, such as humans, rodents or other small animals, perfused organs or tissue samples, by injection, perfusion or oral ingestion. The distribution of the probe is detected over a period of time and displayed by imaging.

[0429] Products

[0430] In another embodiment of the present invention, an article or "kit" containing substances for treating the above-mentioned conditions is provided. The article comprises a container and a label or package insert on or connected to the container. Suitable containers include, for example, bottles, vials, syringes, blister packs, etc., and the container can be formed of various materials, such as glass or plastic. The container can hold an antibody-drug conjugate (ADC) composition that is effective in treating the disease and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can penetrate a hypodermic needle). At least one active agent in the composition is ADC. The label or package insert indicates that the composition is used to treat a selected disease, such as cancer. Alternatively or in addition, the article may further contain a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other substances required from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0431] Example

[0432] Example 1 - Preparation of biotinylated ThioFab phage

[0433] ThioFab-phage (5x10 12 Phage particles) were mixed with 150-fold excess of biotin-PEO-maleimide ((+)-biotinyl-3-maleimidopropionamidyl-3,6

[0434] -dioxaoctainediamine), Oda et al. (2001) Nature Biotechnology 19:379-382, Pierce Biotechnology, Inc.) at room temperature for 3 hours. Excess biotin-PEO-maleimide was removed from the biotin-coupled phage by repeated PEG precipitation (3-4 times). Other commercially available biotinylation reagents with electrophilic groups that react with cysteine ​​thiols can be used, including biotin-BMCC, PEO-iodoacetyl biotin, iodoacetyl-LC-biotin, and biotin-HPDP (Pierce Biotechnology, Inc.) and N α -(3-Maleimidopropionyl)biocytin (N α-(3-maleimidylpropionyl)biocytin) (MPB, Molecular Probes, Eugene, OR). Commercial sources of other biotinylated bifunctional and multifunctional linker reagents include Molecular Probes, Eugene, OR and Sigma, St. Louis, MO.

[0435]

[0436] Biotin-PEO-maleimide

[0437] Example 2 - PHESELECTOR Assay

[0438] Bovine serum albumin (BSA), erbB2 extracellular domain (HER2) and streptavidin (100 μl of 2 μg / ml) were coated on Maxisorp 96-well plates. After blocking with 0.5% Tween-20 (in PBS), biotinylated and non-biotinylated hu4D5Fabv8-ThioFab-phage (2×10 10 phage particles) for 1 hour, followed by incubation with horseradish peroxidase (HRP)-labeled secondary antibody (anti-M13 phage envelope protein, pVIII protein antibody). Figure 8 The PHESELECTOR assay is illustrated by diagrams depicting binding of Fab or ThioFab to HER2 (top) and biotinylated ThioFab to streptavidin (bottom).

[0439] A standard HRP reaction was performed and the absorbance was measured at 450 nm. The OD of streptavidin was calculated by 450 OD of HER2 450 Thiol reactivity was determined by the ratio of 1 to 2. A thiol reactivity value of 1 indicates complete biotinylation of cysteine ​​thiols. For Fab protein binding assay, hu4D5Fabv8 (2-20 ng) was used and then incubated with HRP-labeled goat polyclonal anti-Fab antibody.

[0440] Example 3a - Expression and purification of ThioFab

[0441] ThioFab was expressed upon induction in the non-repressive E. coli strain 34B8 (Baca et al. (1997) Journal Biological Chemistry 272(16):10678-84). The collected cell pellet was resuspended in PBS (phosphate buffered saline), passed through a microfluidizer for complete cell lysis and purified by using protein G SEPHAROSETM ThioFab was purified by affinity chromatography using Amersham.

[0442] ThioFab L-V15C, L-V110C, H-A88C and H-A121C were expressed and SEPHAROSE TM Purification by column chromatography. Oligo-Fab was present in fractions 26-30, and the majority of the monomeric form was present in fractions 31-34. Fractions consisting of the monomeric form were collected and analyzed by SDS-PAGE along with wild-type hu4D5Fabv8, and analyzed with SDS-PAGE gels under reducing (with DTT or BME) and non-reducing (without DTT or BME) conditions. Gel filtration fractions of A121C-ThioFab were analyzed using non-reducing SDS-PAGE.

[0443] ThioFab was coupled to biotin-PEO-maleimide as described above and stained with Superdex-200. TM Biotinylated-ThioFab was further purified by (Amersham) gel filtration chromatography, which eliminated free biotin-PEO-maleimide and oligomeric fractions of ThioFab. Wild-type hu4D5Fabv8 and hu4D5Fabv8 A121C-ThioFab (0.5 mg) were incubated with 100-fold molar excess of biotin-PEO-maleimide at room temperature for 3 hours and loaded onto a Superdex-200 gel filtration column to separate free biotin and oligomeric Fab from monomeric forms.

[0444] Example 3b - Analysis of ThioFab

[0445] The enzymatic digestion fragments of biotinylated hu4D5Fabv8 (A121C) ThioFab and wild-type hu4D5Fabv8 were analyzed by liquid chromatography electrospray ionization mass spectrometry (LS-ESI-MS). The difference between the original mass of biotinylated hu4D5Fabv8 (A121C) 48294.5 and the original mass of wild-type hu4D5Fabv8 47737.0 was 557.5 mass units. This fragment indicated the presence of a single biotin-PEO-maleimide moiety (C 23 H 36 Table 8 shows the fragmentation values, which confirm the sequence. Table 8 LC-ESI-MS analysis of biotinylated hu4D5Fabv8 ThioFab A121C after trypsin digestion

[0446]

[0447]

[0448] Biotinylated ABP-hu4D5Fabv8-A121C, biotinylated ABP-hu4D5Fabv8-V110C, biotinylated bis-Cys ABP-hu4D5Fabv8-(V110C-A88C), and biotinylated bis-Cys ABP-hu4D5Fabv8-(V110C-A121C) were analyzed by SDS-PAGE gel with and without DTT or BME reduction before and after Superdex-200 gel filtration.

[0449] Mass spectrometry analysis (MS / MS) of hu4D5Fabv8-(V110C)-BMPEO-DM1 (after Superdex-200 gel filtration purification): Fab+1 51607.5; Fab 50515.5. The data indicate 91.2% coupling. MS / MS analysis of hu4D5Fabv8-(V110C)-BMPEO-DM1 (reduced): LC 23447.2; LC+1 24537.3; HC (Fab) 27072.5. The data indicate that all DM1 coupling is on the light chain of Fab.

[0450] Example 4 - Preparation of ABP-hu4D5Fabv8-(V110C) by coupling ABP-hu4D5Fabv8-(V110C) and MC-MMAE (V110C)-MC-MMAE

[0451] According to US 7521541, US 7659241, and US 7498298, the drug linker reagent maleimidocaproyl-monomethyl auristatin E (MMAE), ie MC-MMAE, dissolved in DMSO was diluted with known concentrations of acetonitrile and water and added to cooled ABP-hu4D5Fabv8-(V110C)ThioFab in phosphate buffered saline (PBS). After about 1 hour, an excess of maleimide was added to quench the reaction and cap any unreacted antibody thiols. The reaction mixture was concentrated by centrifugal ultrafiltration and ABP-hu4D5Fabv8-(V110C)-MC-MMAE was purified and desalted by elution through G25 resin in PBS, filtered through a 0.2 μm filter under sterile conditions and frozen for storage.

[0452] Example 5 - Preparation of ABP-hu4D5Fabv8- by coupling ABP-hu4D5Fabv8-(LC V110C) and MC-MMAF (LC V110C)-MC-MMAF

[0453] According to the procedure of Example 4, ABP-hu4D5Fabv8-(LC V110C)-MC-MMAF was prepared by coupling ABP-hu4D5Fabv8-(LC V110C)ThioFab and MC-MMAF.

[0454] Example 6 - Preparation of ABP-HC by coupling A121C-ThioFab and MC-val-cit-PAB-MMAE A121C-ThioFab-MC-val-cit-PAB-MMAE

[0455] According to the procedure of Example 4, ABP-hu4D5Fabv8-(HC A121C)-MC-val-cit-PAB-MMAE was prepared by coupling ABP-hu4D5Fabv8-(HC A121C) and MC-val-cit-PAB-MMAE.

[0456] Example 7 - Preparation of ABP-HC by coupling A121C-ThioFab and MC-val-cit-PAB-MMAF A121C-ThioFab-MC-val-cit-PAB-MMAF

[0457] According to the procedure of Example 4, ABP-hu4D5Fabv8-(HC A121C)-MC-val-cit-PAB-MMAF was prepared by coupling ABP-hu4D5Fabv8-(HC A121C) and MC-val-cit-PAB-MMAF.

[0458]

[0459] Example 8 - Preparation of hu4D5Fabv8-(LCV110C)ThioFab-BMPEO-DM1

[0460] The free cysteines on hu4D5Fabv8-(V110C)ThioFab were modified with the bis-maleimide reagent BM(PEO)4 (Pierce Chemical), leaving unreacted maleimide groups on the surface of the antibody. This process was accomplished by dissolving BM(PEO)4 in a 50% ethanol / water mixture to a concentration of 10 mM and adding a 10-fold molar excess of about 1.6 mg / ml (10 micromoles) of BM(PEO)3 to a solution containing hu4D5Fabv8-(V110C)ThioFab in phosphate buffered saline and allowing it to react for 1 hour. Excess BM(PEO)3 was removed by gel filtration in 30 mM citrate, pH 6 containing 150 mM NaCl buffer (HiTrap column, Pharmacia). About a 10-fold molar excess of DM1 dissolved in dimethylacetamide (DMA) was added to the hu4D5Fabv8-(LC V110C)ThioFab-BMPEO intermediate. Dimethylformamide (DMF) can also be used to dissolve the drug moiety reagent. The reaction mixture is allowed to react overnight, after which it is gel filtered or dialyzed into PBS to remove unreacted drug. Gel filtration on an S200 column in PBS is used to remove high molecular weight aggregates and obtain purified hu4D5Fabv8-(LC V110C)ThioFab-BMPEO-DM1.

[0461] hu4D5Fabv8(HC A121C)ThioFab-BMPEO-DM1 was prepared by the same protocol.

[0462] Example 9 - In vitro cell proliferation assay

[0463] ADC efficacy was determined by a cell proliferation assay (CellTiter Glo Luminiscent Cell Viability Assay, Promega Corp. Technical Bulletin TB288; Mendoza et al. (2002) Cancer Res. 62:5485-5488) using the following protocol:

[0464] 1. Make the culture medium contain about 10 4 Aliquots of 100 μl of cell culture of 10 cells (SKBR-3, BT474, MCF7 or MDA-MB-468) were deposited in each well of a 96-well opaque-walled plate.

[0465] 2. Prepare control wells containing culture medium but no cells.

[0466] 3. Add ADC to the experimental wells and incubate for 3-5 days.

[0467] 4. Allow the plate to equilibrate to room temperature for approximately 30 minutes.

[0468] 5. Add a volume of CellTiter-Glo Reagent equal to the volume of cell culture medium present in each well.

[0469] 6. Mix the contents on an orbital shaker for 2 minutes to induce cell lysis.

[0470] 7. Incubate the plate at room temperature for 10 minutes to stabilize the luminescent signal.

[0471] 8. Luminescence was recorded and reported in the graphs as RLU = relative luminescence units.

[0472] Some cells were seeded on 96-well plates with 1000-2000 / hole (PC3 system) or 2000-3000 / hole (OVCAR-3), 50uL / hole. After 1 (PC3) or 2 (OVCAR-3) days, the ADC added in 50 μ L volume to a final concentration of 9000, 3000, 1000, 333, 111, 37, 12.4, 4.1 or 1.4 ng / mL, wherein the "no ADC" control well only receives culture medium. Conditions are duplicate or triplicate. After 3 (PC3) or 4-5 (OVCAR-3) days, 100 μ L / hole Cell TiterGlo II (based on luciferase test; Proliferation determined according to ATP levels) was added and cell counts were determined using a luminometer. Data were plotted as the luminescent mean values ​​of each group of replicates, wherein standard deviation bar graphs were used. This protocol is a modification of the CellTiter Glo Luminiscent Cell Viability Assay (Promega):

[0473] 1. Plate the cells with 1000 cells / well of PC3 / Muc16, PC3 / neo (in 50 μL / well) of culture medium. Ovcar3 cells should be plated with 2000 cells / well (in 50 μL) of their culture medium (the formula of the culture medium is as follows). Allow the cells to attach overnight.

[0474] 2. Serially dilute the ADC 1:3 in culture medium starting at a working concentration of 18 μg / ml (this results in a final concentration of 9 μg / ml). Add 50 μL of diluted ADC to the 50 μL of cells and culture medium already in the well.

[0475] 3. Incubate for 72-96 hours (the standard is 72 hours, but when the cells reach 85-95% confluence, a concentration of 0 ug / mL is observed and the assay is terminated).

[0476] 4. Add 100 μL / well of Promega Cell Titer Glo reagent, shake for 3 minutes and read on a luminometer.

[0477] Media: PC3 / neo and PC3 / MUC16 were grown in 50 / 50 / 10% FBS / glutamine / 250 μg / mL G-418OVCAR-3 was grown in RPMI / 20% FBS / glutamine.

[0478] Example 10 - In vivo efficacy of tumor growth inhibition in transgenic explant mice that highly express HER2

[0479] Animals suitable for transgenic experiments can be obtained from standard commercial sources, such as Taconic (Germantown, NY). Many strains are suitable, but FVB female mice are preferred because they are highly susceptible to tumor formation. FVB male mice are used for mating and vasectomized CD.1 breeders are used to stimulate pseudopregnancy. Vasectomized mice can be obtained from any commercial supplier. Founders are bred using FVB mice or 129 / BL6 x FVB p53 heterozygous mice. Mice heterozygous for the p53 allele are used to potentially increase tumor formation. However, it has been shown that this is unnecessary. Therefore, some F1 tumors have mixed strains. The tumors of the founders are only FVB. Six founders with some degree of tumorigenesis but no litters were obtained.

[0480] Animals bearing tumors (allogeneic transplants from Fo5 mmtv transgenic mice) were treated with single or multiple doses of ADC by IV injection. Tumor volume was evaluated at various time points after injection.

[0481] Tumors readily develop in transgenic mice that express a mutant activated form of neu, a homolog of rat HER2, but not mutated HER2 overexpressed in human breast cancer, and tumor formation is much lower than in transgenic mice overexpressing unmutated HER2 (Webster et al. (1994) Semin. Cancer Biol. 5:69-76).

[0482] To improve tumor formation with unmutated HER2, transgenic mice were generated using HER2 cDNA plasmids in which the upstream of the ATG was deleted to prevent translation initiation at such upstream ATG codons, which would otherwise reduce the frequency of translation initiation from the downstream true start codon of HER2 (e.g., see Child et al. (1999) J. Biol. Chem. 274: 24335-24341). In addition, a chimeric intron was added to the 5' end, which should also increase expression levels 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 is derived from the Promega vector, i.e., the Pci-neo mammalian expression vector (bp 890-1022). The cDNA 3'-end is located at the flank of human growth hormone exon 4 and 5 and the polyadenylation sequence. In addition, FVB mice are used because the strain is more susceptible to tumorigenesis. The promoter from MMTV-LTR is used to ensure tissue-specific HER2 expression in the mammary gland. Animals are fed AIN76A diet to increase the susceptibility to tumor formation (Rao et al. (1997) Breast Cancer Res. and Treatment 45: 149-158).

[0483] Example 11 - Reduction / oxidation of ThioMab for conjugation

[0484] Full-length cysteine ​​engineered monoclonal antibody (ThioMab) expressed in CHO cells was reduced with approximately 50-fold excess of TCEP (tris(2-carboxyethyl)phosphine hydrochloride; Getz et al. (1999) Anal. Biochem. Vol 273:73-80; Soltec Ventures, Beverly, MA) at 37°C for 3 hours. The reduced ThioMab ( Fig.11 ) 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 eluted reduced ThioMab was treated with 200 nM aqueous copper sulfate (CuSO4) overnight at room temperature. Dehydroascorbic acid (DHAA) and ambient air oxidation are also effective oxidants.

[0485] Example 12 - Conjugation of ThioMab

[0486] The reoxidized ThioMabs from Example 11, including thio-trastuzumab (HC A121C), thio-2H9 (A121C) and thio-3A5 (A121C), were combined with a 10-fold excess of the drug-linker intermediate BM (PEO) 3-DM1, mixed and kept at room temperature for about 1 hour to couple and form ThioMab antibody-drug conjugates, including thio-trastuzumab (HC A121C)-BMPEO-DM1, thio-2H9 (HC A121C)-BMPEO-DM1 and thio-3A5 (HC A121C)-BMPEO-DM1. The coupling mixture was gel filtered or loaded onto a column and eluted through a HiTrap S column to remove excess drug-linker intermediates and other impurities.

[0487] The present invention is not limited to the scope of the specific embodiments disclosed in the examples, which are used to illustrate several aspects of the present invention and any embodiments that are functionally equivalent belong to the scope of the present invention. In fact, in addition to what is shown and described herein, various modifications of the present invention are also apparent to those skilled in the art and belong to the scope of the claims appended hereto.

[0488] All patents, patent applications, and references cited throughout this specification are expressly incorporated by reference in their entirety for all purposes.

Claims

1. An isolated cysteine ​​engineered antibody comprising a free cysteine ​​amino acid and a sequence selected from SEQ ID NO: 50-98 in the heavy chain or a sequence selected from SEQ ID NO: 99-147 in the light chain, wherein the cysteine ​​in the sequence is a free cysteine ​​amino acid.

2. The cysteine ​​engineered antibody of claim 1, wherein the cysteine ​​engineered antibody binds to one or more of receptors (1)-(36): (1) BMPR1B (bone morphogenetic protein receptor type IB); (2) E16 (LAT1, SLC7A5); (3) STEAP1 (six-transmembrane epithelial cell antigen of the prostate); (4) 0772P (CA125, MUC16); (5) MPF (MPF, MSLN, SMR, megakaryocyte enhancer factor, mesothelin); (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b); (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B); (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA2700050C12 gene); (9) ETBR (endothelin type B receptor); (10) MSG783 (RNF124, putative protein FLJ20315); (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-associated gene 1, prostate cancer-associated protein 1, six-transmembrane epithelial cell antigen of the prostate 2, six-transmembrane prostate protein); (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4); (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratoma-derived growth factor); (14) CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein-Barr virus receptor) or Hs.73792); (15) CD79b (CD79B, CD79β, IGb (immunoglobulin-associated β), B29); (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchoring protein 1a), SPAP1B, SPAP1C); (17) HER2; (18) NCA; (19) MDP; (20)IL20Rα; (21) Brevican; (22) EphB2R; (23)ASLG659; (24) PSCA; (25)GEDA; (26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3); (27) CD22 (B-cell receptor CD22-B isoform); (28) CD79a (CD79A, CD79α, immunoglobulin-related α, B cell-specific protein); (29) CXCR5 (Burkitt lymphoma receptor 1, G protein-coupled receptor); (30) HLA-DOB (MHC class II molecule β subunit (Ia antigen)); (31) P2X5 (purinergic receptor P2X ligand-gated ion channel 5); (32) CD72 (B-cell differentiation antigen CD72, Lyb-2); (33) LY64 (lymphocyte antigen 64 (RP105), a type I membrane protein of the leucine-rich repeat (LRR) family); (34) FcRH1 (Fc receptor-like protein 1); (35) IRTA2 (immunoglobulin superfamily receptor translocation-associated protein 2); and (36) TENB2 (putative transmembrane proteoglycan).

3. The cysteine ​​engineered antibody of claim 1, wherein the antibody is covalently attached to a capture label, a detection label, a drug moiety, or a solid support.

4. The cysteine ​​engineered antibody of claim 3, wherein the antibody is covalently attached to a drug moiety selected from the group consisting of a maytansinoid, an auristatin, a dolastatin, a trichothecene, CC1065, a calicheamicin, an enediyne antibiotic, a taxane, and an anthracycline antibiotic to form an antibody-drug conjugate having Formula I: Ab-(L-D) p I wherein Ab is an antibody, L is a linker, D is a drug moiety, and p is 1, 2, 3, or 4.

5. The cysteine ​​engineered antibody of claim 4, wherein the antibody-drug conjugate has the following structure:

6. The cysteine ​​engineered antibody of claim 4, wherein D is a maytansinoid having the following structure: The wavy line indicates the covalent attachment of the sulfur atom of D to the linker; R is independently selected from H, 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; and m is 1, 2, or 3.

7. The cysteine ​​engineered antibody of claim 6, wherein D is selected from the following structures:

8. The cysteine ​​engineered antibody of claim 7, having the following structure: Where n is 0, 1, or 2.

9. The cysteine ​​engineered antibody of claim 4, wherein D is a monomethyl auristatin drug moiety MMAE or MMAF having the following structure:

10. The cysteine ​​engineered antibody of claim 9, wherein the antibody-drug conjugate is selected from the following structures: wherein Val is valine; Cit is citrulline; and p is 1, 2, 3 or 4.

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