Cysteine-modified antibodies and conjugates

Abstract

To provide novel cysteine engineered antibodies comprising a free cysteine amino acid in a heavy chain or light chain and to provide conjugates.SOLUTION: A cysteine engineered antibody comprising a free cysteine amino acid in a heavy chain or light chain is prepared by: mutagenizing a nucleic acid sequence of a parent antibody so as to encode the cysteine engineered antibody; replacing one or more amino acid residues by cysteine; expressing the cysteine engineered antibody; and isolating the cysteine engineered antibody.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 050,022, filed on 12 September 2014, which is incorporated herein by reference in its entirety.

[0003] This invention generally relates to antibodies engineered with reactive cysteine ​​residues, and more specifically, to antibodies having therapeutic or diagnostic applications. Cysteine-engineered antibodies can be conjugated with affinity ligands such as chemotherapeutic agents, toxins, and biotin, and detection labels such as fluorophores. This invention also relates to methods of using antibodies and antibody-drug conjugate compounds for the diagnosis or treatment of mammalian cells or related pathological conditions in vitro, in sights, and in vivo. [Background technology]

[0004] Antibody-drug conjugates (ADCs) are promising targeted chemotherapy molecules because they combine the ideal properties of both antibodies and cytotoxic agents by targeting the antigen-expressing tumor cells of potent cytotoxic drugs, thereby enhancing their antitumor activity. Successful ADC development for a given target antigen depends on optimizing antibody selection, linker stability, cytotoxic agent potency, and the mechanism of linker-drug conjugation to the antibody.

[0005] Conventional methods of attaching drug moieties to antibodies, i.e., covalent bonding, generally result in a heterogeneous mixture of molecules in which the drug moieties are attached to many sites on the antibody. For example, cytotoxic drugs are typically conjugated to antibodies via lysine residues, which are abundant on antibodies, producing a heterogeneous antibody-drug conjugate mixture. Depending on the reaction conditions, the heterogeneous mixture usually contains dispersed antibodies with 0 to approximately 8 or more drug moieties attached. In addition, within each subgroup of conjugates where the ratio of drug moieties to antibody is a specific integer ratio, there are potentially heterogeneous mixtures in which the drug moieties are attached to various sites on the antibody. Analytical and preparation methods are inadequate for classifying and characterizing the antibody-drug conjugate species molecules contained in heterogeneous mixtures obtained by conjugation reactions. Antibodies are large, complex, and structurally diverse biomolecules, often possessing numerous reactive functional groups. Their reactivity with linker reagents and drug-linker intermediates depends on factors such as pH, concentration, salt concentration, and cosolvent. Furthermore, multi-step conjugation processes may be irreproducible due to the difficulty in controlling reaction conditions and characterizing reactants and intermediates.

[0006] Unlike most amines, which are protonated around pH 7 and have low nucleophilicity, cysteinethiols react at neutral pH. Because free thiol (RSH, sulfhydryl) groups are relatively reactive, proteins containing cysteine ​​residues often exist in their oxidized form as disulfide-bonded oligomers, or the disulfide groups are internally cross-linked. The cysteinethiol group of antibodies is generally more reactive to electrophilic conjugation reagents than the amine or hydroxyl group of the antibody, i.e., it is more nucleophilic. Manipulating the cysteinethiol group by mutating various amino acid residues of proteins to cysteine ​​amino acids can be problematic, especially with unpaired (free Cys) residues or residues that are relatively prone to reaction or oxidation. In concentrated protein solutions, whether in the periplasm of E. coli, in the culture supernatant, or in partially or completely purified proteins, unpaired Cys residues on the protein surface pair up and oxidize to form intermolecular disulfides, thereby creating protein dimers or polymers. The formation of disulfide dimers renders the new Cys groups inactive for conjugation to drugs, ligands, or other labels. Furthermore, if the protein forms intermolecular disulfides between newly manipulated Cys residues and existing Cys residues, neither Cys group can be utilized for active site involvement and interaction. Additionally, this protein may become inactive or nonspecific due to misfolding or loss of tertiary structure (Zhang et al (2002) Anal. Biochem. 311:1-9).

[0007] Antibodies having cysteine ​​substitutions at sites where modified cysteine ​​can be used for conjugation (THIOMAB® antibodies), however, do not disrupt immunoglobulin folding and assembly, nor alter antigen binding or effector function (Junutula, et al., 2008b Nature Biotech., 26(8):925-932, Dornan et al (2009) Blood 114(13):2721-2729, US Publication No. 7521541, US Publication No. 7723485, International Publication No. WO2009 / 052249). These THIOMAB® antibodies can then be conjugated with cytotoxic drugs via modified cysteinethiol groups to obtain THIOMAB® antibody-drug conjugates (TDCs) with homogeneous stoichiometry (e.g., up to two drugs per antibody for antibodies with a single modified cysteine ​​site). Studies using multiple antibodies against different antigens have shown that TDCs are as potent as conventional ADCs in xenograft models and tolerate higher doses in relevant preclinical models. THIOMAB® antibodies are engineered to bind to drugs at different sites on the antibody (e.g., specific amino acid positions (i.e., sites) within the light chain-Fab, heavy chain-Fab, and heavy chain-Fc). The in vitro and in vivo stability, efficacy, and PK properties of THIOMAB® antibodies offer unique advantages over conventional ADCs due to their homogeneity and site-specific conjugation with cytotoxic drugs. [Overview of the Initiative]

[0008] This application includes disclosure of a novel isolated cysteine-manipulated antibody (THIOMAB® antibody) containing a free cysteine ​​amino acid in its heavy or light chain.

[0009] One aspect of the present invention is a process for preparing an isolated cysteine-modified antibody (THIOMAB® antibody) by inducing mutations in the nucleic acid sequence of a parent antibody by replacing one or more amino acid residues with cysteine ​​to encode a cysteine-modified antibody, expressing the cysteine-modified antibody, and isolating the cysteine-modified antibody.

[0010] Another aspect of the present invention is an antibody conjugate, wherein an isolated cysteine-modified antibody (THIOMAB® antibody) is covalently bound to a capture label, detection label, drug moiety, or solid support.

[0011] In certain embodiments, the present invention is a cysteine-manipulated antibody comprising a cysteine ​​mutation selected from the cysteine ​​mutations specified in any of Tables 1 to 4, preferably Table 1 or 2. In specific embodiments, the cysteine ​​mutation is a free cysteine ​​amino acid. In certain embodiments, the cysteine ​​mutation in the heavy chain is selected from the cysteine ​​mutations specified in Table 2 or 3. In certain embodiments, the cysteine ​​mutation in the light chain is selected from the cysteine ​​mutations specified in Table 1 or 4. In certain embodiments, the cysteine ​​mutation is selected from the group consisting of HC-I195C, HC-S420C, HC-Y432C, and LC-G64C (according to Kabat numbering). In certain embodiments, the cysteine ​​mutation is selected from the group consisting of HC-Y432C and LC-G64C (according to Kabat numbering). In certain embodiments, the cysteine ​​mutation is a heavy chain mutation and is selected from the group consisting of Y33C, G162C, V184C, I195C, S420C, Y432C, and Q434C (according to Kabat numbering). In certain embodiments, the cysteine ​​mutation is a heavy chain mutation and is selected from the group consisting of R19C, E46C, T57C, Y59C, A60C, M100cC, W103C, G162C, I195C, V258C, S420C, H425C, and N430C (according to Kabat numbering). In certain embodiments, the cysteine ​​mutation is a heavy chain mutation and is selected from the group consisting of Y33C, G162C, V184C, and I195C (according to Kabat numbering). In a particular embodiment, the cysteine ​​mutation is a heavy chain mutation selected from the group consisting of R19C, E46C, Y59C, A60C, M100cC, W103C, V258C, H425C, and N430C (according to Kabat numbering).

[0012] In certain embodiments, the cysteine ​​mutation is a light chain mutation and is selected from the group consisting of Y55C, G64C, T85C, T180C, and N430C (according to Kabat numbering). In certain embodiments, the cysteine ​​mutation is a light chain mutation and is selected from the group consisting of T31C, S52C, G64C, R66C, A193C, and N430C (according to Kabat numbering). In certain embodiments, the cysteine ​​mutation is a light chain mutation and is selected from the group consisting of G64C, T85C, T180C, and N430C (according to Kabat numbering). In certain embodiments, the cysteine ​​mutation is a light chain mutation and is selected from the group consisting of S52C, G64C, R66C, and A193C, N430C (according to Kabat numbering). In specific embodiments, the cysteine ​​mutation in the light chain is selected from a group of cysteine ​​mutations including LC-I106C, LC-R108C, LC-R142C, and LC-K149C based on Kabat numbering (see Figure 1a and Table 1). In preferred embodiments, the cysteine ​​mutation in the light chain is LC-K149C based on Kabat numbering (see Figure 1a). Table 1. Exemplary light chain cysteine ​​mutations JPEG0007924744000001.jpg46170

[0013] In a preferred embodiment, the cysteine ​​mutation in the heavy chain is selected from the group of cysteine ​​mutations including HC-T114C, HC-A140C, HC-L174C, HC-L179C, HC-T187C, HC-T209C, HC-V262C, HC-G371C, HC-Y373C, HC-E382C, HC-S424C, HC-N434C, and HC-Q438C according to EU numbering (see Figure 1b, Table 2). In a preferred embodiment, the cysteine ​​mutation in the heavy chain is HC-A143C according to Kabat numbering (i.e., HC-A140C according to EU numbering) (see Figures 1b and 21, Table 2). In a preferred embodiment, the cysteine ​​mutation in the heavy chain is HC-A174C according to EU numbering (see Figures 1b and 21, Table 2). Table 2. Exemplary heavy chain cysteine ​​mutations JPEG0007924744000002.jpg107170

[0014] In certain embodiments, the cysteine-modified antibody described herein is (i) Mutation generation in the nucleic acid sequence of the parent antibody by replacing one or more amino acid residues with cysteine ​​to encode a cysteine-modified antibody, (ii) Expressing the cysteine-modified antibody, (iii) Prepared by a process including isolating a cysteine-modified antibody.

[0015] In certain embodiments, the cysteine-modified antibody described herein is a fusion protein comprising an albumin-binding peptide (ABP). In specific embodiments, the ABP is a) CDKTHTGGGSQRLMEDICLPRWGCLWEDDF (Sequence ID 144), b) QRLMEDICLPRWGCLWEDDF (Sequence ID 145), c) QRLIEDICLPRWGCLWEDDF (Sequence ID 146), d) RLIEDICLAPRWGCLWEDD (Sequence ID 147), or e) Contains an array selected from DICLPRWGCLW (sequence number 148).

[0016] In certain embodiments, the cysteine-modified antibodies described herein are selected from monoclonal antibodies, antibody fragments, bispecific antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In specific embodiments, the antibody fragment is a Fab fragment.

[0017] In certain embodiments, the cysteine-modified antibody described herein is an anti-HER2 antibody. In certain embodiments, the cysteine-modified antibody described herein is an anti-MUC16 antibody. In certain embodiments, the cysteine-modified antibody described herein is an anti-STEAP1 antibody. In certain embodiments, the cysteine-modified antibody described herein is an anti-CD79b antibody. In certain embodiments, the cysteine-modified antibody described herein is an anti-CD22 antibody. In certain embodiments, the cysteine-modified antibody described herein is an anti-B7H4 antibody. In certain embodiments, the cysteine-modified antibody described herein is an anti-Ly6E antibody. In certain embodiments, the cysteine-modified antibody described herein is an anti-NaPi2b antibody.

[0018] In certain embodiments, the cysteine-modified antibodies described herein bind to one or more of the following receptors (1) to (53): (1) BMPR1B (Bone morphogenetic protein receptor IB type), (2) E16 (LAT1, SLC7A5), (3) STEAP1 (prostate 6-transmembrane epithelial antigen), (4) 0772P (CA125, MUC16), (5) MPF (MPF, MSLN, SMR, megakaryocyte-enhancing factor, mesothelin), (6) Napi3b (also known as NaPi2b) (NAPI-3B, NPTIIb, SLC34A2, solute transporter 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, 7 thrombospongin 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 cDNA 2700050C12 gene), (9) ETBR (endothelin type B receptor), (10) MSG783 (RNF124, hypothetical protein FLJ20315), (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-related gene 1, prostate cancer-related protein 1, six-transmembrane epithelial 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, growth factor derived from teratoma), (14) CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein-Barr virus receptor) or Hs.73792), (15) CD79b (CD79B, CD79β, IGb (immunoglobulin-associated beta), B29), (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain-containing phosphatase anchor 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 activator receptor, BlyS receptor 3, BR3, (27) CD22 (B cell receptor CD22-B isoform), (28) CD79a (CD79A, CD79α, immunoglobulin-associated alpha, B cell-specific protein), (29) CXCR5 (Burkitt lymphoma receptor 1, G protein-bound receptor), (30) HLA-DOB (beta subunit of MHC class II molecule (Ia antigen), (31) P2X5 (purine receptor P2X ligand open 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 related 2), (36) TENB2 (presumably a transmembrane proteoglycan), (37) PMEL17 (silver homologue, SILV, D12S53E, PMEL17, SI, SIL), (38) TMEFF1 (a transmembrane protein 1 having an EGF-like domain and two follistatin-like domains, tomoregulin 1), (39) GDNF-Ra1 (GDNF family receptor alpha-1, GFRA1, GDNFR, GDNFRA, RETL1, TRNR1, RET1L, GDNFR-alpha-1, GFR-alpha-1), (40) Ly6E (Lymphocyte antigen 6 complex, gene locus E, Ly67, RIG-E, SCA-2, TSA-1), (41)TMEM46 (shisa homolog 2), (42) Ly6G6D (Lymphocyte antigen 6 complex, gene locus G6D, Ly6-D, MEGT1), (43) LGR5 (Leucine-rich repeated-containing G protein-bound receptor 5, GPR49, GPR67), (44) RET (ret proto-oncogene, MEN2A, HSCR1, MEN2B, MTC1, PTC, CDHF12, Hs.168114, RET51, RET-ELE1), (45) LY6K (Lymphocyte antigen 6 complex, locus K, LY6K, HSJ001348, FLJ35226), (46) GPR19 (G protein-coupled receptor 19, Mm.4787), (47) GPR54 (KISS1 receptor, KISS1R, GPR54, HOT7T175, AXOR12), (48) ASPHD1 (Aspartate beta-hydroxylase domain containing 1, LOC253982), (49) Tyrosinase (TYR, OCAIA, OCA1A, tyrosinase, SHEP3), (50) TMEM118 (ring finger protein, transmembrane 2, RNFT2, FLJ14627), (51) GPR172A (G protein-coupled receptor 172A, GPCR41, FLJ11856, D15Ertd747e), (52) CD33, and (53) CLL-1 (CLEC12A, MICL, and DCAL2).

[0019] In certain specific embodiments, the cysteine-engineered antibodies described herein are covalently bound to a capture label, a detection label, a drug moiety, or a solid support. In specific embodiments, the antibody is covalently bound to a biotin capture label. In specific embodiments, the antibody is covalently bound to a fluorescent dye detection label. In specific embodiments, the fluorescent dye is selected from fluorescein-type, rhodamine-type, dansyl, lissamine, cyanine, phycoerythrin, Texas Red, and analogs thereof. In specific embodiments, the antibody is 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 The antibody is covalently bound to a radionuclide detection label selected from Bi. In a specific embodiment, the antibody is covalently bound to the detection label by a chelate ligand. In a specific embodiment, the chelate ligand is selected from DOTA, DOTP, DOTMA, DTPA, and TETA.

[0020] In certain embodiments, the cysteine-modified antibodies described herein are covalently bound to a drug moiety selected from mytansinoids, auristatin, drastatin, trichothecene, CC1065, calicheamicin, enediine antibiotics, taxanes, and anthracyclines, thereby forming a compound of formula I (i.e., Ab-(LD)). p The antibody-drug conjugate has the following structure: TIFF0007924744000003.tif33169

[0021] In specific embodiments, the drug (D) of formula I conjugated to the cysteine-modified antibody described herein is a mytansinoid having the following structure: TIFF0007924744000004.tif67170

[0022] In the formula, the dashed line indicates a covalent bond between the sulfur atom of D and 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.

[0023] In a specific embodiment, the drug (D) of formula I conjugated to the cysteine-modified antibody described herein has the following structure: Selected from TIFF0007924744000005.tif239170.

[0024] In a specific embodiment, the drug (D) of formula I conjugated to the cysteine-modified antibody described herein has the following structure: The expression has TIFF0007924744000006.tif73170, where n is 0, 1, or 2.

[0025] In a specific embodiment, the drug (D) of formula I conjugated to the cysteine-modified antibody described herein has the following structure: The monomethyl auristatin drug part MMAE or MMAF has the code TIFF0007924744000007.tif64170.

[0026] In a particular embodiment of the present invention, the cysteine-modified antibody described herein has the following structure: The formula has one of the following: TIFF0007924744000008.tif146170, where Val is valine, Cit is citrulline, and p is 1, 2, 3, or 4. In certain embodiments of the present invention, the cysteine-modified antibody described herein has the following structure: TIFF0007924744000009.tif27170

[0027] In certain embodiments, the cysteine-modified antibodies described herein are conjugated to drugs belonging to one of the following classes, which are described in more detail in later sections herein: auristatin, drastatin, trichothecene, CC1065, calicheamicin, engine antibiotics, taxanes, pyrrolobenzodiazepines (PBD), 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI) dimers, CBI-PBD heterodimers, and anthracyclines.

[0028] In certain embodiments, the linker (L) of the cysteine-modified antibody described herein comprises a thiol-reactive substance. In specific embodiments, the linker (L) is selected from the group consisting of maleimide, iodoacetamide, and pyridyl disulfide. In specific embodiments, the linker (L) is pyridyl disulfide. In specific embodiments, the linker (L) is pyridyl disulfide and drug (D) is MMAE.

[0029] In a particular embodiment, the present invention includes a method for preparing an antibody-drug conjugate, the method comprising reacting at least one free cysteine ​​of a cysteine-modified antibody (Ab) with a linker-drug (LD) reagent to form the formula (i.e., Ab-(LD) pThe present invention comprises forming an antibody-drug conjugate having the formula (i.e., Ab-(LD)), where Ab is a cysteine-manipulated antibody, L is a linker, D is a drug moiety, and p is 1, 2, 3, or 4, wherein the cysteine-manipulated antibody comprises one or more free cysteine ​​amino acids, at least one free cysteine ​​amino acid selected from cysteine ​​mutations identified in either Table 1 or 2, or from alternative stable cysteine ​​mutations identified in Table 3 or 4. In certain embodiments, the present invention comprises a method for preparing an antibody-drug conjugate, the method comprising reacting at least one free cysteine ​​of a cysteine-manipulated antibody (Ab) with a linker-drug (LD) reagent to form the formula (i.e., Ab-(LD) p The process involves forming an antibody-drug conjugate having the formula, where Ab is a cysteine-modified antibody, L is a linker, D is a drug moiety, and p is 1, 2, 3, or 4, wherein the cysteine-modified antibody comprises one or more free cysteine ​​amino acids, at least one of which is preferably selected from the cysteine ​​mutations identified in Tables 1 and 2, or from alternative stable cysteine ​​mutations identified in Table 3 or 4.

[0030] In specific embodiments, the present method can be used to produce cysteine-manipulated antibodies, where the cysteine ​​mutation is located in the heavy chain and selected from the cysteine ​​mutations specified in Table 2 or 3. In specific embodiments, the present method can be used to produce cysteine-manipulated antibodies, where the cysteine ​​mutation is located in the light chain and selected from the cysteine ​​mutations specified in Tables 1 and 4. In preferred embodiments, the present method can be used to produce cysteine-manipulated antibodies, where the cysteine ​​mutation is selected from the cysteine ​​mutations specified in Figure 21. In other embodiments, the present method can be used to produce cysteine-manipulated antibodies, where the cysteine ​​mutation is selected from the cysteine ​​mutations specified in Table 5. In certain embodiments, the present method can be used to produce cysteine-manipulated antibodies, where the cysteine ​​mutation is selected from the group consisting of HC-I195C, HC-S420C, HC-Y432C, and LC-G64C (according to Kabat numbering). In certain embodiments, the present method can be used to produce cysteine-modified antibodies, where the cysteine ​​mutation is selected from the group consisting of HC-Y432C and LC-G64C (according to Kabat numbering). In certain embodiments, the present method can be used to produce cysteine-modified antibodies, where the cysteine ​​mutation is a heavy chain mutation and is selected from the group consisting of Y33C, G162C, V184C, I195C, S420C, Y432C, and Q434C (according to Kabat numbering). In certain embodiments, the present method can be used to produce cysteine-manipulated antibodies, where the cysteine ​​mutation is a heavy chain mutation selected from the group consisting of R19C, E46C, T57C, Y59C, A60C, M100cC, W103C, G162C, I195C, V258C, S420C, H425C, and N430C (according to Kabat numbering). In certain embodiments, the present method can be used to produce cysteine-manipulated antibodies, where the cysteine ​​mutation is a heavy chain mutation selected from the group consisting of Y33C, G162C, V184C, and I195C (according to Kabat numbering).In certain embodiments, the present method can be used to produce a cysteine-manipulated antibody, where the cysteine ​​mutation is a heavy chain mutation and is selected from the group consisting of R19C, E46C, Y59C, A60C, M100cC, W103C, V258C, H425C, and N430C (according to Kabat numbering). In certain embodiments, the present method can be used to produce a cysteine-manipulated antibody, where the cysteine ​​mutation is in the light chain and is selected from the group consisting of Y55C, G64C, T85C, and T180C (according to Kabat numbering). In certain embodiments, the present method can be used to produce a cysteine-manipulated antibody, where the cysteine ​​mutation is in the light chain and is selected from the group consisting of T31C, S52C, G64C, R66C, and A193C (according to Kabat numbering). In certain embodiments, the present method can be used to produce cysteine-modified antibodies, where the cysteine ​​mutation is located in the light chain and is selected from the group consisting of G64C, T85C, and T180C (according to Kabat numbering). In certain embodiments, the present method can be used to produce cysteine-modified antibodies, where the cysteine ​​mutation is located in the light chain and is selected from the group consisting of S52C, G64C, R66C, and A193C (according to Kabat numbering).

[0031] In a preferred embodiment, a cysteine-manipulated antibody can be produced using this method, where the cysteine ​​mutation in the light chain is selected from a group of cysteine ​​mutations including LC-I106C, LC-R108C, LC-R142C, and LC-K149C according to Kabat numbering (see Figures 1a and 21). In a preferred embodiment, a cysteine-manipulated antibody can be produced using this method, where the cysteine ​​mutation in the light chain is LC-K149C according to Kabat numbering (see Figures 1a and 21 and Table 1).

[0032] In a preferred embodiment, a cysteine-manipulated antibody can be produced using this method, where the cysteine ​​mutation in the heavy chain is selected from the group of cysteine ​​mutations including HC-T114C, HC-A140C, HC-L174C, HC-L179C, HC-T187C, HC-T209C, HC-V262C, HC-G371C, HC-Y373C, HC-E382C, HC-S424C, HC-N434C, and HC-Q438C according to EU numbering (see Figures 1b and 21 and Table 2). In a preferred embodiment, a cysteine-manipulated antibody can be produced using this method, where the cysteine ​​mutation in the heavy chain is HC-A140C according to EU numbering (i.e., HC-A136C according to Kabat numbering) (see Figures 1b and 21 and Table 2). In a preferred embodiment, a cysteine-modified antibody can be prepared using this method, where the cysteine ​​mutation in the heavy chain is HC-L174C according to EU numbering (see Figures 1b and 21 and Table 2).

[0033] In a particular embodiment, a cysteine-modified antibody can be prepared using this method, where the cysteine-modified antibody is (i) Mutation generation in the nucleic acid sequence of the parent antibody by replacing one or more amino acid residues with cysteine ​​to encode a cysteine-modified antibody, (ii) Expressing the cysteine-modified antibody, (iii) Prepared by a process including isolating a cysteine-modified antibody.

[0034] In certain embodiments, the present method can be used to produce a cysteine-modified antibody, which is a fusion protein containing an albumin-binding peptide (ABP). In specific embodiments, the ABP is: a) CDKTHTGGGSQRLMEDICLPRWGCLWEDDF (Sequence ID 144), b) QRLMEDICLPRWGCLWEDDF (Sequence ID 145), c) QRLIEDICLPRWGCLWEDDF (Sequence ID 146), d) RLIEDICLAPRWGCLWEDD (Sequence ID 147), or e) Contains an array selected from DICLPRWGCLW (sequence number 148).

[0035] In certain embodiments, this method can be used to produce cysteine-modified antibodies, which are selected from monoclonal antibodies, antibody fragments, bispecific antibodies, human antibodies, and humanized antibodies. In specific embodiments, the antibody fragment is a Fab fragment.

[0036] In certain embodiments, a cysteine-modified antibody can be produced using this method, where the cysteine-modified antibody is an anti-HER2 antibody. In certain embodiments, the cysteine-modified antibody described herein is an anti-MUC16 antibody. In certain embodiments, the cysteine-modified antibody described herein is an anti-STEAP1 antibody. In certain embodiments, the cysteine-modified antibody described herein is an anti-CD79b antibody. In certain embodiments, the cysteine-modified antibody described herein is an anti-CD22 antibody. In certain embodiments, the cysteine-modified antibody described herein is an anti-B7H4 antibody. In certain embodiments, the cysteine-modified antibody described herein is an anti-Ly6E antibody. In certain embodiments, the cysteine-modified antibody described herein is an anti-NaPi2b antibody.

[0037] In a particular embodiment, the present method can be used to produce a cysteine-modified antibody, which binds to one or more of the receptors (1) to (53): (1) BMPR1B (Bone morphogenetic protein receptor IB type), (2) E16 (LAT1, SLC7A5), (3) STEAP1 (prostate 6-transmembrane epithelial antigen), (4) 0772P (CA125, MUC16), (5) MPF (MPF, MSLN, SMR, megakaryocyte-enhancing factor, mesothelin), (6) Napi3b (also known as NaPi2b) (NAPI-3B, NPTIIb, SLC34A2, solute transporter 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, 7 thrombospongin 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 cDNA 2700050C12 gene), (9) ETBR (endothelin type B receptor), (10) MSG783 (RNF124, hypothetical protein FLJ20315), (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-related gene 1, prostate cancer-related protein 1, six-transmembrane epithelial 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, growth factor derived from teratoma), (14) CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein-Barr virus receptor) or Hs.73792), (15) CD79b (CD79B, CD79β, IGb (immunoglobulin-associated beta), B29), (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain-containing phosphatase anchor 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 activator receptor, BlyS receptor 3, BR3, (27) CD22 (B cell receptor CD22-B isoform), (28) CD79a (CD79A, CD79α, immunoglobulin-associated alpha, B cell-specific protein), (29) CXCR5 (Burkitt lymphoma receptor 1, G protein-bound receptor), (30) HLA-DOB (beta subunit of MHC class II molecule (Ia antigen), (31) P2X5 (purine receptor P2X ligand open 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 related 2), (36) TENB2 (presumably a transmembrane proteoglycan), (37) PMEL17 (silver homologue, SILV, D12S53E, PMEL17, SI, SIL), (38) TMEFF1 (a transmembrane protein 1 having an EGF-like domain and two follistatin-like domains, tomoregulin 1), (39) GDNF-Ra1 (GDNF family receptor alpha-1, GFRA1, GDNFR, GDNFRA, RETL1, TRNR1, RET1L, GDNFR-alpha-1, GFR-alpha-1), (40) Ly6E (Lymphocyte antigen 6 complex, gene locus E, Ly67, RIG-E, SCA-2, TSA-1), (41)TMEM46 (shisa homolog 2), (42) Ly6G6D (Lymphocyte antigen 6 complex, gene locus G6D, Ly6-D, MEGT1), (43) LGR5 (Leucine-rich repeated-containing G protein-bound receptor 5, GPR49, GPR67), (44) RET (ret proto-oncogene, MEN2A, HSCR1, MEN2B, MTC1, PTC, CDHF12, Hs.168114, RET51, RET-ELE1), (45)LY6K (lymphocyte antigen 6 complex, gene locus K, LY6K, HSJ001348, FLJ35226), (46) GPR19 (G protein-bound receptor 19, Mm. 4787), (47) GPR54 (KISS1 receptor, KISS1R, GPR54, HOT7T175, AXOR12), (48) ASPHD1 (containing aspartate beta-hydroxylase domain 1, LOC253982), (49) Tyrosinase (TYR, OCAIA, OCA1A, tyrosinase, SHEP3), (50) TMEM118 (ring finger protein, transmembrane 2, RNFT2, FLJ14627), (51) GPR172A (G protein-bound receptor 172A, GPCR41, FLJ11856, D15Ertd747e), (52) CD33, and (53) CLL-1 (CLEC12A, MICL, and DCAL2).

[0038] In certain embodiments, the present method can be used to produce cysteine-modified antibodies, where the cysteine-modified antibody is covalently bound to a capture label, detection label, drug moiety, or solid support. In specific embodiments, the antibody is covalently bound to a biotin capture label. In certain embodiments, the antibody is covalently bound to a fluorescent dye detection label. In certain embodiments, the fluorescent dye is selected from fluoroceine type, rhodamine type, dansyl, lysamine, cyanine, phycoerythrin, Texas Red, and their analogues. In certain embodiments, the antibody is 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 The antibody is covalently bound to a radionuclide detection label selected from Bi. In a specific embodiment, the antibody is covalently bound to the detection label by a chelate ligand. In a specific embodiment, the chelate ligand is selected from DOTA, DOTP, DOTMA, DTPA, and TETA.

[0039] In certain embodiments, the present method can be used to produce a cysteine-modified antibody, which is covalently bonded to a drug moiety selected from mytansinoids, auristatin, drastatin, trichothecene, CC1065, calicheamicin, enediine antibiotics, taxanes, and anthracyclines, to form a compound of formula I (i.e., Ab-(LD)). p The formula has the following characteristics, forming an antibody-drug conjugate where Ab is the antibody, L is the linker, D is the drug moiety, and p is 1, 2, 3, or 4.

[0040] In a specific embodiment, this method can be used to produce a cysteine-modified antibody, where the cysteine-modified antibody has the following structure: It has TIFF0007924744000010.tif32170.

[0041] In a specific embodiment, this method can be used to produce a cysteine-modified antibody, where the drug (D) of the cysteine-modified antibody has the following structure: TIFF0007924744000011.tif67170

[0042] A mytansinoid having the formula, where the dashed line indicates a covalent bond between the sulfur atom of D and 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.

[0043] In a specific embodiment, this method can be used to produce a cysteine-modified antibody, where drug (D) has the following structure: Selected from TIFF0007924744000012.tif239170.

[0044] In a specific embodiment, this method is used to construct: TIFF0007924744000013.tif72170 (where n is 0, 1, or 2) or A cysteine-modified antibody containing TIFF0007924744000014.tif27170 can be produced.

[0045] In a specific embodiment, this method can be used to produce cysteine-modified antibodies, where D is the structure: The monomethyl auristatin drug part MMAE or MMAF has the code TIFF0007924744000015.tif64170.

[0046] In a specific embodiment, this method is used to construct: Cysteine-modified antibodies can be prepared from TIFF0007924744000016.tif148170, where Val is valine, Cit is citrulline, and p is 1, 2, 3, or 4.

[0047] In specific embodiments, the present method can be used to produce cysteine-manipulated antibodies conjugated to drugs belonging to any of the following classes, which are described in more detail in later sections herein: auristatin, drastatin, trichothecene, CC1065, calicheamicin, engine antibiotics, taxanes, pyrrolobenzodiazepines (PBD), 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI) dimers, CBI-PBD heterodimers, and anthracyclines.

[0048] In specific embodiments, the present method can be used to produce cysteine-modified antibodies, where the linker (L) comprises a thiol-reactive substance. In specific embodiments, the linker (L) is selected from the group consisting of maleimide, iodoacetamide, and pyridyl disulfide. In specific embodiments, the linker (L) is pyridyl disulfide. In specific embodiments, the linker (L) is pyridyl disulfide and drug (D) is MMAE.

[0049] In certain embodiments, the method can be used to produce a cysteine-manipulated antibody, where the cysteine-manipulated antibody is an isolated cysteine-manipulated antibody. In certain embodiments, the method can be used to produce an isolated cysteine-manipulated antibody, where the light chain mutation of this isolated cysteine ​​is selected from a group of cysteine ​​mutations including LC-I106C, LC-R108C, LC-R142C, and LC-K149C according to Kabat numbering (see Figures 1a and 21). In certain embodiments, the method can be used to produce an isolated cysteine-manipulated antibody, where the light chain mutation of this isolated cysteine ​​is LC-K149C according to Kabat numbering (see Figures 1a and 21 and Table 1). In certain embodiments, the method can be used to produce an isolated cysteine-manipulated antibody, where the mutation in the heavy chain of the isolated cysteine ​​is selected from the group of cysteine ​​mutations including HC-T114C, HC-A140C, HC-L174C, HC-L179C, HC-T187C, HC-T209C, HC-V262C, HC-G371C, HC-Y373C, HC-E382C, HC-S424C, HC-N434C, and HC-Q438C according to EU numbering (see Figures 1b and 21 and Table 2). In certain embodiments, the method can be used to produce an isolated cysteine-manipulated antibody, where the mutation in the heavy chain of the isolated cysteine ​​is HC-A140C according to EU numbering (i.e., HC-A136C according to Kabat numbering) (see Figures 1b and 21 and Table 2).

[0050] In a preferred embodiment, the cysteine-manipulated antibodies described herein have one of the following cysteine ​​mutations: LC-K149C by Kabat numbering and HC-A140C by EU numbering (see Tables 1 and 2 and Figures 1a and 1b).

[0051] In certain embodiments of the present invention, any cysteine-modified antibody described herein has a thiol reactivity value of 0.8 to 1.0. In certain embodiments of the present invention, any cysteine-modified antibody described herein has a thiol reactivity value of 0.9 to 1.0. In certain embodiments of the present invention, any cysteine-modified antibody described herein has a thiol reactivity value of 0.6 to 0.9. In certain embodiments of the present invention, any cysteine-modified antibody described herein has a thiol reactivity value of 0.5 to 0.7. In certain embodiments of the present invention, any cysteine-modified antibody described herein has a thiol reactivity value of 0.4 to 0.6. In certain embodiments of the present invention, any cysteine-modified antibody described herein has a thiol reactivity value of 0.3 to 0.5. In certain embodiments of the present invention, any cysteine-modified antibody described herein has a thiol reactivity value of 0.2 to 0.4.

[0052] In certain embodiments, the present invention is a pharmaceutical composition comprising any cysteine-modified antibody described herein. [Brief explanation of the drawing]

[0053] [Figure 1A] Figure 1a shows the Kabat numbering scheme for the 4D5 light chain. Figure 1b shows the sequential numbering scheme (left column) of the 4D5 antibody from the N-terminus, compared with the Kabat numbering scheme (center column) and EU numbering (right column). [Figure 1B-1] Figure 1a shows the Kabat numbering scheme for the 4D5 light chain. Figure 1b shows the sequential numbering scheme (left column) of the 4D5 antibody from the N-terminus, compared with the Kabat numbering scheme (center column) and EU numbering (right column). [Figure 1B-2] Figure 1a shows the Kabat numbering scheme for the 4D5 light chain. Figure 1b shows the sequential numbering scheme (left column) of the 4D5 antibody from the N-terminus, compared with the Kabat numbering scheme (center column) and EU numbering (right column). [Figure 1B-3]Figure 1a shows the Kabat numbering scheme for the 4D5 light chain. Figure 1b shows the sequential numbering scheme (left column) of the 4D5 antibody from the N-terminus, compared with the Kabat numbering scheme (center column) and EU numbering (right column). [Figure 2A] This figure shows exemplary sites of cysteine ​​mutagenesis and drug conjugation in antibodies. Bold and underlined residues are exemplary sites of cysteine ​​mutagenesis. Underlined residues are further exemplary sites of cysteine ​​mutagenesis. Figure 2A shows exemplary and further exemplary residues of cysteine ​​mutagenesis in the heavy chain. Figure 2B shows exemplary and further exemplary residues of cysteine ​​mutagenesis in the light chain. Preferred sites for cysteine ​​mutagenesis are indicated by gray shading and include EU-numbered HC-T114C, HC-A140C, HC-L174C, HC-L179C, HC-T187C, HC-T209C, HC-V262C, HC-G371C, HC-Y373C, HC-E382C, HC-S424C, HC-N434C, and HC-Q438C (Figure 2A), as well as Kabat-numbered LC-I106C, LC-R108C, LC-R142C, and LC-K149C (Figure 2B). Preferred EU-numbered HC-A140C (Kabat-numbered HC-A136C) and Kabat-numbered LC-K149C are enclosed in squares and highlighted in large font. [Figure 2B]This figure shows exemplary sites of cysteine ​​mutagenesis and drug conjugation in antibodies. Bold and underlined residues are exemplary sites of cysteine ​​mutagenesis. Underlined residues are further exemplary sites of cysteine ​​mutagenesis. Figure 2A shows exemplary and further exemplary residues of cysteine ​​mutagenesis in the heavy chain. Figure 2B shows exemplary and further exemplary residues of cysteine ​​mutagenesis in the light chain. Preferred sites for cysteine ​​mutagenesis are indicated by gray shading and include EU-numbered HC-T114C, HC-A140C, HC-L174C, HC-L179C, HC-T187C, HC-T209C, HC-V262C, HC-G371C, HC-Y373C, HC-E382C, HC-S424C, HC-N434C, and HC-Q438C (Figure 2A), as well as Kabat-numbered LC-I106C, LC-R108C, LC-R142C, and LC-K149C (Figure 2B). Preferred EU-numbered HC-A140C (Kabat-numbered HC-A136C) and Kabat-numbered LC-K149C are enclosed in squares and highlighted in large font. [Figure 3] This plot shows the peaks of aggregates and monomers used in the analysis and calculation of aggregation. The large peak at 8-10 minutes is the monomer peak, and the small peak at 8 minutes is the aggregate peak. [Figure 4A] The UV280 LC / MS results for the THIOMAB® antibody are shown. Figure 4a shows the conjugation peaks detected by UV280 LC / MS. Figure 4b shows the conjugation peaks for DAR0 (naked antibody), DAR1, and DAR2. [Figure 4B] The UV280 LC / MS results for the THIOMAB® antibody are shown. Figure 4a shows the conjugation peaks detected by UV280 LC / MS. Figure 4b shows the conjugation peaks for DAR0 (naked antibody), DAR1, and DAR2. [Figure 5]This document describes a protocol for performing total antibody cysteine ​​screening. Total antibody screening was performed using PDS-MMAE and MC-vc-MMAE conjugates. Therefore, 648 PDS-MMAE and 648 MC-vc-MMAE (1296 total) THIOMAB® antibodies were prepared and tested. [Figure 6A] The following are representative stability graphs of MC-vc-MMAE and PDS-MMAE conjugates. Figure 6a shows a representative stability graph of the PDS-MMAE LC-R142C THIOMAB® antibody using LC / MS analysis at 0, 48, and 96 hours. Figure 6b shows a representative stability graph of the MC-vc-MMAE LC-R142C THIOMAB® antibody using LC / MS analysis at 0, 48, and 96 hours. [Figure 6B] The following are representative stability graphs of MC-vc-MMAE and PDS-MMAE conjugates. Figure 6a shows a representative stability graph of the PDS-MMAE LC-R142C THIOMAB® antibody using LC / MS analysis at 0, 48, and 96 hours. Figure 6b shows a representative stability graph of the MC-vc-MMAE LC-R142C THIOMAB® antibody using LC / MS analysis at 0, 48, and 96 hours. [Figure 7] This graph shows the stability of different PDS-MMAE THIOMAB™ drugs as evaluated by drug load percentage. [Figure 8] This graph shows the stability of different MC-vc-MMAE THIOMAB™ formulations as evaluated by drug load percentage. [Figure 9] The graphs show the mean DAR over time for LC-K149C, LC-V205C, and HC-A114C THIOMAB® anti-HER2 and anti-CD33 antibodies. [Figure 10A]The structures of exemplary THIOMAB® antibodies are shown. Figure 10a shows the structures of thio-Her2-hu7C2-HC-A118C-disulfide-PBD and thio-Her2-hu7C2-LC-K149C-disulfide-PBD THIOMAB® antibodies. Figure 10b shows the structure of the thio-Her2-hu7C2-LC-K149C-CBI dimer. Figure 10c shows the structure of thio-Her2-hu7C2-LC-K149C-disulfide-CBI-PBD. Figure 10d shows the structure of thio-Her2-hu7C2-LC-K149C-disulfide-PNU. Figure 10e shows the structures of thio-Her2-hu7C2-HC-A118C-maleimide-PNU and thio-Her2-hu7C2-LC-K149C-maleimide-PNU. [Figure 10B] The structures of exemplary THIOMAB® antibodies are shown. Figure 10a shows the structures of thio-Her2-hu7C2-HC-A118C-disulfide-PBD and thio-Her2-hu7C2-LC-K149C-disulfide-PBD THIOMAB® antibodies. Figure 10b shows the structure of the thio-Her2-hu7C2-LC-K149C-CBI dimer. Figure 10c shows the structure of thio-Her2-hu7C2-LC-K149C-disulfide-CBI-PBD. Figure 10d shows the structure of thio-Her2-hu7C2-LC-K149C-disulfide-PNU. Figure 10e shows the structures of thio-Her2-hu7C2-HC-A118C-maleimide-PNU and thio-Her2-hu7C2-LC-K149C-maleimide-PNU. [Figure 10C]The structures of exemplary THIOMAB® antibodies are shown. Figure 10a shows the structures of thio-Her2-hu7C2-HC-A118C-disulfide-PBD and thio-Her2-hu7C2-LC-K149C-disulfide-PBD THIOMAB® antibodies. Figure 10b shows the structure of the thio-Her2-hu7C2-LC-K149C-CBI dimer. Figure 10c shows the structure of thio-Her2-hu7C2-LC-K149C-disulfide-CBI-PBD. Figure 10d shows the structure of thio-Her2-hu7C2-LC-K149C-disulfide-PNU. Figure 10e shows the structures of thio-Her2-hu7C2-HC-A118C-maleimide-PNU and thio-Her2-hu7C2-LC-K149C-maleimide-PNU. [Figure 10D] The structures of exemplary THIOMAB® antibodies are shown. Figure 10a shows the structures of thio-Her2-hu7C2-HC-A118C-disulfide-PBD and thio-Her2-hu7C2-LC-K149C-disulfide-PBD THIOMAB® antibodies. Figure 10b shows the structure of the thio-Her2-hu7C2-LC-K149C-CBI dimer. Figure 10c shows the structure of thio-Her2-hu7C2-LC-K149C-disulfide-CBI-PBD. Figure 10d shows the structure of thio-Her2-hu7C2-LC-K149C-disulfide-PNU. Figure 10e shows the structures of thio-Her2-hu7C2-HC-A118C-maleimide-PNU and thio-Her2-hu7C2-LC-K149C-maleimide-PNU. [Figure 10E]The structures of exemplary THIOMAB® antibodies are shown. Figure 10a shows the structures of thio-Her2-hu7C2-HC-A118C-disulfide-PBD and thio-Her2-hu7C2-LC-K149C-disulfide-PBD THIOMAB® antibodies. Figure 10b shows the structure of the thio-Her2-hu7C2-LC-K149C-CBI dimer. Figure 10c shows the structure of thio-Her2-hu7C2-LC-K149C-disulfide-CBI-PBD. Figure 10d shows the structure of thio-Her2-hu7C2-LC-K149C-disulfide-PNU. Figure 10e shows the structures of thio-Her2-hu7C2-HC-A118C-maleimide-PNU and thio-Her2-hu7C2-LC-K149C-maleimide-PNU. [Figure 11] A diagram (not to exact scale) of the MC-vc-MMAE THIOMAB(trademark) antibody is shown. [Figure 12] A diagram of the PDS-MMAE THIOMAB(trademark) antibody (not to exact scale) is shown. [Figure 13] The diagram shows the enzymatic modification of a specific drug conjugated to an LC-K149C cysteine-modified antibody. The modifications were as follows: cryptophycin underwent amide cleavage, tubulysin underwent acetyl removal (i.e., deacetylation), the CBI-PBD heterodimer linker-drug intermediate underwent carbamate removal, and the taxoid was unstable and showed multiple enzymatic cleavage. [Figure 14A] Figure 14a shows that HC-A140C provides protection of the acetyl group of tubulicin compared to LC-K149C. Specifically, LC-MS showed that when HC-A140C was used as the binding site, it was less degraded than LC-K149C after 24 hours of incubation. Figures 14b and 14c show that HC-A140C was more stable than LC-K149C after 24 hours of incubation using a novel whole blood assay. [Figure 14B]Figure 14a shows that HC-A140C provides protection of the acetyl group of tubulicin compared to LC-K149C. Specifically, LC-MS showed that when HC-A140C was used as the binding site, it was less degraded than LC-K149C after 24 hours of incubation. Figures 14b and 14c show that HC-A140C was more stable than LC-K149C after 24 hours of incubation using a novel whole blood assay. [Figure 14C] Figure 14a shows that HC-A140C provides protection of the acetyl group of tubulicin compared to LC-K149C. Specifically, LC-MS showed that when HC-A140C was used as the binding site, it was less degraded than LC-K149C after 24 hours of incubation. Figures 14b and 14c show that HC-A140C was more stable than LC-K149C after 24 hours of incubation using a novel whole blood assay. [Figure 15] Multiple WB assays demonstrated that acetyl removal was dramatically reduced with HC-A140C compared to LC-K149C. This confirmed that HC-A140C rescues acetyl removal modifications. [Figure 16A] Figure 16a shows that HC-A140C provides protection for the carbamate group of the CBI-PBD heterodimer linker-drug intermediate. Specifically, LC-MS showed that HC-A140C was less degraded than LC-K149C after 24 hours of incubation when used as the binding site (Figure 16a). Figures 16b and 16c show that HC-A140C was more stable than LC-K149C after 24 hours of incubation using a novel whole blood assay. [Figure 16B]Figure 16a shows that HC-A140C provides protection for the carbamate group of the CBI-PBD heterodimer linker-drug intermediate. Specifically, LC-MS showed that HC-A140C was less degraded than LC-K149C after 24 hours of incubation when used as the binding site (Figure 16a). Figures 16b and 16c show that HC-A140C was more stable than LC-K149C after 24 hours of incubation using a novel whole blood assay. [Figure 16C] Figure 16a shows that HC-A140C provides protection for the carbamate group of the CBI-PBD heterodimer linker-drug intermediate. Specifically, LC-MS showed that HC-A140C was less degraded than LC-K149C after 24 hours of incubation when used as the binding site (Figure 16a). Figures 16b and 16c show that HC-A140C was more stable than LC-K149C after 24 hours of incubation using a novel whole blood assay. [Figure 17] This specification demonstrates that HC-A140C was more stable than LC-K149C using the novel whole blood assay described herein. [Figure 18A] Figure 18a shows that HC-A140C provides protection from taxoid modification. Specifically, LC-MS demonstrated that when HC-A140C was used as the binding site, it was less degraded than LC-K149C after 24 hours of incubation. Figures 18b and 18c show that HC-A140C was more stable than LC-K149C after 24 hours of incubation using a novel whole blood assay. [Figure 18B] Figure 18a shows that HC-A140C provides protection from taxoid modification. Specifically, LC-MS demonstrated that when HC-A140C was used as the binding site, it was less degraded than LC-K149C after 24 hours of incubation. Figures 18b and 18c show that HC-A140C was more stable than LC-K149C after 24 hours of incubation using a novel whole blood assay. [Figure 18C] Figure 18a shows that HC-A140C provides protection from taxoid modification. Specifically, LC-MS demonstrated that when HC-A140C was used as the binding site, it was less degraded than LC-K149C after 24 hours of incubation. Figures 18b and 18c show that HC-A140C was more stable than LC-K149C after 24 hours of incubation using a novel whole blood assay. [Figure 19] Using a novel whole blood assay, we demonstrate that HC-A140C was more stable than LC-K149C (for example, in terms of protecting against taxoid drug removal). [Figure 20] This demonstrates that HC-A140C protects cryptophycin from amide and ester cleavage compared to LC-K149C. [Figure 21] The antibody diagram shows the preferred HC and LC cysteine ​​mutations for the PDS and -vc linkers, as mapped. [Modes for carrying out the invention]

[0054] The following describes in detail certain embodiments of the present invention, examples of which are illustrated in the attached structures and formulas. While the present invention is described in conjunction with the listed embodiments, it should be understood that they are not intended to limit the invention to these embodiments. Conversely, the present invention is intended to include all alternative forms, modifications, and equivalents, which may fall within the scope of the invention as defined by the claims.

[0055] Those skilled in the art will understand that there are numerous methods and materials similar to or equivalent to those described herein that can be used in carrying out the present invention. The present invention is by no means limited to the methods and materials described herein.

[0056] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art in the field to which this invention pertains, and are consistent with 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) Immunobiology, 5th Ed., Garland Publishing, New York.

[0057] definition

[0058] Unless otherwise indicated, the following terms and phrases used herein are intended to have the meanings set forth below.

[0059] When a trademark name is used herein, the applicants intend to independently include the product formulations, generic drugs, and the active pharmaceutical components(s) of the product(s) of the trademark name.

[0060] In this specification, the term “antibody” is used in its broadest sense and includes, in particular, monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, insofar as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854-4861). Antibodies may be derived from mouse, human, humanized, chimeric, or 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 numerous binding sites (also called epitopes) that are recognized by the CDRs of multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. Antibodies include full-length immunoglobulin molecules, or molecules containing an immunologically active portion of a full-length immunoglobulin molecule, i.e., an antigen-binding site that immune-specifically binds to a target antigen or a portion thereof, such targets include, but are not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein may be any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. Immunoglobulins may originate from any species. However, in one embodiment, the immunoglobulins are of human, mouse, or rabbit origin.

[0061] An "antibody fragment" is a portion of a full-length antibody, generally including its antigen-binding region 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 produced by Fab expression libraries, anti-idiotype (anti-Id) antibodies, CDRs (complementarity-determining regions), and epitope-binding fragments that immunospecifically bind to cancer cell antigens, viral antigens, or microbial antigens, as well as single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0062] In certain embodiments, the antibodies provided herein are multispecific antibodies, for example, bispecific antibodies. As used herein, the term “multispecific antibody” refers to an antibody that has multiple epitope specificity (i.e., an antigen-binding domain that can bind to two or more different epitopes on one molecule or to epitopes on two or more different molecules).

[0063] In some embodiments, a multispecific antibody is a monoclonal antibody (such as a bispecific antibody) that has binding specificity to at least two different antigen-binding sites. In some embodiments, the first and second antigen-binding domains of a multispecific antibody can bind to two epitopes within the exact same molecule (intramolecular binding). For example, the first and second antigen-binding domains of a multispecific antibody can bind to two different epitopes on the same molecule. In certain embodiments, the two different epitopes to which the multispecific antibody binds are epitopes to which a single monospecific antibody, such as a conventional antibody, or a single variable domain of an immunoglobulin, would not normally bind simultaneously. In some embodiments, the first and second antigen-binding domains of a multispecific antibody can bind to epitopes located within two different molecules (intermolecular binding). For example, the first antigen-binding domain of a multispecific antibody can bind to a certain epitope on one molecule, while the second antigen-binding domain of the multispecific antibody can bind to another epitope on a different molecule, thereby cross-linking the two molecules.

[0064] In some embodiments, the antigen-binding domain of a multispecific antibody (e.g., a bispecific antibody) comprises two VH / VL units, where the first VH / VL unit binds to a first epitope and the second VH / VL unit binds to a second epitope, and each VH / VL unit comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). Such multispecific antibodies include, but are not limited to, full-length antibodies, antibodies having two or more VL and VH domains, and antibody fragments (e.g., Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies and triabodies, antibody fragments linked by covalent or non-covalent bonds). A VH / VL unit further comprising at least a portion of the heavy chain variable region and / or at least a portion of the light chain variable region may also be referred to as an "arm," "hemimer," or "half-antibody." In some embodiments, the hemimer contains a heavy chain variable region portion sufficient to enable the formation of an intramolecular disulfide bond with a second hemimer. In some embodiments, the hemimer includes a knob mutation or a hole mutation that enables heterodimerization with a second hemimer or half-antibody containing a complementary hole mutation or knob mutation, for example. Knob mutations and hole mutations are discussed in more detail below.

[0065] In certain embodiments, the multispecific antibodies provided herein may be bispecific antibodies. As used herein, the term “bispecific antibody” refers to a multispecific antibody containing an antigen-binding domain capable of binding to two different epitopes on one molecule or to epitopes on two different molecules. Bispecific antibodies may also be referred to as having “dual specificity” or being “dual specific.” Exemplary bispecific antibodies may bind to both molecules and any other antigen. In certain embodiments, one of the binding specificities is to the molecule and the other is to CD3. See, for example, U.S. Patent No. 5,821,337. In certain embodiments, a bispecific antibody may bind to two different epitopes on the same molecule. In certain embodiments, a bispecific antibody may bind to two different epitopes on two different molecules. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing the molecule of interest. Bispecific antibodies may be prepared as full-length antibodies or antibody fragments.

[0066] Techniques for producing multispecific antibodies include the recombinant simultaneous expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983), International Publication No. WO93 / 08829, and Traunecker et al., EMBO J.10:3655 (1991)), and the "knob-in-hole" operation (e.g., U.S. Patent No. 5,731,168, International Publication No. WO2009 / 089004, U.S. Publication No. 2009 / 0182127, U.S. Publication No. 2011 / 0287009, Marvin and Zhu, Acta Pharmacol. Sin. (2005) 26(6):649-658, and Kontermann (2005) Acta Pharmacol. Examples include, but are not limited to, Sin., 26:1-9. As used herein, the terms “knob-in-hole” or “KnH” technology refer to techniques that induce two polypeptides to pair together in vitro or in vivo by introducing a bump (knob) into one polypeptide and a cavity (hole) into the other polypeptide at the interface in which they interact. For example, KnH has been introduced at the Fc:Fc binding interface, CL:CH1 interface, or VH / VL interface of an antibody (see, e.g., US Publication 2011 / 0287009, US2007 / 0178552, International Publication WO96 / 027011, WO98 / 050431, Zhu et al., 1997, Protein Science 6:781-788, and WO2012 / 106587). In some embodiments, KnH facilitates the pairing of two different heavy chains during the production of multispecific antibodies. For example, a multispecific antibody having KnH within an Fc region may further include a single variable domain linked to each Fc region, or may further include different heavy chain variable domains that pair with similar or different light chain variable domains.KnH technology can also be used to pair the extracellular domains of two different receptors together, or to pair any other polypeptide sequence containing different target recognition sequences (including, for example, affibodies, peptide bodies, and other Fc fusions).

[0067] As used herein, the term “knob mutation” refers to a mutation that introduces a bump (knob) into a polypeptide at an interface where one polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a whole mutation.

[0068] As used herein, the term “hole mutation” refers to a mutation that introduces a cavity (hole) into a polypeptide at an interface where one polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a knob mutation. A brief, non-limiting consideration is provided below.

[0069] A “protrusion” refers to at least one amino acid side chain that can stabilize a heteromultimer and thereby protrude from the interface of the first polypeptide, for example, to prioritize heteromultimerization over homomultimerization, and thus be positioned within a complementary cavity at the adjacent interface (i.e., the interface of the second polypeptide). Protrusions may exist within the original interface or may be introduced synthetically (e.g., by altering the nucleic acid encoding the interface). In some embodiments, the nucleic acid encoding the interface of the first polypeptide is altered to encode a protrusion. To achieve this, the nucleic acid encoding at least one “prototype” amino acid residue at the interface of the first polypeptide is replaced with a nucleic acid encoding at least one “imported” amino acid residue having a larger side chain volume than the prototype amino acid residue. It will be understood that there may be more than one prototype residue and corresponding imported residues. Side chain volumes of various amino residues are shown, for example, in Table 1 of U.S. Publication No. 2011 / 0287009. Mutations for introducing “protrusions” are sometimes referred to as “knob mutations.”

[0070] In some embodiments, the introduced residue for ridge formation is a native amino acid residue selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). In some embodiments, the introduced residue is tryptophan or tyrosine. In some embodiments, the prototype residue for ridge formation has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.

[0071] A “cavity” refers to at least one amino acid side chain that is recessed from the interface of the second polypeptide and thus accommodates a corresponding bulge on the interface of the adjacent first polypeptide. The cavity may exist within the original interface or may be introduced synthetically (e.g., by altering the nucleic acid encoding the interface). In some embodiments, the nucleic acid encoding the interface of the second polypeptide is altered to encode the cavity. To achieve this, the nucleic acid encoding at least one “prototype” amino acid residue at the interface of the second polypeptide is replaced with DNA encoding at least one “imported” amino acid residue having a smaller side chain volume than the prototype amino acid residue. It will be understood that there may be more than one prototype residue and corresponding imported residues. In some embodiments, the imported residue for cavity formation is a native amino acid residue selected from alanine (A), serine (S), threonine (T), and valine (V). In some embodiments, the imported residue is serine, alanine, or threonine. In some embodiments, the prototype residue for cavity formation has a large side-chain volume, such as tyrosine, arginine, phenylalanine, or tryptophan. Mutations that introduce a "cavity" are sometimes referred to as "hole mutations."

[0072] The ridges are "positionable" within the cavities, meaning that the spatial position of the ridges and cavities on the interface of the first and second polypeptides, respectively, as well as the size of the ridges and cavities, are such that the ridges can be positioned within the cavities without significantly disrupting the normal association of the first and second polypeptides at the interface. Since ridges such as Tyr, Phe, and Trp typically do not extend perpendicular to the interface axis and do not have a desirable three-dimensional structure, the alignment of the ridges with the corresponding cavities may, in some cases, depend on modeling the ridge / cavity pair based on three-dimensional structure, such as that obtained by X-ray crystallography or nuclear magnetic resonance (NMR). This can be achieved using techniques widely accepted in the art.

[0073] In some embodiments, the knob mutation within the IgG1 constant region is T366W (EU numbering). In some embodiments, the hole mutation within the IgG1 constant region includes one or more mutations selected from T366S, L368A, and Y407V (EU numbering). In some embodiments, the hole mutation within the IgG1 constant region includes T366S, L368A, and Y407V (EU numbering).

[0074] In some embodiments, the knob mutation within the IgG4 constant region is T366W (EU numbering). In some embodiments, the hole mutation within the IgG4 constant region includes one or more mutations selected from T366S, L368A, and Y407V (EU numbering). In some embodiments, the hole mutation within the IgG4 constant region includes T366S, L368A, and Y407V (EU numbering).

[0075] Multispecific antibodies can also be produced by manipulating the electrostatic steering effect to create antibody Fc heterodimer molecules (International Publication No. WO2009 / 089004A1), crosslinking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)), producing bispecific antibodies using leucine zippers (see, e.g., Kostelny et al., J.Immunol., 148(5):1547-1553 (1992)), producing bispecific antibody fragments using "diabody" technology (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)), and using single-stranded Fv(sFv) dimers (see, e.g., Gruber et al. They can be prepared by preparing a triplicate antibody, as described in, for example, Tutt et al. J.Immunol. 147:60 (1991), and also by preparing a triplicate antibody as described in, for example, Tutt et al. J.Immunol. 147:60 (1991).

[0076] Manipulated antibodies having three or more functional antigen-binding sites, including "Octopus antibody" or "Dual Variable Domain Immunoglobulin" (DVD), are also included herein (see, for example, U.S. Publication No. 2006 / 0025576A1 and Wu et al. Nature Biotechnology (2007)). Antibodies or fragments herein also include "Dual Acting FAb" or "DAF" which contain a molecule and an antigen-binding site that binds to another different antigen (see, for example, U.S. Publication No. 2008 / 0069820).

[0077] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies in that population are identical, excluding the possibility of natural variations that may exist in small amounts. Monoclonal antibodies are directed to a single antigenic site and are highly specific. Furthermore, in contrast to polyclonal antibody preparations, which contain different antibodies directed to different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage of being able to be synthesized without contamination by other antibodies. The modifier “monoclonal” indicates a characteristic of the antibody that it is obtained from a substantially homogeneous population of antibodies, and is not to be interpreted as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies used according to the present invention may be prepared by the hybridoma method first described in Kohler et al (1975) Nature 256:495, or by the recombinant DNA method (see, for example, U.S. Patent Nos. 4,816,567 and 5,807,715). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, Clackson et al (1991) Nature, 352:624-628 and Marks et al (1991) J. Mol. Biol., 222:581-597.

[0078] The monoclonal antibodies described herein include, in particular, “chimeric” antibodies in which a portion of the heavy chain and / or light chain is identical or identical to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or identical to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, insofar as they exhibit the desired biological activity (U.S. Patent No. 4,816,567 and Morrison et al (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). The chimeric antibodies of interest include “primatized” antibodies that contain a variable domain antigen-binding sequence derived from a non-human primate (e.g., Old World monkeys, apes, etc.) and a human constant region sequence.

[0079] In this specification, “intact antibody” includes a VL domain and a VH domain, as well as a light chain constant domain (CL) and heavy chain constant domains CH1, CH2, and CH3. The constant domains may be the constant domain of the natural sequence (e.g., the constant domain of the natural sequence of humans) or amino acid sequence variants thereof. An intact antibody may have one or more “effector functions,” which refer to biological activity that may be attributable to the antibody’s Fc constant region (the sequence Fc region of the natural sequence or the Fc region of an amino acid sequence variant). Examples of antibody effector functions include C1q binding, complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and downregulation of cell surface receptors such as B cell receptors and BCRs.

[0080] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to generate an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0081] In certain embodiments, the present invention envisions antibody mutants that, by possessing some, but not all, effector functions, are desirable candidates for applications where the in vivo half-life of the antibody is important, but certain effector functions (e.g., complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to confirm that the antibody lacks FcγR binding (and therefore is likely to lack ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only Fc(RIII), while monocytes express Fc(RI), Fc(RII, and Fc(RIII). FcR expression in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of target molecules are given in U.S. Patent No. 5,500,362 (see, for example, Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA This is described in 82:1499-1502 (1985) and U.S. Patent No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be used (see, for example, ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, CA) and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.Alternatively or additionally, the ADCC activity of the molecule of interest may be evaluated in vivo in an animal model, such as the one disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). Alternatively, a C1q binding assay may be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in international publications WO2006 / 029879 and WO2005 / 100402. To evaluate complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996), Cragg, MS et al., Blood 101:1045-1052 (2003), and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life can also be determined using methods known in the art (see, for example, Petkova, S B et al., Int'l.Immunol. 18(12):1759-1769 (2006)).

[0082] In some embodiments, one or more amino acid modifications may be introduced into the Fc portion of the antibody provided herein to increase the binding of IgG to the fetal Fc receptor. In certain embodiments, the antibody contains three mutations by EU numbering, namely M252Y, S254T, and T256E ("YTE mutations") (see also U.S. Patent No. 8,697,650, Dall'Acqua et al., Journal of Biological Chemistry 281(33):23514-23524 (2006)). In certain embodiments, the YTE mutation does not affect the antibody's ability to bind to its homologous antigen. In certain embodiments, the YTE mutation increases the serum half-life of the antibody compared to a natural (i.e., non-YTE mutant) antibody. In some embodiments, the YTE mutation triples the serum half-life of the antibody compared to a natural (i.e., non-YTE mutant) antibody. In some embodiments, the YTE mutation triples the serum half-life of the antibody compared to a natural (i.e., non-YTE mutant) antibody. The YTE mutation doubles the serum half-life of the antibody. In some embodiments, the YTE mutation quadruples the serum half-life of the antibody compared to a natural (i.e., non-YTE mutant) antibody. In some embodiments, the YTE mutation increases the serum half-life of the antibody by at least five times compared to a natural (i.e., non-YTE mutant) antibody. In some embodiments, the YTE mutation increases the serum half-life of the antibody by at least ten times compared to a natural (i.e., non-YTE mutant) antibody. See also, for example, U.S. Patent No. 8,697,650, Dall'Acqua et al., Journal of Biological Chemistry 281(33):23514-23524 (2006).

[0083] In certain embodiments, the YTE variant provides a means to modulate the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of an antibody. In certain embodiments, the YTEO variant provides a means to modulate the ADCC activity of a human antigen-targeted humanized IgG antibody. See also, for example, U.S. Patent No. 8,697,650, and Dall'Acqua et al., Journal of Biological Chemistry 281(33):23514-23524 (2006).

[0084] In certain embodiments, YTE variants enable simultaneous regulation of serum half-life, tissue distribution, and antibody activity (e.g., ADCC activity of IgG antibodies). See also, for example, U.S. Patent No. 8,697,650, and Dall'Acqua et al., Journal of Biological Chemistry 281(33):23514-23524 (2006).

[0085] Antibodies with reduced effector function include those having one or more substitutions at EU-numbered Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include the so-called "DANA" Fc variant (U.S. Patent No. 7,332,581) having substitutions to alanine at EU-numbered residues 265 and 297 (i.e., EU-numbered D265A and N297A), and Fc variants having substitutions at two or more EU-numbered amino acid positions 265, 269, 270, 297, and 327. In certain embodiments, the Fc variant comprises the following two amino acid substitutions, namely D265A and N297A.

[0086] In certain embodiments, proline at position 329 (EU numbering) of the wild-type human Fc region (P329) is replaced with glycine or arginine, or with an amino acid residue large enough to disrupt the proline sandwich at the Fc / Fcγ receptor interface formed between P329 of Fc and the tryptophan residues W87 and W110 of FcgRIII (Sondermann et al.: Nature 406, 267-273 (20 July 2000)). In further embodiments, the at least one further amino acid substitution in the Fc variant is S228P, E233P, L234A, L235A, L235E, N297A, N297D, or P331S, and in yet another embodiment, the at least one further amino acid substitution is L234A and L235A in the human IgG1 Fc region or S228P and L235E in the human IgG4 Fc region, all of which are EU numbered (U.S. Patent No. 8,969,526, which is incorporated in whole by reference).

[0087] In certain embodiments, the polypeptide comprises an Fc variant of the wild-type human IgG Fc region, wherein P329 of the human IgG Fc region is substituted with glycine, and the Fc variant comprises at least two further amino acid substitutions in L234A and L235A of the human IgG1 Fc region or S228P and L235E of the human IgG4 Fc region, the numbering of these residues according to EU numbering (U.S. Patent No. 8,969,526, which is incorporated in whole by reference). In certain embodiments, the polypeptide comprising substitutions of P329G, L234A, and L235A (EU numbering) exhibits reduced affinity for human FcγRIIIA and FcγRIIA, downmodulating ADCC to at least 20% of ADCC induced by the polypeptide comprising the wild-type human IgG Fc region, and / or downmodulating ADCP (U.S. Patent No. 8,969,526, which is incorporated in whole by reference).

[0088] In specific embodiments, the polypeptide comprising an Fc variant of the wild-type human Fc polypeptide comprises a triple mutation, namely an amino acid substitution at position Pro329 according to EU numbering, the L234A mutation, and the L235A mutation (P329 / LALA) (U.S. Patent No. 8,969,526, which is incorporated in whole by reference). In specific embodiments, this polypeptide comprises the following amino acid substitutions, namely P329G, L234A, and L235A according to EU numbering.

[0089] Specific antibody variants exhibiting improved or reduced binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056, International Publication No. WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604(2001)).

[0090] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that enhance ADCC, for example, substitutions at positions 298, 333, and / or 334 (EU numbering) of the Fc region.

[0091] In some embodiments, changes are made to the Fc region that result in altered (i.e., either increased or decreased) C1q binding and / or complement-dependent cytotoxicity (CDC), as described, for example, in U.S. Patent No. 6,194,551, International Publication No. WO99 / 51642, and Idusogie et al. J.Immunol. 164:4178-4184 (2000).

[0092] Antibodies with an extended half-life and improved binding affinity to the fetal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J.Immunol. 117:587 (1976) and Kim et al., J.Immunol. 24:249 (1994)), are described in U.S. Publication No. US2005 / 0014934A1 (Hinton et al.). These antibodies contain an Fc region with one or more substitutions internally that improve the binding affinity of the Fc region to FcRn. Such Fc variants include those with substitutions in one or more of the EU-numbered Fc region residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, for example, substitution of Fc region residue 434 (U.S. Patent No. 7,371,826). For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patents No. 5,648,260, U.S. Patent No. 5,624,821, and International Publication No. WO94 / 29351.

[0093] Depending on the amino acid sequence of the heavy chain constant domain, intact antibodies can be assigned different "classes." Intact immunoglobulin antibodies have five major classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. The morphology of Ig includes hinged or unhinged forms (Roux et al (1998) J.Immunol. 161:4083-4090, Lund et al (2000) Eur. J.Biochem. 267:7246-7256, US Publication No. 2005 / 0048572, US Publication No. 2004 / 0229310).

[0094] A "cysteine-modified antibody" or "cysteine-modified antibody variant" is an antibody in which one or more residues are substituted with cysteine ​​residues. The thiol group(s) of a cysteine-modified antibody can be conjugated with a drug moiety (e.g., via a linker) to form a THIOMAB® antibody (i.e., a THIOMAB® antibody-drug conjugate (TDC)). In certain embodiments, the substituted residues occur at readily available sites on the antibody. By substituting these residues with cysteine, a reactive thiol group is positioned at a readily available site on the antibody, and this reactive thiol group can be used to conjugate the antibody to a drug moiety or other moiety, such as a linker-drug moiety, as further described herein, to create an immunoconjugate. For example, THIOMAB® antibodies are caused by a single mutation in a non-cysteine ​​native residue in the light chain to cysteine ​​(e.g., Kabat numbering G64C, I106C, R108C, K149C, or R142C) or in the heavy chain (e.g., Kabat numbering HC-D101C, HC-V184C, or HC-T205C, or EU numbering HC-T114C, H Antibodies may have C-A140C, HC-L174C, HC-L179C, HC-T187C, HC-T209C, HC-V262C, HC-G371C, HC-Y373C, HC-E382C, HC-S424C, HC-N434C, and HC-Q438C (i.e., HC-A136C according to Kabat numbering is HC-A140C according to EU numbering) (see Figure 1b). In a specific example, THIOMAB® antibodies have a single cysteine ​​mutation in either the heavy chain or the light chain, so that each full-length antibody (i.e., an antibody with two heavy chains and two light chains) has two manipulated cysteine ​​residues.

[0095] The ErbB receptor is a receptor protein tyrosine kinase belonging to the ErbB receptor family, and members of this family are important mediators of cell growth, differentiation, and survival. The ErbB receptor family includes four distinct members: epidermal growth factor receptor (EGFR, ErbB1, HER1), HER2 (ErbB2 or p185neu), HER3 (ErbB3), and HER4 (ErbB4 or tyro2). A panel of anti-ErbB2 antibodies was characterized using the human breast tumor cell line SKBR3 (Hudziak et al (1989) Mol. Cell. Biol. 9(3):1165-1172). Maximum inhibition was achieved using an antibody designated 4D5, which inhibited cell proliferation by 56%. Other antibodies in this panel reduced cell proliferation to a lower degree in this assay. Antibody 4D5 was further found to make breast tumor cell lines overexpressing ErbB2 sensitive to the cytotoxic effects of TNF-α (U.S. Patent No. 5677171).The anti-ErbB2 antibodies discussed in Hudziak et al. (1990) Cancer Research 50:1550-1558, Kotts et al. (1990) In Vitro 26(3):59A, Sarup et al. (1991) Growth Regulation 1:72-82, Shepard et al. J. (1991) Clin. Immunol. 11(3):117-127, Kumar et al. (1991)Mol. Cell. Biol. 11(2):979-986, Lewis et al. (1993)Cancer Immunol. Immunother. 37:255-263, Pietras et al. (1994)Oncogene 9:1829-1838, Vitetta et al. (1994) Cancer Research The ErbB receptor is characterized in 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. The ErbB receptor will generally contain an extracellular domain capable of binding to an ErbB ligand, a lipophilic transmembrane domain, a conserved intracellular tyrosine kinase domain, and a carboxyl-terminal signaling domain with multiple phosphorylated tyrosine residues. The ErbB receptor may be the “naturally occurring” ErbB receptor or an “amino acid sequence variant” thereof. Preferably, the ErbB receptor is the naturally occurring human ErbB receptor. Therefore, “members of the ErbB receptor family” include EGFR(ErbB1), ErbB2, ErbB3, and ErbB4.

[0096] The term "amino acid sequence variant" refers to a polypeptide that has an amino acid sequence that differs to some extent from that of the naturally occurring polypeptide. Amino acid sequence variants have substitutions, deletions, and / or insertions at specific positions within the amino acid sequence of the natural sequence. Amino acids are represented by their conventional names, one-letter, and three-letter codes.

[0097] For example, in a particular embodiment, the amino acid sequence variant has at least about 70% sequence identity with at least one receptor-binding domain of a native ErbB ligand or at least one ligand-binding domain of a native ErbB receptor, and preferably the sequence is at least about 80%, more preferably about 90%, homologous to such receptor or ligand-binding domain.

[0098] "Sequence identity" is defined as the percentage of amino acid sequence variant residues that are identical after aligning sequences and introducing gaps as necessary to achieve the maximum sequence identity percentage. Alignment methods and computer programs are well known in the art. One such computer program is "Align 2," developed by Genentech, Inc., which was filed with the United States Copyright Office, Washington, DC 20559, on December 10, 1991, along with user documentation.

[0099] "Natural antibodies" are typically heterotetrameric glycoproteins with approximately 150,000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is attached to a heavy chain by one disulfide covalent bond, although the number of disulfide bonds varies depending on the heavy chain of a different immunoglobulin isotype. Each heavy and light chain also has regularly separated interchain disulfide bridges. Each heavy chain has a variable domain (V) at one end. H Each light chain has a variable domain (V) at one end. LIt has a constant domain at the other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. Certain amino acid residues are thought to form the boundary between the light chain variable domain and the heavy chain variable domain.

[0100] The term "variable" refers to the fact that certain parts of the variable domain have significantly different sequences depending on the antibody, and these are used for the binding and specificity of each particular antibody to a particular antigen. However, variability is not evenly distributed throughout the variable domain of an antibody. It is concentrated in three segments called hypervariable regions in both the light and heavy chain variable domains. The more highly conserved parts of the variable domain are called framework regions (FRs). The variable domains of the natural heavy and light chains each contain four FRs, which primarily take the form of β-sheet structures, and these are linked by three hypervariable regions, thereby forming loops that link the β-sheet structures, or in some cases, loops that form part of them. The hypervariable regions in each chain are held in close proximity to the hypervariable regions of the other chain by the FRs, contributing to the formation of the antibody's antigen-binding site (see Kabat et al (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD). The constant domain does not directly participate in the binding of the antibody to the antigen, but it exhibits various effector functions, such as involvement in the antibody-dependent cytotoxicity (ADCC) of that antibody.

[0101] As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody responsible for antigen binding. The hypervariable region generally includes amino acid residues from the "complementarity-determining region" or "CDR" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain, and residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain, Kabat et al. (see above)), and / or residues from the "hypervariable loop" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain, and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain, Chothia and Lesk (1987) J.Mol. Biol., 196:901-917). A "framework region" or "FR" residue is a variable domain residue other than the hypervariable region residues as defined herein.

[0102] Papain degradation of the antibody yields two identical antigen-binding fragments called "Fab," each having a single antigen-binding site, and the remaining "Fc" fragment. Pepsin treatment yields an F(ab')2 fragment with two antigen-binding sites that can still crosslink to an antigen.

[0103] "Fv" is the smallest antibody fragment containing a complete antigen recognition and antigen-binding site. This region consists of a dimer in which one heavy chain and one light chain variable domain are tightly linked by non-covalent bonds. The three hypervariable regions of each variable domain interact to form V H -V L It is in this configuration that the antigen-binding site is determined on the surface of the dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of Fv containing only the three antigen-specific hypervariable regions) has the ability to recognize and bind to the antigen, albeit with lower affinity than the entire binding site.

[0104] The Fab fragment also contains a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in the addition of several residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the hinge region of the antibody. Fab'-SH is the herein notation for Fab' having at least one free thiol group in the cysteine ​​residue(s) of the constant domain. The F(ab')2 antibody fragment was originally produced as a pair with the Fab' fragment, which has a hinge cysteine ​​in between. Other chemical bindings of antibody fragments are also known.

[0105] The "light chains" of antibodies derived from any vertebrate species can be assigned one of two distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant region.

[0106] "Single-stranded Fv" or "scFv" antibody fragments are the V of the antibody. H and V L The scFv polypeptide contains domains, which are present in a single polypeptide chain. Preferably, the Fv polypeptide further contains a polypeptide linker between the VH and VL domains, which allows the scFv to form a structure desirable for antigen binding. For further information on scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). The anti-ErbB2 antibody scFv fragment is described in International Publication No. WO93 / 16185, U.S. Patent No. 5571894, and U.S. Patent No. 5587458.

[0107] The "humanization" of non-human (e.g., rodent) antibodies is a chimeric antibody containing a minimal amount of sequences derived from non-human immunoglobulins. Humanization is a method for transferring mouse antigen-binding information into non-immunogenic human antibody acceptors, resulting in numerous therapeutically useful drugs. The humanization method generally begins by transferring all six mouse complementarity-determining regions (CDRs) into a human antibody framework (Jones et al, (1986) Nature 321:522-525). Antibodies grafted with these CDRs generally do not retain their original affinity for antigen binding, and in fact, affinity is often severely impaired. In addition to the CDRs, selected non-human antibody framework residues also need to be incorporated to maintain the appropriate CDR configuration (Chothia et al, (1989) Nature 342:877). To support the structural arrangement of grafted CDRs, it has been shown that antigen binding and affinity can be restored by transferring key mouse framework residues to human acceptors (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). Humanized antibodies are generally human immunoglobulins (recipient antibodies), in which residues from the hypervariable region of the recipient antibody are replaced with residues from the hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, that possess the desired specificity, affinity, and functionality. In some cases, framework (FR) residues of human immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in the recipient or donor antibody. These modifications may further improve the performance of the antibody.Generally, humanized antibodies will contain substantially all of at least one, typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of non-human immunoglobulins, and all or substantially all of the FRs being human immunoglobulin sequences. Humanized antibodies may optionally contain at least a portion of the immunoglobulin constant region (Fc), typically that of human immunoglobulins. For further details, see U.S. Patent No. 6407213, Jones et al (1986) Nature, 321:522-525, Riechmann et al (1988) Nature 332:323-329, and Presta, (1992) Curr. Op. Struct. Biol., 2:593-596.

[0108] "Free cysteine ​​amino acids" refer to cysteine ​​amino acids that have been modified in the parent antibody, possess a thiol functional group (-SH), and are not paired as intramolecular or intermolecular disulfide bridges.

[0109] The "thiol reactivity value" is a quantitative characterization of the reactivity of free cysteine ​​amino acids. The thiol reactivity value is the proportion of free cysteine ​​amino acids in a cysteine-manipulated antibody that reacts with a thiol-reactive reagent, and is converted to a maximum value of 1. For example, free cysteine ​​amino acids on a cysteine-manipulated antibody that react with a thiol-reactive reagent such as a biotin-maleimide reagent in 100% yield have a thiol reactivity value of 1.0. Another cysteine ​​amino acid manipulated on the same or a different parent antibody that reacts with a thiol-reactive reagent in 90% yield has a thiol reactivity value of approximately 0.9. Another cysteine ​​amino acid manipulated on the same or a different parent antibody that reacts with a thiol-reactive reagent in 80% yield has a thiol reactivity value of approximately 0.8. Another cysteine ​​amino acid manipulated on the same or a different parent antibody that reacts with a thiol-reactive reagent in 70% yield has a thiol reactivity value of approximately 0.7. Another cysteine ​​amino acid manipulated with the same or a different parent antibody that reacts with a thiol-reactive reagent in 60% yield has a thiol reactivity value of approximately 0.6. Another cysteine ​​amino acid manipulated with the same or a different parent antibody that reacts with a thiol-reactive reagent in 50% yield has a thiol reactivity value of approximately 0.5. Another cysteine ​​amino acid manipulated with the same or a different parent antibody that reacts with a thiol-reactive reagent in 40% yield has a thiol reactivity value of approximately 0.4. Another cysteine ​​amino acid manipulated with the same or a different parent antibody that reacts with a thiol-reactive reagent in 30% yield has a thiol reactivity value of approximately 0.3. Another cysteine ​​amino acid manipulated with the same or a different parent antibody that reacts with a thiol-reactive reagent in 20% yield has a thiol reactivity value of approximately 0.2. Another cysteine ​​amino acid manipulated with the same or a different parent antibody that reacts with a thiol-reactive reagent in 10% yield has a thiol reactivity value of approximately 0.1. Another cysteine ​​amino acid manipulated with the same or a different parent antibody that does not react with the thiol-reactive reagent at all has a thiol reactivity value of 0. The thiol reactivity value of a particular cysteine ​​can be determined by ELISA assay, mass spectrometry, liquid chromatography, autoradiography, or other quantitative analysis tests.

[0110] A "parent antibody" is an antibody containing an amino acid sequence derived from a sequence in which one or more amino acid residues are replaced by one or more cysteine ​​residues. Parent antibodies may contain natural or wild-type sequences. Parent antibodies may have existing amino acid sequence modifications (such as additions, deletions, and / or substitutions) compared to other natural, wild-type, or modified forms of the antibody. Parent antibodies may target a target antigen of interest, such as a biologically important polypeptide. Antibodies targeting non-polypeptide antigens (such as tumor-associated glycolipid antigens; see U.S. Patent No. 5091178) are also intended.

[0111] Exemplary parental antibodies include antibodies that possess affinity and selectivity for cell surface and transmembrane receptors and tumor-associated antigens (TAAs).

[0112] "Isolated" antibodies are those identified, separated, and / or recovered from components of their natural environment. Contaminations from their natural environment are materials that interfere with the diagnostic and therapeutic use of the antibody, and these may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody is purified to the following extent: (1) to more than 95% by weight of the antibody as determined by the Lowry method, most preferably more than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spinning cup sequenator; or (3) to homogenize by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue, or preferably silver staining. Isolated antibodies contain insights of the antibody within recombinant cells because at least one component of the antibody's natural environment is absent. However, typically, isolated antibodies will be prepared by at least one purification step.

[0113] An antibody that "binds" to a target molecular target or antigen, such as the ErbB2 antigen, is one that can bind to that antigen with sufficient affinity to be useful in targeting cells expressing the antigen. If the antibody binds to ErbB2, it may typically preferentially bind to ErbB2 rather than other ErbB receptors and not significantly cross-react with other proteins such as EGFR, ErbB3, or ErbB4. In such embodiments, the degree of binding of the antibody to these non-ErbB2 proteins (e.g., cell surface binding to endogenous receptors) would be less than 10% as determined by fluorescence-activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA). Anti-ErbB2 antibodies may not significantly cross-react with rat neu proteins, as described, for example, in Schecter et al. (1984) Nature 312:513 and Drebin et al (1984) Nature 312:545-548.

[0114] Molecular targets of antibodies included in the present invention include CD proteins and their ligands, for example, but are 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 alpha-v / beta-3 integrins (including either their alpha or beta subunits (e.g., anti-CD11a, anti-CD18, or anti-CD11b antibodies)), (iv) VEGF, IgE, blood group antigens, flk2 / flt3 receptors, obesity (OB) receptors, mpl receptors, CTLA-4, protein C, BR3, c-met, tissue factor and other growth factors, and (v) cell surface and transmembrane tumor-associated antigens (TAAs).

[0115] The terms “anti-Ly6E antibody” and “antibody that binds to Ly6E” refer to an antibody that can bind to Ly6E with sufficient affinity to be useful for targeting Ly6E as a diagnostic and / or therapeutic agent. In one embodiment, the degree of binding of the anti-Ly6E antibody to unrelated non-Ly6E proteins is less than about 10% of the binding of this antibody to Ly6E, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to Ly6E is less than 1 μM, less than 100 nM, less than 10 nM, less than 5 nm, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.1 nM, less than 0.01 nM, or less than 0.001 nM (e.g., 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 It has a dissociation constant (Kd) of M). In certain embodiments, the anti-Ly6E antibody binds to Ly6E epitopes that are conserved among Ly6E from different species.

[0116] The terms “anti-STEAP1 antibody” and “antibody that binds to STEAP1” refer to an antibody that can bind to STEAP1 with sufficient affinity to be useful for targeting STEAP1 as a diagnostic and / or therapeutic agent. In one embodiment, the degree of binding of the anti-STEAP1 antibody to unrelated non-STEAP1 proteins is less than about 10% of the binding of this antibody to STEAP1, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to STEAP1 may have concentrations of 1 μM or less, 100 nM or less, 10 nM or less, 5 nm or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 It has a dissociation constant (Kd) of M). In certain embodiments, the anti-STEAP1 antibody binds to an epitope of STEAP1 that is conserved among STEAP1 from different species.

[0117] The terms “anti-CD79b antibody” and “CD79b-binding antibody” refer to an antibody that can bind to CD79b with sufficient affinity to be useful for targeting CD79b as a diagnostic and / or therapeutic agent. In one embodiment, the degree of binding of the anti-CD79b antibody to unrelated non-CD79b proteins is less than about 10% of the binding of this antibody to CD79b, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to CD79b may have concentrations of 1 μM or less, 100 nM or less, 10 nM or less, 5 nm or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 It has a dissociation constant (Kd) of M). In certain embodiments, the anti-CD79b antibody binds to an epitope of CD79b that is conserved among CD79b from different species.

[0118] The terms “anti-MUC16 antibody” and “antibody that binds to MUC16” refer to an antibody that can bind to MUC16 with sufficient affinity to be useful for targeting MUC16 as a diagnostic and / or therapeutic agent. In one embodiment, the degree of binding of the anti-MUC16 antibody to unrelated non-MUC16 proteins is less than about 10% of the binding of this antibody to MUC16, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to MUC16 may have concentrations of ≤1 μM, ≤100 nM, ≤10 nM, ≤5 nm, ≤4 nM, ≤3 nM, ≤2 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example 10 -9 M~10 -13It has a dissociation constant (Kd) of M). In certain embodiments, the anti-MUC16 antibody binds to an epitope of MUC16 that is conserved among MUC16 from different species.

[0119] The terms “anti-HER2 antibody” and “HER2-binding antibody” refer to an antibody that can bind to HER2 with sufficient affinity to be useful for targeting HER2 as a diagnostic and / or therapeutic agent. In one embodiment, the degree of binding of the anti-HER2 antibody to unrelated non-HER2 proteins is less than about 10% of the binding of this antibody to HER2, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to HER2 may be ≤1 μM, ≤100 nM, ≤10 nM, ≤5 nm, ≤4 nM, ≤3 nM, ≤2 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 It has a dissociation constant (Kd) of M). In certain embodiments, the anti-HER2 antibody binds to HER2 epitopes that are conserved among HER2s from different species.

[0120] The terms “anti-CD22 antibody” and “CD22-binding antibody” refer to an antibody that can bind to CD22 with sufficient affinity to be useful for targeting CD22 as a diagnostic and / or therapeutic agent. In one embodiment, the degree of binding of the anti-CD22 antibody to unrelated non-CD22 proteins is less than about 10% of the binding of this antibody to CD22, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to CD22 may have concentrations of 1 μM or less, 100 nM or less, 10 nM or less, 5 nm or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example 10 -9 M~10-13 It has a dissociation constant (Kd) of M. In certain embodiments, the anti-CD22 antibody binds to an epitope of CD22 that is conserved among CD22 from different species.

[0121] The terms “anti-CD79b antibody” and “CD79b-binding antibody” refer to an antibody that can bind to CD79b with sufficient affinity to be useful for targeting CD79b as a diagnostic and / or therapeutic agent. In one embodiment, the degree of binding of the anti-CD79b antibody to unrelated non-CD79b proteins is less than about 10% of the binding of this antibody to CD79b, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to CD79b may have concentrations of 1 μM or less, 100 nM or less, 10 nM or less, 5 nm or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 It has a dissociation constant (Kd) of M). In certain embodiments, the anti-CD79b antibody binds to an epitope of CD79b that is conserved among CD79b from different species.

[0122] The terms “anti-NaPi2b antibody” and “antibody that binds to NaPi2b” refer to an antibody that can bind to NaPi2b with sufficient affinity to be useful for targeting NaPi2b as a diagnostic and / or therapeutic agent. In one embodiment, the degree of binding of the anti-NaPi2b antibody to unrelated non-NaPi2b proteins is less than about 10% of the binding of this antibody to NaPi2b, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to NaPi2b is less than 1 μM, less than 100 nM, less than 10 nM, less than 5 nm, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.1 nM, less than 0.01 nM, or less than 0.001 nM (e.g., 10 -8 M or less, for example, 10 -8 M~10-13 M, for example 10 -9 M to 10 -13 M). In certain specific embodiments, the anti-NaPi2b antibody binds to an epitope of NaPi2b that is conserved among NaPi2b derived from different species.

[0123] Unless otherwise indicated, the term "monoclonal antibody 4D5" refers to an antibody that comprises the antigen-binding residues of mouse 4D5 antibody (ATCC CRL10463) or antigen-binding residues derived therefrom. For example, monoclonal antibody 4D5 may be mouse 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, HERCEPTIN®), as set forth in U.S. Patent No. 5,821,337.

[0124] Unless otherwise indicated, the term "monoclonal antibody 7C2" or "7C2" refers to an antibody that comprises the antigen-binding residues of 7C2.v2.2.LA antibody or antigen-binding residues derived therefrom. The 7C2 antibody is an anti-HER2 antibody.

[0125] "hu7C2.v.2.2.LA Antibody-Drug Conjugate" (hu7C2 ADC) refers to a humanized 7C2 antibody conjugated to a drug. In a specific embodiment, it is a humanized 7C2 antibody conjugated to a drug via an engineered cysteine and a linker. In specific embodiments, the humanized 7C2 ADC is co-administered with one or more additional therapeutic agents selected from trastuzumab (Herceptin®), T-DM1 (Kadcyla®), and pertuzumab (Perjeta®). In some embodiments, hu7C2 ADC is co-administered with trastuzumab. In some embodiments, hu7C2 ADC is co-administered with T-DM1. In some embodiments, hu7C2 ADC is co-administered with pertuzumab. In some embodiments, hu7C2 ADC is co-administered with trastuzumab and pertuzumab. In some embodiments, hu7C2 ADC is co-administered with T-DM1 and pertuzumab.

[0126] The terms "treat" and "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, the objective of which is to prevent or delay (alleviate) an undesirable physiological change or disorder, for example, the development or spread of cancer. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, amelioration or temporary palliation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to the survival expected if the patient did not receive treatment. Those in need of treatment include those who already have the condition or disorder, as well as those prone to have the condition or disorder, or those in need of preventing the condition or disorder.

[0127] The term "therapeutic dose" refers to the amount of a drug effective in treating a disease or disorder in a mammal. In the case of cancer, a therapeutic dose of a drug can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., delay and preferably halt) the invasion of cancer cells into peripheral organs; inhibit (i.e., delay and preferably halt) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate to some extent one or more of the symptoms associated with cancer. A drug can be cell proliferation inhibitory and / or cytotoxic insofar as it can prevent the growth of existing cancer cells and / or kill them. With respect to cancer therapy, efficacy can be measured, for example, by evaluating the time to tumor progression (TTP) and / or determining the rate of response (RR).

[0128] The terms “cancer” and “malignant” refer to or describe a physiological condition in mammals typically characterized by uncontrolled cell growth. A “tumor” includes one or more cancerous cells. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias or malignant lesions of the lymphatic system. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell cancer), lung cancer including small cell lung cancer, non-small cell lung cancer ("NSCLC"), adenocarcinoma of the lung, and squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer.

[0129] "ErbB-expressing cancer" refers to cells in which the ErbB protein is present on the cell surface. "ErbB2-expressing cancer" refers to cells that produce sufficient levels of ErbB2 on their cell surface, so that anti-ErbB2 antibodies can bind to them and have a therapeutic effect against cancer.

[0130] Cancers that "overexpress" antigenic receptors have significantly higher levels of receptors, such as ErbB2, on their cell surface compared to non-cancerous cells of the same tissue type. Such overexpression can be caused by gene amplification or increased transcription or translation. Receptor overexpression can be determined by diagnostic or prognostic assays by assessing the increased levels of receptor protein present on the cell surface (e.g., by immunohistochemical assay, IHC). Alternatively or additionally, levels of nucleic acids encoding the receptor within cells may be measured by fluorescence insight hybridization (FISH, International Publication WO98 / 45479), Southern blotting, or polymerase chain reaction (PCR) techniques, such as real-time quantitative PCR (RT-PCR).

[0131] "Chemotherapy" refers to the use of chemotherapy agents that are useful in treating cancer.

[0132] A “chemotherapeutic agent” is any compound useful for the treatment of cancer, regardless of its mechanism of action. As used herein, “drug” or “drug portion” is an example of a chemotherapeutic agent. Therefore, the THIOMAB® antibody described herein comprises a cysteine-modified antibody, a linker, and a drug, which may be any of the chemotherapeutic agents described herein.

[0133] Classes of chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, spindle toxin plant alkaloids, cytotoxic / antitemocyte antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Examples of chemotherapy agents include erlotinib (TARCEVA®, Genentech / OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS number 51-21-8), gemcitabine (GEMZAR®, Lilly), PD-0325901 (CAS number 391210-10-9, Pfizer), cisplatin (cis-diamine, dichloroplatinum(II), CAS number 15663-27-1), carboplatin (CAS number 41575-94-4), and paclitaxel (TAXOL®, Bristol-Myers Squibb). Examples include Oncology (Princeton, NJ), temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide, CAS number 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen ((Z)-2-[4-(1,2-diphenylbuta-1-enyl)phenoxy]-N,N-dimethylethanamine, NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®), Akti-1 / 2, HPPD, and rapamycin.

[0134] Further examples of chemotherapy agents include oxaliplatin (ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent (SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib methylate (GLEEVEC®, Novartis), XL-518 (MEK inhibitor, Exelixis, International Publication No. WO2007 / 044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, Astra Zeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), and PTK787 / ZK. 222584 (Novartis), Fulvestrant (FASLODEX®, AstraZeneca), Leucovorin (folic acid), Rapamycin (Sirolimus, RAPAMUNE®, Wyeth), Lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), Ronafarnib (SARASAR®, SCH 66336, Schering Plough), Sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11, Pfizer), tipifarnib (ZARNESTRA®, Johnson & Johnson), ABRAXANE® (cremohol-free), albumin-modified nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Il), vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chlorambucil, AG1478, AG1571 (SU5271, Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib (GlaxoSmithKline), camphospamide (TELCYTA®, Telik), thiotepa and cyclosphosphamide (CYTOXAN®, NEOSAR®); alkyl sulfonic acids, e.g., busulfan, improsulfan, and pigosulfan; aziridines, e.g., benzodopa, carbocone, meturedopa, and uredopa; ethyleneimines and methylamelamines, e.g., altoretamine, triethylenemelamine, triethylenethiophosphoramide, and trimethylomellamine; acetogenins (especially , bratacin and bratacinone); camptothecin (including its synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogs azozeresin, karzeresin, and bizeresin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dorastatin; duocalmycin (including its synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictiin; spongistatin; nitrogen mustard, e.g., chlorambucil, chlornafadin, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride. (oxide hydrochloride), melphalan, novembichin, phenesterine, prednimustine, trophosphamide, uracil mustard; nitrosourea, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, e.g., engine antibiotics (e.g., calicheamicin, calicheamicin gamma 1I, calicheamicin omega I1 (Angew Chem. Intl. Ed.)Engl. (1994) 33:183-186); Dynemicin, Dynemicin A; Bisphosphonates, e.g., clodronate; Esperamicin; and neocardinostatin chromophores and related pigment proteins (endiin antibiotic chromophores), acranomycin, actinomycin, autramycin, azaserin, bleomycin, kakutinomycin, carabicin, carminomycin, cardinophilin, chromomycin, Dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, e.g., mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, porphyry Romycin, puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folate analogs, e.g., denopterin, methotrexate, pteropterin, trimethrexate; purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiprine (t hiamiprine), thioguanine; pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenal agents, e.g., aminoglutethimide, mitotane, trilostane; folic acid replenishers, e.g., frolinic acid; acegraton; aldophosphamide glycosideglycoside; aminolevulinic acid; enyluracil; amsacrine; bestrabucil; bistrianthrene; edatraxate; defofamine; demecoltin; diaziquan; elfornithine; eriptinium acetate; epotilone; etogluside; gallium nitrate; hydroxyurea; lentinan; lonidainine; mytansinoids, e.g., mytansin and anthamitosin; mitogluazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; fenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK (registered trademark) polysaccharide complex (JHS Natural Products, Eugene, OR); Lazoxane; Rhizoxin; Schizophyllan; Spirogermanium; Tenuazonic Acid; Triadiquan; 2,2',2”-Trichlorotriethylamine; Trichothecene (T-2 toxin, Beraclin A, Loridine A, and Anguidin); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacytosine; Arabinoside (Ara-C); Cyclophosphamide; Thiote Examples include: 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, e.g., cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (NAVELBINE®); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®, Roche); ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, e.g., retinoic acid; and pharmaceutically acceptable salts, acids, and derivatives of any of the above.

[0135] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. A chemotherapeutic agent (i.e., a "drug" or "drug moiety") may be a cytotoxic agent. Accordingly, the THIOMAB™ antibodies described herein comprise a cysteine-engineered antibody, a linker, and a drug, wherein the drug may be any of the cytotoxic agents described herein. The term includes radioisotopes (e.g., 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 60 C, and Lu radioisotopes), 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.

[0136] "Phage display" is a technique in which mutant polypeptides are presented as fusion proteins with the coat protein on the surface of phages, such as filamentous phage particles. One of the advantages of phage display is that it allows for the rapid and efficient classification of large libraries of random protein mutants in terms of sequences that bind to target molecules with high affinity. Display of peptide and protein libraries on phages has been used to screen millions of polypeptides for those with specific binding properties. Multivalent phage display methods have been used to display small random peptides and small proteins, in particular, through fusion to either pIII or pVIII of filamentous phages (Wells and Lowman, (1992) Curr. Opin. Struct. Biol., 3:355-362 and references cited therein). Monovalent phage display libraries are fused to the phage coat protein or a portion thereof and expressed at low levels in the presence of wild-type protein. Because their binding activity is reduced compared to multivalent phages, classification is based on intrinsic ligand affinity, and phagemide vectors are used, which simplifies DNA manipulation. (Lowman and Wells, Methods: A companion to Methods in Enzymology, 3:205-0216 (1991)). Phage display includes techniques for producing antibody-like molecules (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immunobiology, 5th Ed., Garland Publishing, New York, pp. 627-628, Lee et al).

[0137] A "phagemide" is a plasmid vector containing a bacterial replication origin, e.g., Co1E1, and a copy of the intergenetic region of a bacteriophage. Phagemids can be used with any known bacteriophage, including filamentous and lambdoid bacteriophages. Plasmids will also typically contain selective markers for antibiotic resistance. Segments of DNA cloned into these vectors can be grown as plasmids. When cells containing these vectors are provided with all the genes necessary for phage particle production, the plasmid replication mode changes to rolling circle replication, producing single-stranded copies of the plasmid DNA and packaged phage particles. Phagemids can form infectious or non-infectious phage particles. The term includes phagemides containing a phage coat protein gene or a fragment thereof, bound to a heterologous polypeptide gene as a gene fusion, so that the heterologous polypeptide is displayed on the surface of the phage particle.

[0138] "Linker," "linker unit," or "link" refers to a chemical moiety that includes a covalent bond or a chain of atoms that covalently bond an antibody to the drug moiety. In various embodiments, the linker is denoted as L. The linker may contain divalent radicals, such as alkyldiyl, arylene, heteroarylene, and -(CR2). n O(CR2) n Examples include repeating units of alkyloxy (e.g., polyethyleneoxy, PEG, polymethyleneoxy) and alkylamino (e.g., polyethyleneamino, Jeffamine®), as well as esters and amides of divalent acids, including succinates, succinamides, diglycolates, malonates, and caproamides.

[0139] The term "label" means any part that can covalently bind to an antibody and functions to (i) provide a detectable signal, (ii) interact with a second label to alter a detectable signal provided by a first or second label, e.g., FRET (fluorescence resonance energy transfer), (iii) stabilize the affinity for interaction with or binding to an antigen or ligand, (iv) affect mobility, e.g., electrophoretic mobility or cell permeability, by charge, hydrophobicity, shape, or other physical parameters, or (v) provide a capture portion for regulating ligand affinity, antibody / antigen binding, or ionic complex formation.

[0140] The stereochemical definitions and conventions used herein generally follow SP. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984), McGraw-Hill Book Company, New York, and Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds (1994), John Wiley & Sons, Inc., New York. Many organic compounds exist in optically active forms, that is, they have the ability to rotate the plane of plane polarization. When describing optically active compounds, the prefixes D and L, or R and S, are used to indicate the absolute configuration of the molecule around its chiral center(s). The prefixes d and l, or (+) and (-), are used to specify the rotation of plane polarization by the compound, with (-) or l meaning that the compound is levorotatory. Compounds with the prefix (+) or d are dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. Certain stereoisomers are also called enantiomers, and mixtures of such isomers are sometimes called enantiomer mixtures. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, and these can occur when there is no stereoselectivity or stereospecificity in a chemical reaction or process. The terms “racemic mixture” and “racemate” refer to equimolar mixtures of two enantiomer species that lack optical activity.

[0141] As used herein, the term “pharmaceutically acceptable salt” refers to a pharmaceutically acceptable organic or inorganic salt of ADC. Exemplary salts include, but are not limited to, sulfates, citrates, acetates, oxalates, hydrochlorides, bromides, iodides, nitrates, bisulfates, phosphates, acidic phosphates, isonicotinates, lactates, salicylates, acidic citrates, tartrates, oleates, tannic acids, pantothenic acids, bistartrates, ascorbic acid, succinates, maleates, gentisinates, fumarates, glucons, sugars, formates, benzoates, glutamates, methanesulfons, ethanesulfons, benzenesulfons, p-toluenesulfons, and pamonates (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)). A pharmaceutically acceptable salt may involve the inclusion of another molecule, such as an acetate ion, a succinate ion, or another counterion. The counterion can be any organic or inorganic part that stabilizes the charge in the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. If multiple charged atoms are part of the pharmaceutically acceptable salt, it may have multiple counterions. Therefore, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0142] A "pharmaceutically acceptable solvate" refers to the bonding of one or more solvent molecules with an 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.

[0143] Cysteine-modified antibodies

[0144] The compounds of the present invention include cysteine-modified antibodies in which one or more amino acids of the wild-type or parent antibody are replaced with cysteine ​​amino acids (i.e., "modified cysteine") (i.e., "substituted" or "mutated"). Any form of antibody can be modified, i.e., mutated in this way. For example, a parent monoclonal antibody can be modified to form a "THIOMAB® antibody". One example of a THIOMAB antibody is an antibody fragment (i.e., Fab) having modified cysteine. This Fab THIOMAB® antibody may be referred to as "ThioFab". Note that a single-site mutation results in a single modified cysteine ​​residue in ThioFab, but a single-site mutation results in two modified cysteine ​​residues in a THIOMAB® antibody due to the dimerization of the IgG antibody. The reactivity of the newly introduced modified cysteine ​​thiol group was evaluated in mutants having modified cysteine ​​(Cys) residues. The thiol reactivity value is a relative numerical value in the range of 0 to 1.0 and can be measured for any cysteine-modified antibody. The thiol reactivity value of the cysteine-modified antibody of the present invention is in the range of 0.0 to 1.0. Specifically, the thiol reactivity value of the cysteine-modified antibody of the present invention is in the range of 0.1 to 1.0. In certain embodiments, the thiol reactivity value of the cysteine-modified antibody of the present invention is in the range of 0.0 to 0.1, 0.1 to 0.5, 0.1 to 0.6, 0.1 to 0.7, 0.1 to 0.8, 0.1 to 0.9, or 0.1 to 1.0. In certain embodiments, the thiol reactivity value of the cysteine-modified antibody of the present invention is in the range of 0.2 to 1.0, 0.3 to 1.0, 0.4 to 1.0, 0.5 to 1.0, 0.6 to 1.0, 0.7 to 1.0, or 0.8 to 1.0. In certain embodiments, the thiol reactivity value of the cysteine-modified antibody of the present invention is in the range of 0.6 to 1.0. In certain embodiments, the thiol reactivity value of the cysteine-modified antibody of the present invention is in the range of 0.7 to 1.0. In certain embodiments, the thiol reactivity value of the cysteine-modified antibody of the present invention is in the range of 0.8 to 10. In certain embodiments, the thiol reactivity value of the cysteine-modified antibody of the present invention is in the range of 0.5 to 0.8.In certain embodiments, the thiol reactivity value of the cysteine-modified antibody of the present invention is in the range of 0.5 to 0.9. In certain embodiments, the thiol reactivity value of the cysteine-modified antibody of the present invention is in the range of 0.5 to 0.7. In certain embodiments, the thiol reactivity value of the cysteine-modified antibody of the present invention is in the range of 0.5 to 1.0.

[0145] The design, selection, and preparation methods of the present invention enable cysteine-manipulated antibodies that react with electrophilic functional groups. These methods further enable antibody conjugate compounds, such as antibody-drug conjugate (ADC) compounds, which have a drug molecule at a specified and designed selective site. Reactive cysteine ​​residues on the antibody surface allow for specific conjugation to the drug moiety via thiol-reactive groups such as maleimide or haloacetyl. The nucleophilic reactivity of the thiol functional group of a Cys residue to a maleimide group is approximately 1000 times higher than that of any other amino acid functional group in a protein, such as the amino group or N-terminal amino group of a lysine residue. While thiol-specific functional groups in iodoacetyl and maleimide reagents can react with amino groups, they require a higher pH (above 9.0) and longer reaction times (Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London).

[0146] Preferably, the cysteine-engineered antibodies of the present invention retain the antigen-binding ability of their wild-type parent antibody counterparts. Accordingly, the cysteine-engineered antibodies are preferably capable of specifically binding to an antigen. Examples of such antigens include tumor-associated antigens (TAA), cell surface receptor proteins and other cell surface molecules, transmembrane proteins, signal transduction proteins, cell survival regulators, cell growth regulators, molecules associated with (e.g., known or suspected to functionally contribute to) tissue development or differentiation, lymphokines, cytokines, molecules involved in cell odor control, molecules involved in angiogenesis-like processes, and molecules associated with (e.g., known or suspected to functionally contribute to) angiogenesis. The tumor-associated antigen can be a cluster of differentiation factor (i.e., a CD protein). The antigen that can be bound by the cysteine-engineered antibody may be a member of one subset of the above classification, wherein the other subset(s) of said classification comprise molecules / antigens having different characteristics (with respect to the antigen of interest).

[0147] The parent antibody may also be a humanized antibody selected from huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (Trastuzumab, HERCEPTIN®) described in Table 3 of U.S. Pat. No. 5,821,337, which is expressly incorporated herein by reference, humanized 520C9 (International Publication WO93 / 21319), and the humanized 2C4 antibody described herein.

[0148] The cysteine-modified antibody of the present invention can bind to a thiol-reactive reagent in a site-specific and efficient manner. The thiol-reactive reagent may be a polyfunctional linker reagent, a capture-i.e., affinity-labeling reagent (e.g., biotin-linker reagent), a detection label (e.g., a fluorescent reagent), a solid-phase immobilization reagent (e.g., SEPHAROSE®, polystyrene, or glass), or a drug-linker intermediate. An example of a thiol-reactive reagent is N-ethylmaleimide (NEM). In exemplary embodiments, a biotinylated THIOMAB® antibody is obtained by reaction of a THIOMAB® antibody with a biotin-linker reagent, which can be used to detect and measure the presence and reactivity of a modified cysteine ​​residue. A THIOMAB® antibody is obtained by reaction of a THIOMAB® antibody with a polyfunctional linker reagent, which may be further reacted with a drug partial reagent or other label. A THIOMAB® antibody-drug conjugate is obtained by reaction of a THIOMAB® antibody with a drug-linker intermediate. In certain embodiments, the THIOMAB® antibody is ThioFab.

[0149] The exemplary methods described herein can generally be applied to the identification and production of antibodies, and more generally to other proteins, through the application of the design and screening steps described herein.

[0150] Such approaches can be applied to the conjugation of other reactive substances, where the reactive group is, for example, maleimide, iodoacetamide, pyridyl disulfide, or other thiol-reactive conjugation 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 may be a cytotoxic agent (e.g., doxorubicin or pertussis toxin), a fluorophore such as a fluorescent dye like fluorescein or rhodamine, a chelating agent for metals used in imaging and radiotherapy, a peptidyl or non-peptidyl label or detection tag, or a clearance modifier such as various isotopes of polyethylene glycol, a peptide that binds to the third component, or another carbohydrate or lipophilic substance.

[0151] The sites identified in the exemplary antibody hu4D5 herein are primarily located in the constant domain of the antibody and are well-conserved across all antibody species. These sites should be broadly applicable to other antibodies without requiring further structural design or knowledge of specific antibody structures, and without interfering with the antigen-binding properties inherent to the antibody's variable domain.

[0152] Cysteine-modified antibodies that may be useful in the treatment of cancer include, but are not limited to, antibodies against cell surface receptors and tumor-associated antigens (TAAs). Such antibodies can be used as naked antibodies (not conjugated with a drug or labeling portion) or as antibody-drug conjugates (ADCs) of formula I. Tumor-associated antigens are known in the art and can be prepared for use in generating antibodies using methods and information well known in the art. With the aim of discovering effective cellular targets for the diagnosis and treatment of cancer, researchers have sought to identify transmembrane polypeptides or tumor-associated polypeptides that are specifically expressed on the surface of one or more specific cancer cell types compared to one or more normal non-cancerous cells. Often, such tumor-associated polypeptides are more abundantly expressed on the surface of cancer cells compared to the surface of non-cancerous cells. Identification of such tumor-associated cell surface antigen polypeptides has given rise to the ability of antibody-based therapies to specifically target cancer cells for destruction.

[0153] Examples of TAAs include, but are not limited to, the TAA(1)–(53) listed below. For convenience, information relating to these antigens (all known in the art) is listed below, including the name, alternative name, Genbank accession number, and primary reference(s), followed by the convention of identifying nucleic acid and protein sequences from the National Center for Biotechnology Information (NCBI). The nucleic acid and protein sequences corresponding to TAA(1)–(53) are available in public databases such as GenBank. Tumor-associated antigens targeted by antibodies include all amino acid sequence variants and isotopes having at least 70%, 80%, 85%, 90%, or 95% sequence identity compared to the sequences identified in the cited references, or exhibiting substantially the same biological properties or characteristics as TAAs having sequences found in the cited references. For example, TAAs having variant sequences can generally bind specifically to antibodies that specifically bind to TAAs having the corresponding sequences listed. Sequences and disclosures in references specifically cited herein are expressly incorporated by reference.

[0154] Tumor-associated antigens (1)~(53):

[0155] (1) BMPR1B (Bone morphogenetic protein 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)), International Publication No. WO2004063362 (Claim 2), International Publication No. WO2003042661 (Claim 12), US Publication No. 2003134790-A1 (pp. 38-39), International Publication No. WO2002102235 (Claim 13, p. 296), International Publication No. WO2003055443 (pp. 91-92), International Publication No. WO200299122 (Example 2, pp. 528-530), International Publication No. WO2003029421 (Claim 6) International Publication No. WO2003024392 (Claim 2, Figure 112), International Publication No. WO200298358 (Claim 1, p. 183), International Publication No. WO200254940 (pp. 100-101), International Publication No. WO200259377 (pp. 349-350), International Publication No. WO200230268 (Claim 27, p. 376), International Publication No. WO200148204 (Examples, Figure 4), NP_001194 osteomorphonesis receptor, type IB, / pid=NP_001194.1. Cross-reference: MIM:603248, NP_001194.1, AY065994

[0156] (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), International Publication No. WO2004048938 (Example 2), International Publication No. WO2004032842 (Example IV), International Publication No. WO2003042661 (Claim 12), International Publication No. WO2003016475 (Claim 1), International Publication No. WO200278524 (Example 2), International Publication No. WO200299074 (Claim 19, pp. 127-129), International Publication No. WO200286443 (Claim 27, (pp. 222, 393), International Publication No. WO2003003906 (Claim 10, p. 293), International Publication No. WO200264798 (Claim 33, pp. 93-95), International Publication No. WO200014228 (Claim 5, pp. 133-136), US Publication No. 2003224454 (Figure 3), International Publication No. WO2003025138 (Claim 12, p. 150), NP_003477 Solute Transporter Family 7 (Cationic Amino Acid Transporter, y+ System), Member 5 / pid=NP_003477.3- Homo sapiens, Cross-reference: MIM:600182, NP_003477.3, NM_015923, NM_003486_1

[0157] (3) STEAP1 (six-transmembrane epithelial antigen of the 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), International Publication No. WO2004065577 (Claim 6), International Publication No. WO2004027049 (Figure 1L), European Patent No. EP1394274 (Example 11), International Publication No. WO2004016225 (Claim 2), International Publication No. WO2003042661 (Claim 12), US Publication No. 200315 Publication No. 7089 (Example 5), Publication No. 2003185830 (Example 5), Publication No. 2003064397 (Figure 2), International Publication No. WO200289747 (Example 5, pp. 618-619), Publication No. WO2003022995 (Example 9, Figure 13A, Example 53, p. 173, Example 2, Figure 2A), NP_036581 Six-transmembrane epithelial antigen of the prostate Cross references: MIM:604415, NP_036581.1, NM_012449_1

[0158] (4)0772P (CA125, MUC16, Genbank accession number AF361486), J. Biol. Chem. 276(29):27371-27375(2001)), International Publication No. WO2004045553 (Claim 14), International Publication No. WO200292836 (Claim 6, Figure 12), International Publication No. WO200283866 (Claim 15, pp. 116-121), US Publication No. 2003124140 (Example 16), Cross-reference: GI:34501467, AAK74120.3, AF361486_1

[0159] (5) MPF (MPF, MSLN, SMR, megakaryocyte enhancement factor, mesothelin, Genbank accession number NM_005823) Yamaguchi, N., et al Biol. Chem. 269(2), 805-808(1994), Proc. Natl. Acad. Sci. USA96 (20):11531-11536(1999), Proc. Natl. Acad. Sci. USA93(1):136-140(1996), J.Biol. Chem. 270(37):21984-21990(1995)), International Publication No. WO2003101283 (Claim 14), (International Publication No. WO2002102235 (Claim 13, pp. 287-288), International Publication No. WO2002101075 (Claim 4, pp. 308-309), International Publication No. WO200271928 (pp. 320-321), International Publication No. WO9410312 (pp. 52-57), Cross-reference: MIM:601051, NP_005814.2, NM_005823_1

[0160] (6) Napi3b (also known as NaPi2b) (NAPI-3B, NPTIIb, SLC34A2, solute transporter 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), Field, JA, et al (1999) Biochem. Biophys. Res.Commun. 258(3):578-582), International Publication No. WO2004022778 (Claim 2), European Patent No. EP1394274 (Example 11), International Publication No. WO2002102235 (Claim 13, p. 326), European Patent No. EP875569 (Claim 1, pp. 17-19), International Publication No. WO200157188 ​​(Claim 20, p. 329), International Publication No. WO2004032842 (Example IV), International Publication No. WO200175177 (Claim 24, pp. 139-140), Cross-reference: MIM:604217, NP_006415.1, NM_006424_1

[0161] (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, semadomain, 7 thrombospongin 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), International Publication No. WO2004000997 (Claim 1), International Publication No. WO2003003984 (Claim 1), International Publication No. WO200206339 (Claim 1, p. 50), International Publication No. WO200188133 (Claim 1, pp. 41-43, 48-58), International Publication No. WO2003054152 (Claim 20), International Publication No. WO2003101400 (Claim 11), Commissioned: Q9P283, EMBL, AB040878, BAA95969.1. Genew, HGNC:10737

[0162] (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, US Publication No. 2003129192 (Claim 2), US Publication No. 2004044180 (Claim 12), US Publication No. 2004044179 (Claim 11), US Publication No. 2003096961 (Claim 11), US Publication No. 2003232056 (Example 5), International Publication No. WO2003105758 (Claim 12), US Publication No. 2003206918 (Example 5), European Patent No. EP1347046 (Claim 1), International Publication No. WO2003025148 (Claim 20), Cross-reference: GI:37182378, AAQ88991.1, AY358628_1

[0163] (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 N.A., 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 R.L., et al Proc. Natl. Acad. Sci. U.S.A. 99, 16899-16903, 2002, Bourgeois C., et al J. Clin. Endocrinol. Metab. 82, 3116-3123, 1997, Okamoto Y., et al Biol. Chem. 272, 21589-21596, 1997, Verheij J.B., et al Am. J. Med. Genet. 108, 223-225, 2002, Hofstra R.M.W., et al Eur. J. Hum. Genet. 5, 180-185, 1997, Puffenberger E.G., et al Cell 79, 1257-1266, 1994, Attie T., et al, Hum. Mol. Genet. 4, 2407-2409, 1995, Auricchio A., et al Hum. Mol. Genet. 5: 351-354, 1996, Amiel J., et al Hum. Mol. Genet. 5, 355-357, 1996, Hofstra R.M.W.,et al Nat. Genet. 12,445-447,1996, Svensson 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, International Publication No. WO2004045516 (Claim 1), WO2004048938 (Example 2), WO2004040000 (Claim 151), WO2003087768 (Claim 1), WO2003016475 (Claim 1), WO2003016475 (Claim 1), WO200261087 (Figure 1), WO2003016 Patent No. 494 (Figure 6), Patent No. WO2003025138 (Claim 12, p. 144), Patent No. WO200198351 (Claim 1, pp. 124-125), European Patent No. EP522868 (Claim 8, Figure 2), International Publication No. WO200177172 (Claim 1, pp. 297-299), US Publication No. 2003109676, US Patent No. 6518404 (Figure 3), US Patent No. 5773223 (Claim 1a, Col 31-34), International Publication No. WO2004001004.

[0164] (10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession number NM_017763), International Publication No. WO2003104275 (Claim 1), International Publication No. WO2004046342 (Example 2), International Publication No. WO2003042661 (Claim 12), International Publication No. WO2003083074 (Claim 14, p. 61), International Publication No. WO2003018621 (Claim 1), International Publication No. WO2003024392 (Claim 2, Figure 93), International Publication No. WO200166689 (Example 6), Cross-reference: LocusID:54894, NP_060233.2, NM_017763_1

[0165] (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-related gene 1, prostate cancer-related protein 1, six-transmembrane epithelial antigen of the prostate 2, six-transmembrane prostate protein, Genbank accession number AF455138), Lab. Invest. 82(11):1573-1582(2002)), International Publication No. WO2003087306, US Publication No. 2003064397 (Claim 1, Figure 1), International Publication No. WO200272596 (Claim 13, pp. 54-55), US Publication No. WO200172962 (Claim 1, Figure 4B), US Publication No. WO2003104270 (Claim 11), US Publication No. WO2003104270 (Claim 16) , U.S. Publication No. 2004005598 (Claim 22), International Publication No. WO2003042661 (Claim 12), U.S. Publication No. 2003060612 (Claim 12, Figure 10), International Publication No. WO200226822 (Claim 23, Figure 2), International Publication No. WO200216429 (Claim 12, Figure 10), Cross-reference: GI:22655488, AAN04080.1, AF455138_1

[0166] (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)), US Publication No. 2003143557 (Claim 4), International Publication No. WO200040614 (Claim 14, pp. 100-103), Ibid. WO200210382 (Claim 1, Figure 9A), Ibid. WO2003042661 (Claim 12), Ibid. WO200230268 (Claim 27, p. 391), US Publication No. 2003219806 (Claim 4), International Publication No. WO200162794 (Claim 14, Figures 1A-D), Cross-reference: MIM:606936, NP_060106.2, NM_017636_1

[0167] (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, growth factor derived from teratoma, 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)), U.S. Publication No. 2003224411 (Claim 1), International Publication No. WO2003083041 (Example 1), WO2003034984 (Claim 12), WO200288170 (Claim 2, pp. 52-53), WO2003024392 (Claim 2, Figure 58), WO200216413 (Claim 1, pp. 94-95, 105), WO200222808 (Claim 2, Figure 1), U.S. Patent No. 5854399 (Example 2, Col 17-18), U.S. Patent No. 5,792,616 (Figure 2), cross-reference: MIM:187395, NP_003203.1, NM_003212_1

[0168] (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. USA84,9194-9198,1987, Barel M., et al Mol. Immunol. 35,1025-1031,1998, Weis JJ, et al Proc. Natl. Acad. Sci. USA83,5639-5643,1986, Sinha SK, et al(1993)J.Immunol. 150,5311-5320, International Publication No. WO2004045520 (Example 4), US Publication No. 2004005538 (Example 1), International Publication No. WO2003062401 (Claim 9), Ibid. WO2004045520 (Example 4), Ibid. WO9102536 (Figures 9.1-9.9), Ibid. WO2004020595 (Claim 1), Accession numbers: P20023, Q13866, Q14212, EMBL, M26004, AAA35786.1.

[0169] (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), International Publication No. WO2004016225 (Claim 2, Figure 140), International Publication No. WO2003087768, U.S. Publication No. 2004101874 (Claim 1, p. 102), International Publication No. WO2003062401 (Claim 9), International Publication No. WO200278524 (Example 2), U.S. Publication No. 2002150573 (Claim 5, p. 15), U.S. Japanese Patent No. 5644033, International Publication No. WO2003048202 (Claim 1, pp. 306 and 309), International Publication No. WO99 / 558658, U.S. Patent No. 6534482 (Claim 13, Figure 17A / B), International Publication No. WO200055351 (Claim 11, pp. 1145-1146), Cross-reference: MIM:147245, NP_000617.1, NM_000626_1

[0170] (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain-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), Blood 99(8):2662-2669 (2002), Proc. Natl. Acad. Sci. USA 98(17):9772-9777 (2001), Xu, MJ, et al (2001) Biochem. Biophys. Res. Commun. 280(3):768-775, International Publication No. WO2004016225 (Claim 2), International Publication No. WO2003077836, International Publication No. WO200138490 (Claim 5, Figures 18D-1~18D-2), International Publication No. WO2003097803 (Claim 12), International Publication No. WO2003089624 (Claim 25), Cross-reference: MIM:606509, NP_110391.2, NM_030764_1

[0171] (17) HER2 (ErbB2, Genbank Accession No. M11730), Coussens L., et al Science (1985) 230(4730): 1132-1139), Yamamoto T., et al Nature 319, 230-234, 1986, Semba K., et al Proc. Natl. Acad. Sci. U.S.A. 82, 6497-6501, 1985, Swiercz J.M., et al J. Cell Biol. 165, 869-880, 2004, Kuhns J.J., et al J. Biol. Chem. 274, 36422-36427, 1999, Cho H.-S., et al Nature 421, 756-760, 2003, Ehsani A., et al (1993) Genomics 15, 426-429, International Publication No. WO2004048938 (Example 2), International Publication No. WO2004027049 (Figure 1I), International Publication No. WO2004009622, International Publication No. WO2003081210, International Publication No. WO2003089904 (Claim 9), International Publication No. WO2003016475 (Claim 1), U.S. Publication No. 2003118592, International Publication No. WO2003008537 (Claim 1), International Publication No. WO2003055439 (Claim 29, Figures 1A-B), International Publication No. WO2003025228 (Claim 37, Figure 5C), International Publication No. WO200222636 (Example 13, pages 95-107), International Publication No. WO200212341 (Claim 68, Figure 7), International Publication No. WO200213847 (pages 71-74), International Publication No. WO200214503 (pages 114-117), International Publication No. WO200153463 (Claim 2, pages 41-46), International Publication No. WO200141787 (page 15), International Publication No. WO200044899 (Claim 52, Figure 7), International Publication No. WO200020579 (Claim 3, Figure 2), U.S. Patent No. 5869445 (Claim 3, Col 31-38), International Publication No. WO9630514 (Claim 2, pages 56-61), European Patent No. EP1439393 (Claim 7), International Publication No. WO2004043361 (Claim 7), International Publication No. WO2004022709, International Publication No. WO200100244 (Example 3, Figure 4), Accession No.: P04626, EMBL, M11767, AAA35808.1, EMBL, M11761, AAA35808.1

[0172] (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. USA99:16899-16903, 2002, International Publication No. WO2004063709, European Patent No. EP1439393 (Claim 7), International Publication No. WO2004044178 (Example 4), International Publication No. WO2004031238, International Publication No. WO2003042661 (Claim 12), International Publication No. WO200278524 (Example 2), International Publication No. WO200286443 (Claim 27, p. 427), International Publication No. WO200260317 (Claim 2), Accession Number: P40199, Q14920, EMBL, M29541, AAA59915.1. EMBL, M18728

[0173] (19) MDP (DPEP1, Genbank accession number BC017023), Proc. Natl. Acad. Sci. USA99(26):16899-16903(2002)), International Publication No. WO2003016475 (Claim 1), International Publication No. WO200264798 (Claim 33, pp. 85-87), Japanese Patent No. JP05003790 (Figures 6-8), International Publication No. WO9946284 (Figure 9), Cross-reference: MIM:179780, AAH17023.1, BC017023_1

[0174] (20)IL20Rα (IL20Ra, ZCYTOR7, Genbank Trustee No. 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, European Patent No. EP1394274 (Example 11), US Publication No. 2004005320 (Example 5), International Publication No. WO2003 No. 029262 (pages 74~75), same as WO2003002717 (claim 2, page 63), same as WO200222153 (pages 45~47), U.S. Publication No. 20 No. 02042366 (pages 20-21), International Publication No. WO200146261 (pages 57-59), No. WO200146232 (pages 63-65), No. WO9837193 (request item 1, pages 55-59), entrusted number: Q9UHF4, Q6UWA9, Q96SH8, EMBL, AF184971, AAF01320.1.

[0175] (21) Brevican (BCAN, BEHAB, Genbank Request No. 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, U.S. Publication No. 2003186372 (Request 11), Ibid. 2003186373 (Request 11), Ibid. 2003119131 (Request 1, Figure 52), Ibid. 2003119122 (Request 1, Figure 52), Ibid. 2003119126 (Request 1), Ibid. 200311 Request No. 9121 (Request 1, Figure 52), Request No. 2003119129 (Request 1), Request No. 2003119130 (Request 1), Request No. 2003119128 (Request 1, Figure 52), Request No. 2003119125 (Request 1), International Publication No. WO2003016475 (Request 1), Request No. WO200202634 (Request 1)

[0176] (22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank commissioned number NM_004442), Chan, J. and Watt, VM, Oncogene 6(6), 1057-1061 (1991) Oncogene 10(5): 897-905 (1995), Annu. Rev. Neurosci. 21: 309-345 (1998), Int. Rev. Cytol. 196:177-244(2000)), International Publication No. WO2003042661 (Request 12), Ibid. No. WO200053216 (Request 1, page 41), Ibid. No. WO2004065576 (Request 1), Ibid. No. WO2004020583 (Request 9), Ibid. No. WO2003004529 (pages 128-132), Ibid. No. WO200053216 (Request 1, page 42), Cross-reference: MIM:600997, NP_004433.2, NM_004442_1

[0177] (23) ASLG659 (B7h, Genbank accession number AX092328), U.S. Publication No. 20040101899 (Claim 2), International Publication No. WO2003104399 (Claim 11), International Publication No. WO2004000221 (Figure 3), U.S. Publication No. 2003165504 (Claim 1), International Publication No. 2003124140 (Example 2), International Publication No. 2003065143 (Figure 6 0), International Publication No. WO2002102235 (Claim 13, p. 299), U.S. Publication No. 2003091580 (Example 2), International Publication No. WO200210187 (Claim 6, Figure 10), Ibid. WO200194641 (Claim 12, Figure 7b), Ibid. WO200202624 (Claim 13, Figures 1A-1B), U.S. Publication No. 2002034749 (Claim 54, pp. 45-46), International Publication No. WO200206317 (Example 2, pp. 320-321, Claim 34, pp. 321-322), International Publication No. WO200271928 (pp. 468-469), International Publication No. WO200202587 (Example 1, Figure 1), International Publication No. WO2001402 No. 69 (Example 3, pp. 190-192), No. WO200036107 (Example 2, pp. 205-207), No. WO2004053079 (Claim 12), No. WO2003004989 (Claim 1), No. WO200271928 (pp. 233-234, pp. 452-453), No. WO0116318

[0178] (24) PSCA (prostate stem cell antigen precursor, Genbank accession number AJ297436), Reiter RE, et al Proc. Natl. Acad. Sci. USA95,1735-1740,1998, Gu Z.,et al Oncogene 19,1288-1296,2000, Biochem. Biophys. Res.Commun. (2000)275(3):783-788, International Publication No. WO2004022709, European Patent No. EP1394274 (Example 11), US Publication No. 2004018553 (Claim 17), International Publication No. WO2003008537 (Claim 1), International Publication No. WO200281646 (Claim 1, p. 164), International Publication No. WO2003003906 (Claim 10, p. 288), International Publication No. WO2001403 Publication No. 09 (Example 1, Figure 17), U.S. Publication No. 2001055751 (Example 1, Figure 1b), International Publication No. WO200032752 (Claim 18, Figure 1), International Publication No. WO9851805 (Claim 17, p. 97), International Publication No. WO9851824 (Claim 10, p. 94), International Publication No. WO9840403 (Claim 2, Figure 1B), Accession Number: O43653, EMBL, AF043498, AAC39607.1

[0179] (25) GEDA (Genbank accession number AY260763), AAP14954 lipoma HMGIC fusion partner-like protein / pid=AAP14954.1-Homo sapiens (human), International Publication No. WO2003054152 (Claim 20), International Publication No. WO2003000842 (Claim 1), International Publication No. WO2003023013 (Example 3, Claim 20), US Publication No. 2003194704 (Claim 45), Cross-reference: GI:30102449, AAP14954.1, AY260763_1

[0180] (26) BAFF-R (B cell activator receptor, BlyS receptor 3, BR3, Genbank accession number AF116456), BAFF receptor / pid=NP_443177.1-Homo sapiens: Thompson, JS, et al Science 293(5537), 2108-2111(2001), International Publication No. WO2004058309, International Publication No. WO2004011611, International Publication No. WO2003045422 (Examples, pp. 32-33), International Publication No. WO2003014294 (Claim 35, Figure 6B), International Publication No. WO2003035846 (Claim 70, pp. 615-616), International Publication No. WO200294852 (Col 136-137), International Publication No. WO200238766 (Claim 3, p. 133), International Publication No. WO200224909 (Example 3, Figure 3), Cross-reference: MIM:606269, NP_443177.1, NM_052945_1, AF132600

[0181] (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, International Publication No. WO2003072036 (Claim 1, Figure 1), Cross-reference: MIM:107266, NP_001762.1, NM_001771_1

[0182] (28) CD79a (a B cell-specific protein that interacts covalently with CD79A, CD79α, immunoglobulin-associated alpha, and Ig beta (CD79B), forming a complex with IgM molecules on its surface and transmitting signals involved in B cell differentiation), pI: 4.84, Molecular weight: 25028, TM: 2[P]Gene, Chromosome: 19q13.2, Genbank accession number NP_001774.10), International Publication No. WO2003088808, US Publication No. 20030228319, International Publication No. WO2003062401 (Claim 9), US Publication No. 2002150573 (Claim 4, pp. 13-14), International Publication No. WO9958658 (Claim 13, Figure 16), International Publication No. WO9207574 (Figure 1), US Patent No. 5644033, 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.

[0183] (29) CXCR5 (Burkitt lymphoma receptor 1, a G protein-conjugated receptor activated by the CXCL13 chemokine, which functions in lymphocyte migration and humoral defense and plays a role in HIV-2 infection and possibly in the development of AIDS, lymphoma, myeloma, and leukemia), 372 amino acids, pI: 8.54, Molecular weight: 41959, TM: 7[P]Gene, Chromosome: 11q23.3, Genbank accession number NP_001707.1), International Publication No. WO2004040000, International Publication No. WO2004015426, U.S. Publication No. 2003105292 (Example 2), U.S. Patent No. 6555339 (Example 2), International Publication No. WO200261087 (Figure 1), International Publication No. WO200157188 ​​(Claim 20, p. 269), International Publication No. WO200172830 (pp. 12-13), International Publication No. WO200022129 (Example 1, pp. 152-153, Example 2, pp. 254-256), International Publication No. WO9928468 (Claim 1, p. 38), U.S. Patent No. 5440021 (Example 2, col 49-52), International Publication No. 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

[0184] (30) HLA-DOB (beta subunit (Ia antigen) of MHC class II molecules that binds to peptides and presents them to CD4+ T lymphocytes), 273 amino acids, pI: 6.56, molecular weight: 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, International Publication No. WO9958658 (Claim 13, Figure 15), US Patent No. 6153408 (Col 35-38), US Patent No. 5976551 (Col 168-170), US Patent No. 6011146 (Col 145-146), Kasahara et al (1989) Immunogenetics 30(1):66-68, Larhammar et al (1985) J. Biol. Chem. 260(26):14111-14119

[0185] (31) P2X5 (purine receptor P2X ligand open-type ion channel 5, which may be involved in synaptic transmission and neurogenesis, is an ion channel that is opened and closed by extracellular ATP, and its deficiency may contribute to the pathophysiology of idiopathic detrusor instability), 422 amino acids), pI: 7.63, molecular weight: 47206 TM: 1[P]Gene chromosome: 17p13.3, Genbank accession number NP_002552.2), Le et al (1997) FEBS Lett. 418(1-2):195-199, International Publication No. WO2004047749, International Publication No. WO2003072035 (Claim 10), Touchman et al (2000) Genome Res.10:165-173, International Publication No. WO200222660 (Claim 20), WO2003093444 (Claim 1), WO2003087768 (Claim 1), WO2003029277 (p. 82)

[0186] (32)CD72 (B cell differentiation antigen CD72, Lyb-2), 359 amino acids, pI: 8.66, molecular weight: 40225, TM: 1[P]Gene chromosome: 9p13.3, Genbank accession number NP_001773.1), International Publication No. WO2004042346 (claim 65), International Publication No. WO2003026493 (pages 51-52, pages 57-58), International Publication No. WO200075655 (pages 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.

[0187] (33)LY64 (Lymphocyte antigen 64 (RP105), a type I membrane protein of the leucine-rich repeat (LRR) family, regulates B cell activation and apoptosis, and loss of function is associated with increased disease activity in patients with systemic lupus erythematosus), 661 amino acids, pI: 6.20, molecular weight: 74147 TM: 1[P]Gene chromosome: 5q12, Genbank accession number NP_005573.1), US Publication No. 2002193567, International Publication No. WO9707198 (Claim 11, pp. 39-42), Miura et al (1996) Genomics 38(3):299-304, Miura et al (1998) Blood 92:2815-2822, International Publication No. WO2003083047, International Publication No. WO9744452 (Claim 8, pp. 57-61), International Publication No. WO200012130 (pp. 24-26)

[0188] (34) FcRH1 (Fc receptor-like protein 1, putative receptor for immunoglobulin Fc domain containing C2 Ig-like and ITAM domains, potentially playing a role in B-lymphocyte differentiation), 429 amino acids, pI: 5.28, molecular weight: 46925 TM: 1[P]Gene chromosome: 1q21-1q22, Genbank accession number NP_443170.1), International Publication No. WO2003077836, International Publication No. WO200138490 (Claim 6, Figures 18E-1~18-E-2), Davis et al (2001) Proc. Natl. Acad. Sci USA 98(17):9772-9777, International Publication No. WO2003089624 (Claim 8), European Patent No. EP1347046 (Claim 1), International Publication No. WO2003089624 (Claim 7)

[0189] (35) IRTA2 (Immunoglobulin superfamily receptor translocation-related 2, a putative immune receptor with a potential role in B cell growth and lymphoma formation; deregulation of the gene due to translocation occurs in some B cell malignancies), 977 amino acids, pI: 6.88, molecular weight: 106468, TM: 1[P]Gene, chromosome: 1q21, Genbank accession numbers: Human: AF343662, AF343663, AF343664, AF343665, AF369794, AF397453, AK090423, AK090475, AL834187, AY358085, Mouse: AK089756, AY158090, AY506558, NP_112571.1, International Publication No. WO2003024392 (Claim 2, Figure 97), Nakayama et al. al(2000) Biochem. Biophys. Res.Commun. 277(1):124-127, International Publication No. WO2003077836, International Publication No. WO200138490 (Claim 3, Figures 18B-1~18B-2)

[0190] (36)TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembrane proteoglycan, related to the EGF / heregulin family of growth factors and follistatin), 374 amino acids, NCBI accession numbers: AAD55776, AAF91397, AAG49451, NCBI reference sequence: NP_057276, NCBI gene: 23671, OMIM: 605734, SwissProt Q9UIK5, Genbank accession number AF179274, AY358907, CAF85723, CQ782436, International Publication No. WO2004074320, Japanese Patent No. JP2004113151, International Publication No. WO2003042661, International Patent No. WO2003009814, European Patent No. EP1295944 (pp. 69-70), International Publication No. WO2002 Publication No. 30268 (page 329), Publication No. WO200190304, U.S. Publication No. 2004249130, U.S. Publication No. 2004022727, International Publication No. WO2004063355, U.S. Publication No. 2004197325, U.S. Publication No. 2003232350, U.S. Publication No. 2004005563, U.S. Publication No. 2003124579, Horie et al(2000)Genomics 67:146-152, Uchida et al(1999)Biochem. 15;94(2):178-84.(37)PMEL17 (silver homolog, SILV, D12S53E, PMEL17, SI, SIL), ME20, gp100) BC001414, BT007202, M32295, M77348, NM_006928, McGlinchey,RPet al(2009)Proc. Natl. Acad. Sci. USA106(33),13731-13736, Kummer, MPet al(2009) J.Biol. Chem. 284(4),2296-2306;

[0191] (38) TMEFF1 (transmembrane protein 1 with an EGF-like domain and two follistatin-like domains, tomoreglin 1), H7365, C9orf2, C9ORF2, U19878, X83961, NM_080655, NM_003692, Harms, PW (2003) Genes Dev. 17(21), 2624-2629, Gery, S. et al (2003) Oncogene 22(18):2723-2727;

[0192] (39) GDNF-Ra1 (GDNF family receptor alpha-1, GFRA1, GDNFR, GDNFRA, RETL1, TRNR1, RET1L, GDNFR-alpha-1, GFR-ALPHA-1), U95847, BC014962, NM_145793 NM_005264, Kim, M. et al (2009) Mol. Cell. Biol. 29(8), 2264-2277, Treanor, J. et al (1996) Nature 382(6586):80-83;

[0193] (40)Ly6E (lymphocyte antigen 6 complex, locus E, Ly67, RIG-E, SCA-2, TSA-1), NP_002337.1, NM_002346.2, de Nooij-van Dalen,AGet al(2003)Int. J.Cancer 103(6),768-774, Zammit,DJet al(2002)Mol. Cell. Biol. 22 (3):946-952;

[0194] (41) TMEM46 (shisa homolog 2 (African clawed frog), SHISA2), NP_001007539.1, NM_001007538.1, Furushima, K. et al (2007) Dev. Biol. 306(2), 480-492, Clark, HF et al (2003) Genome Res. 13(10): 2265-2270;

[0195] (42)Ly6G6D (lymphocyte antigen 6 complex, loci G6D, Ly6-D, MEGT1), NP_067079.2, NM_021246.2, Mallya, M. et al (2002) Genomics 80(1):113-123, Ribas, G. et al (1999) J. Immunol. 163(1):278-287;

[0196] (43) LGR5 (Leucine-rich repeat-containing G protein-bound receptor 5, GPR49, GPR67), NP_003658.1, NM_003667.2, Salanti, G. et al (2009) Am. J. Epidemiol. 170(5):537-545, Yamamoto, Y. et al (2003) Hepatology 37(3):528-533;

[0197] (44)RET (ret proto-oncogene, MEN2A, HSCR1, MEN2B, MTC1, PTC, CDHF12, Hs.168114, RET51, RET-ELE1), NP_066124.1, NM_020975.4, Tsukamoto, H. et al (2009) Cancer Sci. 100(10):1895-1901, Narita, N. et al (2009) Oncogene 28(34):3058-3068;

[0198] (45) LY6K (lymphocyte antigen 6 complex, gene locus K, LY6K, HSJ001348, FLJ35226), NP_059997.3, NM_017527.3, Ishikawa, N. et al (2007) Cancer Res. 67(24):11601-11611, de Nooij-van Dalen, AGet al (2003) Int. J. Cancer 103(6):768-774;

[0199] (46) GPR19 (G protein-bound receptor 19, Mm. 4787), NP_006134.1, NM_006143.2, Montpetit, A. and Sinnett, D. (1999) Hum. Genet. 105(1-2):162-164, O'Dowd, B. Fet al (1996) FEBS Lett. 394(3):325-329;

[0200] (47) GPR54 (KISS1 receptor, KISS1R, GPR54, HOT7T175, AXOR12), NP_115940.2, NM_032551.4, Navenot, JMet al (2009) Mol. Pharmacol. 75(6):1300-1306, Hata, K. et al (2009) Anticancer Res.29(2):617-623;

[0201] (48) ASPHD1 (containing aspartate beta-hydroxylase domain 1, LOC253982), NP_859069.2, NM_181718.3, Gerhard, DSet al (2004) Genome Res. 14(10B):2121-2127;

[0202] (49) Tyrosinase (TYR, OCAIA, OCA1A, tyrosinase, SHEP3), NP_000363.1, NM_000372.4, Bishop, D. et al (2009) Nat. Genet. 41(8):920-925, Nan, H. et al (2009) Int. J. Cancer 125(4):909-917;

[0203] (50) TMEM118 (ring finger protein, transmembrane 2, RNFT2, FLJ14627), NP_001103373.1, NM_001109903.1, Clark, HF et al (2003) Genome Res. 13(10):2265-2270, Scherer, SE et al (2006) Nature 440(7082):346-351

[0204] (51) GPR172A (G protein-bound receptor 172A, GPCR41, FLJ11856, D15Ertd747e), NP_078807.1, NM_024531.3, Ericsson, TA et al (2003) Proc. Natl. Acad. Sci. USA 100(11):6759-6764, Takeda, S. et al (2002) FEBS Lett. 520(1-3):97-101.

[0205] (52) CD33 is a 67 kDa glycosylated transmembrane protein, a member of the immunoglobulin-like lectin family that binds to sialic acid. CD33 is expressed in most myeloid leukemia cells and monocytic leukemia cells, in addition to committed myelomonocyte precursor cells and erythrocyte precursor cells. It is not found in the earliest pluripotent stem cells, mature granulocytes, lymphoid cells, or non-hematopoietic cells (Sabbath et al., (1985) J. Clin. Invest. 75:756-56, Andrews et al., (1986) Blood 68:1030-5). CD33 contains two tyrosine residues in its cytoplasmic tail, each of which is followed by a hydrophobic residue similar to the immunoreceptor tyrosine system inhibitory motif (ITIM) found in many inhibitory receptors.

[0206] (53) CLL-1 (CLEC12A, MICL, and DCAL2) encodes members of the C-type lectin / C-type lectin-like domain (CTL / CTLD) superfamily. Members of this family share common protein folding and have diverse functions, including roles in adhesion, intercellular signaling, glycoprotein turnover, and inflammation and immune responses. The protein encoded by this gene is a negative regulator of granulocyte and monocyte function. Several alternative splice transcript variants of this gene have been described, but the full-length properties of some of these variants are unknown. This gene is closely related to other CTL / CTLD superfamily members within the natural killer gene complex region on chromosome 12p13 (Drickamer K (1999) Curr. Opin. Struct. Biol. 9(5):585-90, van Rhenen A, et al., (2007) Blood 110(7):2659-66, Chen CH, et al. (2006) Blood 107(4):1459-67, Marshall AS, et al. (2006) Eur. J. Immunol. 36(8):2159-69, Bakker AB, et al (2005) Cancer Res. 64(22):8443-50, Marshall AS, et al (2004) J. Biol. Chem. 279(15):14792-802). CLL-1 has been shown to be a type II transmembrane receptor containing a single C-type lectin-like domain (not predicted to bind to either calcium or sugar), a stalk region, a transmembrane domain, and a short cytoplasmic tail containing an ITIM motif.

[0207] The parent antibody may also be a fusion protein containing 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, International Publication No. WO01 / 45746). The antibodies of the present invention include fusion proteins with ABP sequences taught in (i) Dennis et al (2002) J Biol Chem. 277:35035-35043 at Tables III and IV, page 35038, (ii) U.S. Publication No. 20040001827

[0076] , and (iii) International Publication No. WO01 / 45746 (pages 12-13), all of which are incorporated herein by reference.

[0208] Mutation generation

[0209] DNA encoding amino acid sequence variants of the start polypeptide can be prepared by various methods known in the art. These methods include, but are not limited to, site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and preparation by cassette mutagenesis of DNA encoding a pre-prepared polypeptide. Mutagenesis of recombinant antibodies can also be constructed by restriction fragment manipulation or overlap extension PCR with synthetic oligonucleotides. Mutagenesis primers encode cysteine ​​codon substitutions. DNA encoding such mutant cysteine-manipulated antibodies can be generated using standard mutagenesis techniques. General guidelines can be found in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York, NY, 1993.

[0210] Site-directed mutagenesis is a method for preparing substitutional mutants, i.e., mutant proteins. This technique is well known in the art (see, for example, Carter (1985) et al Nucleic Acids Res. 13:4431-4443, Ho et al (1989) Gene (Amst.) 77:51-59, and Kunkel et al (1987) Proc. Natl. Acad. Sci. USA 82:488). In short, when performing site-directed mutagenesis of DNA, the start DNA is first modified by hybridizing an oligonucleotide encoding the desired mutation into a single strand of such start DNA. After hybridization, the hybridized oligonuclease is used as a primer, and the entire second strand is synthesized by DNA polymerase using the single strand of start DNA as a template. Thus, the oligonucleotide encoding the desired mutation is incorporated into the resulting double-stranded DNA. Site-directed mutagenesis can be performed within the gene expressing the protein to be mutated in an expression plasmid, and the resulting plasmid can be sequenced to confirm the introduction of the desired cysteine ​​substitution mutation (Liu et al (1998) J. Biol. Chem. 273:20252-20260). Site-directed protocols and formats are commercially available, such as the QuikChange® Multi Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA).

[0211] PCR mutagenesis is also suitable for generating amino acid sequence variants of initiating polypeptides. See Higuchi, (1990) in PCR Protocols, pp. 177-183, Academic Press, Ito et al (1991) Gene 102: 67-70, Bernhard et al (1994) Bioconjugate Chem. 5: 126-132, and Vallette et al (1989) Nuc. Acids Res. 17: 723-733. In short, when using a small amount of template DNA as a starting material in PCR, it is possible to generate a relatively large amount of specific DNA fragments in which only the position where the primer differs from the template differs from the template sequence, by using primers that have a slightly different sequence from the corresponding region of the template DNA.

[0212] Another method for preparing mutants, cassette mutagenesis, is based on the technique described by Wells et al (1985) Gene 34:315-323. The starting material is a plasmid (or other vector) containing the starting polypeptide DNA to be mutageneised. The codon(s) in the starting DNA to be mutageneised are identified. Specific restriction endonucleases are required on both sides of the identified mutation site(s). If such restriction sites are not present, they can be generated at appropriate locations on the starting polypeptide DNA using the oligonucleotide-mediated mutagenesis method described above. The plasmid DNA is cleaved at these locations and linearized. Double-stranded oligonucleotides containing the desired mutation(s) encoding the DNA sequence between the restriction sites are synthesized using a standard procedure, where the two strands of the oligonucleotide are synthesized separately and then hybridized together using a standard technique. Oligonucleotides are prepared by a phosphoramidite synthesis method (U.S. Patent No. 4,415,732, 4,458,066; Beaucage, S. and Iyer, R. (1992) "Advances in the synthesis of oligonucleotides by the phosphoramidite approach", Tetrahedron 48:2223-2311). These double-stranded oligonucleotides are referred to as cassettes. These cassettes are designed to have 5' and 3' ends that are compatible with the ends of linear plasmids and therefore can be directly ligated to the plasmid. This plasmid contains a mutant DNA sequence. The mutant DNA, including the encoded cysteine ​​substitution, can be identified by DNA sequencing.

[0213] Single mutations are generated by oligonucleotide-directed mutagenesis, using double-stranded plasmid DNA as a template and PCR-based mutagenesis (Sambrook and Russel, (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; Zoller et al (1983) Methods Enzymol. 100:468-500; Zoller, M. Jand Smith, M. (1982) Nucl. Acids Res. 10:6487-6500).

[0214] In this invention, hu4D5 presented 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 in the experiments. Cysteine ​​mutations were introduced into hu4D5-phage, hu4D5, and ABP-hu4D5 constructs. The hu4D5-THIOMAB® antibody was prepared using the polyethylene glycol (PEG) precipitation method described above (Lowman, Henry B. (1998) Methods in Molecular Biology (Totowa, New Jersey) 87 (Combinatorial Peptide Library Protocols) 249-264).

[0215] PHESELECTOR assay

[0216] The PHESELECTOR (Phage ELISA for Reactive Thiol Selection) assay enables the detection of reactive cysteine ​​groups in antibodies during ELISA phage formation. See U.S. Patent No. 7,521,541 and U.S. Patent Publication No. 20110301334, which are incorporated in their entirety by reference. Specifically, the PHESELECTOR assay involves coating the well surface with the protein of interest (e.g., an antibody), followed by incubation with phage particles, and then incubation with an HRP-labeled secondary antibody, followed by detection of absorbance. Mutant proteins presented on the phage can be screened in a rapid, robust, and high-throughput manner. A library of cysteine-manipulated antibodies can be generated and subjected to binding selection using the same approach to identify suitable reactive sites for free cysteine ​​incorporation from a random protein-phage library of antibodies or other proteins. This technique involves reacting the cysteine ​​mutant proteins presented on the phage with an affinity reagent or reporter group (which is also thiol-reactive).

[0217] In a particular embodiment, the PHESELECTOR assay includes the following steps: 1) Separately coating Maxisorp 96-well plates with bovine serum albumin (BSA), part or all of the target protein (e.g., erbB2 extracellular domain (HER2)), and streptavidin (100 μl at 2 μg / ml); 2) Blocking with 0.5% Tween-20 (in PBS), followed by biotinylated and non-biotinylated THIOMAB® antibody-phage (2 × 10⁻¹⁶). 101) Incubate the phage particles at room temperature for 1 hour (e.g., hu4D5-THIOMAB(trademark) antibody-phage if the target protein is the erbB2 extracellular domain (HER2)), 2) After incubation with the phage, incubate with horseradish peroxidase (HRP) labeled secondary antibody (anti-M13 phage coated protein, pVIII protein antibody), 3) Perform a standard HRP reaction and measure absorption at 450 nm, 4) Add streptavidin OD so that a thiol reactivity value of 1 indicates complete biotinylation of cysteinethiol. 450 / OD of target protein (e.g., HER2) 450 Thiol reactivity is measured by calculating [a certain value].

[0218] Protein expression and purification

[0219] The DNA encoding cysteine-modified antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of mouse antibodies). Hybridoma cells serve as a source of such DNA. After isolation, the DNA can be placed into an expression vector, which is then transfected into host cells that do not normally produce antibody proteins, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, HEK293T cells, or other mammalian host cells, such as myeloma cells (U.S. Patent No. 5807715, U.S. Publication No. 2005 / 0048572, U.S. Publication No. 2004 / 0229310), to achieve the synthesis of monoclonal antibodies in recombinant host cells. In many cases, the yield of cysteine-modified antibodies was similar to that of wild-type antibodies. Articles discussing the recombinant expression of antibody-encoding DNA in bacteria include Skerra et al (1993) Curr. Opinion in Immunol. 5:256-262 and Pluckthun (1992) Immunol. Revs. 130:151-188.

[0220] After design and selection, cysteine-modified antibodies having highly reactive unpaired Cys residues, such as THIOMAB® antibodies, can be produced by (i) expression in bacterial systems, such as Escherichia coli or mammalian cell culture systems (International Publication No. WO01 / 00245), such as Chinese hamster ovary (CHO) cells or HEK293 cells (e.g., HEK293T cells), and (ii) purification using general purification techniques (Lowman et al (1991) J. Biol. Chem. 266(17):10982-10988). In a specific embodiment of the present invention, THIOMAB® antibodies were expressed in a mammalian cell expression system. In a specific embodiment, the mammalian cell system was HEK293T cells.

[0221] THIOMAB® antibodies are full-length antibodies containing native cysteine ​​residues that form disulfide bonds within the antibody. Therefore, these native cysteine ​​residues do not have any reactive thiol groups that conjugate with drug-maleimides (unless treated with a reducing agent). Thus, newly engineered Cys residues may remain unpaired or react with electrophilic linker reagents or drug-linker intermediates, such as drug-maleimides, i.e., they can be conjugated.

[0222] The structural positions of the manipulated Cys residues in the heavy and light chains are numbered according to a sequential numbering system. This sequential numbering system correlates with the Kabat numbering system (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD) for 4D5 antibodies. 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 FR or CDR in the variable domain. The cysteine ​​manipulated heavy chain and light chain mutant sites are identified by sequential numbering and Kabat numbering in Figures 1A and 1B.

[0223] Thiol reactivity can also be generalized to specific domains of an 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 approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 0.95 or higher can be made to the heavy chain constant domains α, δ, ε, γ, and μ of intact antibodies IgA, IgD, IgE, IgG, and IgM (including IgG subclasses IgG1, IgG2, IgG3, IgG4, IgA, and IgA2), respectively.

[0224] Labeled cysteine-modified antibody

[0225] The cysteine-modified antibodies of the present invention can be conjugated with any labeling moiety that can be covalently bound to the antibody via a reactive cysteinethiol group (Singh et al (2002) Anal. Biochem. 304:147-15, Harlow E. and Lane, D. (1999) Using Antibodies: A Laboratory Manual, Cold Springs Harbor Laboratory Press, Cold Spring Harbor, NY, Lundblad RL (1991) Chemical Reagents for Protein Modification, 2nd ed. CRC Press, Boca Raton, FL). The bound label may function to (i) provide a detectable signal, (ii) interact with a second label to alter the detectable signal provided by the first or second label, e.g., perform FRET (fluorescence resonance energy transfer), (iii) stabilize the affinity for interaction with or binding to an antigen or ligand, (iv) affect mobility, e.g., electrophoretic mobility or cell permeability, by charge, hydrophobicity, shape, or other physical parameters, or (v) provide a capture site for regulating ligand affinity, antibody / antigen binding, or ionic complex formation.

[0226] Labeled cysteine-modified antibodies can be useful in diagnostic assays, for example, to detect the expression of a target antigen in specific cells, tissues, or serum. For diagnostic applications, antibodies will typically be labeled at a detectable portion. Numerous labels are available, which can generally be categorized as follows:

[0227] (a) Radioactive isotopes (radionuclides), for example, 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 213 Bi. Radioisotope-labeled antibodies are useful in receptor-targeting imaging experiments. Antibodies can be labeled with a ligand reagent (which reacts with the antibody's operating cysteinethiol) that conjugates, chelates, or otherwise forms a complex with a radioisotope metal, using the techniques described in Current Protocols in Immunology, (1991) Volumes 1 and 2, Coligen et al, Ed. Wiley-Interscience, New York, NY, Pubs. Examples of chelate ligands that form complexes with metal ions include DOTA, DOTP, DOTMA, DTPA, and TETA (Macrocyclics, Dallas, TX). Radionuclides can be targeted via complex formation with the antibody-drug conjugate of the present invention (Wu et al (2005) Nature Biotechnology 23(9):1137-1146). DOTA-maleimide reagents react with the free cysteine ​​amino acid of cysteine-manipulated antibodies, yielding a metal that forms a ligand complex on the antibody (Lewis et al (1998) Bioconj. Chem. 9:72-86). Chelate linker-labeled 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-targeted imaging using radionuclide-labeled antibodies can provide markers of pathway activation by detecting and quantifying the progressive accumulation of antibodies in tumor tissue (Albert et al (1998) Bioorg. Med. Chem. Lett. 8:1207-1210).

[0228] Metal-chelate complexes suitable as antibody labels for imaging experiments (US Publication No. 2010 / 0111856, U.S. Patent No. 5342606, U.S. Patent No. 5428155, U.S. Patent No. 5316757, U.S. Patent No. 5480990, U.S. Patent No. 5462725, U.S. Patent No. 5428139, U.S. Patent No. 5385893, U.S. Patent No. 5739294, U.S. Patent No. 5750660, U.S. Patent No. 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 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). .

[0229] (b) Fluorescent labels, e.g., rare earth chelates (europium chelate), fluorescein species including FITC, 5-carboxyfluoroceine, 6-carboxyfluoroceine; rhodamine species including TAMRA; dansyl; lysamine; cyanine; phycoerythrin; Texas Red; and analogs thereof. Fluorescent labels can be conjugated to antibodies using techniques disclosed, for example, in Current Protocols in Immunology (above). Fluorescent dyes and fluorescent labeling reagents include those commercially available from Invitrogen / Molecular Probes (Eugene, OR) and Pierce Biotechnology, Inc. (Rockford, IL).

[0230] 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 a detectable signal and are generally applicable to labeling antibodies, preferably having the following characteristics: (i) the labeled antibody should produce a very high signal with low background so that small amounts of antibody can be detected with high sensitivity in both cell-free and cell-based assays, and (ii) the labeled antibody needs to be photostable so that the fluorescent signal can be observed, monitored, and recorded without significant photobleaching. For applications involving cell surface binding of labeled antibodies to membranes or cell surfaces, particularly to living cells, it is preferable that the label (iii) has good water solubility to achieve effective conjugation concentrations and detection sensitivity, and (iv) is non-toxic to living cells so as not to disrupt normal metabolic processes of cells or cause immature cell death.

[0231] (c) Various enzyme-substrate labels are available or disclosed (U.S. Patent No. 4275149). Enzymes generally catalyze chemical modifications of chromogenic substrates, which can be measured using a variety of techniques. For example, an enzyme may catalyze a change in the color of a substrate, which can be measured by spectrophotometry. Alternatively, an enzyme may alter the fluorescence or chemiluminescence of a substrate. Techniques for quantifying changes in fluorescence are described above. A chemiluminescent substrate may become electronically excited by a chemical reaction and then emit light, which may be measured (e.g., using a chemiluminometer) or which transfers energy to a fluorescence acceptor. Examples of enzyme labeling include luciferases (e.g., firefly luciferase and bacterial luciferase, U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, malate dehydrogenase, urease, horseradish peroxidase (HRP) and other peroxidases, alkaline phosphatase (AP), β-galactosidase, glucoamylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, and microperoxidase. Techniques for conjugating enzymes into antibodies are described in O'Sullivan et al (1981) “Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay”, in Methods in Enzym. (ed J. Langone & H. Van Vunakis), Academic Press, New York, 73:147-166.

[0232] Examples of enzyme-substrate combinations (U.S. Patent Nos. 4,275,149 and 4,318,980) include the following:

[0233] (i) Horseradish peroxidase (HRP) and hydrogen peroxidase as a substrate (hydrogen peroxidase oxidizes the pigment precursor (e.g., orthophenylenediamine (OPD) or 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB))),

[0234] (ii) Alkaline phosphatase (AP) and para-nitrophenyl phosphate as a chromogenic substrate,

[0235] (iii) β-D-galactosidase (β-D-Gal) and a chromogenic substrate (e.g., p-nitrophenyl-β-D-galactosidase) or a fluorescent substrate 4-methylumbelliferyl-β-D-galactosidase.

[0236] The label can be indirectly conjugated with a cysteine-modified antibody. For example, the antibody can be conjugated with biotin, and any of the broad categories of labels described above may be conjugated with avidin or streptavidin, and vice versa. Since biotin selectively binds to streptavidin, this label can be conjugated to the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the label with a polypeptide variant, the polypeptide variant is conjugated with a low molecular weight hapten (e.g., digoxin), and any of the various types of labels described above is conjugated with an anti-hapten polypeptide variant (e.g., an anti-digoxin antibody). In this way, indirect conjugation of the label with a polypeptide variant can be achieved (Hermanson, G. (1996) in Bioconjugate Techniques, Academic Press, San Diego).

[0237] 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.).

[0238] Detection labels can be useful for localizing, visualizing, and quantifying binding or recognition events. The labeled antibodies of the present invention can detect cell surface receptors. Another application of detectably labeled antibodies is a bead-based immunocapsulation method, which involves conjugating beads with a fluorescently labeled antibody and detecting the fluorescent signal upon ligand binding. Similar binding detection techniques utilize surface plasmon resonance (SPR) to measure and detect antibody-antigen interactions.

[0239] The labeled cysteine-modified antibody of the present invention is useful as an imaging biomarker and probe for various biomedical and molecular imaging methods and techniques, including (i) MRI (magnetic resonance imaging), (ii) MicroCT (computed 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, U.S. Patent Application No. US2010 / 0111856, (v) bioluminescence, (vi) fluorescence, and (vii) ultrasound. Immunoscintigraphy is an imaging procedure in which an antibody labeled with a radioactive substance is administered to an animal or human patient, and an image is obtained at the site in the body where the antibody is localized (U.S. Patent No. US6528624). Imaging biomarkers can be objectively measured and evaluated as indicators of normal biological processes, pathological processes, or pharmacological responses to therapeutic interventions. Biomarkers can be of several types: Type 0 markers are markers of the natural course of disease and correlate over the long term with known clinical indicators, e.g., MRI assessment of synovial inflammation in rheumatoid arthritis; Type I markers capture the effect of interventions according to their mechanism of action, although their mechanism of action may not be related to clinical prognosis; and Type II markers function as surrogate endpoints, predicting the clinical benefit of "validating" targeted responses, such as bone erosion in rheumatoid arthritis measured by CT, through changes in or signals from the biomarker. Thus, imaging biomarkers can provide pharmacodynamic (PD) therapeutic information regarding (i) the expression of target proteins, (ii) the binding of therapeutic agents to target proteins, i.e., selectivity, and (iii) clearance and pharmacokinetic data. Compared to laboratory-based biomarkers, the advantages of in vivo imaging biomarkers include non-invasiveness, quantification, systemic evaluation, repeated administration and evaluation (i.e., multiple time points), and the potential for efficacy transfer from preclinical results (small animals) to clinical results (humans).For some applications, bioimaging can eliminate or minimize the number of animal experiments required in preclinical research.

[0240] Peptide labeling methods are well known. Haugland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc., Brinkley, 1992, Bioconjugate Chem. 3:2, Garman, (1997) Non-Radioactive Labeling: A Practical Approach, Academic Press, London, Means (1990) Bioconjugate Chem. 1:2, Glazer et al (1975) Chemical Modification of Proteins. Laboratory Techniques in Biochemistry and Molecular Biology (TSWork and E. Work, Eds.) American Elsevier Publishing Co., New York, Lundblad, RLand Noyes, CM (1984) Chemical Reagents for Protein Modification, Vols. I and II, CRC Press, New York, Pfleiderer, G. (1985) “Chemical Modification of Proteins”, Modern Methods. See *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*, and Mier et al (2005) *Bioconjugate Chem. 16:240-237*.

[0241] Peptides and proteins labeled with two moieties, a fluorescent reporter and a quencher, are subjected to fluorescence resonance energy transfer (FRET) at sufficient proximity. The reporter group is typically a fluorescent dye that is excited by light of a specific wavelength, transfers energy to an acceptor or quencher group, and has a Stokes shift suitable for emission at maximum brightness. Fluorescent dyes include highly aromatic molecules such as fluorescein and rhodamine, and their derivatives. The fluorescent reporter can be partially or fully quenched by the quencher in the intact peptide. The increase in detectable fluorescence can be measured by cleavage of the peptide by a peptidase or protease (Knight, C. (1995) “Fluorimetric Assays of Proteolytic Enzymes”, Methods in Enzymology, Academic Press, 248:18-34).

[0242] The labeled antibody of the present invention may also be used as an affinity purifier. In this process, the labeled antibody is immobilized on a solid phase such as Sephadex resin or filter paper using methods well known in the art. The immobilized antibody is contacted with a sample containing the antigen to be purified, and then the support is washed with a suitable solvent to remove substantially all material from the sample except for the antigen to be purified that is bound to the immobilized polypeptide variant. Finally, the support is washed with another suitable solvent such as glycine buffer at pH 5.0, thereby separating the antigen from the polypeptide variant.

[0243] Labeling reagents typically have a reactive functional group that can (i) react directly with cysteinethiol of a cysteine-manipulated antibody to form a labeled antibody, (ii) react with a linker reagent to form a linker-labeling intermediate, or (iii) react with a linker antibody to form a labeled antibody. Examples of reactive functional groups for labeling reagents include maleimide, haloacetyliodoacetamidosuccinimidyl ester (e.g., NHS, N-hydroxysuccinimidyl), isothiocyanate, sulfonyl chloride, 2,6-dichlorotriazinyl, pentafluorophenyl ester, and phosphoramidite, but other functional groups can also be used.

[0244] Conjugation of linker drugs to THIOMAB® antibodies

[0245] To demonstrate the conjugation efficiency of the manipulated cysteine ​​at each site within the heavy and light chains, the THIOMAB® antibody described herein was conjugated to two different linker drugs.

[0246] THIOMAB® antibodies were generated by manipulating cysteine ​​residues at various positions in the representative antibody Hu anti-Her2 4D5 ("4D5 antibody"). Each THIOMAB® antibody was separately conjugated to two linker drugs (MC-vc-PAB-MMAE and PDS-MMAE). Conjugation was performed in a 96-well filter plate (2 ml volume 0.45 μm polypropylene filter from EK Scientific). The buffer solution flowed only when the plate was centrifuged at 500 x g for 2 minutes. 450 μl of MabSelect SuRe resin (GE Healthcare) was added to each well as a 50% slurry in 20% ethanol. The resin was washed three times and equilibrated in 50 mM Tris pH 8.0, 150 mM NaCl, and 2 mM EDTA (buffer A).

[0247] Each THIOMAB® antibody (1.5 mg) was added to each well and bound to the resin on a plate shaker at 600 RPM at room temperature for 30 minutes. After 30 minutes, the plate was centrifuged to remove excess buffer. The THIOMAB® antibody was reduced overnight at room temperature in the presence of 0.9 ml of 2 mM dithiothreitol (in buffer A) with stirring. The reducing agent and any cysteine ​​or glutathione block were purified and removed by washing twice with buffer A. The plate was saturated with the oxidizing agent by washing three times with 1 ml of 1 mM dehydroascorbic acid (DHAA) (in buffer A). After adding 0.9 ml of 1 mM DHAA (in buffer A) as a final step, the THIOMAB® antibody was oxidized again on a plate shaker at room temperature for 3 hours.

[0248] The oxidizing agent was removed by centrifugation. A 2x molar excess (relative to the available thiol groups) of linker drug (MC-vc-PAB-MMAE or PDS-MMAE), dissolved in 10% DMA (in buffer A), was added to each well and incubated with THIOMAB® antibody on a plate shaker at room temperature for 2 hours. Excess linker drug was purified and removed by washing the plate six times with equilibrium buffer. The conjugated THIOMAB® antibody was eluted with 0.1 M glycine buffer pH 2.7 on a plate shaker at room temperature for 30 minutes. The THIOMAB® antibody was immediately neutralized with 15% 0.5 M Tris, pH 8.0. The number of conjugated drug molecules per mAb was quantified by LC / MS analysis. Conjugation was evaluated by size exclusion chromatography.

[0249] Analysis by mass spectrometry

[0250] Liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) was used to accurately determine the molecular weight of the conjugated THIOMAB® antibody (Cole, RB Electro Spray Ionization Mass Spectrometry: Fundamentals, Instrumentation And Applications. (1997) Wiley, New York).

[0251] LC / MS analysis was performed using a 6224 Mass Time-of-Flight (TOF) LC / MS (Agilent Technologies). Samples were subjected to chromatography using a PRLP-S column heated to 80°C, 1000 Å, 8 μm (50 mm × 2.1 mm, Agilent Technologies). A linear gradient of B (solvent A, 0.05% TFA (in water), solvent B, 0.04% TFA (in acetonitrile)) with a flow rate of 0.7 ml / min for 3 minutes was used, and the eluent was directly ionized using an electrospray source. Data were collected and deconvoluted using Agilent Mass Hunter qualitative analysis software. The drug-to-antibody ratio (DAR) was calculated using the numerous deconvoluted peaks present in the LC / MS chromatogram.

[0252] Agglutination analysis of THIOMAB® antibodies

[0253] Size exclusion chromatography was performed using an 1100 series HPLC (Agilent Technologies). Chromatography was performed on the sample using a Shodex KW 802.5 column. The conjugate was eluted using an isocratic method with a mobile phase of 0.75 ml / min for 15 minutes, using 0.2 M potassium phosphate, 0.25 potassium chloride, and pH 6.2. The aggregation percentage was calculated from the integrated area of ​​the aggregation and monomer peaks in the UV 280 nm chromatogram.

[0254] Manipulation of thioIgG variants of trastuzumab

[0255] Cysteine ​​was introduced into all residues of the heavy and light chains of the full-length monoclonal antibody trastuzumab (HERCEPTIN®, Genentech Inc.) (each naturally occurring non-cysteine ​​residue in SEQ ID NO: 1 (4D5 heavy chain) and SEQ ID NO: 2 (4D5 light chain) was mutated to cysteine). The heavy chain of a typical 4D5 antibody has 450 amino acids, consisting of 12 cysteine ​​residues and 438 non-cysteine ​​residues. The light chain of a typical 4D5 antibody has 214 amino acids, consisting of 6 cysteine ​​residues and 208 non-cysteine ​​residues. Specifically, a single residue was mutated from its naturally occurring amino acid to cysteine, thereby obtaining a full-length antibody with two manipulated cysteine ​​residues. Each cysteine ​​mutation was conjugated to PDS-MMAE and MC-vc-MMAE. This resulted in a total of 648 THIOMAB® antibodies: 648 PDS-MMAE THIOMAB® antibodies and 648 MC-vc-MMAE THIOMAB® antibodies. These cysteine-modified antibodies (THIOMAB® antibodies) were expressed in HEK293T cells in culture medium. EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 18 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence ID 19

[0256] In a preferred embodiment, the THIOMAB® antibody contains one or more heavy chain mutations from the Kabat-numbered heavy chain mutations in Table 2. In a preferred embodiment, the THIOMAB® antibody contains a modified cysteine ​​at a position equivalent to those listed in Table 2 by 4D5 sequence. In a preferred embodiment, the modified cysteine ​​specified in Table 2 or at an equivalent position contained in the THIOMAB® antibody is free cysteine. In a preferred embodiment, the THIOMAB® antibody contains a modified cysteine ​​at position HC-A136C by Kabat numbering (i.e., HC-A140C by EU numbering) (see Example 11).

[0257] In a preferred embodiment, the THIOMAB® antibody contains one or more heavy chain mutations as assigned EU numbers in Figure 21. Specifically, the heavy chain cysteine ​​mutation in the cysteine-manipulated antibody is selected from the group of sites consisting of HC-T110C, HC-A140C, HC-L174C, HC-L179C, HC-T187C, HC-T209C, HC-V262C, HC-G371C, HC-Y373C, HC-E382C, HC-S242C, HC-N434C, and Q438C, as assigned EU numbers. In a preferred embodiment, the THIOMAB® antibody comprises a cysteine-modified antibody conjugated to the drug moiety by a PDS linker in a modified cysteine ​​selected from the group consisting of HC-A140C, HC-L174C, HC-L179C, HC-G371C, HC-Y373C, HC-S424C, and HC-Q438C by EU numbering. In a preferred embodiment, the THIOMAB® antibody comprises a cysteine-modified antibody conjugated to the drug moiety by a vc- (i.e., maleimide) linker in a modified cysteine ​​selected from the group consisting of HC-T110C, HC-T187C, HC-T209C, HC-V262C, HC-G371C, HC-E382C, and HC-N434C.

[0258] In certain embodiments, the THIOMAB® antibody contains one or more heavy chain mutations as numbered in Table 3 by Kabat. In certain embodiments, the THIOMAB® antibody contains a modified cysteine ​​at a position equivalent to those listed in Table 3 by 4D5 sequence. For example, if an antibody contains natural alanine (A) at position 5 by Kabat numbering in its heavy chain (compared to natural valine (V) in 4D5), this alanine can be mutated to cysteine ​​to obtain the HC-A5C THIOMAB® antibody. In certain embodiments, the modified cysteine ​​specified in Table 3 or at an equivalent position in the THIOMAB® antibody is free cysteine. Table 3. Heavy chain cysteine ​​mutations according to Kabat numbering. JPEG0007924744000017.jpg255170JPEG0007924744000018.jpg208170

[0259] In certain embodiments, the THIOMAB® antibody contains the sequences listed in Table 3. In certain embodiments, the THIOMAB® antibody having the mutations and / or sequences listed in Table 3 contains a PDS linker. In certain embodiments, the THIOMAB® antibody having the mutations and / or sequences listed in Table 3 contains a maleimide (e.g., -vc) linker. In specific embodiments, a heavy chain site (according to Kabat numbering) selected from the following group is mutated to cysteine ​​to form a cysteine-modified antibody, which is then conjugated to a drug using a PDS linker: V2C, T29C, Y30C, A37C, E43C, Y77C, W96C, G97C, D105C, T139C, N159C, A162C, G166C, G178C, L179C, V188C, I199C, N203C, S207C, E388C, K414C, Q418C, S242C, Y436C, T437C, Q438C, L443C, and M104C. In specific embodiments, a heavy chain site (according to Kabat numbering) selected from the following group is mutated to cysteine ​​to form a cysteine-modified antibody, which is then conjugated to a drug using a -vc (i.e., maleimide) linker: V2C, L1C, V2C, L8C, R16C, F24C, I26C, Y30C, Q36C, A37C, K40C, L42C, E43C, T51C, G53C, T55C, R56C, Y57C, A58C, T66C, N74C, Q79C, W107C, T120C, K121C A140C, G166C, G178C, T187C, I199C, T209C, F243C, M252C, E258C, V262C, N276C, V282C, L309C, T335C, S337C, R344C, Q347C, K360C, G371C, E382C, P387C, E388C, S403C, K414C, Q418C, G420C, N421C, S424C, N434C, Y436C, T437C, Q438C, K439C, S442C, L443C, M104C, and N81C.

[0260] In specific embodiments, a cysteine-manipulated antibody containing a heavy chain cysteine ​​mutation selected from the mutations listed in Table 2 has an average DAR of 1.0 to 2.0 using the Affinity-Capture LC-MS Assays for Stability Determination Assay described herein (see Examples 12 and 13). In specific embodiments, a cysteine-manipulated antibody containing a heavy chain cysteine ​​mutation selected from the mutations listed in Table 2 has an average DAR of 1.1 to 1.3. In specific embodiments, a cysteine-manipulated antibody containing a heavy chain cysteine ​​mutation selected from the mutations listed in Table 2 has an average DAR of 1.3 to 1.5. In specific embodiments, a THIOMAB® antibody having a cysteine ​​mutation selected from the list in Table 1 has an average DAR of 1.5 to 1.8. In specific embodiments, a cysteine-manipulated antibody containing a heavy chain cysteine ​​mutation selected from the mutations listed in Table 2 has an average DAR of 1.8 to 2.0.

[0261] In a preferred embodiment, the cysteine-modified antibody contains one or more heavy chain mutations from the Kabat-numbered heavy chain mutations in Table 2. In a preferred embodiment, the cysteine-modified antibody contains modified cysteine ​​at a position equivalent to those listed in Table 2 by 4D5 sequence. For example, if an antibody contains natural lysine (K) at position 19 by Kabat numbering in its heavy chain (compared to natural arginine (R) in 4D5), this lysine can be mutated to cysteine ​​to obtain the K19C THIOMAB® antibody. In a preferred embodiment, the modified cysteine ​​specified in Table 2 or at an equivalent position in the THIOMAB® antibody is free cysteine. Table 4. Light chain cysteine ​​mutations according to Kabat numbering. JPEG0007924744000019.jpg255170JPEG0007924744000020.jpg154170

[0262] In a preferred embodiment, the THIOMAB® antibody contains one or more light chain mutations from the Kabat-numbered light chain mutations listed in Table 1. In a preferred embodiment, the THIOMAB® antibody contains a modified cysteine ​​at a position equivalent to those listed in Table 1 by 4D5 sequence. In a preferred embodiment, the modified cysteine ​​specified in Table 1 or at an equivalent position contained in the THIOMAB® antibody is free cysteine. In a preferred embodiment, the THIOMAB® antibody contains a modified cysteine ​​at position LC-K149C by Kabat numbering (see Examples 2 and 7). In a preferred embodiment, the LC-K149C antibody is conjugated to the drug moiety via a PDS linker. In a preferred embodiment, the LC-K149C antibody is conjugated to the drug moiety via a -vc (i.e., maleimide) linker.

[0263] In certain embodiments, the THIOMAB® antibody contains one or more light chain mutations from the Kabat-numbered light chain mutations shown in Figure 21. Specifically, the light chain cysteine ​​mutation in the cysteine-manipulated antibody is selected from the Kabat-numbered light chain cysteine ​​mutations LC-T22C, LC-K39C, LC-Y49C, LC-Y55C, LC-T85C, LC-T97C, LC-I106C, LC-R108C, LC-R142C, LC-K149C, and LC-V205C. In preferred embodiments, the THIOMAB® antibody contains a cysteine-manipulated antibody conjugated to the drug moiety by a PDS linker in a manipulated cysteine ​​selected from the group consisting of Kabat-numbered LC-I106C, LC-R108C, and LC-V205C. In a preferred embodiment, the THIOMAB® antibody comprises a cysteine-modified antibody conjugated to the drug moiety by a vc-(i.e., maleimide) linker in a modified cysteine ​​selected from the group consisting of Kabat-numbered LC-T22C, LC-K39C, LC-Y49C, LC-Y55C, LC-T85C, LC-T97C, LC-R142C, and LC-K149C.

[0264] In specific embodiments, a cysteine-manipulated antibody containing a light chain cysteine ​​mutation selected from the mutations listed in Table 1 has an average DAR of 1.0 to 2.0 using the Affinity-Capture LC-MS Assays for Stability Determination Assay described herein (see Examples 12 and 13). In specific embodiments, a cysteine-manipulated antibody containing a light chain cysteine ​​mutation selected from the mutations listed in Table 1 has an average DAR of 1.1 to 1.3. In specific embodiments, a cysteine-manipulated antibody containing a light chain cysteine ​​mutation selected from the mutations listed in Table 1 has an average DAR of 1.3 to 1.5. In specific embodiments, a cysteine-manipulated antibody having a cysteine ​​mutation selected from the list in Table 1 has an average DAR of 1.5 to 1.8. In specific embodiments, a cysteine-manipulated antibody containing a heavy chain cysteine ​​mutation selected from the mutations listed in Table 1 has an average DAR of 1.8 to 2.0. In a specific embodiment, a cysteine-modified antibody containing a heavy chain cysteine ​​mutation selected from the mutations listed in Table 1 has an average DAR of 0.8 to 1.4.

[0265] In a preferred embodiment, the THIOMAB® antibody contains the sequences listed in Table 1. In a preferred embodiment, the THIOMAB® antibody having the mutations and / or sequences listed in Table 1 contains a PDS linker. In a preferred embodiment, the THIOMAB® antibody having the mutations and / or sequences listed in Table 1 contains a -vc linker.

[0266] In certain embodiments, the THIOMAB® antibody contains the sequences listed in Table 4. In certain embodiments, the THIOMAB® antibody having the mutations and / or sequences listed in Table 4 contains a PDS linker. In certain embodiments, the THIOMAB® antibody having the mutations and / or sequences listed in Table 4 contains a -vc linker.

[0267] In certain embodiments, a cysteine-manipulated antibody containing a light chain cysteine ​​mutation selected from the mutations listed in Table 4 has an average DAR of 1.0 to 2.0 using the Affinity-Capture LC-MS Assays for Stability Determination Assay described herein (see Examples 12 and 13). In specific embodiments, a THIOMAB® antibody having a cysteine ​​mutation selected from the list in Table 4 has an average DAR of 1.3 to 1.9. In specific embodiments, a THIOMAB® antibody having a cysteine ​​mutation selected from the list in Table 4 has an average DAR of 1.3 to 1.8. In specific embodiments, a THIOMAB® antibody having a cysteine ​​mutation selected from the list in Table 4 has an average DAR of 1.5 to 1.9. In specific embodiments, a THIOMAB® antibody having a cysteine ​​mutation selected from the list in Table 4 has an average DAR of 0.8 to 1.0.

[0268] In a preferred embodiment, the THIOMAB® antibody contains one or more light chain mutations according to EU numbering as shown in Figure 21. Specifically, the light chain cysteine ​​mutation in the cysteine-manipulated antibody is selected from the group consisting of EU-numbered sites LC-T22C, LC-K39C, LC-Y49C, LC-Y55C, LC-T85C, LC-T97C, LC-I106C, LC-R108C, LC-R142C, LC-K149C, and LC-V205C. In a preferred embodiment, the THIOMAB® antibody contains a cysteine-manipulated antibody conjugated to the drug moiety by a PDS linker in a manipulated cysteine ​​selected from the group consisting of EU-numbered sites LC-T22C, LC-K39C, LC-Y49C, LC-Y55C, LC-T85C, LC-T97C, LC-R142C, and LC-K149C. In a preferred embodiment, the THIOMAB® antibody comprises a cysteine-modified antibody conjugated to the drug moiety by a vc-(i.e., maleimide) linker in a modified cysteine ​​selected from the group consisting of LC-I106C, LC-R108C, and LC-V205C.

[0269] In certain embodiments, the THIOMAB® antibody contains the sequences listed in Table 5. In certain embodiments, the THIOMAB® antibody contains a modified cysteine ​​at a position equivalent to those listed in Table 5 by 4D5 sequence. For example, if an antibody contains a natural serine (S) at position 40 by Kabat numbering in its heavy chain (compared to a natural alanine (A) in 4D5), this serine can be mutated to cysteine ​​to obtain the S40C THIOMAB® antibody. In certain embodiments, the modified cysteine ​​specified in Table 5 or a modified cysteine ​​at an equivalent position contained in the THIOMAB® antibody is free cysteine. Table 5. Heavy and light chain cysteine ​​mutations according to Kabat numbering. JPEG0007924744000021.jpg38170

[0270] In certain embodiments, the THIOMAB® antibody contains the sequences listed in Table 5. In certain embodiments, the THIOMAB® antibody having the mutations and / or sequences listed in Table 5 contains a -vc or PDS linker.

[0271] In specific embodiments, THIOMAB® antibodies having cysteine ​​mutations selected from the list in Table 5 have an average DAR of 1.0 to 2.0. In specific embodiments, THIOMAB® antibodies having cysteine ​​mutations selected from the list in Table 5 have an average DAR of 1.3 to 1.9. In specific embodiments, THIOMAB® antibodies having cysteine ​​mutations selected from the list in Table 5 have an average DAR of 1.3 to 1.8. In specific embodiments, THIOMAB® antibodies having cysteine ​​mutations selected from the list in Table 5 have an average DAR of 1.5 to 1.9.

[0272] According to another embodiment, the THIOMAB® antibody comprises a modified cysteine ​​specified in any one of Tables 1 to 5. In a preferred embodiment, the THIOMAB® antibody comprises a modified cysteine ​​specified in any one of Tables 1 and 2. According to another embodiment, the THIOMAB® antibody may be designed for any target antigen.

[0273] Design and manipulation of THIOMAB® antibodies

[0274] Single cysteine ​​substitutions were introduced at the respective positions of the heavy and light chains of the anti-HER2 hu4D5 antibody (i.e., a total of 648 antibodies with PDS linkers and a total of 648 antibodies with -vc linkers were produced) (Example 1). Single cysteine ​​substitutions were also introduced at select positions of anti-CD33 antibody, anti-STEAP1 antibody, anti-MUC16 antibody, anti-NaPi2b antibody, anti-Ly6E antibody, anti-CD22 antibody, anti-CD79b antibody, anti-B7H4 antibody, and additional anti-HER2 antibodies. All heavy and light chain variants were prepared according to the methods described herein. Kabat numbering system. The heavy and light chain sequences were numbered according to the Kabat numbering system. Positional equivalences by sequential numbering and EU numbering of each Kabat position are shown in Figures 1A and 1B. In the light chain, Kabat, sequential, and EU numbering indicate the same number.

[0275] Heavy and light chain 4D5 THIOMAB® antibodies were conjugated to drugs via PDS or vc linkers (i.e., a total of 648 antibodies with PDS linkers and a total of 648 antibodies with vc linkers were produced). Each THIOMAB® antibody was screened for mean drug-to-antibody ratio (DAR), concentration (mg / mL), agglutination (total %), and stability (Example 2). Accordingly, each THIOMAB® antibody had measured values ​​for DAR, concentration, agglutination, and stability for both the PDS and vc conjugates. The PDS conjugate and measured values ​​are shown in the tables provided herein, and the vc conjugate and measured values ​​are shown in the tables provided herein. Table 6. Mean DAR, concentration, agglutination, and stability (ELISA rat plasma stability) of cysteine-modified antibodies from Tables 3 and 4 conjugated with PDS-linkers. JPEG0007924744000022.jpg255170JPEG0007924744000023.jpg255170JPEG0007924744000024.jpg255170Table 7. Mean DAR, concentration, aggregation, and stability (mass spectrometry rat plasma stability) of cysteine-modified antibodies from Tables 3 and 4 conjugated with PDS-linkers. JPEG0007924744000025.jpg249170JPEG0007924744000026.jpg255170JPEG0007924744000027.jpg255170JPEG0007924744000028.jpg143170Table 8. Mean DAR, concentration, agglutination, and stability (ELISA rat plasma stability) of cysteine-manipulated antibodies from Tables 3 and 4 conjugated with -vc linker. JPEG0007924744000029.jpg249170JPEG0007924744000030.jpg255170JPEG0007924744000031.jpg255170JPEG0007924744000032.jpg217170Table 9. Mean DAR / stability (mass spectrometry rat plasma stability) of cysteine-modified antibodies from Tables 3 and 4 conjugated with -vc linker. JPEG0007924744000033.jpg248170JPEG0007924744000034.jpg255170JPEG0007924744000035.jpg255170JPEG00079247440 00036.jpg255170JPEG0007924744000037.jpg255170JPEG0007924744000038.jpg255170JPEG0007924744000039.jpg109170

[0276] The PDS-MMAE THIOMAB(trademark) antibodies identified in Tables 6 and 7 were analyzed at starting concentrations of 1 mg / mL or higher of the raw material (i.e., PDS-MMAE THIOMAB(trademark) antibody). The DAR calculation values ​​for all PDS-MMAE THIOMAB(trademark) antibodies identified in Tables 6 and 7 were DAR ≥ 1. All PDS-MMAE THIOMAB(trademark) antibodies identified in Tables 6 and 7 exhibited aggregation and reoxidation of 50% or less. The PDS-MMAE THIOMAB(trademark) antibodies identified in Table 6 showed stability of at least 77% over time in the rat matrix against repeated ELISA tests. The same samples analyzed in Table 6 were used in the experiments performed in Table 7. Table 7 shows the confirmation of the ELISA stability results in Table 6 by LCSM.

[0277] The MC-VC-MMAE THIOMAB® antibodies identified in Tables 8 and 9 were analyzed at starting concentrations of 1 mg / mL or higher of the raw material (i.e., MC-VC-MMAE THIOMAB® antibodies). The DAR calculation values ​​for all MC-VC-MMAE THIOMAB® antibodies identified in Tables 8 and 9 were DAR ≥ 1. All MC-VC-MMAE THIOMAB® antibodies identified in Tables 8 and 9 exhibited aggregation and reoxidation of 50% or less. The MC-VC-MMAE THIOMAB® antibodies identified in Table 8 showed stability of at least 77% over time in the rat matrix for repeated ELISA tests. Table 9 shows confirmation of the ELISA stability results for Table 8 by LCSM.

[0278] Table 10. Mean DAR, concentration, and aggregation of preferred cysteine-modified antibodies from Tables 1 and 2 (same samples as those shown in Tables 11 and 12) JPEG0007924744000040.jpg248170JPEG0007924744000041.jpg63170Table 11. ELISA and MS stability results of preferred cysteine-modified antibodies from Tables 1 and 2 (same samples as those shown in Tables 10 and 12) JPEG0007924744000042.jpg246170JPEG0007924744000043.jpg251170JPEG0007924744000044.jpg252170JPEG0007924744000045.jpg232170Table 12. Results of cysteine ​​reduction assays of preferred cysteine-manipulated antibodies from Tables 1 and 2 (same samples as those shown in Tables 10 and 11) JPEG0007924744000046.jpg245170JPEG0007924744000047.jpg255170JPEG0007924744000048.jpg255170JPEG00079247440 00049.jpg255170JPEG0007924744000050.jpg255170JPEG0007924744000051.jpg255170JPEG0007924744000052.jpg191170

[0279] The DAR calculations for all PDS-MMAE and -vc-MMAE THIOMAB® antibodies identified in Tables 8-10 were above DAR0. The PDS-MMAE THIOMAB® antibodies identified in Table 8 exhibited at least 80% stability in the rat matrix for at least one of the two ELISA repeats.

[0280] Interestingly, DAR, concentration, aggregation, and stability were not the same for vc-conjugated cysteine-modified antibodies (i.e., THIOMAB® antibody) and PDS-conjugated antibodies. For example, the preferred cysteine-modified antibodies (PDS linkers) in Tables 6 and 7 were not identical to those in Tables 8 and 9 (vc linkers). For example, preferred superior stability sites for conjugation to the drug moiety by PDS linkers were LC-T22C, LC-K39C, LC-Y49C, LC-Y55C, LC-T85C, LC-T97C, LC-R142C, LC-K149C, HC-A140C, HC-L174C, HC-L179C, HC-G371C, HC-Y373C, and HC-S424C. On the other hand, the preferred superior stability sites for conjugation of the drug moiety with the -vc linker are LC-I106C, LC-R108C, LC-V205C, HC-T110C (Kabat numbering), HC-T187C (EU numbering), HC-T209C, HC-V262C, HC-G371C, HC-E382C, and HC-N434C (see Figure 21). Therefore, in this screening, only HC-G371C was a superior site for both PDS and -vc. Consequently, determining which site is optimal in terms of stability when conjugating the drug moiety to a cysteine-modified antibody using either the PDS or -vc linker could not be predicted and required detailed experiments.

[0281] THIOMAB (trademark) antibody, later all-reactive The thiol reactivity of full-length IgG cysteine-modified antibodies (THIOMAB® antibodies) can be measured by biotinylation and streptavidin conjugation, as described in U.S. Patent No. 7,521,541 (which is incorporated in whole by reference). Specifically, a Western blot assay can be constructed to screen for THIOMAB® antibodies specifically conjugated with biotin-maleimide. In this assay, the antibody can be analyzed by reductive SDS-PAGE, and the presence of biotin is specifically probed by incubation with streptavidin-HRP. The streptavidin-HRP interaction may be observed on either the heavy or light chain, depending on where the modified cys mutant is used, and this conjugation can be compared to the biotin-streptavidin interaction of wild-type IgG without modified cysteine, thereby indicating which THIOMAB® antibodies specifically conjugate to biotin compared to any background conjugation of wild-type antibodies.

[0282] Antibody-drug conjugates

[0283] The cysteine-modified antibodies of the present invention can be conjugated with any therapeutic agent, i.e., a drug moiety, which may be covalently bound to the antibody via a reactive cysteinethiol group. For illustrative purposes, the cysteine-modified antibodies disclosed in the table provided herein were conjugated with mytansinoid drugs, specifically MMAEs.

[0284] An exemplary embodiment of an antibody-drug conjugate (ADC) compound comprises a cysteine-modified antibody (Ab) and a drug moiety (D), where the antibody has one or more free cysteine ​​amino acids, and the antibody is conjugated to D by a linker moiety (L) via one or more free cysteine ​​amino acids, and this composition is of formula I: Ab-(LD)p I

[0285] The formula comprises, where p is 1, 2, 3, or 4. The number of drug moieties that can be conjugated to the antibody molecule via the thiol-reactive linker moiety is limited by the number of cysteine ​​residues introduced by the method described herein. Thus, an exemplary ADC of formula I comprises an antibody having 1, 2, 3, or 4 manipulated cysteine ​​amino acids.

[0286] Another exemplary embodiment of an antibody-drug conjugate compound (ADC) comprises a cysteine-modified antibody (Ab), an albumin-conjugated peptide (ABP), and a drug moiety (D), where the antibody is conjugated to the drug moiety by a linker moiety (L), and the antibody is conjugated to the albumin-conjugated peptide by an amide bond or a second linker moiety, and this composition is given by formula Ia: ABP-Ab-(LD) p Ia

[0287] The formula has such that p is 1, 2, 3, or 4.

[0288] The ADC compounds of the present invention include those that have usefulness with respect to anticancer activity. Specifically, the compounds contain a cysteine-modified antibody conjugated, i.e., covalently bound, to a drug moiety, i.e., a toxin, by a linker. When the drug moiety is not conjugated to the antibody, the drug has cytotoxic or cell proliferation inhibitory effects. The biological activity of the drug moiety is therefore regulated by conjugation to the antibody. The antibody-drug conjugates (ADCs) of the present invention can selectively deliver an effective volume of a cytotoxic agent to tumor tissue, thereby achieving higher selectivity, i.e., a lower effective dose.

[0289] Drug portion

[0290] The drug portion (D) of an antibody-drug conjugate (ADC) includes any compound, moiety, or group having cytotoxic or cell proliferation inhibitory effects. Examples of drug portions include (i) chemotherapeutic agents (which may function as microtubulin inhibitors, mitotic inhibitors, topoisomerase inhibitors, or DNA intercalators), (ii) protein toxins (which may function enzymatically), and (iii) radioisotopes.

[0291] Examples of drug moieties include, but are not limited to, mytansinoids, auristatin, drastatin, trichothecene, CC1065, calicheamicin, and other engine antibiotics, taxanes, pyrrolobenzodiazepines (PBD), 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI) dimers, CBI-PBD heterodimers, anthracyclines, and their stereoisomers, homologs, analogs, or derivatives.

[0292] Suitable mytansinoid compounds for use as the mytansinoid drug portion are well known in the art and may be isolated from natural sources by known methods or produced using genetic engineering techniques (Yu et al (2002) PROC. NAT. ACAD. SCI. (USA) 99:7968-7973), or mytansinol and mytansinol analogs may be prepared by synthesis according to known methods.

[0293] Examples of mytansinoid drug moieties include, but are not limited to, those having modified aromatic rings such as C-19-dechloro (U.S. Patent No. 4,256,746) (prepared by reduction of ansamytocin P2 with lithium aluminum hydride), C-20-hydroxy (or C-20-demethyl)+ / -C-19-dechloro (U.S. Patents No. 4,361,650 and 4,307,016) (prepared by demethylation using streptomyces or actinomyces or dechlorination using LAH), and C-20-demethoxy, C-20-acyloxy (-OCOR),+ / -dechloro (U.S. Patent No. 4,294,757) (prepared by acylation using acyl chloride), as well as those having modifications at other positions.

[0294] Furthermore, exemplary mytansinoid drug moieties include C-9-SH (US Patent No. 4424219) (prepared by reaction of mytansinol with H2S or P2S5); C-14-alkoxymethyl (demethoxy / CH2OR) (US Patent No. 4331598); C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (US Patent No. 4450254) (prepared from Norcadia); C-15-hydroxy / acyloxy (US Patent No. 4364866) (prepared by conversion of mytansinol by Streptomyces); C-15-methoxy (US Patents No. 4313946 and 4315929) (isolated from Trewia nudlflora); C-18-N-demethyl (US Examples include those with modifications such as (US Patent No. 4362663 and No. 4322348) (prepared by demethylation of mytansinol with Streptomyces) and 4,5-deoxy (US Patent No. 4371533) (prepared by titanium trichloride / LAH reduction of mytansinol). Numerous positions on the mytansin compound are known to be useful as bonding sites depending on the type of bond. For example, for the formation of ester bonds, the C-3 position with a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with a hydroxyl group, and the C-20 position with a hydroxyl group are all suitable.

[0295] The drug portion (D) of the antibody-drug conjugate (ADC) of formula I has the following structure: TIFF0007924744000053.tif69170

[0296] The formula contains a mytansinoid having the following characteristics, where the dashed line indicates a covalent bond between the sulfur atom of D and the linker (L) of the antibody-drug conjugate (ADC). R can independently be H, or a C1-C6 alkyl selected from methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl, 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, and 3,3-dimethyl-2-butyl. The alkylene chain, in which an amide group is bonded to a sulfur atom, can be methanyl, etanyl, or propyl, i.e., m is 1, 2, or 3.

[0297] Mytansin compounds inhibit cell proliferation by inhibiting the formation of microtubules during mitosis through the inhibition of tubulin polymerization, a microtubule protein (Remillard et al (1975) Science 189:1002-1005). Although mytansins and mytansinoids are highly cytotoxic, their clinical use in cancer therapy has been severely limited, mainly due to severe systemic side effects resulting from low selectivity for tumors. Clinical trials of mytansins have been discontinued due to serious side effects on the central nervous system and gastrointestinal system (Issel et al (1978) Can. Treatment. Rev.5:199-207).

[0298] Mytansinoid drug moieties are promising drug moieties in antibody-drug conjugates because they are: (i) relatively easy to prepare by fermentation or chemical modification and derivatization of fermentation products; (ii) suitable for derivatization using functional groups suitable for conjugation to antibodies via non-disulfide linkers; (iii) stable in plasma; and (iv) effective against a variety of tumor cell lines (US Publication No. 2005 / 0169933, International Publication No. WO2005 / 037992, U.S. Patent No. 5208020).

[0299] As with other drug moieties, any stereoisomer of the mytansinoid drug, i.e., any combination of R and S configurations at the chiral carbon of D, is intended for the compound of the present invention. In one embodiment, the mytansinoid drug moiety (D) will have the following stereochemistry. TIFF0007924744000054.tif72170

[0300] Exemplary embodiments of the mytansinoid drug portion include: DM1, (CR2) m =CH2CH2;DM3, (CR2) m =CH2CH2CH(CH3); and DM4, (CR2) m =CH2CH2C(CH3)2, these have the following structure. TIFF0007924744000055.tif230170

[0301] Depending on the type of bond, the linker can be attached to the mytansinoid molecule at various positions. For example, an ester bond may be formed by a reaction with a hydroxyl group using conventional coupling techniques. The reaction can occur at the C-3 position with a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with a hydroxyl group, and the C-20 position with a hydroxyl group. In a preferred embodiment, the bond is formed at the C-3 position of mytansinol or a mytansinol analog.

[0302] The drug portion (D) of the antibody-drug conjugate (ADC) of formula I also includes drastatin and its peptide analogs and derivatives, auristatin (U.S. Patent No. 5,635,483, 5,780,588). Drastatin and auristatin 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 to possess anticancer activity (U.S. Patent No. 5,663,149) and antifungal activity (Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). Various forms of drastatin or auristatin drug moieties can be covalently bound to antibodies via the N (amino) or C (carboxyl) terminus of the peptide drug moiety (International Publication No. WO02 / 088172, Doronina et al (2003) Nature Biotechnology 21(7):778-784, Francisco et al (2003) Blood 102(4):1458-1465).

[0303] The drug portion includes drastatin, auristatin (U.S. Patent Nos. 5,635,483, 5,780,588, 5,767,237, and 6,124,431), as well as their analogs and derivatives. Drastatin and auristatin 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 to possess anticancer activity (U.S. Patent No. 5,663,149) and antifungal activity (Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). The drastatin or auristatin drug moiety can bind to antibodies via the N (amino) or C (carboxyl) terminus of the peptide drug moiety (International Publication No. WO02 / 088172).

[0304] Exemplary embodiments of auristatin include the N-terminally linked monomethyl auristatin drug moieties DE and DF disclosed in U.S. Patent No. 7,498,298 and No. 7,659,241, respectively, the disclosures of each of these patents expressly incorporated in their entirety by reference.

[0305] The drug portion (D) of the antibody-drug conjugate (ADC) of formula I contains monomethyl auristatin drug portions MMAE and MMAF that bind to the antibody via the N-terminus, and these have the following structures. TIFF0007924744000056.tif64170

[0306] Exemplary MMAE ADCs are shown in Figures 11 and 12. Typically, peptide-based drug moieties can be prepared by forming peptide bonds between two or more amino acids and / or peptide fragments. Such peptide bonds can be prepared, for example, by liquid-phase synthesis methods well known in the field of peptide chemistry (e.g., E. Schroder and K. Lubke, “The Peptides”, volume 1, pp. 76-136, 1965, Academic Press).

[0307] The drug portion includes calicheamicin and its analogs and derivatives. The calicheamicin family of antibiotics can cause double-stranded DNA cleavage at concentrations below picomoles. For the preparation of calicheamicin family conjugates, see U.S. Patents 5,712374, 5,714586, 5739116, 5767285, 5770701, 5770710, 5773001, and 5877296. A structural analog of calicheamicin that can be used is γ1 I , α2 I , α3 I N-acetyl-γ1 I , PSAG, and θ I1. Examples include, but are not limited to, Hinman et al. Cancer Research 53:3336-3342 (1993) and Lode et al. Cancer Research 58:2925-2928 (1998).

[0308] In some embodiments, the ADC contains a pyrrolobenzodiazepine (PBD). In some embodiments, the PBD dimer recognizes and binds to a specific DNA sequence. The natural product anthramycin, which is a PBD, was first reported in 1965 (Leimgruber, et al., (1965) J.Am. Chem. Soc., 87:5793-5795, Leimgruber, et al., (1965) J.Am. Chem. Soc., 87:5791-5793). Since then, numerous PBDs have been reported, both natural and analogous, including dimers of the tricyclic PBD scaffold (US Patent Nos. 6,884,799, 7049,311, 7067,511, 7265,105, 7511,032, 7528,126, and 7557099) (Thurston, et al., (1994) Chem. Rev. 1994, 433-465). While not intended to be bound by any particular theory, it is thought that the dimer structure provides a three-dimensional shape suitable for isohelicity with the minor groove of type B DNA, allowing for a snug fit at the binding site (Kohn, In Antibiotics III. Springer-Verlag, New York, pp.3-11 (1975), Hurley and Needham-VanDevanter, (1986) Acc. (Chem. Res., 19:230-237). Dimeric PBD compounds with C2 aryl substituents have been shown to be useful as cytotoxic agents (Hartley et al (2010) Cancer Res. 70(17):6849-6858, Antonow (2010) J. Med. Chem. 53(7):2927-2941, Howard et al (2009) Bioorganic and Med. Chem. Letters 19(22):6463-6466).

[0309] In some embodiments, PBD compounds can be used as prodrugs by protecting them at the N10 position with nitrogen protecting groups that can be removed in vivo (International Publication Nos. WO00 / 12507 and WO2005 / 023814).

[0310] In some embodiments, the immunoconjugate comprises antibodies conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics and their analogues can induce double-strand DNA cleavage at sub-picomole concentrations (Hinman et al., (1993) Cancer Research 53:3336-3342, Lode et al., (1998) Cancer Research 58:2925-2928). Although calicheamicin has an intracellular site of action, in certain cases, it does not readily cross the cell membrane. Therefore, intracellular uptake of these drugs through antibody-mediated internal transfer can significantly enhance their cytotoxic effects in some embodiments. Non-limiting exemplary methods for preparing antibody-drug conjugates using calicheamicin drug moieties are described, for example, in U.S. Patents 5,712,374, 5,714,586, 5,739,116, and 5,767,285.

[0311] In some embodiments, the calicheamycin drug portion conjugated to the antibody is given by formula: The formula has TIFF0007924744000057.tif49170, where X is Br or I, L stands for linker, R for hydrogen, C for hydrogen. 1-6 Alkyl, or -C(=O)C 1-6 It is alkyl, R a is hydrogen or C 1-6 It is alkyl. In some embodiments, X is Br and R a R is hydrogen, and R is isopropyl. In other embodiments, X is Br and R aR is hydrogen, and R is ethyl. In other embodiments, X is I and R a R is hydrogen, and R is isopropyl. In other embodiments, X is I and R a R is hydrogen, and R is ethyl. In some embodiments, X is Br and R a is hydrogen, and R is -C(=O)CH3. In other embodiments, X is I and R a is hydrogen, and R is -C(=O)CH3. In other embodiments, X is I and R a is ethyl, and R is -C(=O)CH3. In other embodiments, X is Br and R a is ethyl, and R is -C(=O)CH3.

[0312] PBD dimers have been conjugated to antibodies, and the resulting ADCs have been shown to possess anticancer properties (US Publication 2010 / 0203007). Non-limiting exemplary binding sites of PBD dimers include five-membered pyrrolo rings, tethers between PBD units, and N10-C11 imine groups (International Publication WO2009 / 016516, US Publication 2009 / 304710, US Publication 2010 / 047257, US Publication 2009 / 036431, US Publication 2011 / 0256157, International Publication WO2011 / 130598).

[0313] In some embodiments, the ADC contains a pyrrolobenzodiazepine (PBD). In some embodiments, the PBD dimer recognizes and binds to a specific DNA sequence. The natural product anthramycin, which is a PBD, was first reported in 1965 (Leimgruber, et al., (1965) J.Am. Chem. Soc., 87:5793-5795, Leimgruber, et al., (1965) J.Am. Chem. Soc., 87:5791-5793). Since then, numerous PBDs have been reported, both natural and analogous, including dimers of the tricyclic PBD scaffold (US Patent Nos. 6,884,799, 7049,311, 7067,511, 7265,105, 7511,032, 7528,126, and 7557099) (Thurston, et al., (1994) Chem. Rev. 1994, 433-465). While not intended to be bound by any particular theory, it is thought that the dimer structure provides a three-dimensional shape suitable for isohelicity with the minor groove of type B DNA, allowing for a snug fit at the binding site (Kohn, In Antibiotics III. Springer-Verlag, New York, pp.3-11 (1975), Hurley and Needham-VanDevanter, (1986) Acc. (Chem. Res., 19:230-237). Dimeric PBD compounds with C2 aryl substituents have been shown to be useful as cytotoxic agents (Hartley et al (2010) Cancer Res. 70(17):6849-6858, Antonow (2010) J. Med. Chem. 53(7):2927-2941, Howard et al (2009) Bioorganic and Med. Chem. Letters 19(22):6463-6466).

[0314] In some embodiments, PBD compounds can be used as prodrugs by protecting them at the N10 position with nitrogen protecting groups that can be removed in vivo (International Publication Nos. WO00 / 12507 and WO2005 / 023814).

[0315] PBD dimers have been conjugated to antibodies, and the resulting ADCs have been shown to possess anticancer properties (US Publication 2010 / 0203007). Non-limiting exemplary binding sites of PBD dimers include five-membered pyrrolo rings, tethers between PBD units, and N10-C11 imine groups (International Publication WO2009 / 016516, US Publication 2009 / 304710, US Publication 2010 / 047257, US Publication 2009 / 036431, US Publication 2011 / 0256157, International Publication WO2011 / 130598).

[0316] An example of a non-limiting PBD dimer component of ADC is given by formula A: The substance of TIFF0007924744000058.tif42170, as well as its salts and solvates, in which, The wavy line indicates the covalent bond portion to the linker. The dotted line indicates the presence of any double bond between C1 and C2 or between C2 and C3. R 2 These are independently H, OH, =O, =CH2, CN, R, OR, =CH-R D , =C(R D )2, O-SO 2- Selected from R, CO2R, and COR, and possibly further selected from halo or dihalo, in the formula, R D These are independently selected from R, CO2R, COR, CHO, CO2H, and Halogen. R 6 and R 9 These are independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', NO2, Me3Sn, and Halo. R 7 These are independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', NO2, Me3Sn, and Halo. Q is independently selected from O, S, and NH. R 11 is either H or R, or if Q is O, it is SO3M, where M is a metal cation. R and R' are each independently of any substitution of C. 1-8 Alkyl, C 1-12 Alkyl, C 3-8 Heterocyclyl, C 3-20 Heterogeneous rings, and C 5-20 Selected from aryl groups, and possibly in association with NRR' groups, R and R', together with the nitrogen atoms to which they are bonded, form optionally substituted 4-membered, 5-membered, 6-membered, or 7-membered heterocyclic rings. R 12 , R 16 , R 19 , and R 17 These are, respectively, R 2 , R 6 , R 9 , and R 7 As defined with respect to, R″ is C 3-12 The alkylene group, whose chain may be interrupted by one or more heteroatoms, such as O, S, N(H), NMe, and / or aromatic rings, such as benzene or pyridine, and these rings may be optionally substituted. X and X' are independently selected from O, S, and N(H).

[0317] In some embodiments, R and R' are each independently substituted with an optionally replaced C. 1-12 Alkyl, C 3-20 Heterogeneous rings, and C 5-20 Selected from aryl groups, and possibly in association with an NRR' group, R and R', together with the nitrogen atom to which they are bonded, form optionally substituted 4-membered, 5-membered, 6-membered, or 7-membered heterocyclic rings.

[0318] In some embodiments, R 9 and R 19 H is H.

[0319] In some embodiments, R 6 and R 16 H is H.

[0320] In some embodiments, R7 and R 17 All of these are OR 7A And in the formula, R 7A is an arbitrarily substituted C 1-4 It is alkyl. In some embodiments, R 7A is Me. In some embodiments, R 7A The formula is Ch2Ph, where Ph represents a phenyl group.

[0321] In some embodiments, X is O.

[0322] In some embodiments, R 11 H is H.

[0323] In some embodiments, a double bond exists between C2 and C3 in each monomer unit.

[0324] In some embodiments, R 2 and R 12 These are independently selected from H and R. In some embodiments, R 2 and R 12 R is independent of R. In some embodiments, R 2 and R 12 This is an independently and arbitrarily substituted C 5-20 Aryl or C 5-7 Aryl or C 8-10 It is an arrow. In some embodiments, R 2 and R 12 R is independently an optionally substituted fenliu, thienyl, naphthyl, pyridyl, quinolinyl, or isoquinolinyl. In some embodiments, R 2 and R 12 These are independently =O, =CH2, and =CH-R D , and =C(R D ) Selected from 2. In some embodiments, R 2 and R 12 These are, respectively, =CH2. In some embodiments, R 2 and R 12 These are H, respectively. In some embodiments, R2 and R 12 Each of these is = O. In some embodiments, R 2 and R 12 Each of these is =CF2. In some embodiments, R 2 and / or R 12 These are independently, =C(R D )2. In some embodiments, R 2 and / or R 12 These are independently, =CH-R D That is the case.

[0325] In some embodiments, R 2 and / or R 12 ga = CH-R D When this is the case, each base independently has one of the following stereoconfigurations. TIFF0007924744000059.tif42170 In some embodiments, =CH-R D It is arranged in a three-dimensional configuration (I).

[0326] In some embodiments, R″ is a C3 alkylene group or a C5 alkylene group.

[0327] In some embodiments, an exemplary PBD dimer component of the ADC is given by formula A(I): It has the structure TIFF0007924744000060.tif35170, where n is 0 or 1.

[0328] In some embodiments, an exemplary PBD dimer component of the ADC is given by formula A(II): It has the structure TIFF0007924744000061.tif36170, where n is 0 or 1.

[0329] In some embodiments, an exemplary PBD dimer component of the ADC is given by formula A(III): It has the structure of TIFF0007924744000062.tif34170, in the formula, R E and R E”These are, independently, H or R D Selected from, in the formula, R D It is defined as described above, In the formula, n is either 0 or 1.

[0330] In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, R E and / or R E” H is H. In some embodiments, R E and R E” H is H. In some embodiments, R E and / or R E” R D And in the formula, R D is an arbitrarily substituted C 1-12 It is alkyl. In some embodiments, R E and / or R E” R D And in the formula, R D It is methyl.

[0331] In some embodiments, an exemplary PBD dimer component of the ADC is given by formula A(IV): The structure of TIFF0007924744000063.tif36170 is, in the formula, Ar 1 and Ar 2 Each of these is independently and arbitrarily substituted C 5-20 It is an aryl, and in the formula, Ar 1 and Ar 2 They may be the same or different. In the formula, n is either 0 or 1.

[0332] In some embodiments, an exemplary PBD dimer component of the ADC is given by formula A(V): It has the structure of TIFF0007924744000064.tif36170, in which Ar 1 and Ar 2 Each of these is independently and arbitrarily substituted C 5-20 It is an aryl, and in the formula, Ar 1 and Ar 2They may be the same or different. It has the structure shown in the formula, where n is 0 or 1.

[0333] In some embodiments, Ar 1 and Ar 2 Each is independently selected from optionally substituted phenyl, furanyl, thiophenyl, and pyridyl. In some embodiments, Ar 1 and Ar 2 Each is independently an optionally substituted phenyl. In some embodiments, Ar 1 and Ar 2 These are, independently, optionally substituted chain-2-yl or chain-3-yl. In some embodiments, Ar 1 and Ar 2 Each of these is independently an optionally substituted quinolinyl or isoquinolinyl group. The quinolinyl or isoquinolinyl group can be bonded to the PBD core through any available ring position. For example, quinolinyl can be quinoline-2-yl, quinoline-3-yl, quinoline-4-yl, quinoline-5-yl, quinoline-6-yl, quinoline-7-yl, and quinoline-8-yl. In some embodiments, quinolinyl is selected from quinoline-3-yl and quinoline-6-yl. Isoquinolinyl can be isoquinoline-1-yl, isoquinoline-3-yl, isoquinoline-4-yl, isoquinoline-5-yl, isoquinoline-6-yl, isoquinoline-7-yl, and isoquinoline-8-yl. In some embodiments, isoquinolinyl is selected from isoquinoline-3-yl and isoquinoline-6-yl.

[0334] Further non-limiting exemplary PBD dimer components of ADC are given by formula B: The substance of TIFF0007924744000065.tif34170, as well as its salts and solvates, in which, The wavy line indicates the covalent bond portion to the linker. The wavy line connected to OH indicates an S or R stereochemical configuration. R V1 and R V2These are independently H, methyl, ethyl, and phenyl (wherein phenyl may be optionally substituted with fluoro, particularly at the 4-position), and C 5-6 Selected from heterocyclines, in the formula, R V1 and R V2 They may be the same or different. n is either 0 or 1.

[0335] In part, the embodiment, R V1 and R V2 This is independently selected from H, phenyl, and 4-fluorophenyl.

[0336] In some embodiments, the linker may bind to one of several sites on the PBD dimer drug moiety, including the N10 imine of the B ring, the C-2 endo / exo position of the C ring, or the tether unit connecting the A ring (see structures C(I) and C(II) below).

[0337] Non-restrictive exemplary PBD dimer components of ADC include the following formulas C(I) and C(II): TIFF0007924744000066.tif68170

[0338] Formulas C(I) and C(II) are shown in their N10-C11 imine forms. The exemplary PBD drug portions also include the carbinolamine and protected carbinolamine forms shown in the table below. TIFF0007924744000067.tif35170In formula, X is CH2 (n=1~5), N, or O. Z and Z' are independently selected from OR and NR2, respectively, where R is a primary, secondary, or tertiary alkyl chain containing 1 to 5 carbon atoms. R1, R'1, R2, and R'2 are each independently H, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, and C 5-20 Aryl (including substituted aryls), C 5-20Selected from heteroaryl groups, -NH2, -NHMe, -OH, and -SH, in some embodiments the alkyl, alkenyl, and alkynyl chains contain up to 5 carbon atoms. R3 and R'3 are independently selected from H, OR, NHR, and NR2, where R is a primary, secondary, or tertiary alkyl chain containing 1 to 5 carbon atoms. R4 and R'4 are independently selected from H, Me, and OMe. R5 is C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C 5-20 Aryls (including aryls substituted with halo, nitro, cyano, alkoxy, alkyl, and heterocyclyl compounds), and C 5-20 Selected from heteroaryl groups, in some embodiments, alkyl, alkenyl, and alkynyl chains contain up to 5 carbon atoms. R 11 This is H, C1-C8 alkyl, or a protecting group (such as acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ), 9-fluorenylmethyleneoxycarbonyl (Fmoc), or a moiety containing a self-destructing unit such as valine-citrulline-PAB), R 12 is H, C1-C8 alkyl, or protecting group, R1, R'1, R2, R'2, R5, or R 12 A hydrogen atom in one of the A rings, or -OCH2CH2(X) between the A rings n The hydrogen atoms in the CH2CH2O- spacer are replaced by linker-connecting bonds in the ADC.

[0339] Examples of PDB dimer portions of ADCs include, but are not limited to, the following (the dashed lines indicate covalent bonding sites to the linker): TIFF0007924744000068.tif33170

[0340] A non-limiting exemplary embodiment of an ADC containing a PBD dimer has the following structure: TIFF0007924744000069.tif173170In the formula, n is between 0 and 12. In some embodiments, n is between 2 and 10. In some embodiments, n is between 4 and 8. In some embodiments, n is selected from 4, 5, 6, 7, and 8.

[0341] In some embodiments, ADCs containing the PBD dimer described herein can be prepared by conjugating a linker-drug intermediate containing a pyridine leaving group to the cysteine ​​thiol of the antibody via a sulfur atom to form a disulfide bond. Furthermore, in some embodiments, ADCs containing the PBD dimer described herein can be prepared by conjugating a linker-drug intermediate containing a thiopyridyl leaving group, where the pyridine group is substituted with one or more nitro groups. In some embodiments, the pyridyl ring is monosubstituted with -NO2. In some embodiments, the -NO2 monosubstituted is para relative to the disulfide. In some embodiments, the PBD dimer is conjugated via the N10 position. For example, a non-limiting exemplary ADC containing the PBD dimer can be prepared by conjugating an antibody with monomethylethylpyridyl disulfide (shown below), which is an N10-conjugated PBD linker intermediate. TIFF0007924744000070.tif43170

[0342] The linkers of the PBD dimer-val-cit-PAB-Ab and PBD dimer-Phe-Lys-PAB-Ab can be cleaved by proteases, but the PBD dimer-maleimide-acetal is acid-unstable.

[0343] PBD dimers and ADCs containing PBD dimers can be prepared according to methods known in the art. See, for example, International Publication WO2009 / 016516, U.S. Publications 2009 / 304710, 2010 / 047257, 2009 / 036431, 2011 / 0256157, International Publication WO2011 / 130598, and WO2013 / 055987.

[0344] In some embodiments, ADCs contain anthracyclines. Anthracyclines are antimicrobial compounds that exhibit cytotoxic activity. While not intended to be bound by any particular theory, studies have shown that anthracyclines may act to kill cells through several different mechanisms, including 1) inhibition of DNA-dependent nucleic acid synthesis by intercalation of the drug molecule into the cell's DNA, 2) generation of free radical drugs that react with cellular macromolecules to damage cells, and / or 3) interaction between the drug molecule and the cell membrane (see, for example, C. Peterson et al., “Transport And Storage Of Anthracycline In Experimental Systems And Human Leukemia” in Anthracycline Antibiotics In Cancer Therapy, NRBachur, “Free Radical Damage” id. at pp.97-102). Due to their potential cytotoxicity, anthracyclines have been used to treat numerous cancers, including leukemia, breast cancer, lung cancer, ovarian adenocarcinoma, and sarcoma (see, for example, PH-Wiernik, in Anthracycline: Current Status And New Developments, p. 11).

[0345] Examples of non-exclusive anthracyclines include doxorubicin, epirubicin, idarubicin, daunomycin, nemorubicin, and their derivatives. Immunoconjugates and prodrugs of daunorubicin and doxorubicin have been prepared and studied (Kratz et al (2006) Current Med. Chem. 13:477-523, Jeffrey et al (2006) Bioorganic & Med. Chem. Letters 16:358-362, Torgov et al (2005) Bioconj. Chem. 16:717-721, Nagy et al (2000) Proc. Natl. Acad. Sci. USA 97:829-834, Dubowchik et al (2002) Bioorg. & Med. Chem. Letters 12:1529-1532, King et al (2002) J.Med. Chem. (45:4336-4343, European Publication No. EP0328147, U.S. Patent No. 6630579). The antibody-drug conjugate BR96-doxorubicin specifically reacts with the tumor-associated antigen Lewis-Y and has been evaluated in Phase I and Phase II studies (Saleh et al (2000) J. Clin. Oncology 18:2282-2292, Ajani et al (2000) Cancer Jour. 6:78-81, Tolcher et al (1999) J. Clin. Oncology 17:478-484).

[0346] PNU-159682 is a potent metabolite (or derivative) of nemorubicin (Quintieri, et al. (2005) Clinical Cancer Research 11(4):1608-1617). Nemorubicin is a semi-synthetic analog of doxorubicin, possessing a 2-methoxymorpholino group in its glycoside amino acid, and has undergone clinical evaluation, including in Phase II / III clinical trials for hepatocellular carcinoma (Grandi et al (1990) Cancer Treat. Rev. 17:133, Ripamonti et al (1992) Brit. J. Cancer 65:703) (Sun et al (2003) Proceedings of the American Society for Clinical Oncology 22, Abs 1448, Quintieri (2003) Proceedings of the American Association of Cancer Research, 44: 1st Ed, Abs 4649, Pacciarini et al (2006) Jour. Clin. Oncology 24: 14116).

[0347] Non-limiting exemplary ADCs containing nemorubicin or nemorubicin derivatives are given by formula Ia: Represented in TIFF0007924744000071.tif64170, where R1 is a hydrogen atom, a hydroxyl group, or a methoxy group, and R2 is a C1-C5 alkoxy group or a pharmaceutically acceptable salt thereof. L1 and Z together are linkers (L) as described herein. T is an antibody (Ab) as described herein, m is between 1 and approximately 20. In some embodiments, m is between 1 and 10, 1 and 7, 1 and 5, or 1 and 4.

[0348] In some embodiments, both R1 and R2 are methoxy(-OMe).

[0349] Further non-limiting exemplary ADCs, including nemorubicin or nemorubicin derivatives, are given by formula Ib As shown in TIFF0007924744000072.tif67170, in the formula R1 is a hydrogen atom, a hydroxyl group, or a methoxy group, and R2 is a C1-C5 alkoxy group or a pharmaceutically acceptable salt thereof. L2 and Z together are linkers (L) as described herein. T is an antibody (Ab) as described herein, m is between 1 and approximately 20. In some embodiments, m is between 1 and 10, 1 and 7, 1 and 5, or 1 and 4.

[0350] In some embodiments, both R1 and R2 are methoxy(-OMe).

[0351] In some embodiments, the nemorubicin component of the nemorubicin-containing ADC is PNU-159682. In some such embodiments, the drug portion of the ADC has the following structure: It may have one of the following: TIFF0007924744000073.tif145170, where the tilde indicates coupling to linker(L).

[0352] Anthracyclines, including PNU-159682, can be conjugated to antibodies via multiple binding sites and various linkers (including those described herein) (US Publication 2011 / 0076287, INTERNATIONAL Publication WO2009 / 099741, US Publication 2010 / 0034837, INTERNATIONAL Publication WO2010 / 009124).

[0353] Examples of ADCs containing nemorubicin include, but are not limited to, the following: TIFF0007924744000074.tif236170TIFF0007924744000075.tif114170 (in the formula, R1 and R2 are independently selected from H and C1-C6 alkyl groups, and TIFF0007924744000076.tif70170

[0354] The linker of PNU-159682 maleimide-Ab is acid-unstable, but PNU-159682-val-cit-PAB-Ab, PNU-159682-val-cit-PAB-spacer-Ab, and PNU-159682-val-cit-PAB-spacer(R 1 R 2 The linker of )-Ab can be cleaved by proteases.

[0355] An exemplary PNU ADC is shown in Figures 10D and 10E.

[0356] In some embodiments, the ADC contains 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI). DNA subgroove alkylating agents of the 5-amino-1-(chloromethyl)-1,2-dihydro-3H-benz[e]indole (aminoCBI) class are potent cytotoxins (Atwell, et al (1999) J. Med. Chem., 42:3400) and are used as effector units in several classes of prodrugs designed for cancer therapy. These include antibody conjugates (Jeffrey, et al. (2005) J. Med. Chem., 48:1344), prodrugs for gene therapy based on nitrobenzyl carbamate (Hay, et al (2003) J. Med. Chem. 46:2456), and corresponding nitro-CBI derivatives such as hypoxia-activated prodrugs (Tercel, et al (2011) Angew. Chem., Int. Ed., 50:2606-2609). The pharmacophores of CBI and pyrrolo[2,1-c][1,4]benzodiazepine (PBD) are linked together by an alkyl chain (Tercel et al (2003) J. Med. Chem 46:2132-2151).

[0357] In some embodiments, the ADC comprises a 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI) dimer (International Publication No. WO2015 / 023355).

[0358] An exemplary CBI dimer ADC is shown in Figure 10B.

[0359] In some such embodiments, the dimer is a heterodimer in which half of the dimer is the CBI portion and the other half is the PBD portion.

[0360] An exemplary CBI-PBD heterodimer ADC is shown in Figure 10C.

[0361] In some embodiments, the CBI dimer is given by formula: It has TIFF0007924744000077.tif49170, and in the formula, R 1 H, P(O)3H2, C(O)NR a R b , or selected from joining to linker (L), R 2 H, P(O)3H2, C(O)NR a R b , or selected from joining to linker(L), R a and R b These are independently selected from C1-C6 alkyl groups which may be optionally substituted with H and one or more F, or R a and R b It forms a 5-membered or 6-membered heterocyclyl group, T is C3-C 12 A tether group selected from alkylene, Y, (C1-C6 alkylene)-Y-(C1-C6 alkylene), (C1-C6 alkylene)-Y-(C1-C6 alkylene)-Y-(C1-C6 alkylene), (C2-C6 alkenylene)-Y-(C2-C6 alkenylene), and (C2-C6 alkynylene)-Y-(C2-C6 alkynylene), In the formula, Y is independently O, S, NR 1 Selected from aryl and heteroaryl, Alkylenes, alkenylenes, aryls, and heteroaryls may be independently substituted with F, OH, O(C1-C6 alkyl), NH2, NHCH3, N(CH3)2, OP(O)3H2, and C1-C6 alkyls (where the alkyl is substituted with one or more Fs). Alternatively, alkylenes, alkenylenes, aryls, and heteroaryls may be substituted independently and by bonding to L. D' is, The drug portion is selected from TIFF0007924744000078.tif126170, where the wavy line indicates the binding site to T and X. 1 and X 2 These are, independently, O and NR 3 Selected from, in the formula, R 3 is selected from C1-C6 alkyl groups which may be substituted with H and one or more F, and R 4 R is selected from bonding to H, CO2R, or linker (L), where R is a C1-C6 alkyl or benzyl, and R 5 It is H or C1-C6 alkyl.

[0362] In some embodiments, the immunoconjugate comprises antibodies conjugated to one or more amatoxin molecules. Amatoxins are cyclic peptides composed of eight amino acids. They can be isolated from the mushroom Amanita phalloides or prepared synthetically. Amatoxins specifically inhibit DNA-dependent RNA polymerase II in mammalian cells, thereby specifically inhibiting transcription and protein biosynthesis in affected cells. By inhibiting transcription in cells, they cause cessation of growth and proliferation. For example, see Moldenhauer et al. JNCI 104:1-13 (2012), International Publications WO2010115629, WO2012041504, WO2012119787, WO2014043403, WO2014135282, and WO2012119787, which are incorporated herein by reference in their entirety. In some embodiments, one or more amatoxin molecules are one or more α-amanitin molecules.

[0363] Other drug molecules include protein toxins, such as diphtheria A chain, unbound active fragments of diphtheria toxin, exotoxin A chain (derived from Pseudomonas erginosa), lysine A chain (Vitetta et al (1987) Science, 238:1098), abrin A chain, modessin A chain, alpha-sarcin, Aleurites fordi protein, dianthin protein, Phytolacca americana protein (PAPI, PAPII, and PAP-S), Momordica charantia inhibitors, curcin, crotin, Sapoaonaria officinalis inhibitors, geronin, mitogenin, restrictosin, phenomycin, enomycin, and tricothecene (International Publication No. WO93 / 21232).

[0364] When more than one nucleophilic group reacts with a drug-linker intermediate or linker reagent, the resulting product is a mixture of ADC compounds in which more than one drug moiety is dispersed and bound to the antibody. The average drug number per antibody (DAR) can be calculated from this mixture by a dual ELISA antibody assay that is specific to both the antibody and the drug. Individual ADC molecules can be identified in the mixture by mass spectrometry and separated by HPLC, for example, hydrophobic interaction chromatography (e.g., McDonagh et al (2006) Prot. Engr. Design & Selection 19(7):299-307, Hamblet et al (2004) Clin. Cancer Res. 10:7063-7070, Hamblet, KJ, et al. “Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate,” Abstract No. 624, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004, Alley, SC, et al. “Controlling the location of drug attachment in antibody-drug conjugates,” Abstract No. 627, American Association for Cancer See Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004. In certain embodiments, homogeneous ADCs having a single loading value may be isolated from the conjugation mixture by electrophoresis or chromatography.

[0365] The antibody-drug conjugates 51-58 in Table 18 (see Example 16) can be prepared by coupling the drug portion with a linker reagent and conjugating it with any of the antibodies described herein, including cysteine-modified antibodies, according to the procedures in International Publications WO2013 / 055987, WO2015 / 023355, WO2010 / 009124, and WO2015 / 095227. Specific antibody-drug conjugates are listed in Table 19 (see Example 16).

[0366] The drug portion also includes compounds with nucleic acid degradation activity (e.g., ribonucleases or DNA endonucleases).

[0367] As therapeutic radioactive isotopes, 32 P, 33 P, 90 Y, 125 I, 131 I, 131 In, 153 Sm, 186 Re, 188 Re, 211 At, 212 Bi, 212 Examples include radioactive isotopes of Pb and Lu.

[0368] Radioactive isotopes or other labels can be incorporated into conjugates using known methods (Fraker et al (1978) Biochem. Biophys. Res.Commun. 80:49-57, “Monoclonal Antibodies in Immunoscintigraphy” Chatal, CRC Press 1989). Carbon-14 labeled 1-isothiocyanate benzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclides into antibodies (International Publication No. WO94 / 11026).

[0369] Further specific examples of ADCs are presented in Example 16.

[0370] Linker

[0371] A "linker" (L) is a bifunctional or polyfunctional moiety that can be used to conjugate one or more drug moieties (D) to an antibody (Ab) to form an antibody-drug conjugate (ADC) of formula I. In some embodiments, the antibody-drug conjugate (ADC) can be prepared using a linker having a reactive functional group for covalent bonding to the drug and the antibody. For example, in some embodiments, the cysteinethiol of the antibody (Ab) can form a bond with the reactive functional group of the linker or drug-linker intermediate to form the ADC.

[0372] In one embodiment, the linker has a functional group that can react with free cysteine ​​present in the antibody to form a covalent bond. Non-limiting examples of such reactive functional groups include maleimides, haloacetamides, α-haloacetyls, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. See, for example, the conjugation method on page 766 of Bioconjugate Chemistry 15(4):765-773 by Klussman et al (2004) and the examples herein.

[0373] In some embodiments, the linker has a functionality that allows it to react with electrophilic groups present in the antibody. Examples of such electrophilic groups include, but are not limited to, aldehydes and ketone carbonyl groups. In some embodiments, the heteroatoms of the reactive functional groups of the linker can react with the electrophilic groups of the antibody to form a covalent bond with the antibody unit. Examples of such reactive functional groups, not limited to those mentioned above, include, but are not limited to, hydrazides, oximes, aminos, hydrazines, thiosemicarbazones, hydrazine carboxylates, and arylhydrazides.

[0374] A linker may contain one or more linker components. Exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), and 4-(N-maleimidomethyl)cyclohexane-1 carboxylate ("MCC"). Various linker components are known in the art, some of which are listed below.

[0375] The linker may be a “cleavable linker” that facilitates the release of the drug. Examples of cleavable linkers that are not limited to these include acid-unstable linkers (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, photodegradable linkers, or disulfide-containing linkers (Chari et al., Cancer Research 52:127-131 (1992), U.S. Patent No. 5208020).

[0376] In a particular embodiment, the linker is given by the following equation II: The formula has TIFF0007924744000079.tif11170, where A is a “stretcher unit”, a is an integer from 0 to 1, W is an “amino acid unit”, w is an integer from 0 to 12, Y is a “spacer unit”, y is 0, 1, or 2, and Ab, D, and p are defined above with respect to Formula I. An exemplary embodiment of such a linker is described in U.S. Patent No. 7,498,298, which is expressly incorporated herein by reference.

[0377] In some embodiments, the linker component includes a “stretcher unit” that binds the antibody to another linker component or drug moiety. A non-limiting, exemplary stretcher unit is shown below (where the wavy line indicates the site of covalent binding to the antibody, drug, or additional linker component): In some embodiments, the linker may be a peptide-mimicking linker, such as those described in International Publication No. WO2015 / 095227, WO2015 / 095124, or WO2015 / 095223, which are incorporated herein by reference in their entirety.

[0378] In one embodiment, the linker has a reaction site having an electrophilic group that reacts with nucleophilic cysteine ​​present in the antibody. The cysteine ​​thiol of the antibody reacts with the electrophilic group of the linker and forms a covalent bond with the linker. Useful electrophilic groups include, but are not limited to, maleimide and haloacetamide groups.

[0379] Cysteine-modified antibodies react with linker reagents or drug-linker intermediates, maleimides, or electrophilic functional groups such as α-halocarbonyls, according to the conjugation method and the protocol in Example 4 on page 766 of Klussman, et al (2004), Bioconjugate Chemistry 15(4):765-773.

[0380] 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 the free cysteinethiol of the antibody. Examples of thiol-reactive functional groups include, but are not limited to, maleimide, α-haloacetyl, activated esters such as succinimide, 4-nitrophenyl, pentafluorophenyl, tetrafluorophenyl, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates.

[0381] In another embodiment, the linker may be a dendritic linker for covalently attaching more than one drug moiety to the antibody via a branched, polyfunctional linker moiety (Sun et al (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215, Sun et al (2003) Bioorganic & Medicinal Chemistry 11:1761-1768, King (2002) Tetrahedron Letters 43:1987-1990). The dendritic linker can increase the drug-to-antibody molar ratio, i.e., the loading, which is related to the potency of the ADC. Therefore, if a cysteine-modified antibody has only one reactive cysteinethiol group, multiple drug moieties can be attached via the dendritic linker.

[0382] The linker may contain a plurality of amino acid residues that conjugate the antibody (Ab) and the drug moiety (D) of the cysteine-manipulated antibody-drug conjugate (ADC) of the present invention. These amino acid residues may form dipeptides, tripeptides, tetrapeptides, pentapeptides, hexapeptides, heptapeptides, octapeptides, nonapeptides, decapeptides, undecapeptides, or dodecapeptide units. The amino acid residues include naturally occurring amino acids as well as trace amino acids and naturally occurring amino acid analogs, such as citrulline.

[0383] Useful amino acid residue units can be designed to optimize selectivity by being enzymatically cleaved by specific enzymes, such as tumor-associated proteases, to release the active drug moiety. In one embodiment, an amino acid residue unit such as valine-citrulline (vc or val-cit) is one whose cleavage is catalyzed by cathepsins B, C, and D, or plasmin proteases.

[0384] The linker unit may be a self-destructing type, such as a p-aminobenzylcarbamoyl (PAB) unit, where ADC is an exemplary structure: TIFF0007924744000081.tif32170

[0385] The formula has the following characteristics, where Q is -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro, or -cyano, m is an integer in the range of 0 to 4, and p is in the range of 1 to 4.

[0386] Other examples of self-destructing spacers include, but are not limited to, aromatic compounds that are electronically similar to the PAB group, such as 2-aminoimidazole-5-methanol derivatives (U.S. Patent No. 7375078, Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and other ortho or para-aminobenzyl acetals. Spacers that are cyclized by 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 amide (Amsberry, et al (1990) J. Org. Chem. 55:5867). Elimination of amine-containing drugs that substitute for glycine (Kingsbury et al (1984) J.Med. Chem. 27:1447) is another example of a self-destructing spacer useful in ADCs.

[0387] In another embodiment, linker L may be a dendritic linker for covalently binding one or more drug moieties to the antibody via branched, polyfunctional linker moieties (Sun et al (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215, Sun et al (2003) Bioorganic & Medicinal Chemistry 11:1761-1768). The dendritic linker can increase the drug-to-antibody molar ratio, i.e., the loading, which is related to the potency of the ADC. Therefore, if a cysteine-modified antibody has only one reactive thiol group, multiple drug moieties can be bound via dendritic linkers (International Publication No. WO2004 / 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).

[0388] Embodiments of the antibody-drug conjugate compound of formula Ia include (val-cit), (MC-val-cit), and (MC-val-cit-PAB). TIFF0007924744000082.tif168170

[0389] Another exemplary embodiment of the antibody-drug conjugate compound of formula Ia has the following structure: TIFF0007924744000083.tif87170

[0390] In the formula, X is, TIFF0007924744000084.tif95170

[0391] R is independently H or C1-C6 alkyl, and n is 1 to 12.

[0392] In another embodiment, the linker has a reactive functional group having a nucleophilic group that reacts with an electrophilic group present in the antibody. Useful electrophilic groups on the antibody include, but are not limited to, aldehydes and ketone carbonyl groups. The heteroatom of the nucleophilic group of the linker can react with the electrophilic group of the antibody to form a covalent bond with the antibody unit. Useful nucleophilic groups on the linker include, but are not limited to, hydrazides, oximes, aminos, hydrazines, thiosemicarbazones, hydrazine carboxylates, and arylhydrazides. The electrophilic group of the antibody provides a site favorable for binding to the linker.

[0393] Typically, peptide linkers can be prepared by forming peptide bonds between two or more amino acids and / or peptide fragments. Such peptide bonds can be prepared, for example, by liquid-phase synthesis methods well known in the field of peptide chemistry (E. Schroder and K. Lubke (1965) “The Peptides”, volume 1, pp. 76-136, Academic Press).

[0394] In another embodiment, the linker may be substituted with a group whose solubility or reactivity is controlled, for example, a sulfonate (-SO3 - ) or charged substituents such as ammonium can improve the water solubility of the linker reagent and promote the coupling reaction between the linker reagent and the antibody or drug portion, or, depending on the synthetic route used in the preparation of the ADC, promote the coupling reaction between Ab-L (antibody-linker intermediate) and D or DL ​​(drug-linker intermediate) and Ab.

[0395] The compounds of the present invention expressly intend to include, but are not limited to, ADCs prepared using 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, as well as SVSB (succinimidyl-(4-vinylsulfone)benzoate), and ADCs prepared 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, POBox 117, Rockford, IL. 61105 USA, 1-800-874-3723, International +815-968-0747. See pages 467-498 of the 2003-2004 Applications Handbook and Catalog. Bis-maleimide reagents allow for the sequential or simultaneous attachment of thiol groups of cysteine-modified antibodies to thiol-containing drug moieties, labels, or linker intermediates. Other functional groups that react with thiol groups, drug moieties, labels, or intermediates of cysteine-modified antibodies include iodoacetamide, bromoacetamide, vinylpyridine, disulfide, pyridyl disulfide, isocyanates, and isothiocyanates. TIFF0007924744000085.tif41170

[0396] Useful linker reagents can also be obtained from other commercial sources such as Molecular Biosciences Inc. (Boulder, CO), or synthesized according to the procedures described in Toki et al (2002) J. Org. Chem. 67:1866-1872, Walker, MA (1995) J. Org. Chem. 60:5352-5355, Frisch et al (1996) Bioconjugate Chem. 7:180-186, U.S. Publication No. 6,214,345, International Publication No. WO02 / 088172, U.S. Publication No. 2003130189, U.S. Publication No. 2003096743, International Publication No. WO03 / 026577, U.S. Publication No. WO03 / 043583, and U.S. Publication No. WO04 / 032828.

[0397] An exemplary valine-citrulline (val-cit or vc) dipeptide linker reagent having a maleimide stretcher and a para-aminobenzylcarbamoyl (PAB) self-destructing spacer has the following structure: TIFF0007924744000086.tif50170

[0398] The formula has the following properties, where Q is -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro, or -cyano, and m is an integer in the range of 0 to 4.

[0399] An exemplary phe-lys(Mtr) dipeptide linker reagent having maleimide stretcher units and p-aminobenzyl self-destructing spacer units can be prepared according to Dubowchik, et al. (1997) Tetrahedron Letters, 38:5257-60, and this linker reagent has the following structure: TIFF0007924744000087.tif42170

[0400] The formula comprises, where Mtr is mono-4-methoxytrityl, Q is -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro, or -cyano, and m is an integer in the range of 0 to 4.

[0401] Exemplary antibody-drug conjugate compounds of the present invention include the following: TIFF0007924744000088.tif135170

[0402] In the formula, Val is valine, Cit is citrulline, p is 1, 2, 3, or 4, and Ab is a cysteine-modified antibody. Another exemplary antibody-drug conjugate, in which the mytansinoid drug portion DM1 is conjugated to the thiol group of trastuzumab via a BMPEO linker, has the following structure: TIFF0007924744000089.tif71170

[0403] The formula is such that Ab is a cysteine-modified antibody, n is 0, 1, or 2, and p is 1, 2, 3, or 4.

[0404] Preparation of antibody-drug conjugates

[0405] The ADC of formula I can be prepared by several routes, including the following, using organic chemical reactions, conditions, and reagents known to those skilled in the art: (1) reacting the cysteine ​​group of a cysteine-modified antibody with a linker reagent to form an antibody-linker intermediate Ab-L by covalent bonding, followed by reaction with the 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 by covalent bonding, followed by reaction with the cysteine ​​group of the cysteine-modified antibody. Conjugation methods (1) and (2) can be performed using various cysteine-modified antibodies, drug moieties, and linkers to prepare antibody-drug conjugates of formula I.

[0406] The cysteinethiol group of the antibody is nucleophilic and can react with electrophilic groups of linker reagents and drug-linker intermediates to form covalent bonds, including (i) active esters, such as NHS esters, HOBt esters, haloformic acids, and acid halides; (ii) alkyl and benzyl halides, such as haloacetamides; (iii) aldehyde, ketone, carboxyl, and maleimide groups; and (iv) disulfides, including pyridyl disulfide, obtained by sulfide exchange. Examples of nucleophilic groups of the drug moiety include, but are not limited to, amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups, which can react with electrophilic groups of the linker moiety and linker reagent to form covalent bonds.

[0407] Mytansin can be converted, for example, to May-SSCH3, which can then be reduced to the free thiol May-SH and react with a modified antibody (Chari et al (1992) Cancer Research 52:127-131) to produce a mytansinoid-antibody immunoconjugate with a disulfide linker. Antibody-mytansinoid conjugates with disulfide linkers have been reported (International Publication No. WO04 / 016801, U.S. Publication No. 6884874, U.S. Publication No. 2004 / 039176A1, International Publication No. WO03 / 068144, U.S. Publication No. 2004 / 001838A1, U.S. Publication No. 6441163, U.S. Publication No. 5208020, U.S. Publication No. 5416064, International Publication No. WO01 / 024763). Disulfide linker SPP is composed of the linker reagent N-succinimidyl 4-(2-pyridylthio)pentanoate.

[0408] Under certain conditions, cysteine-modified antibodies can be made reactive to conjugate with linker reagents by treatment with reducing agents such as DTT (Clealand Reagent, dithiothreitol) or TCEP (tris(2-carboxyethyl)phosphine hydrochloride, Getz et al (1999) Anal. Biochem. Vol 273:73-80, Soltec Ventures, Beverly, MA). A full-length cysteine-modified monoclonal antibody (THIOMAB® antibody) expressed in CHO cells was reduced with approximately 50-fold excess TCEP at 37°C for 3 hours to reduce the disulfide bond that may form between the newly introduced cysteine ​​residue and the cysteine ​​present in the culture medium. The reduced THIOMAB® antibody was diluted, packed into a HiTrap S column with 10 mM sodium acetate, pH 5, and eluted with PBS containing 0.3 M sodium chloride. By placing the compound in a diluted (200 nM) aqueous copper sulfate solution (CuSO4) overnight at room temperature, the disulfide bonds between cysteine ​​residues present in the parent Mab were reconstructed. Other oxidizing agents and conditions known in the art may be used. Oxidation by ambient air is also effective. This mild partial oxidation step efficiently forms intrachain disulfides with high fidelity. Approximately 10-fold excess of a drug-linker intermediate, e.g., BM(PEO)4-DM1, was added, mixed, and allowed to stand at room temperature for about 1 hour to achieve conjugation and form an antibody-drug conjugate (i.e., a conjugated THIOMAB® antibody). The conjugation mixture was gel filtered and eluted through a HiTrap S column to remove excess drug-linker intermediate and other impurities.

[0409] For example, U.S. Patent Publication No. 20110301334 (in its entirety by reference) describes a general process for preparing cysteine-manipulated antibodies expressed from cell cultures for conjugation. The cysteine ​​adduct, along possibly with various interchain disulfide bonds, is cleaved by reduction to obtain the reduced form of the antibody. The interchain disulfide bonds between paired cysteine ​​residues are reformed under partial oxidation conditions, such as exposure to ambient air. The newly introduced, manipulated unpaired cysteine ​​residues remain available to react with a linker reagent or drug-linker intermediate to form the antibody conjugate of the present invention. THIOMAB® antibodies expressed in mammalian cell lines result in Cys adducts externally conjugated to manipulated Cys through the formation of -SS- bonds. Therefore, to obtain reactive THIOMAB® antibodies, purified THIOMAB® antibodies must be treated with reduction and oxidation procedures. These THIOMAB® antibodies are conjugated with maleimides containing cytotoxic agents, fluorophores, and other labels.

[0410] Exemplary THIOMAB® antibody conjugates have been prepared, which can be found in the table provided herein.

[0411] In vitro cell proliferation assay

[0412] Generally, the cytotoxic or cell proliferation inhibitory activity of antibody-drug conjugates (ADCs) is measured by exposing mammalian cells possessing a receptor protein, such as HER2, to an antibody of the ADC in cell culture medium, culturing the cells for approximately 6 hours to 5 days, and measuring cell viability. Using cell-based in vitro assays, the viability (proliferation), cytotoxicity, and induction of apoptosis (caspase activation) of the ADCs of the present invention were measured.

[0413] The in vitro efficacy of antibody-drug conjugates can be measured by cell proliferation assays. For example, the CellTiter-Glo® Luminescent Cell Viability Assay is a commercially available (Promega Corp., Madison, WI) allogeneic assay method based on recombinant expression of coleopteran luciferase (U.S. Patents 5583024, 5674713, and 5700670). This cell proliferation assay determines the number of viable cells in a culture based on the quantification of ATP present, an indicator of metabolically active cells (Crouch et al (1993) J.Immunol. Meth. 160:81-88, U.S. Publication No. 6602677). The CellTiter-Glo® assay is performed in a 96-well format and is suitable for automated high-throughput screening (HTS) (Cree et al (1995) AntiCancer Drugs 6:398-404). The allogeneic assay procedure involves directly adding a single reagent (CellTiter-Glo® reagent) to cells cultured in serum-supplemented medium. Cell washing, medium removal, and multiple pipetting steps are not required. This system, in a 384-well format, detects small amounts of cells—approximately 15 per well—within 10 minutes after reagent addition and mixing. Cells may be continuously treated with an ADC, or treated and then separated from the ADC. Generally, cells treated for a short period, i.e., 3 hours, exhibit the same potency as cells treated continuously.

[0414] This homogeneous "add-mix-measure" method induces cell lysis, leading to the generation of a luminescence signal proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in the culture. The CellTiter-Glo® assay produces a "glow-type" luminescence signal generated by the luciferase reaction, which typically has a half-life exceeding 5 hours depending on the cell type and culture medium used. Viable cells are reflected in relative luminescence units (RLU). The substrate, beetle luciferin, undergoes oxidative removal of its carboxyl group by recombinant firefly luciferase, simultaneously accelerating the conversion of ATP to AMP and generating photons.

[0415] In vivo efficacy

[0416] The in vivo efficacy of the THIOMAB® antibodies described herein can be measured using a high-expression transgenic explant mouse model (for example, the THIOMAB® antibodies provided in the table herein, prepared from anti-HER2 4D5 antibodies, can be measured using a high-expression HER2 transgenic explant mouse model). Allogeneic grafts may be bred from Fo5 mmtv transgenic mice that do not respond well to HERCEPTIN® therapy. Subjects may be treated once with an anti-HER2 4D5 THIOMAB® antibody and a placebo PBS buffered control (vehicle), and the subjects may be monitored for 3 weeks to measure the time to tumor doubling, log cell elimination, and tumor shrinkage.

[0417] Administration of antibody-drug conjugates

[0418] The antibody-drug conjugate (ADC) of the present invention can be administered via any route suitable for the condition to be treated. Typically, ADCs will be administered parenterally, i.e., by infusion, subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and epidural.

[0419] Pharmaceutical formulations

[0420] The pharmaceutically acceptable antibody-drug conjugates (ADCs) of the present invention are typically prepared in injectable unit dosage forms with a pharmaceutically acceptable parenteral vehicle for parenteral administration, i.e., bolus, intravenous, or intratumoral injection. Antibody-drug conjugates (ADCs) of the desired degree of purity are optionally mixed with a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.) in the form of a lyophilized formulation or aqueous solution.

[0421] The pharmaceutically acceptable formulations of cysteine-modified antibodies described herein can be prepared by mixing such antibodies (i.e., THIOMAB® antibodies) having the desired degree of purity, in the form of lyophilized formulations or aqueous solutions, with one or more optionally selected pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers generally do not toxic to the recipient at the dosage and concentration used, and include buffers, e.g., phosphoric acid, citrate, and other organic acids; antioxidants, e.g., ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride: hexamethonium chloride: benzalkonium chloride: benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens, e.g., methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, e.g. Examples of pharmaceutically acceptable carriers herein include, but are not limited to, serum albumin, gelatin, immunoglobulins; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids, e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents, e.g., EDTA; sugars, e.g., sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, e.g., sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, e.g., polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein also include, for example, interstitial drug dispersants, e.g., soluble neutral-active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, e.g., rHuPH20 (HYLENEX® Baxter International, Inc.).Certain exemplary sHASEGPs containing rHuPH20 and methods of use are described in U.S. Patent Publications 2005 / 0260186 and 2006 / 0104968. In one embodiment, sHASEGP is combined with one or more additional glycosaminoglycansases, such as chondroitinases.

[0422] Exemplary lyophilized antibody and immunoconjugate formulations are described in U.S. Patent No. 6,267,958, which is incorporated herein by reference in its entirety. Water-soluble antibody or immunoconjugate formulations include those described in U.S. Patent No. 6,171,586 and International Publication No. WO2006 / 044908 (both incorporated herein by reference in their entirety), the latter of which includes histidine acetate buffer.

[0423] The formulations herein may also contain more than one active ingredient, preferably having complementary activity that does not adversely affect each other, as required for the specific indication being treated.

[0424] The active ingredient may be encapsulated in a colloidal drug delivery system (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in a macroemulsion, for example, in microcapsules prepared by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly-(methylmethacylate) microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0425] A sustained-release preparation may be prepared. A suitable example of a sustained-release preparation is a semipermeable matrix of a hydrophobic solid polymer containing an antibody or immunoconjugate, the matrix of which may be in the form of a molded article, such as a film or microcapsules.

[0426] The formulations used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through a sterile filtration membrane.

[0427] Methods and compositions for antibody-drug conjugate therapy

[0428] The antibody-drug conjugates (ADCs) of the present invention can be used to treat a variety of diseases or disorders, such as those characterized by the overexpression of tumor antigens. Exemplary conditions include benign or malignant tumors, and hyperproliferative disorders, including leukemia and lymphoid malignancies. Other examples include immune disorders, including neurological, glial, astrocytic, hypothalamic, glandular, macrophage, epithelial, stromal, blastocyst, inflammatory, angioplastic, and autoimmune disorders.

[0429] Generally, the diseases or disorders to be treated are hyperproliferative diseases such as cancer. Examples of cancers to be treated herein include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemia or malignant lesions of the lymphatic system. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell ca...

Claims

1. An antibody-drug conjugate in which a cysteine-modified antibody containing an L174C-modified cysteine ​​amino acid, assigned an EU number, in its heavy chain is conjugated to a drug moiety via a pyridyl disulfide (PDS) linker bound to the L174C-modified cysteine ​​amino acid of the cysteine-modified antibody.

2. The antibody-drug conjugate according to claim 1, comprising the sequence TFPAVCQSSGL (HC-L174C; SEQ ID NO: 7) in the heavy chain.

3. (i) Mutation generation in the nucleic acid sequence of the parent antibody by replacing one or more amino acid residues with cysteine ​​to encode a cysteine-modified antibody, (ii) Expressing the cysteine-modified antibody, (iii) The antibody-drug conjugate according to claim 1 or 2, which is prepared by a process comprising isolating the cysteine-modified antibody.

4. The antibody-drug conjugate according to any one of claims 1 to 3, wherein the cysteine-modified antibody is selected from a monoclonal antibody, an antibody fragment, a bispecific antibody, a chimeric antibody, a human antibody, and a humanized antibody.

5. The antibody-drug conjugate according to claim 4, wherein the antibody fragment is a Fab fragment.

6. The antibody-drug conjugate according to any one of claims 1 to 5, wherein the cysteine-modified antibody is selected from the group consisting of anti-HER2 antibody, anti-Ly6E antibody, anti-CD79b antibody, anti-MUC16 antibody, anti-STEAP1 antibody, anti-NaPi2b antibody, and anti-CD22 antibody.

7. The antibody-drug conjugate according to claim 6, wherein the cysteine-modified antibody is an anti-HER2 antibody, and the anti-HER2 antibody is trastuzumab.

8. Cysteine-modified antibodies, (i) HVR-H1 containing the amino acid sequence of SEQ ID NO: 179, HVR-H2 containing the amino acid sequence of SEQ ID NO: 180, HVR-H3 containing the amino acid sequence of SEQ ID NO: 181, HVR-L1 containing the amino acid sequence of SEQ ID NO: 176, HVR-L2 containing the amino acid sequence of SEQ ID NO: 177, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 178, or (ii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 175 and light chain variable region containing the amino acid sequence of SEQ ID NO: 174 The antibody-drug conjugate according to claim 6, wherein the anti-Ly6E antibody contains an anti-Ly6E antibody.

9. Cysteine-modified antibodies, (i) HVR-H1 containing the amino acid sequence of SEQ ID NO: 186, HVR-H2 containing the amino acid sequence of SEQ ID NO: 187, HVR-H3 containing the amino acid sequence of SEQ ID NO: 188, HVR-L1 containing the amino acid sequence of SEQ ID NO: 189, HVR-L2 containing the amino acid sequence of SEQ ID NO: 190, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 191, or (ii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 184 and light chain variable region containing the amino acid sequence of SEQ ID NO: 185 The antibody-drug conjugate according to claim 6, wherein the anti-CD79b antibody contains an anti-CD79b antibody.

10. Cysteine-modified antibodies, (i) HVR-H1 containing the amino acid sequence of SEQ ID NO: 152, HVR-H2 containing the amino acid sequence of SEQ ID NO: 153, HVR-H3 containing the amino acid sequence of SEQ ID NO: 154, HVR-L1 containing the amino acid sequence of SEQ ID NO: 149, HVR-L2 containing the amino acid sequence of SEQ ID NO: 150, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 151, or (ii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 156 and light chain variable region containing the amino acid sequence of SEQ ID NO: 157 The antibody-drug conjugate according to claim 6, wherein the anti-MUC16 antibody contains an anti-MUC16 antibody.

11. Cysteine-modified antibodies, (i) HVR-H1 containing the amino acid sequence of SEQ ID NO: 157, HVR-H2 containing the amino acid sequence of SEQ ID NO: 158, HVR-H3 containing the amino acid sequence of SEQ ID NO: 159, HVR-L1 containing the amino acid sequence of SEQ ID NO: 160, HVR-L2 containing the amino acid sequence of SEQ ID NO: 161, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 162, or (ii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 163 and light chain variable region containing the amino acid sequence of SEQ ID NO: 164 The antibody-drug conjugate according to claim 6, wherein the anti-STEAP1 antibody contains an anti-STEAP1 antibody.

12. Cysteine-modified antibodies, (i) HVR-H1 containing the amino acid sequence of SEQ ID NO: 165, HVR-H2 containing the amino acid sequence of SEQ ID NO: 167, HVR-H3 containing the amino acid sequence of SEQ ID NO: 168, HVR-L1 containing the amino acid sequence of SEQ ID NO: 169, HVR-L2 containing the amino acid sequence of SEQ ID NO: 170, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 171, or (ii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 172 and light chain variable region containing the amino acid sequence of SEQ ID NO: 173 The antibody-drug conjugate according to claim 6, wherein the anti-NaPi2b antibody contains an anti-NaPi2b antibody.

13. Cysteine-modified antibodies, The antibody-drug conjugate according to claim 6, comprising an anti-CD22 antibody containing HVR-H1 containing the amino acid sequence of SEQ ID NO: 192, HVR-H2 containing the amino acid sequence of SEQ ID NO: 193, HVR-H3 containing the amino acid sequence of SEQ ID NO: 194, HVR-L1 containing the amino acid sequence of SEQ ID NO: 195, HVR-L2 containing the amino acid sequence of SEQ ID NO: 196, and HVR-L3 containing the amino acid sequence of SEQ ID NO:

197.

14. Cysteine-modified antibodies are used with receptors (1) to (53): (1) BMPR1B (Bone Morphogenesis Protein Receptor type IB), (2) E16 (LAT1, SLC7A5), (3) STEAP1 (six-transmembrane epithelial antigen of the prostate), (4) 0772P (CA125, MUC16), (5) MPF (MPF, MSLN, SMR, megakaryocyte-enhancing factor, mesothelin), (6) Napi3b (NaPi2b, NAPI-3B, NPTIIb, SLC34A2, solute transporter 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, 7 thrombospongin 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 cDNA 2700050C12 gene), (9) ETBR (endothelin type B receptor), (10) MSG783 (RNF124, hypothetical protein FLJ20315), (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-related gene 1, prostate cancer-related protein 1, six-transmembrane epithelial 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, growth factor derived from teratoma), (14) CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein-Barr virus receptor) or Hs. 73792), (15) CD79b (CD79B, CD79β, IGb (immunoglobulin-associated beta), B29), (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain-containing phosphatase anchor 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 activator receptor, BLyS receptor 3, BR3, (27) CD22 (B cell receptor CD22-B isoform), (28) CD79a (CD79A, CD79α, immunoglobulin-associated alpha, B cell-specific protein), (29) CXCR5 (Burkitt lymphoma receptor 1, G protein-bound receptor), (30) HLA-DOB (beta subunit of MHC class II molecule (Ia antigen), (31) P2X5 (purine receptor P2X ligand open 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-related 2), (36) TENB2 (presumably a transmembrane proteoglycan), (37) PMEL17 (silver homolog, SILV, D12S53E, PMEL17, SI, SIL), (38) TMEFF1 (a transmembrane protein 1 having an EGF-like domain and two follistatin-like domains, tomoregulin 1), (39) GDNF-Ra1 (GDNF family receptor alpha-1, GFRA1, GDNFR, GDNFRA, RETL1, TRNR1, RET1L, GDNFR-alpha-1, GFR-alpha-1), (40) Ly6E (Lymphocyte antigen 6 complex, gene locus E, Ly67, RIG-E, SCA-2, TSA-1), (41) TMEM46 (shisa homologue 2), (42) Ly6G6D (lymphocyte antigen 6 complex, gene locus G6D, Ly6-D, MEGT1), (43) LGR5 (Leucine-rich repeat-containing G protein-bound receptor 5, GPR49, GPR67), (44) RET (RET proto-oncogene, MEN2A, HSCR1, MEN2B, MTC1, PTC, CDHF12, Hs.168114, RET51, RET-ELE1), (45) LY6K (lymphocyte antigen 6 complex, gene locus K, LY6K, HSJ001348, FLJ35226), (46) GPR19 (G protein-bound receptor 19, Mm. 4787), (47) GPR54 (KISS1 receptor, KISS1R, GPR54, HOT7T175, AXOR12), (48) ASPHD1 (containing aspartate beta-hydroxylase domain 1, LOC253982), (49) Tyrosinase (TYR, OCAIA, OCA1A, tyrosinase, SHEP3), (50) TMEM118 (ring finger protein, transmembrane 2, RNFT2, FLJ14627), (51) GPR172A (G protein-bound receptor 172A, GPCR41, FLJ11856, D15Ertd747e), (52) CD33, and (53) CLL-1 (CLEC12A, MICL, and DCAL2) An antibody-drug conjugate according to any one of claims 1 to 5, which binds to one or more of the following.

15. An antibody-drug conjugate according to any one of claims 1 to 14, comprising a capture label, a detection label, a drug moiety, or covalently bound to a solid support.

16. The antibody-drug conjugate according to claim 15, which is covalently bound to a biotin capture label.

17. The antibody-drug conjugate according to claim 15, which is covalently bound to a fluorescent dye detection label.

18. The antibody-drug conjugate according to claim 17, wherein the fluorescent dye is selected from fluorothane type, rhodamine type, dansyl, lysamine, cyanine, and phycoerythrin. [

19. ] 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 The antibody-drug conjugate according to claim 15, which is covalently bound to a radionuclide detection label selected from Bi.

20. The antibody-drug conjugate according to claim 15, wherein the detection label is covalently bound by a chelate ligand.

21. The antibody-drug conjugate according to claim 20, wherein the chelate ligand is selected from the group consisting of DOTA, DOTP, DOTMA, DTPA, and TETA.

22. An antibody-drug conjugate according to any one of claims 1 to 14, wherein the antibody-drug conjugate is Formula I Ab-(L-D) ｐ I It has, In the formula, Ab is a cysteine-modified antibody, L is a linker, D is the drug moiety, p is 1 or 2, the cysteine-modified antibody contains L174C modified cysteine ​​amino acids according to EU numbering in its heavy chain, the linker is pyridyl disulfide (PDS), and the drug moiety is conjugated to the modified cysteine ​​amino acid via a linker bound to the L174C modified cysteine ​​amino acid of the cysteine-modified antibody, thus forming an antibody-drug conjugate.

23. The antibody-drug conjugate according to claim 22, wherein the drug portion (D) is a mytansinoid, auristatin, drastatin, trichothecene, CC1065, calicheamicin, engine antibiotic, taxane, pyrrolobenzodiazepine (PBD) dimer, 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI) dimer, CBI-PBD heterodimer, or anthracycline.

24. D is structure: The antibody-drug conjugate according to claim 23, wherein the monomethyl auristatin drug portion MMAE has a tilde in the formula, where the tilde indicates a covalent binding site to the linker.

25. D is structure: A PBD dimer drug having the same, as well as its salts and solvates. The wavy line indicates the covalent bond portion to the linker. The dotted line indicates the presence of any double bond between C1 and C2 or between C2 and C3. R ２ These are independently H, OH, =O, =CH ２ ,CN,R,OR,=CH-R Ｄ , = C(R Ｄ ) ２ O-SO ２－ R, CO ２ R is selected from and COR, and may be further selected from halo or dihydro, where R Ｄ R and CO are independent of each other. ２ R, COR, CHO, CO ２ Selected from H and Halo, R ６ and R ９ These are independently H, R, OH, OR, SH, SR, NH ２ , NHR, NRR', NO ２ Me ３ Selected from Sn and Halo, R ７ These are independently H, R, OH, OR, SH, SR, NH ２ , NHR, NRR', NO ２ Me ３ Selected from Sn and Halo, Q is independently selected from O, S, and NH. R １１ is either H or R, or if Q is O, then SO ３ It is either M or M, where M is a metal cation. R and R' may be substituted independently of each other. １－８ Alkyl, C １－１２ Alkyl, C ３－８ Heterocyclyl, C ３－２０ Heterogeneous rings, and C ５－２０ Selected from aryl groups, and optionally in association with the group NRR', R and R', together with the nitrogen to which they are bonded, form a substituted 4, 5, 6, or 7-membered heterocyclic ring. R １２ , R １６ , R １９ , and R １７ These are R ２ , R ６ , R ９ , and R ７ As defined with respect to, R'' is C ３－１２ The alkylene group is a chain that may be interrupted by one or more heteroatoms, such as O, S, N(H), NMe, and / or aromatic rings, such as benzene or pyridine, and these rings may be optionally substituted. The antibody-drug conjugate according to claim 23, wherein X and X' are independently selected from O, S, and N(H).

26. The structure of the PBD dimer is It includes its salts and solvates, the wavy lines indicate covalent bonding sites to the linker, and the wavy lines connected to OH indicate S or R stereoconfiguration. Ｖ１ and R Ｖ２ These are independently H, methyl, ethyl, and phenyl (wherein phenyl may be optionally substituted with fluoro, particularly at the 4-position), and C ５－６ Selected from heterocyclines, R Ｖ１ and R Ｖ２ The antibody-drug conjugate according to claim 25, wherein n may be the same or different, and n is 0 or 1.

27. D is structure: It is a CBI dimer having, in the formula, R １ H, P(O) ３ H ２ , C(O)NR ａ R ｂ , or selected from joining to a linker (L), R ２ H, P(O) ３ H ２ , C(O)NR ａ R ｂ , or selected from joining to a linker (L), R ａ and R ｂ C may be independently substituted with H and one or more Fs. １ -C ６ Selected from alkyl groups, or R ａ and R ｂ It forms a 5- or 6-membered heterocyclyl group, T is C ３ -C １２ alkylene, Y, (C １ -C ６ alkylene)-Y-(C １ -C ６ alkylene), (C １ -C ６ alkylene)-Y-(C １ -C ６ alkylene)-Y-(C １ -C ６ alkylene), (C ２ -C ６ alkynylene)-Y-(C ２ -C ６ alkynylene), and (C ２ -C ６ alkynylene)-Y-(C ２ -C ６ alkynylene), and T is a linking group. In the formula, Y is independently O, S, NR １ Selected from aryl and heteroaryl, Alkylenes, alkynylenes, aryls, and heteroaryls are independent of F, OH, O(C) １ -C ６ Alkyl), NH ２ , NHCH ３ , N (CH ３ ) ２ , OP(O) ３ H ２ , and C １ -C ６ It may be substituted with alkyl, and the alkyl may be optionally substituted with one or more F. Alternatively, alkylenes, alkynylenes, aryls, and heteroaryls may be substituted independently and by bonding to L. D' is, The drug portion is selected from the formula, and in the formula, the wavy line indicates the binding site to T. X １ and X ２ O and NR are independent of each other. ３ Selected from, R ３ C may be replaced by H and one or more arbitrary Fs. １ -C ６ Selected from alkyl groups, R ４ H, CO ２ It is a bond to R, or linker (L), where R is C １ -C ６ Alkyl or benzyl, R ５ is H or C １ -C ６ The antibody-drug conjugate according to claim 23, wherein the antibody is alkyl.

28. The method involves reacting at least one cysteine ​​of a cysteine-modified antibody (Ab) with a linker-drug intermediate to form an antibody-drug conjugate having formula I. Ab-(LD)pI A method for preparing an antibody-drug conjugate, wherein Ab is a cysteine-modified antibody, L is a linker, D is a drug moiety, p is 1 or 2, the cysteine-modified antibody contains L174C-modified cysteine ​​amino acids according to EU numbering in its heavy chain, the linker is pyridyl disulfide (PDS), and the cysteine-modified antibody is conjugated to the drug moiety via a linker bound to the L174C-modified cysteine ​​amino acids of the cysteine-modified antibody.

29. The antibody-drug conjugate according to claim 22, wherein D is selected from the group consisting of CBI-PBD heterodimer, cryptophycin, taxoid, and tubulysin M.

30. D is a CBI-PBD heterodimer: The antibody-drug conjugate according to claim 29.

31. A pharmaceutical composition comprising an antibody-drug conjugate according to any one of claims 22 to 27 and 29 to 30.

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