HK40003990A - Bioanalytical method for the characterization of site-specific antibody-drug conjugates - Google Patents

Description

Bioanalytical methods for characterizing site-specific antibody-drug conjugates

CROSS-REFERENCE TO RELATED APPLICATIONS

This non-provisional application filed pursuant to 37CFR § 1.53(b) claiming the benefit of U.S. provisional application serial No. 62/342,825 filed 2016, 5, 27, entitled to 35USC § 119(e), which is incorporated herein by reference in its entirety.

Technical Field

The present disclosure relates to methods of capturing, detecting, analyzing, characterizing, and quantifying antibody-drug conjugates, and their fragments and metabolites, in non-biological or biological matrices by chromatography and/or mass spectrometry.

Background

With brentuximab vedotin (b)Seattle Genetics) and ado-trastuzumab emtansine (Genentech), the therapeutic potential of Antibody Drug Conjugates (ADCs) providing targeted delivery of pharmaceutically active drugs or toxin molecules to specific sites of action was confirmed and led to further research and development. ADCs are generally composed of an antibody, a pharmaceutically active small molecule drug or toxin (often referred to as a "drug moiety" or "payload"), and optionally a linker connecting the two. This protein construct links small molecules, highly potent drugs, and macromolecular antibodies that are selected or engineered to target antigens on specific cell types (typically cancer cells). ADCs thus exploit the powerful targeting capabilities of monoclonal antibodies to specifically deliver highly potent conjugated small molecule therapeutics to cancer cells. Small molecule therapeutic payloads are often highly potent cytotoxic molecules that can be too toxic for use in conventional chemotherapy.

As successful ADC candidates emerge from ongoing research and development procedures and move to clinical evaluation and marketing, there is a need for safety and efficacy assays that can effectively evaluate complex chemical compositions created by combining large protein complexes (antibodies or antibody fragments) with typically much smaller but highly potent drug molecules. Characterization of drug-antibody linkage, antibody and drug concentrations, and drug-to-antibody ratios, and stability of these ADC compositions must first be established, and then monitored for consistency, since these properties of ADCs can affect the biological activity, pharmacokinetics, distribution, immunogenicity, safety, and stability profiles of these therapeutic entities. These challenges in ADC characterization are even more difficult when applied to heterogeneous compositions of ADC molecules (which represent many currently available ADCs that may have zero to eight drug molecules per antibody). This heterogeneity is a factor in the inconsistent measurement of pharmacokinetic parameters and in vivo performance of these therapeutic constructs.

Liquid chromatography tandem mass spectrometry (LC-MS/MS) is a powerful tool for protein analysis and quantification in very complex matrices like plasma/serum/tissue samples. Since peptides derived from digestion of the protein of interest and other endogenous proteins may have the same or similar nominal mass, the second dimension of MS fragmentation often provides a unique fragment of the peptide of interest. The combination of a particular parent peptide and a unique fragment ion can be used to selectively monitor the molecule to be quantified. Such an approach is called "multiple reaction monitoring" (MRM), also known as Selective Reaction Monitoring (SRM), which is a common model for protein mass production. But this powerful tool may be compromised by the analysis of complex mixtures of intact ADCs, antibody fragments, and peptide-linked drugs as well as free drug molecules.

Recent development of next generation ADCs has focused on exploring techniques to generate homogeneous ADCs with improved stability, Pharmacokinetics (PK) and therapeutic index. New classes of linkers and toxins with multiple cytotoxic mechanisms are also being explored. These next generation ADCs can pose additional bioanalytical challenges due to the structural complexity of the new payload and the associated complex in vivo biotransformations. For example, affinity capture LC-MS has been used for drug-to-antibody ratio (DAR) and catabolite characterization of intact ADCs. The complete ADC affinity capture LC-MS assay employs PNGaseF to remove N-glycans in the Fc region and thus reduce complexity and heterogeneity in the ADC mass spectrum. However, the complete mass spectrum of the new ADC is more complex and the sensitivity and resolution of this method may not be sufficient to elucidate some structural modifications and accurately characterize the DAR distribution.

Affinity capture liquid chromatography-mass spectrometry (LC-MS) is widely used for direct drug-to-antibody ratio (DAR) and catabolite characterization of antibody-drug conjugates (ADCs). However, the complete mass spectrum of the new ADC (which incorporates the new type of linker and payload in addition to maytansine and auristatin) is more complex than those previously examined. Current methods have shown some limitations in elucidating certain structural modifications.

Summary of The Invention

One aspect of the invention is an analytical approach for Antibody Drug Conjugates (ADCs), such as THIOMAB antibody-drug conjugates (TDCs), in which a linker drug is site-specifically conjugated in the Fab region. Affinity capture LC-MS F (ab')2 assay combines affinity capture via ADC binding to Fab region, followed by IdeS digestion on beads to remove Fc domain specifically and consistently. The resulting F (ab')2 (-100 kDa) fragment contains key ADC structural information such as drug-to-antibody ratio and drug metabolism and is more easily analyzed by electrospray ionization LCMS than intact ADC (-150 kDa). The reduced size of the analyte results in improved sensitivity and resolution of the mass spectrum. In addition, the shortened and optimized sample preparation time (e.g., rapid removal of Fc fragments by IdeS digestion) minimizes the skewed DAR profile and possible assay artifacts of drug metabolism that may result from extended incubation times (e.g., overnight enzyme treatment for Fc deglycosylation). The affinity capture LC-MS F (ab')2 assay provides more detailed and accurate information about the in vivo biotransformation of ADCs, enabling the analysis of low dose, unstable, and complex site-specific ADCs with linker-drugs conjugated in the Fab region.

One embodiment of the invention is a method of anti-Fc capture and IgdE protease digestion using ADCs with site-specifically conjugated drug moieties in the Fc region.

Robust methods are provided for detecting and quantifying antibody protein concentration and antibody-conjugated drug quantity and structural characterization by digestion of the antibody and separation of the drug building blocks of the ADC, followed by chromatographic and/or mass spectrometric analysis of the resulting composition for the combined drug and peptide released from the digested antibody. The methods of the invention utilize ADC fragments containing the drug moiety as a replacement for a complete ADC and thus provide increased assay sensitivity and resolution. The new methods also minimize artifacts of drug metabolism and reduce potential off-set responses to certain drug-to-antibody ratio (DAR) classes. Antibody and ADC analyses were previously performed using limited proteolytic digestion by attempting to control the activity of endoproteases Lys-C or the protease PNGase, followed by reverse phase HPLC and mass spectrometry. Due to limitations in sensitivity, resolution, and offset response to certain DAR species, these methods were found to be insufficient for some next generation ADCs containing recombinantly engineered, specific drug conjugation sites. ADCs, in particular, are such that they are unstable, require much higher MS resolution, and have site-specific conjugation to highly potent DNA damaging agents that are typically administered at low doses and thus at low concentrations in a sample, requiring much higher sensitivity in analytical techniques for characterizing the ADC composition and the structure of metabolites that may be present in a biological sample collected after administration of the ADC to a human or other test subject.

Thus, the present invention provides methods for assessing stability, post-translational and chemical modification consistently, reliably, efficiently, with high resolution and sensitivity during generation, formulation, storage, and administration during the development of site-specific ADCs by combining site-specific and controlled proteolytic digestion matched to the analysis of homogeneous and site-specific ADCs to reduce the size of ADC analytes.

The reduced size of the analyte results in improved mass spectral sensitivity, and resolution, and the difference in observed responses to some intact DARs is reduced. In addition, specific proteolytic digestion eliminates the need for overnight deglycosylation of Fc carbohydrates. In addition, rapid proteolytic digestion minimizes assay artifacts that may result from overnight enzyme treatment. For the analysis of site-specific ADCs with linker-drugs conjugated in the Fab region that may not be feasible using previous methods, the affinity capture LC-MS F (ab')2 assay of the present disclosure provides some detailed and accurate information about the in vivo bioconversion of the ADC.

The present invention provides methods for evaluating an ADC by digesting an ADC containing at least one drug moiety attached to an antibody at a site of recombinant engineering with a protease that cleaves the ADC to form a digested ADC composition comprising at least one peptide fragment not attached to the drug moiety and at least one peptide fragment attached to the drug moiety. The digested ADC composition is then analyzed by High Performance Liquid Chromatography (HPLC) and/or Mass Spectrometry (MS) to detect at least one peptide fragment attached to the at least one drug moiety. The recombinantly engineered specific site at which the drug moiety is attached to the ADC may be a site selected from the group consisting of a cysteine amino acid residue, a selenocysteine amino acid residue, a glutamine amino acid residue, a non-naturally occurring amino acid residue, and a sugar-modified glycan residue. The ADC may be an IgG antibody. The antibody portion of the ADC may be an antibody fragment. The antibody portion of the ADC may be a human or humanized antibody. The ADC may be glycosylated or phosphorylated. The antibody portion of the ADC can specifically bind to one or more tumor associated antigens or cell surface receptors.

The drug moiety may comprise at least one aromatic ring. Exemplary drug moieties include peptides, polyamides, maytansinoids (maytansinoids), dolastatins (dolastatins), auristatins (auristatins), calicheamicins (calicheamicins), Pyrrolobenzodiazepines (PBDs), PNU-159682, anthracyclines (anthracyclines), duocarmycins (duocarmycins), vinca alkaloids (vinca alkoloids), taxanes (taxanes), trichothecenes (trichothecenes), CC1065, duocarmycins (duocarmycins), camptothecins (camptothecins), elenefades (elinafide), antibiotics, fluorophores, radioisotopes, and stereoisomers, isosteres, metabolites, analogs or derivatives of these compounds. The drug moiety may also be linked to the antibody portion of the ADC via a linker.

The protease utilized in these methods may be selected from the group consisting of IdeS protease, IdeZ protease, IgdE protease, SpeB protease, gingipain, endoglycosidase, and combinations thereof. The digestion protocol may comprise incubating the ADC with the protease at a temperature between about 20 ℃ and about 45 ℃, and typically comprises incubating the ADC with the protease at a temperature of about 37 ℃. The digestion may also comprise incubating the ADC with the protease at a pH between about pH 5 and about pH 9, and typically comprises incubating the ADC with the protease at a pH of about pH7. The digestion may also comprise incubating the ADC with the protease for a period of time between about 0.1 hour and about 48 hours, and typically comprises incubating the ADC with the protease for a period of time of about 1 hour.

The analysis may include at least one of RP-LC, RP-LC/MS, and LC-MS/MS analysis.

In these methods, prior to the digesting step, the ADC may be suspended in a matrix selected from the group consisting of buffer, whole blood, serum, plasma, cerebrospinal fluid, saliva, urine, lymph, bile, feces, sweat, vitreous fluid, tears, and tissue. In exemplary embodiments, the ADC is suspended in whole blood, serum, plasma, or tissue of a mammal selected from the group consisting of human, cynomolgus monkey, rat, and mouse. Thus, the ADC may be enriched prior to the digestion step by a technique selected from the group consisting of size exclusion chromatography, dialysis, selective precipitation, differential centrifugation, filtration, gel electrophoresis, liquid chromatography, reverse phase chromatography, immunoprecipitation, SpinTrap columns comprising protein a and protein G, NHS and streptavidin iron or phosphorous or immobilized antibodies or lectins, paramagnetic beads, immuno-subtraction, fractionation, solid phase extraction, phosphopeptide enrichment, polyacrylamide gel electrophoresis, and desalting. Thus, in these methods, the ADC can be bound to an affinity capture medium. The affinity capture media may comprise at least one of a bead or resin supported protein a/G, a target antigen-paramagnetic bead capture media, an anti-idiotypic antibody, an anti-Hu antibody, and an anti-drug antibody. The analytical methods can include washing the ADC bound to the affinity capture medium to reduce non-antibody proteins in contact with the ADC. The methods may further comprise dephosphorylating or deglycosylating the ADC bound to the affinity capture medium. The digestion step may also be performed simultaneously with binding of the ADC to the affinity capture medium. Alternatively, or in addition, the ADC may be eluted from the affinity capture medium prior to the step of digesting the ADC.

These methods are particularly useful in calculating the total antibody concentration of the ADC from an analysis of the digested ADC composition. Alternatively, or in addition, the concentration of antibody-conjugated drug of the ADC is calculated from the analysis of the digested ADC composition. Alternatively, or in addition, an average DAR for the ADC is calculated from an analysis of the digested ADC composition. Alternatively, or in addition, metabolite or catabolite structures may be determined from analysis of the digested ADC composition. Alternatively, or in addition, the protein concentration of the ADC can be calculated from an analysis of the digested ADC composition. Alternatively or additionally, correlating the protein concentration with peak areas of RP-LC and/or MS analysis of at least one Fc fragment from the digested ADC. Alternatively, or in addition, the extinction coefficient of the ADC is calculated from an analysis of the digested ADC composition. Alternatively, or in addition, the average DAR for the ADC, metabolite or catabolite structure, and the protein concentration of the ADC are obtained from RP-LC and/or MS analysis of at least one Fc fragment from the digested ADC.

This summary is not intended to, and should not be construed to, represent the full breadth and scope of the present disclosure. Moreover, references herein to "the present disclosure" or aspects thereof should be understood to mean certain embodiments of the present disclosure and should not necessarily be construed as limiting all embodiments to the specific description. The present disclosure is set forth at various levels of detail in this summary and in the description of the drawings and embodiments, and is not intended to limit the scope of the disclosure by inclusion or non-inclusion of elements, components, etc. in this summary. Further aspects of the disclosure will become apparent from the description of the embodiments, particularly when taken together with the drawings.

Brief Description of Drawings

FIG. 1A depicts cleavage of Thiomab with IdeS protease^TMAntibody fragments generated by drug conjugates (TDCs) in which the linker-drug is site-specifically conjugated to f (ab). Digestion produces F (ab')2 fragments and Fc fragments. The linker-drug may be site-specifically conjugated to either f (ab) or Fc.

FIG. 1B provides a schematic representation of IdeS digestion, generation 2 affinity capture LC-MS.

Figure 1C shows a schematic representation of PNGaseF (passage 1) and IdeS (passage 2) digestions and the advantages of the passage 2 affinity capture LC-MS assay of the present disclosure.

Figure 1D shows a comparison between direct LC-MS (1D-1), generation 2 (1D-2) and generation 1 (1D-3) affinity capture LC-MS analyses of TDC standard mixtures (DAR0: DAR2 ═ 1: 1). MS peaks labeled with x represent DAR with glycans.

FIG. 1E shows a comparison between linker-drug unconjugate (-LD) and the unstable TDC from rat plasma, PNGaseF digestion of TDC-L1, generation 1 (1E-1) and IdeS digestion, generation 2 (1E-2) analysis. The 2 nd generation affinity capture LC-MS assay minimizes artificial partial drug breakdown (-PD) (B). Partial drug decomposition did not affect the efficacy of TDC-L1, resulting in no change in DAR.

Figure 1F shows MS peaks obtained during characterization of complex TDC catabolites in mouse plasma in vivo. The MS peaks of TDC-L2 catabolites were not resolved in the PNGaseF digestion (passage 1) affinity capture LC-MS assay (1F-1) due to the loss of 42Da from the drug molecule, but they were resolved near baseline (1F-2) using IdeS digestion (passage 2 assay). Partial drug decomposition (-PD, 43Da) significantly affected the efficacy of exemplary TDC-L2, resulting in a corresponding reduction in DAR.

FIG. 2A-1 shows RP-LCMS analysis of site-specific ADC digested with IdeS proteolytic enzyme. The Fc/2 fragment elutes first and is resolved from the baseline for the drug containing F (ab') 2. The peak area of the antibody fragment that does not contain the linker-drug is then used to calculate the protein concentration.

FIG. 2A-2 shows the deconvolution mass spectrum of Fc/2 from FIG. 2A-1.

FIG. 2B shows a standard curve (Fc/2 peak area versus concentration; top) (F (ab')2 peak area versus concentration; bottom) over the range of 0.5-20mg/ml generated using trastuzumab digested with IdeS protease. The protein concentration of TDC specifically conjugated at the upper site on f (ab) can be determined using the peak area of Fc/2 of TDC (top curve) and linear regression. Traditional ADCs conjugated on interchain disulfides can also be characterized using this approach, since the Fc/2 fragment in these conjugates is also drug-free. The protein concentration of TDC specifically conjugated at the upper site on Fc can be determined using the peak area of F (ab')2 of TDC (bottom curve) and linear regression.

Figure 2C shows the concentration determination of trastuzumab site-specifically conjugated at engineered cysteine K149C, with linker-drug at engineered cysteine K149C on f (ab). Concentrations were determined from standard curves using Fc/2 peak area (3 replicates) and linear regression.

Figure 2D shows the concentration determination of trastuzumab site-specifically conjugated at engineered cysteine S400C, with linker-drug at engineered cysteine S400C on Fc. Concentrations were determined from standard curves using F (ab')2 peak area (3 replicates) and linear regression.

Figure 2E shows the determination of the concentration of trastuzumab conjugated with linker-drug on interchain disulfide. Concentrations were determined from standard curves using Fc/2 peak area (3 replicates) and linear regression. Since the Fc/2 fragment does not contain a hinge disulfide, it does not contain a linker-drug.

FIG. 2F shows 81 Thiomabs obtained by the IdeS protease digestion method or bicinchoninic acid assay (BCA) protein assay of the present disclosure^TMCorrelation of drug conjugate concentration values.

Figure 3A shows the DAR (drug-to-antibody ratio) profile analysis of TDC (PBD dimer drug, disulfide linker) standard mixtures (DAR0: DAR2 ═ 1:1) by direct LC-MS assay, IdeS digestion, affinity capture LC-MS F (ab')2 assay, and PNGaseF, affinity capture LC-MS whole antibody assay, with standard deviations of 0.13, 0.09, and 0.14 for 3 replicates, respectively.

Figure 3B shows DAR profiling of TDC standard mixtures (DAR0: DAR2 ═ 1:1) by direct LC-MS, affinity capture LC-MS F (ab ')2 assay with IdeS digested for 1 hour, and affinity capture LC-MS F (ab')2 assay with PNGaseF digested overnight.

Detailed Description

The present disclosure relates to a single measurement method for detecting and quantifying antibodies and drug components of Antibody Drug Conjugates (ADCs) that robustly measure the total antibody and antibody conjugated drug quantity from a single sample preparation, thereby providing drug-to-antibody ratio (DAR) calculations and significant time and resource savings.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and are consistent with the following: singleton et al (1994) "Dictionary of microbiology and Molecular Biology", 2 nd edition, J.Wiley & Sons, New York, N.Y.; and Janeway et al (2001) "microbiology", 5 th edition, Garland Publishing, New York. When tradenames are used herein, tradename product formulations, common name drugs, and active pharmaceutical ingredients of the tradename products are also included.

Definition of

The term "biological sample" refers to any component derived or isolated from an animal and includes blood, plasma, serum, cells, urine, cerebrospinal fluid (CSF), milk, bronchial lavage, bone marrow, amniotic fluid, saliva, bile, vitreous humor, tears, or tissue.

The term "digestive enzyme" refers to an enzyme that is capable of cleaving or hydrolyzing a peptide or protein into fragments in a specific or general random manner. The digestive enzyme is capable of forming a digested antibody sample from an antibody, wherein the antibody is a component of the biological sample. Digestive enzymes include proteases such as trypsin, papain, pepsin, endoprotease LysC, endoprotease ArgC, Staphylococcus aureus V8, chymotrypsin, Asp-N, Asn-C, PNGaseF, endoprotease GluC, and LysN.

The term "antibody" is used in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

An "antibody fragment" refers to a molecule distinct from an intact antibody that comprises a portion of an intact antibody and binds to an antigen to which the intact antibody binds. Examples of antibody fragmentsIncluding but not limited to Fv, Fc, Fab, Fab ', Fab ' -SH, F (ab ')₂(ii) a A diabody; a linear antibody; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab ', Fab ' -SH, F (ab ')₂For reviews of certain antibody fragments, see Hudson et al (2003) Nat. Med.9: 129-134. for reviews of scFv fragments, see for example Pluckth ü n, in The Pharmacology of Monoclonal Antibodies, Vol.113, Rosenburg and Moore eds (Springer-Verlag, New York), p.269-315 (1994); WO 93/16185; US5,571,894; US5,587,458. for Fab and F (ab')₂Discussion of fragments (US5,869,046).

Diabodies are antibody fragments with two antigen binding sites, which may be bivalent or bispecific (EP404,097; WO 1993/01161; Hudson et al (2003) nat. Med.9: 129-. Tri-and tetrabodies are also described in Hudson et al (2003) nat. Med.9: 129-.

Single domain antibodies are antibody fragments that comprise all or part of the heavy chain variable domain or all or part of the light chain variable domain of the antibody. In certain embodiments, the single domain antibody is a human single domain antibody (US 6,248,516).

Antibody fragments can be generated by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and production of recombinant host cells (e.g., e.coli or phage), as described herein.

The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxy-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, NIHPublication 91-3242, Bethesda Md. (1991), volumes 1-3.

"framework" or "FR" refers to constant domain residues other than hypervariable region (HVR) residues. Generally, the FR of a constant domain consists of 4 FR domains: FR1, FR2, FR3, and FR 4. Thus, HVR and FR sequences typically occur in the following order in VH (or VL): FR1-H1(L1) -FR2-H2(L2) -FR3-H3(L3) -FR 4.

The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain comprising an Fc region as defined herein.

"human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source using a repertoire of human antibodies or other human antibody coding sequences. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues.

Humanized antibodies and methods for their production have been extensively reviewed in, e.g., Almagro and Fransson (2008) front. biosci.13:1619-1633, and described in, e.g., Riechmann et al (1988) Nature332: 323-329; queen et al (1989) Proc. nat' l Acad. Sci. USA 86: 10029-10033; U.S. Pat. Nos. 5,821,337; U.S. patent nos. 7,527,791; U.S. patent nos. 6,982,321; U.S. patent nos. 7,087,409; kashmiri et al (2005) Methods 36:25-34 (SDR (a-CDR) grafting is described); padlan (1991) mol.Immunol.28:489-498 (described as "resurfacing"); dall' Acqua et al (2005) Methods 36:43-60 (describing "FR shuffling"); and Osbourn et al (2005) Methods 36: 61-68; klimka et al (2000) Br. J. cancer 83:252-260 (describing the "guided selection" method of FR shuffling).

Human framework regions that may be used for humanization include, but are not limited to: framework regions selected using the "best-fit" (best-fit) method (see, e.g., Sims et al (1993) J.Immunol.151: 2296); framework regions derived from consensus sequences of human antibodies from specific subsets of light or heavy chain variable regions (Carter et al (1992) Proc. Natl. Acad. Sci. USA 89: 4285; and Presta et al (1993) J. Immunol.151: 2623); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson (2008) front. biosci.13: 1619-1633); and framework regions derived by screening FR libraries (see, e.g., Baca et al (1997) J.biol.chem.272:10678-10684 and Rosok et al (1996) J.biol.chem.271: 22611-22618).

Generally, human antibodies are described in van Dijk and van de Winkel (2001) curr. opin. pharmacol.5: 368-74; lonberg (2008) curr. Opin. Immunol.20: 450-.

Human antibodies can be made by administering an immunogen to a transgenic animal that has been modified to produce fully human antibodies or fully antibodies with human variable regions in response to an antigenic challenge. Such animals typically contain all or part of a human immunoglobulin locus, which replaces an endogenous immunoglobulin locus, or which exists extrachromosomally or is randomly integrated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin locus has typically been inactivated. For an overview of the method of obtaining human antibodies from transgenic animals, see Lonberg (2005) nat. Biotech.23: 1117-1125. See also, for example, U.S. Pat. Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE^TMA technique; U.S. Pat. No.5,770,429, which describesA technique; U.S. Pat. No.7,041,870, which describes K-MTechnology, and US 2007/0061900, which describesA technique). Can be further modified, for example, by combination with different human constant regionsThe human variable region of an intact antibody produced by such animals.

Human antibodies can also be generated by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the Production of human Monoclonal antibodies have been described (see, e.g., Kozbor (1984) J.Immunol.133: 3001; Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York,1987), and Borner et al (1991) J.Immunol.147: 86). Human antibodies generated via human B-cell hybridoma technology are also described in Li et al (2006) Proc.Natl.Acad.Sci.USA 103: 3557-3562. Other methods include those described in: U.S. Pat. No.7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines); ni (2006) Xiaoandaixiue 26(4):265-268 (which describes a human-human hybridoma). The human hybridoma technique (Trioma technique) is also described by Vollmers and Brandlein (2005) histocogy and Histopathlogy 20(3): 927) 937 and Vollmers and Brandlein (2005) Methods and Findingsin Experimental and Clinical pharmacy 27(3): 185-91.

Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

"human consensus framework" refers to antibody framework regions that represent the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. Typically, the sequence subgroups are, e.g., Kabat et al, supra. In an exemplary embodiment, for VL, the subgroup is subgroup kappa I. In another exemplary embodiment, for VH, the subgroup is subgroup III.

A "humanized" antibody is a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise at least one, and typically two, substantially the entire variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. Optionally, the humanized antibody may comprise at least a portion of an antibody constant region derived from a human antibody. An antibody, e.g., a "humanized form" of a non-human antibody, refers to an antibody that has undergone humanization.

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species. An exemplary "chimeric" antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as monkey) and a human constant region (U.S. Pat. No.4,816,567; Morrison et al (1984) Proc. Natl. Acad. Sci. USA 81: 6851-. Another exemplary chimeric antibody is a "class-switched" antibody, wherein the class or subclass has been altered from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and FRs (or portions thereof) are derived from a human antibody sequence. Optionally, the humanized antibody will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody from which HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Antibodies of the disclosure can be isolated by screening combinatorial libraries for antibodies having a desired activity or activities. For example, various methods for generating phage display libraries and screening such libraries for antibodies possessing desired binding characteristics are known in the art. Such Methods are reviewed, for example, in Hoogenboom et al, in Methods in molecular biology 178:1-37 (O' Brien et al, eds., Human Press, Totowa, N.J.,2001), and further described, for example, in McCafferty et al (1990) Nature 348: 552-; clackson et al (1991) Nature352: 624-; marks et al (1992) J.mol.biol.222: 581-597; marks and Bradbury, Methods in molecular biology248:161-175(Lo eds., Human Press, Totowa, N.J., 2003); sidhu et al (2004) J.mol.biol.338(2): 299-310; lee et al (2004) J.mol.biol.340(5): 1073-; fellouse (2004) Proc. Natl. Acad. Sci. USA 101(34): 12467-; and Lee et al (2004) J. immunological methods 284(1-2): 119-132.

In some phage display methods, the repertoire of VH and VL genes, respectively, are cloned by Polymerase Chain Reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding phages, as described in Winter et al (1994) Ann.Rev.Immunol.12: 433-455. Phage typically display antibody fragments either as single chain fv (scfv) fragments or as Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, the natural repertoire can be cloned (e.g.from humans) to provide a single source of antibodies to a large panel of non-self and also self-antigens in the absence of any immunisation, as described by Griffiths et al (1993) EMBO J12: 725-. Finally, non-rearranged V gene segments can also be synthesized by cloning non-rearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the highly variable CDR3 regions and effecting rearrangement in vitro, as described by Hoogenboom and Winter (1992) J.mol.biol.227: 381-388. Human antibody phage libraries are described in US5,750,373; US7,985,840; US7,785,903; US8,679,490; US8,054,268; and US 2005/0079574; US 2007/0117126; US 2007/0237764; US 2007/0292936. Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments for purposes of this disclosure.

The antibody can be a multispecific antibody, e.g., a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. One of the binding specificities may be for one antigen and the other for a second antigen. Alternatively, bispecific antibodies can bind to two different epitopes of the same antigen. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing the antigen. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (see, e.g., Ortiz-Sanchez et al (2008) Expert Opin. biol. Ther.8(5): 609-632).

Techniques for generating multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello (1983) Nature305: 537; WO 1993/08829; and Traunecker et al (1991) EMBO J.10:3655), and "bump-in-hole" engineering (U.S. Pat. No.5,731,168). Effects can also be manipulated electrostatically by engineering the molecules for the generation of antibody Fc-heterodimers (WO 2009/089004a 1); crosslinking two or more antibodies or fragments (see, e.g., U.S. Pat. No.4,676,980; and Brennan et al (1985) Science 229: 81); the use of leucine zippers for the production of bispecific antibodies (e.g., Kostelny et al (1992) J. Immunol.148(5): 1547-; the "diabody" technique used to generate bispecific antibody fragments is used (see, e.g., Hollinger et al (1993) Proc. Natl. Acad. Sci. USA 90: 6444-; and the use of single chain fv (sFv) dimers (Gruber et al (1994) J. Immunol.152: 5368); and making a trispecific antibody (Tutt et al (1991) J. Immunol.147:60) to generate a multispecific antibody.

Also included herein are engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies" (e.g., US 2006/0025576).

Antibodies or fragments herein also include "dual action fabs" or "DAFs" comprising an antigen binding site that binds an antigen and another, different antigen (e.g., US 2008/0069820).

Antibody variants

Amino acid sequence variants of the antibodies provided herein are also contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, so long as the final construct possesses the desired characteristics, such as antigen binding.

Antibodies include fusion proteins comprising an antibody and a protein, a drug moiety, a label, or some other moiety. Fusion proteins can be generated by recombinant techniques, conjugation, or peptide synthesis to optimize properties such as pharmacokinetics. The human or humanized antibody may also be a fusion protein comprising an Albumin Binding Peptide (ABP) sequence (see Dennis et al (2002) JBiol. chem.277:35035-35043, pages III and IV, 35038, page tables III and IV; U.S. patent publication No.2004/0001827, paragraph [0076], and WO 01/45746, pages 12-13, both of which are incorporated herein by reference).

Substitution, insertion, and deletion variants

Antibody variants having one or more amino acid substitutions are provided for use and analysis in the methods of the present disclosure. Sites of interest for substitutional mutagenesis include HVRs and FRs. Substantial changes are provided in the following table under the heading of "exemplary substitutions" and are described further below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the product screened for a desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved antibody-dependent cell-mediated cytotoxicity (ADCC) or CDC.

According to common side chain properties, amino acids can be grouped as follows:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral, hydrophilic: cys, Ser, Thr, Asn, Gln;

(3) acidic: asp, Glu;

(4) basic: his, Lys, Arg;

(5) residues that influence chain orientation: gly, Pro;

(6) aromatic: trp, Tyr, Phe.

Non-conservative substitutions may entail replacing one of these classes with a member of the other class.

One class of surrogate variants involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further study will have an alteration (e.g., an improvement) in certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody and/or will substantially retain certain biological properties of the parent antibody. Exemplary surrogate variants are affinity matured antibodies, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

Changes (e.g., substitutions) can be made to HVRs, for example, to improve antibody affinity. Such changes can be made to HVR "hot spots", i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury (2008) Methods mol. biol.207: 179. 196), and/or SDR (a-CDRs), where the resulting variant VH or VL is tested for binding affinity. Affinity maturation by construction and re-selection of secondary libraries has been described, for example, in Hoogenboom et al, in Methods in Molecular Biology 178:1-37 (O' Brien et al, eds., Human Press, Totowa, N.J. (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). Then, a secondary library is created. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves an HVR-directed method in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are frequently targeted.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, so long as such changes do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes (e.g., conservative substitutions, as provided herein) may be made to HVRs that do not substantially reduce binding affinity. Such changes may be outside of HVR "hotspots" or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unaltered, or contains no more than 1,2, or 3 amino acid substitutions.

One method that can be used to identify residues or regions of an antibody that can be targeted for mutagenesis is referred to as "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science,244: 1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions may be introduced at amino acid positions that indicate functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the contact points between the antibody and the antigen. As alternative candidates, such contact and adjacent residues may be targeted or eliminated. Variants can be screened to determine if they contain the desired property.

Amino acid sequence insertions include amino and/or carboxy-terminal fusions ranging in length from 1 residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include fusions of the N-or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or a polypeptide that extends the serum half-life of the antibody.

Glycosylation variants

In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree of glycosylation of the antibody. Addition or deletion of glycosylation sites of an antibody can be conveniently achieved by altering the amino acid sequence such that one or more glycosylation sites are created or eliminated.

In the case of antibodies comprising an Fc region, the carbohydrate to which they are attached may be altered. Natural antibodies produced by mammalian cells typically comprise branched, bi-antennary oligosaccharides, which are typically N-linked to Asn297 of the CH2 domain attached to the Fc region (Wright et al (1997) TIBTECH 15: 26-32). Oligosaccharides may include various carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "backbone" of the bi-antennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the invention may be modified to create antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. for example, the amount of fucose in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%, the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297 relative to the sum of all carbohydrate structures attached to Asn297 (e.g., complex, hybrid and high mannose structures), as measured by MALDI-TOF mass spectrometry (see, e.g., WO 2008/077546) Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues), however, Asn may also be located upstream or downstream of position 297 due to minor sequence variations in the antibody by about + -3 amino acids, i.e.between positions 2004 and 300, such glycosylated variants may have improved fucosylation functions (US patent publication No. 2003/0157108; WO 12448; WO 3512: 2001/2004/056312; WO 2004-WO 11: 97; WO 11: WO 11.

Further provided are antibody variants having bisected oligosaccharides, for example, wherein biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Publications describing such antibody variants with bisected oligosaccharides include WO 2003/011878; U.S. Pat. Nos. 6,602,684; and U.S. patent publication 2005/0123546. Antibody variants having at least one galactose residue in an oligosaccharide attached to an Fc region are also provided and may have improved CDC function. Publications describing such galactose residue antibody variants include WO 1997/30087; WO 1998/58964; WO 1999/22764.

Site-specific antibody drug conjugates

As described above, one of the major challenges in ADC design is the homogeneity of currently available ADCs that can have zero to eight drug molecules attached to each antibody or antibody fragment. This heterogeneity in ADC species adversely affects analytical methods for assessing and monitoring the stability, consistency, pharmacokinetics, and in vivo performance of ADC compositions. For this reason, conjugation strategies have been identified that allow chemical installation of the drug onto the antibody at a predefined site to ensure stability of the conjugate after generation and while circulating in vivo. These site-specific ADCs, also known as immunoconjugates, rely on emerging site-specific conjugation strategies, including the use of engineered cysteines (e.g., THIOMABTM), unnatural amino acids, selenocysteine residues, enzymatic conjugation via glucosyltransferase and transglutaminase s1, and other techniques. In particular, THIOMAB-drug conjugates (TDCs) can be controlled to generate homogeneous DAR 2.

1) Cysteine engineered antibody drug conjugates

Cysteine engineered antibodies (e.g., "THIOMAbTM") comprise one or more antibody residues substituted with cysteine. The substituted residues may be present at accessible sites of the antibody such that the reactive thiol groups are located at accessible sites of the antibody, and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create site-specific ADCs. Examples of such THIOMAB include cysteine engineered antibodies in which cysteine may be substituted for any one or more of the following residues: v205 of the light chain (Kabat numbering); a118 of the heavy chain (EU numbering); and S400 of the heavy chain Fc region (EU numbering), and S121 of the light chain, and K149. Methods of generating cysteine engineered antibodies include, but are not limited to, US7,521,541; the process described in US9,000,130.

As such, the methods of the present disclosure can be applied to antibody-drug conjugates comprising cysteine engineered antibodies in which one or more amino acids of the wild type or parent antibody are replaced with a cysteine amino acid (THIOMAB)^TM). Any form of antibody may be so engineered. For example, a parent Fab antibody fragment can be engineered to form a cysteine engineered Fab, and a parent monoclonal antibody can be engineered to form a cysteine engineered monoclonal antibody. It should be noted that single-site processThe mutations produce one engineered cysteine residue in the Fab antibody fragment, while the single-site mutations produce two engineered cysteine residues in the full-length antibody, which is a result of the dimeric nature of the IgG antibody. Mutants with a substituted ("engineered") cysteine (Cys) residue were evaluated for reactivity with the newly introduced engineered cysteine thiol group. Thiol reactivity value is a relative numerical term in the range of 0 to 1.0 and can be measured for any cysteine engineered antibody. The thiol reactivity value of the cysteine engineered antibodies of the invention is between 0.6 and 1.0; 0.7 to 1.0; or in the range of 0.8 to 1.0.

Cysteine amino acids may be engineered at reactive sites in the Heavy (HC) or Light (LC) chains of antibodies that do not form intra-or intermolecular disulfide linkages (Junutula et al (2008) Nature Biotech.,26(8): 925-. The engineered cysteine thiol may be reacted with a linker reagent or linker-drug intermediate having a thiol-reactive electrophilic pyridyl disulfide group of the present invention to form an ADC having a cysteine engineered antibody and a drug moiety. The specific location (i.e., site) of the drug module in these engineered ADCs can be designed, controlled, and known as such. Drug loading can thus be controlled because the engineered cysteine thiol groups typically react with thiol-reactive linker reagents or linker-drug intermediates in high yield. Engineered antibodies, introducing cysteine amino acids by substitution of one site on the heavy or light chain gives two new cysteines on the symmetric antibody. Drug loading (DAR) of close to 2 can be achieved with near complete homogeneity in these site-specifically conjugated ADCs.

2) Antibody drug conjugates engineered with unnatural amino acids

Similar to cysteine engineered antibodies, the incorporation of Unnatural Amino Acids (UAA) into proteins provides a flexible method for site-specifically engineering bioorthogonal (biorthogonal) functionality (see, e.g., Agarwal and Biobertozzi, Bioconjugate chem.2015,26: 176-92; Sochaj et al, Biotech. Advances (2015)33: 775-84). To design and specifically introduce an unnatural amino acid into a protein, such as an antibody or antibody fragment, a mutein encoded by a gene having an amber stop codon (TAG) at the site of the desired UAA can be expressed in a cell along with a corresponding orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pair capable of installing UAA at the amber stop codon site (see, e.g., Liu and Schultz (2010) annu. One unnatural amino acid incorporated in the UAA expression system of E.coli is p-acetylphenylalanine, which has been selected for its bioorthogonal reactivity to ketones (Wang et al (2003) Proc. Natl. Acad. Sci. U.S.A.100: 56-61). This unnatural amino acid was conjugated to aminooxy-auristatin F and the resulting trastuzumab antibody exhibited excellent pharmacokinetic properties in mice (Tian et al, (2014) proc.natl.acad.sci.u.s.a.111: 1766-1771). This site-specific engineering methodology can be extended to include more than one bio-orthogonal functional group in the protein. This approach based on the introduction of one or more unnatural amino acids into proteins can provide antibody-drug conjugates with a specific number of known unnatural amino acid substitutions that are easily and consistently conjugated to a therapeutic moiety, such as an anti-cancer drug, resulting in a very homogeneous ADC composition, with drug conjugation limited to precisely designed and identified sites in the protein.

3) Antibody drug conjugates engineered with selenocysteine

Selenocysteine is a natural but rare amino acid that exists in all kingdoms of life as a building block of selenoproteins (of which only 25 are currently known in mammals). Selenocysteine contains selenium instead of sulfur, which makes it more reactive to electrophiles in acidic conditions than cysteine. This chemical property is exploited for the selective coupling of maleimide and iodoacetamide-containing agents to antibodies containing genetically engineered selenocysteine residues (Hofer et al (2009) Biochemistry 48: 12047-57; Li et al (2014) Methods 65: 133-38). Conjugation of fluorescent probes, biotin and biotin polyethylene glycol (biotin-PEG) to antibodies using selenocysteine, resulting in fully functional conjugates with specific site-limiting sites for drug attachment and stoichiometry, demonstrates the generation of homogeneous ADCs engineered based on selenocysteine residues (see, e.g., Agarwal and Bertozzi (2015) Bioconjugate chem.26: 176-92; Sochaj et al (2015) biotech. advances 33: 775-84).

4) Glycan-modified antibody drug conjugates

Human IgG molecules have a conserved glycosylation site at each N297 residue in the CH2 domain, making these pendant N-glycans convenient targets for site-specific conjugation. This glycosylation site is sufficiently distant from the variable region that conjugation of the drug moiety to the attached glycan should not affect antigen binding. One method of linking therapeutic moieties to these glycans involves oxidative cleavage of adjacent diol moieties contained in these glycans with periodate to generate aldehydes that are capable of reductive amination and conjugation of hydrazides and aminooxy compounds (O' shannesset al (1984) immunol.lett.8: 273-77). Another method involves increasing fucosylation of N-acetylglucosamine residues in these glycans. Oxidation of these fucose residues generates carboxylic acid and aldehyde modules that can be used to attach drugs and fluorophores to these specific sites on antibodies (zubberbuhler et al (2012) chem. Another approach involves modification of sialic acid in these glycans (and increasing the sialic acid content in these glycans), followed by oxidation of sialic acid and conjugation of an aminooxy-drug to generate an oxime-linked conjugate (Zhou et al (2014) Bioconjugate chem.25: 510-20). Alternatively, sialyltransferases may be used to incorporate modified sialic acid residues containing bio-orthogonal functional groups into these glycans. The bio-orthogonal functional groups can then be modified to attach the therapeutic moiety to the site of the glycan (Li et al (2014) angelw chem. int.53: 7179-82). Another approach to modify these glycan sites is to use glycosyltransferases to attach galactose or ketone or azide containing galactose analogs to N-acetylglucosamine in these glycans, and to attach drugs or radionucleotides to the galactose molecules (Khidekel et al (2003) J.am.chem.Soc.125: 16162-63; Clark et al (2008) J.am.chem.Soc.130: 11576-77; Boeggeman et al (2007) Bioconjugate chem.18: 806-14). Another approach relies on the introduction of modified sugars into these glycans when the antibodies are expressed by metabolic oligosaccharide engineering (Campbell et al (2007) mol. biosystem.3: 187-94; agrard et al (2009) acc. chem. res.42: 788-97). This approach has been utilized with the introduction of fucose analogs followed by drug attachment/modification at the fucosylation site (Okeley et al (2013) Bioconjugate chem.24: 1650-1655; Okeley et al (2013) Proc. Natl. Acad. Sci.U.S. A.110: 5404-09).

5) Proantibody (Probody) drug conjugates

Preantibodies (PROBODY)^TMCytomx Therapeutics LLC, South San Francisco, CA) is a recombinant, proteolytically activated antibody prodrug consisting of a protease-cleavable linker at the amino terminus of the antibody light chain and a monoclonal antibody designed to block extension of the antibody binding to antigen by a masking peptide (U.S. patent No.8,563,269; desnoyers et al, Sci trans med.2013, 16; 207, (207) 207ra 144; polu and Lowman, Expert Opin biol. 2014,14(8) 1049-53; wong et al, Biochimie.2016,122: 62-7). Cleavage of the linker by specific tumor-associated proteases results in dissociation of the mask and release of antibodies competent to bind to the antigen in the tumor. The pro-antibodies are designed to take advantage of the substantial deregulation of extracellular protease activity present within the tumor microenvironment relative to healthy tissue, thereby binding only minimally to antigens in healthy tissue where the presence of active protease is insufficient to remove the mask. Within the tumor, in the presence of sufficiently deregulated protease activity, the mask is removed by cleavage of the linker and antigen binding occurs. Pre-antibody drug conjugates (PDC) have been engineered to bind pre-antibodies to the microtubule inhibitor MMAE (Weidle et al, CanGen)&Proteom,2014,11:67-80；Sagert et al.,Abstract 2665,AACR AnnualMeeting2014)。

6) Polymer or peptide conjugates

Antibody drug conjugates are also formed using antibodies or antibody fragments that link hydrophilic polymers or peptides composed of natural amino acids that are themselves capable of attaching to therapeutic peptides, proteins or therapeutic small molecules. As such, the polymer or peptide essentially acts as a linker between the antibody and the therapeutic moiety (drug), but such linker provides a means by which multiple therapeutic moieties will be attached, thereby significantly increasing the DAR per ADC molecule. Using these constructs, DAR of 14-18 or even higher were possible while maintaining the site-specific conjugation properties of site-specific ADCs. Exemplary ADCs comprising such peptide/polymer conjugates include ADCs that are linked to xten (tm) peptide conjugates (amumix, Mountain View, CA) at specific, engineered amino acid residues in the light chain of an antibody, such as the cysteine engineered antibody described above. These peptides are substantially homogeneous polypeptides, useful as conjugation partners, linking one or more payloads via a crosslinker reactant, resulting in XTEN-payload ADC conjugates. These peptide linkers are polypeptides consisting of a non-naturally occurring, substantially non-repeating sequence with low or no secondary or tertiary structure under physiological conditions, and typically have from about 36 to about 3000 amino acids, most or all of which are hydrophilic small amino acids, with a limited number of orthogonal pendant reactive groups conjugated to one or more molecules of a target module that acts as a ligand for a cell surface receptor and one or more molecules of an effector drug (U.S. patent publication 2015/0037359).

7) Fc fusion protein

There are a wide variety of antibody-cytokine fusion proteins that have been developed as biopharmaceutical products and are approved by the FDA for use as pharmaceuticals in the united states. Most of these fusion proteins target tumor antigens in protein constructs fused with full-length antibodies or derivatives thereof with different cytokines (see, e.g., Ortiz-Sanchez et al (2008) expert Opin. biol. Ther.8(5): 609-32; Sochaj et al (2015) Biotechnology Advances 33: 775-84). Each cytokine may be fused at the amino or carboxy terminus of the antibody, depending on the structure of the cytokine and the antibody, thereby retaining the biological activity of both components. The number of different antibody-cytokine fusion proteins is large between an increasing number of antibody derivatives and different cytokines that can be combined with them. In addition, Fc fusion constructs are being developed for non-cancer clinical indications such as autoimmune disorders. These proteins can compete directly with antibodies targeting self-proteins. In general, these Fc fusion protein constructs fall into four groups based on ligand specificity (binding to one or more epitopes on the ligand molecule) and valency (stoichiometry of the binding ligand molecule): bivalent and single ligand specificity; monovalent and multiple ligand specificity; multivalent and single ligand specificity; and monovalent and single ligand specificity.

Unlike Fc fusion protein constructs, these site-specific, engineered immunoconjugates can retain the antigen-binding ability of their wild-type parent antibody counterparts. As such, the site-specific antibody conjugate is capable of binding (preferably specifically) to an antigen. Such antigens include, for example, Tumor Associated Antigens (TAAs), cell surface receptor proteins, and other cell surface molecules, transmembrane proteins, signaling proteins, cell survival regulatory factors, cell proliferation regulatory factors, molecules known or suspected to functionally contribute to tissue development or differentiation, lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in vasculogenesis (vasculogenesis), and molecules known or suspected to functionally contribute to angiogenesis (angiogenesis). The tumor associated antigen may be a cluster differentiation factor (i.e., a CD protein). The antigen to which the cysteine engineered antibody is capable of binding may be a member of a subset of one of the domains described above, wherein other subsets of the domains comprise other molecules/antigens having different characteristics (with respect to the antigen of interest).

Site-specific antibody conjugates used in the methods of the present disclosure include immunoconjugates useful in the treatment of cancer, including, but not limited to, antibodies directed against cell surface receptors and Tumor Associated Antigens (TAAs). 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. In an attempt to find effective cellular targets for cancer diagnosis and treatment, researchers have sought to identify transmembrane or additional tumor-associated polypeptides that are specifically expressed on the surface of one or more specific types of cancer cells as compared to one or more normal non-cancerous cells. Typically, such tumor-associated polypeptides are more abundantly expressed on the surface of cancer cells than on the surface of non-cancerous cells. The identification of such tumor-associated cell surface antigen polypeptides has led to the ability to specifically target cancer cells for destruction via antibody-based therapies.

Examples of tumor associated antigens TAA include, but are not limited to, TAA (1) - (53) listed below. Information on these antigens, well known in the art, is listed below and is defined by the nucleic acid and protein sequence identification provisions of the National Center for Biotechnology Information (NCBI), including names, alternative names, Genbank accession numbers and the main references. Nucleic acid and protein sequences corresponding to TAAs (1) - (53) are available in public databases, such as GenBank. Tumor associated antigens targeted by antibodies include all amino acid sequence variants and isoforms that have at least about 70%, 80%, 85%, 90% or 95% sequence identity relative to the sequences identified in the cited references, or that exhibit substantially the same biological properties or characteristics as TAAs having the sequences found in the cited references. For example, TAAs having variant sequences are generally capable of specifically binding to antibodies to which the TAAs listed for the corresponding sequences specifically bind. The sequences and disclosures in the references specifically cited herein are expressly incorporated by reference.

Tumor associated antigens

(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbank accession No. NM _ 001203); ten Dijke, P., et al Science 264(5155): 101-; WO2004063362 (claim 2); WO2003042661 (claim 12); US2003134790-A1 (pages 38-39); WO2002102235 (claim 13; page 296); WO2003055443 (pages 91-92); WO200299122 (example 2; page 528 and 530); WO2003029421 (claim 6); WO2003024392 (claim 2; FIG. 112); WO200298358 (claim 1; page 183); WO200254940 (page 100-101); WO200259377 (page 349 and 350); WO200230268 (claim 27; page 376); WO200148204 (examples; FIG. 4); NP _001194 bone morphogenetic protein receptor, type IB/pid ═ NP _ 001194.1; cross-referencing: 603248 parts of MIM; NP-001194.1; AY 065994;

(2) e16(LAT1, SLC7A5, Genbank accession NM-003486); biochem Biophys Res Commun.255(2), 283-; WO2004048938 (example 2); WO2004032842 (example IV); WO2003042661 (claim 12); WO2003016475 (claim 1); WO200278524 (example 2); WO200299074 (claim 19; page 127 and 129); WO200286443 (claim 27; page 222, 393); WO2003003906 (claim 10; page 293); WO200264798 (claim 33; pages 93-95); WO200014228 (claim 5; page 133 and 136); US2003224454 (fig. 3); WO2003025138 (claim 12; page 150); NP _003477 solute carrier family 7 (cationic amino acid transporter, y + system), member 5/pid — NP _003477.3-Homo sapiens; cross-referencing: 600182 parts of MIM; NP-003477.3; NM-015923; NM _003486_ 1;

(3) STEAP1 (six transmembrane epithelial antigen of prostate, Genbank accession No. NM _ 012449); cancer Res.61(15), 5857-; WO2004065577 (claim 6); WO2004027049 (fig. 1L); EP1394274 (example 11); WO2004016225 (claim 2); WO2003042661 (claim 12); US2003157089 (example 5); US2003185830 (example 5); US2003064397 (fig. 2); WO200289747 (example 5; page 618 and 619); WO2003022995 (example 9; FIG. 13A, example 53; page 173, example 2; FIG. 2A); NP _036581 six transmembrane epithelial antigen of the prostate. Cross-referencing: 604415 parts of MIM; NP-036581.1; NM _012449_ 1;

(4)0772P (CA125, MUC16, Genbank accession AF 361486); j.biol.chem.276(29):27371-27375 (2001); WO2004045553 (claim 14); WO200292836 (claim 6; FIG. 12); WO200283866 (claim 15; page 116-121); US2003124140 (example 16); US 798959; cross-referencing: 34501467 parts of GI; AAK 74120.3; AF361486_ 1;

(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiator, mesothelin, Genbank accession No. NM-005823); yamaguchi, N.A., et al biol. chem.269(2),805-808(1994), Proc Natl. Acad. Sci.U.S.A.96(20):11531-11536(1999), Proc Natl. Acad. Sci.U.S.A.93(1):136-140(1996), J.biol. chem.270(37):21984-21990 (1995); WO2003101283 (claim 14); (WO2002102235 (claim 13; pp 287-288); WO2002101075 (claim 4; pp 308-309); WO200271928 (pp 320-321); WO9410312 (pp 52-57); cross references MIM: 601051; NP-005814.2; NM-005823-1;

(6) napi3B (Napi-3B, NPTIIb, SLC34a2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3B, Genbank accession No. NM — 006424); j.biol.chem.277(22): 19665-; WO2004022778 (claim 2); EP1394274 (example 11); WO2002102235 (claim 13; page 326); EP875569 (claim 1; pages 17 to 19); WO200157188 (claim 20; page 329); WO2004032842 (example IV); WO200175177 (claim 24; page 139-140); cross-referencing: 604217 parts of MIM; NP-006415.1; NM _006424_ 1;

(7) sema5B (FLJ10372, KIAA1445, mm.42015, Sema5B, SEMAG, semaphorin 5B Hlog, Sema domain, seven thrombospondin repeats (type 1 and type 1 patterns), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession No. AB 040878); nagase T, et al (2000) DNA Res.7(2): 143-; WO2004000997 (claim 1); WO2003003984 (claim 1); WO200206339 (claim 1; page 50); WO200188133 (claim 1; pages 41-43, 48-58); WO2003054152 (claim 20); WO2003101400 (claim 11); accession number: Q9P 283; EMBL; AB 040878; baa95969.1. genew; 10737 parts of HGNC;

(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA2700050C12, rikencd 2700050C12 gene, Genbank accession No. AY 358628); ross et al (2002) Cancer Res.62: 2546-; US2003129192 (claim 2); US2004044180 (claim 12); US2004044179 (claim 11); US2003096961 (claim 11); US2003232056 (example 5); WO2003105758 (claim 12); US2003206918 (example 5); EP1347046 (claim 1); WO2003025148 (claim 20); cross-referencing: 37182378 parts of GI; AAQ 88991.1; AY358628_ 1;

(9) ETBR (endothelin type B receptor, Genbank accession No. AY 275463); nakamuta m., et al biochem.biophysis.res.commun.177, 34-39,1991; ogawa y, et al biochem. biophysis. 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., yanagisawam, et al biochem. biophysis. res. commu.178, 656-663,1991; elshourbagy n.a., et al, aj.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; straussberg r.l., et al proc.natl.acad.sci.u.s.a.99,16899-16903,2002; bourgeois c, et al, alin, endocrinol, meta, 82,3116-3123,1997; okamoto y, et al biol. chem.272,21589-21596,1997; verheij j j.b., et al am.j.med.genet.108,223-225,2002; hofstrar.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., equivalent hum. mol. Genet.5: 351-; amiel J., et al hum.mol.Genet.5,355-357,1996; hofstra r.m.w., et al nat. genet.12,445-447,1996; svensson p.j., et alhum.genet.103,145-148,1998; fuchs s, et al mol. med.7,115-124,2001; pingoult V, et al (2002) hum. Genet.111, 198-206; WO2004045516 (claim 1); WO2004048938 (example 2); WO2004040000 (claim 151); WO2003087768 (claim 1); WO2003016475 (claim 1); WO2003016475 (claim 1); WO200261087 (fig. 1); WO2003016494 (fig. 6); WO2003025138 (claim 12; page 144); WO200198351 (claim 1; page 124-125); EP522868 (claim 8; FIG. 2); WO200177172 (claim 1; page 297-299); US 2003109676; US6518404 (fig. 3); US5773223 (claim 1 a; Col 31-34); WO 2004001004;

(10) MSG783(RNF124, hypothetical protein FLJ20315, Genbank accession No. NM — 017763); WO2003104275 (claim 1); WO2004046342 (example 2); WO2003042661 (claim 12); WO2003083074 (claim 14; page 61); WO2003018621 (claim 1); WO2003024392 (claim 2; FIG. 93); WO200166689 (example 6); cross-referencing: LocusID 54894; NP-060233.2; NM _017763_ 1;

(11) STEAP2(HGNC _8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-associated gene 1, prostate cancer-associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein, Genbank accession No. AF 455138); lab.invest.82(11):1573-1582 (2002); WO 2003087306; US2003064397 (claim 1; FIG. 1); WO200272596 (claim 13; pages 54 to 55); WO200172962 (claim 1; FIG. 4B); WO2003104270 (claim 11); WO2003104270 (claim 16); US2004005598 (claim 22); WO2003042661 (claim 12); US2003060612 (claim 12; fig. 10); WO200226822 (claim 23; FIG. 2); WO200216429 (claim 12; figure 10); cross-referencing: 22655488 parts of GI; AAN 04080.1; AF455138_ 1;

(12) TrpM4(BR22450, FLJ20041, TrpM4, TrpM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession No. NM _ 017636); xu, X.Z., et al Proc. Natl. Acad. Sci. U.S.A.98(19):10692-10697(2001), Cell 109(3):397-407(2002), J.biol. chem.278(33):30813-30820 (2003); US2003143557 (claim 4); WO200040614 (claim 14; page 100-103); WO200210382 (claim 1; FIG. 9A); WO2003042661 (claim 12); WO200230268 (claim 27; page 391); US2003219806 (claim 4); WO200162794 (claim 14; FIGS. 1A-D); cross-referencing: 606936 parts of MIM; NP-060106.2; NM _017636_ 1;

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratoma-derived growth factor, Genbank accession No. NP _003203 or NM _ 003212); ciccodicola, A., et al EMBO J.8(7): 1987-; US2003224411 (claim 1); WO2003083041 (example 1); WO2003034984 (claim 12); WO200288170 (claim 2; pages 52-53); WO2003024392 (claim 2; FIG. 58); WO200216413 (claim 1; pages 94-95, 105); WO200222808 (claim 2; FIG. 1); US5,854,399 (example 2; columns 17-18); US5,792,616 (fig. 2); cross-referencing: 187395 parts of MIM; NP-003203.1; NM _003212_ 1;

(14) CD21(CR2 (complement receptor 2) or C3DR (C3 d/Epstein-Barr virus receptor) or Hs.73792Genbank accession number M26004); fujisaku et al (1989) J.biol.chem.264(4): 2118-2125); weis j.j., et j.exp.med.167,1047-1066,1988; moore m., et al proc.natl.acad.sci.u.s.a.84,9194-9198,1987; barel M., et al mol. Immunol.35,1025-1031,1998; weis j.j., et al proc.natl.acad.sci.u.s.a.83,5639-5643,1986; sinha S.K., et al (1993) J.Immunol.150, 5311-5320; WO2004045520 (example 4); US2004005538 (example 1); WO2003062401 (claim 9); WO2004045520 (example 4); WO9102536 (fig. 9.1-9.9); WO2004020595 (claim 1); accession number: p20023; q13866; q14212; EMBL; m26004; AAA 35786.1;

(15) CD79 56B (CD79B, CD79 β (immunoglobulin related β), B29, Genbank accession No. NM-000626 or 11038674), Proc. Natl. Acad. Sci. U.S.A. (2003)100(7), 4126-4131, Blood (2002)100(9), 3068-3076, Muller et al (1992) Eur. J.Immunol.22(6): 1621-1625; WO2004016225 (claim 2, FIG. 140), WO 20030874, US 2001874 (claim 1, page 102), WO 2003062402401 (claim 9), WO200278524 (example 2), US2002150573 (claim 5, page 15), US 4405633, WO 20030402 (claim 1, page 306 and 309), WO 20027854, WO 8295 (claim 13, WO 768,483; WO 2002158235; WO 41031; WO 11435; WO 4111-NP; WO 4111; WO 11411; WO 41024; WO 4131; WO 4135; WO 4131; No. 11; MIM-11435;

(16) FcRH2(IFGP4, IRTA4, spa 1A (SH 2 domain containing phosphatase dockerin 1a), spa 1B, spa 1C, Genbank accession No. NM _030764, AY 358130); genome Res.13(10): 2265-; WO2004016225 (claim 2); WO 2003077836; WO200138490 (claim 5; FIGS. 18D-1-18D-2); WO2003097803 (claim 12); WO2003089624 (claim 25); cross-referencing: 606509 parts of MIM; NP-110391.2; NM _030764_ 1;

(17) HER2(ErbB2, Genbank accession number M11730); coissens L., et al Science (1985)230(4730): 1132-1139; yamamoto T, et al Nature 319, 230-; semba k, et al alproc.natl.acad.sci.u.s.a.82,6497-6501,1985; swiercz J.M., et al J.CellBiol.165,869-880,2004; kuhns J.J., et al J.biol.chem.274,36422-36427,1999; ChoH. -S., et al Nature 421,756-760, 2003; ehsani A., et al (1993) Genomics 15, 426-429; WO2004048938 (example 2); WO2004027049 (fig. 1I); WO 2004009622; WO 2003081210; WO2003089904 (claim 9); WO2003016475 (claim 1); US 2003118592; WO2003008537 (claim 1); WO2003055439 (claim 29; FIGS. 1A-B); WO2003025228 (claim 37; FIG. 5C); WO200222636 (example 13; pages 95 to 107); WO200212341 (claim 68; FIG. 7); WO200213847 (pages 71-74); WO200214503 (page 114-; WO200153463 (claim 2; pages 41-46); WO200141787 (page 15); WO200044899 (claim 52; FIG. 7); WO200020579 (claim 3; FIG. 2); US5,869,445 (claim 3; columns 31-38); WO9630514 (claim 2; pages 56 to 61); EP1439393 (claim 7); WO2004043361 (claim 7); WO 2004022709; WO200100244 (example 3; FIG. 4); accession number: p04626; EMBL; m11767; AAA 35808.1. EMBL; m11761; AAA 35808.1;

(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 R.L., et al Proc.Natl.Acad.Sci.U.S.A.99: 16899-169903, 2002; WO 2004063709; EP1439393 (claim 7); WO2004044178 (example 4); WO 2004031238; WO2003042661 (claim 12); WO200278524 (example 2); WO200286443 (claim 27; page 427); WO200260317 (claim 2); accession number: p40199; q14920; EMBL; m29541; AAA 59915.1. EMBL; m18728;

(19) MDP (DPEP1, Genbank accession number BC 017023); Proc.Natl.Acad.Sci.U.S.A.99(26): 16899-169903 (2002); WO2003016475 (claim 1); WO200264798 (claim 33; pages 85-87); JP05003790 (fig. 6-8); WO9946284 (fig. 9); cross-referencing: 179780 parts of MIM; AAH 17023.1; BC017023_ 1;

(20) IL20R α (IL20Ra, ZCYTOR7, Genbank accession No. AF184971), Clark H.F., et al genome Res.13,2265-2270,2003, Mungall A.J., et al Nature 425,805-811,2003, Blumberg H., et al Cell 104,9-19,2001, Dumoutier L, et al J.Immunol.167, 19,2001-3549,2001, 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, 2006-3613974 (example 11), US 2005320 (example 5), WO 20030262, WO 2004178-3759, WO 20014659-3759; WO 2001462-3759; WO2001462,9259; WO 2004159; WO 2003759-3759; WO 2003759; WO2,27159; WO 2003759; WO2,2719;

(21) brevican (BCAN, BEHAB, Genbank accession No. AF 229053); gary S.C., et al, gene 256,139-147, 2000; clark H.F., et al Genome Res.13,2265-2270,2003; straussberg r.l., et al proc.natl.acad.sci.u.s.a.99,16899-16903,2002; US2003186372 (claim 11); US2003186373 (claim 11); US2003119131 (claim 1; FIG. 52); US2003119122 (claim 1; FIG. 52); US2003119126 (claim 1); US2003119121 (claim 1; FIG. 52); US2003119129 (claim 1); US2003119130 (claim 1); US2003119128 (claim 1; FIG. 52); US2003119125 (claim 1); WO2003016475 (claim 1); WO200202634 (claim 1);

(22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession No. NM-004442); chan, J.and Watt, V.M., Oncogene 6(6), 1057-; WO2003042661 (claim 12); WO200053216 (claim 1; page 41); WO2004065576 (claim 1); WO2004020583 (claim 9); WO2003004529 (page 128-132); WO200053216 (claim 1; page 42); cross-referencing: 600997 parts of MIM; NP-004433.2; NM _004442_ 1;

(23) ASLG659(B7h, Genbank accession number AX 092328); US20040101899 (claim 2); WO2003104399 (claim 11); WO2004000221 (fig. 3); US2003165504 (claim 1); US2003124140 (example 2); US2003065143 (fig. 60); WO2002102235 (claim 13; page 299); US2003091580 (example 2); WO200210187 (claim 6; FIG. 10); WO200194641 (claim 12; FIG. 7 b); WO200202624 (claim 13; FIGS. 1A-1B); US2002034749 (claim 54; pages 45 to 46); WO200206317 (example 2; page 320-321, claim 34; page 321-322); WO200271928 (page 468 and 469); WO200202587 (example 1; FIG. 1); WO200140269 (example 3; page 190-192); WO200036107 (example 2; page 205-207); WO2004053079 (claim 12); WO2003004989 (claim 1); WO200271928 (pages 233-; WO 0116318;

(24) PSCA (prostate stem cell antigen precursor, Genbank accession No. AJ 297436); reiter r.e., et al proc.natl.acad.sci.u.s.a.95,1735-1740,1998; gu Z, et al Oncogene 19,1288-1296, 2000; biochem, biophysis, res, commun, (2000)275(3) 783-788; WO 2004022709; EP1394274 (example 11); US2004018553 (claim 17); WO2003008537 (claim 1); WO200281646 (claim 1; page 164); WO2003003906 (claim 10; page 288); WO200140309 (example 1; FIG. 17); US2001055751 (example 1; FIG. 1 b); WO200032752 (claim 18; FIG. 1); WO9851805 (claim 17; page 97); WO9851824 (claim 10; page 94); WO9840403 (claim 2; FIG. 1B); accession number: o43653; EMBL; AF 043498; AAC 39607.1;

(25) GEDA (Genbank accession number AY 260763); AAP14954 lipoma HMGIC fusion partner-like protein/pid AAP14954.1-Homo sapien species: homo sapiens (human); WO2003054152 (claim 20); WO2003000842 (claim 1); WO2003023013 (example 3, claim 20); US2003194704 (claim 45); cross-referencing: 30102449 parts of GI; AAP 14954.1; AY260763_ 1;

(26) BAFF-R (B cell activating factor receptor, BLyS receptor 3, BR3, Genbank accession AF 116456); BAFF receptor/pid NP _443177.1-Homo sapiens; thompson, J.S., et al Science 293(5537),2108-2111 (2001); WO 2004058309; WO 2004011611; WO2003045422 (examples; pages 32-33); WO2003014294 (claim 35; FIG. 6B); WO2003035846 (claim 70; page 615 and 616); WO200294852 (column 136 and 137); WO200238766 (claim 3; page 133); WO200224909 (example 3; FIG. 3); cross-referencing: 606269 parts of MIM; NP-443177.1; NM _052945_ 1; AF 132600;

(27) CD22(B cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814, Genbank accession No. AK 026467); wilson et al (1991) J.Exp.Med.173: 137-146; WO2003072036 (claim 1; FIG. 1); cross-referencing: 107266 parts of MIM; NP-001762.1; NM _001771_ 1;

(28) CD79a (CD79A, CD79 α, immunoglobulin-related α), B cell-specific protein which interacts covalently with Ig β (CD79B) and forms complexes with IgM molecules on the surface, transducing signals involved in B cell differentiation, pI: 4.84; MW: 25028; TM: 2[ P ] gene chromosome: 19q13.2, Genbank accession No. NP 001774.10; WO2003088808, US 20030228319; WO 2003062402401 (claim 9); US2002150573 (claim 4, pages 13-14); WO9958658 (claim 13, FIG. 16); WO 07574 (FIG. 1); US5,644,033; Ha (1992) J.Immunol.148(5 1526-1531; Mueller al (1992) Eur. J.Biom.22: 1621-5; Mumoto et 3478; Munich J.1621: 1621; Munich J.35; Munich No. 35; 19835; EMB J.35; 19835; Experimental J.7; Experimental 3662; Experimental No. 35; Experimental J.35; Experimental 3662; Experimental J.);

(29) CXCR5 (burkitt's lymphoma receptor 1, a G protein-coupled receptor activated by CXCL13 chemokines, functioning in lymphocyte migration and humoral defense, functioning in HIV-2 infection and possibly AIDS, lymphoma, myeloma, and leukemia development); 372 aa; pI: 8.54 of; MW: 41959; TM: 7[ P ] Gene chromosome: 11q23.3, Genbank accession No. NP _ 001707.1); WO 2004040000; WO 2004015426; US2003105292 (example 2); US6,555,339 (example 2); WO200261087 (fig. 1); WO200157188 (claim 20, page 269); WO200172830 (pages 12-13); WO200022129 (example 1, page 152-153, example 2, page 254-256); WO9928468 (claim 1, page 38); US5440021 (example 2, columns 49-52); WO9428931 (pages 56 to 58); WO9217497 (claim 7, fig. 5); dobner et al (1992) Eur.J.Immunol.22: 2795-; barella et al (1995) biochem.J.309: 773-779;

(30) HLA-DOB (the β subunit of MHC class II molecules (Ia antigen) that bind peptides and present them to CD4+ T lymphocytes; 273 aa; pI: 6.56; MW: 30820; TM: 1[ P ] gene chromosome: 6P21.3, Genbank accession NP-002111.1; Tonnelle et al (1985) EMBO J.4(11): 2839. multidot. 2847; Jonsson et al (1989) Immunogenetics 29(6): 411. multidot. 413; Beck et al (1992) J.mol.228: 433. multidot. 441; Strausberg et al (2002) Proc. Natl.Acad.Sci USA 99: 16899. multidot. 169903; Chenius et al (1987) J.biol.chem.262: 8759; Beaus. multidot. 8766. J.2002. multidot. 134: 141255. multidot. 20. multidot. 19835; WO 11. multidot. 20. multidot. 28; 3635. multidot. 20. multidot. 3635; Cheng. multidot. 28; Cheng. 28; 19835. multidot. 20; Chen. 28; Chen. multidot.

(31) P2X5 (purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, and defects may contribute to pathophysiological conditions of idiopathic detrusor instability); 422 aa; pI: 7.63; MW: 47206; TM: 1[ P ] Gene chromosome: 17p13.3, Genbank accession No. NP _ 002552.2; le et al (1997) FEBS Lett.418(1-2): 195-199; WO 2004047749; WO2003072035 (claim 10); touchman et al (2000) Genome Res.10: 165-173; WO200222660 (claim 20); WO2003093444 (claim 1); WO2003087768 (claim 1); WO2003029277 (page 82);

(32) CD72(B cell differentiation antigen CD72, Lyb-2), pI: 8.66 of; MW: 40225; TM: 1[ P ] Gene chromosome: 9p13.3, Genbank accession No. NP _ 001773.1; WO2004042346 (claim 65); WO2003026493 (pages 51-52, 57-58); WO200075655 (page 105-106); von Hoegen et al (1990) J.Immunol.144(12): 4870-4877; strausberg et al (2002) Proc.Natl.Acad.Sci USA 99: 16899-;

(33) LY64 (lymphocyte antigen 64(RP105), type I membrane protein of the Leucine Rich Repeat (LRR) family, regulates B cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosus); 661 aa; pI: 6.20; MW: 74147, respectively; TM: 1[ P ] Gene chromosome: 5q12, Genbank accession number NP-005573.1; US 2002193567; WO9707198 (claim 11, pages 39-42); miura et al (1996) Genomics 38(3) 299-304; miura et al (1998) Blood 92: 2815-2822; WO 2003083047; WO9744452 (claim 8, pages 57-61); WO200012130 (pages 24to 26);

(34) FcRH1(Fc receptor-like protein 1, a receptor for putative immunoglobulin Fc domains containing Ig-like and ITAM domains of C2 type, likely to have a role in B lymphocyte differentiation); 429 aa; pI: 5.28; MW: 46925, respectively; TM: 1[ P ] Gene chromosome: 1q21-1q22, Genbank accession No. NP _ 443170.1; WO 2003077836; WO200138490 (claim 6, fig. 18E-1-18-E-2); davis et al (2001) Proc. Natl. Acad. Sci USA 98(17) 9772-9777; WO2003089624 (claim 8); EP1347046 (claim 1); WO2003089624 (claim 7);

(35) IRTA2 (immunoglobulin superfamily receptor translocation related 2, putative immunoreceptors that may have a role in B cell development and lymphomata; dysregulation of genes by translocation occurs in some B cell malignancies); 977 aa; pI: 6.88; MW: 106468, respectively; TM: 1[ P ] Gene chromosome: 1q21, Genbank accession No.: AF343662, AF343663, AF343664, AF343665, AF369794, AF 39453, AK090423, AK090475, AL834187, AY 358085; mice: AK089756, AY158090, AY 506558; NP-112571; WO2003024392 (claim 2, fig. 97); nakayama et al (2000) biochem. Biophys. Res. Commun.277(1): 124-127; WO 2003077836; WO200138490 (claim 3, fig. 18B-1-18B-2);

(36) TENB2(TMEFF2, tomorgulin, TPEF, HPP1, TR, putative transmembrane proteoglycans, growth factors involved in the EGF/heregulin family and follistatin); 374 aa; NCBI accession number: AAD55776, AAF91397, AAG49451, NCBI RefSeq: NP-057276; NCBI Gene: 23671, respectively; OMIM: 605734, respectively; SwissProt Q9UIK 5; genbank accession No. AF 179274; AY358907, CAF85723, CQ 782436; WO 2004074320; JP 2004113151; WO 2003042661; WO 2003009814; EP1295944 (pages 69-70); WO200230268 (page 329); WO 200190304; US 2004249130; US 2004022727; WO 2004063355; US 2004197325; US 2003232350; US 2004005563; US 2003124579; horie et al (2000) Genomics 67: 146-; uchida et al (1999) biochem. Biophys. Res. Commun.266: 593-602; liang et al (2000) Cancer Res.60: 4907-12; Glynne-Jones et al (2001) Int J cancer. Oct 15; 94(2) 178-84;

(37) PMEL17(silver homolog; SILV; D12S 53E; PMEL 17; SI; SIL); ME 20; gp100) BC 001414; BT 007202; m32295; m77348; NM-006928; McGlinchey, R.P.et al (2009) Proc.Natl.Acad.Sci.USA.106(33), 13731-; kummer, M.P.et al (2009) J.biol.chem.284(4), 2296-;

(38) TMEFF1 (transmembrane protein 1 with EGF-like and two follistatin-like domains; Tomoregulin-1); h7365; c9orf 2; c9ORF 2; u19878; x83961; NM-080655; NM-003692; harms, P.W, (2003) Genes Dev.17(21), 2624-2629; gery, S.et al (2003) Oncogene 22(18), 2723-2727;

(39) GDNF-Ra1(GDNF family receptor α 1; GFRA 1; GDNFR; GDNFRA; RETL 1; TRNR 1; RET 1L; GDNFR- α 1; GFR-ALPHA-1), U95847; BC 014962; NM-145793 NM-005264; Kim, M.H.et al (2009) mol.cell.biol.29(8), 2264. sup. 2277; Treano, J.J.et al (1996) Nature 382(6586): 80-83);

(40) ly6E (lymphocyte antigen 6 complex, locus E; Ly67, RIG-E, SCA-2, TSA-1); NP-002337.1; NM-002346.2; de Nooij-van Dalen, A.G.et al (2003) int.J.cancer 103(6), 768-; zammit, D.J.et al (2002) mol.cell.biol.22(3): 946-952;

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

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

(43) LGR5 (leucine rich repeat-containing G protein-coupled receptor 5; GPR49, GPR 67); 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;

(44) RET (RET proto-oncogene; MEN 2A; HSCR 1; MEN 2B; MTC 1; PTC; CDHF 12; Hs.168114; RET 51; RET-ELE 1); NP-066124.1; NM-020975.4; tsukamoto, H.et al (2009) Cancer Sci.100(10): 1895-; narita, N.et al (2009) Oncogene 28(34), 3058-3068;

(45) LY6K (lymphocyte antigen 6 complex, locus K; LY 6K; HSJ 001348; FLJ 35226); NP-059997.3; NM-017527.3; ishikawa, N.et al (2007) Cancer Res.67(24): 11601-; deNooij-van Dalen, A.G.et al (2003) int.J.cancer 103(6) 768-774;

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

(47) GPR54(KISS1 receptor; KISS 1R; GPR 54; HOT7T 175; AXOR 12); NP-115940.2; NM-032551.4; nanvenot, J.M.et al (2009) mol.Pharmacol.75(6): 1300-; hata, K.et al (2009) Anticancer Res.29(2): 617-623;

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

(49) tyrosinase (TYR; OCAIA; OCA 1A; tyrosinase; SHEP 3); NP-000363.1; NM-000372.4; bishop, D.T.et al (2009) nat. Genet.41(8): 920-; nan, H.et al (2009) int.J.cancer125(4): 909-917;

(50) TMEM118 (Ring finger protein, transmembrane 2; RNFT 2; FLJ 14627); NP-001103373.1; NM-001109903.1; clark, H.F.et al (2003) Genome Res.13(10): 2265-; scherer, S.E.et al (2006) Nature 440(7082) 346-;

(51) GPR172A (G protein-coupled receptor 172A; GPCR 41; FLJ 11856; D15Ertd747 e); NP-078807.1; NM-024531.3; ericsson, T.A.et al (2003) Proc.Natl.Acad.Sci.U.S.A.100(11): 6759-6764; takeda, S.et al (2002) FEBS Lett.520(1-3): 97-101;

(52) CD33, sialic acid binding, a member of the immunoglobulin-like lectin family, is a 67kDa glycosylated transmembrane protein. In addition to committed myeloid monocytes and erythroid progenitors, CD33 is expressed on most myeloid and monocytic leukemia cells. It was not seen on 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 on its cytoplasmic tail, each of which is followed by a hydrophobic residue, similar to the tyrosine-based inhibitory motif (ITIM) of the immunoreceptor seen in many inhibitory receptors.

(53) CLL-1(CLEC12A, MICL, and DCAL2), encodes a member of the C-type lectin/C-type lectin-like domain (CTL/CTLD) superfamily. Members of this family share a common protein fold and have diverse functions, such as cell adhesion, cell-cell signaling, glycoprotein turnover, and roles in inflammation and immune response. The proteins encoded by such genes are negative regulators of granulocyte and monocyte function. Several alternatively spliced transcript variants of this gene have been described, but the full-length nature of some of these variants has not been determined. This gene is tightly linked to other CTL/CTLD superfamily members in 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): 92. 147802). CLL-1 has been shown to be a type II transmembrane receptor comprising a single C-type lectin-like domain (predicted to not bind calcium or sugars), a stem region, and a transmembrane domain and a short cytoplasmic tail containing an ITIM motif.

Antibody derivatives

The antibodies provided herein can be further modified to contain additional non-proteinaceous moieties known in the art and readily available. Suitable moieties for derivatization of the antibody include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyaminoacids (homopolymers or random copolymers), and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in production due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative is to be used in a therapy under specified conditions, and the like.

Conjugates of the antibody and the nonproteinaceous moiety can be formed by exposure to radiation and selective heating. The non-proteinaceous moiety of such conjugates may be carbon nanotubes (Kam et al (2005) Proc. Natl. Acad. Sci. USA 102: 11600-. The radiation can be of any wavelength and includes, but is not limited to, wavelengths that are not damaging to normal cells, but heat the non-proteinaceous moiety to a temperature at which cells in the vicinity of the antibody-non-proteinaceous moiety are killed.

As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain which is either mutated in sequence and/or forms structurally defined loops ("hypervariable loops"). Generally, a native four-chain antibody comprises six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and/or from "complementarity determining regions" (CDRs) that have the highest sequence variability and/or are involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32(L1), 50-52(L2), 91-96(L3), 26-32(H1), 53-55(H2), and 96-101(H3) (Chothia and Lesk (1987) J.mol.biol.196: 901-917). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3 (Kabat numbering). In addition to CDR1 in VH, the CDRs generally comprise amino acid residues that form hypervariable loops. CDRs also contain "specificity determining residues" or "SDRs," which are residues that contact the antigen. SDR is contained in the CDR regions called shortened CDRs or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3 (Almagro and Fransson (2008) front. biosci.13: 1619-. Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domains are numbered herein according to Kabat et al, supra.

An "isolated" antibody refers to an antibody that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. For a review of methods for assessing antibody purity, see, e.g., Flatman et al (2007) J.Chromatogr.B 848: 79-87.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except, for example, for possible variant antibodies containing naturally occurring mutations or occurring during the production of a monoclonal antibody preparation, such variants are typically present in very small amounts. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody within a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be generated by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods that utilize transgenic animals containing all or part of a human immunoglobulin locus.

"naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel or fluorophore. The naked antibody may be present in a pharmaceutical formulation.

"Natural antibody" refers to a naturally occurring immunoglobulin molecule having a different structure. For example, a native IgG antibody is an heterotetrameric glycan protein of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains that are disulfide-bonded. From N to C-terminus, each heavy chain has one variable region (VH), also called variable or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH 3). Similarly, from N-to C-terminus, each light chain has a variable region (VL), also known as the variable light domain or light chain variable domain, followed by a Constant Light (CL) domain. Antibody light chains can be classified into one of two types, called kappa (κ) and lambda (λ), based on their constant domain amino acid sequences.

Multispecific antibodies

The antibodies provided herein can be multispecific antibodies, e.g., bispecific antibodies. As used herein, the term "multispecific antibody" refers to an antibody comprising an antigen binding domain with polyepitopic specificity (i.e., capable of binding to two or more different epitopes on one molecule or capable of binding to epitopes on two or more different molecules).

In exemplary embodiments, the multispecific antibody is a monoclonal antibody (such as a bispecific antibody) having binding specificities for at least two different antigen binding sites. The first antigen-binding domain and the second antigen-binding domain of the multispecific antibody may bind to two epitopes within one and the same molecule (intramolecular binding). For example, the first antigen-binding domain and the second antigen-binding domain of a multispecific antibody may bind to two different epitopes on the same molecule. In certain embodiments, the two different epitopes bound by the multispecific antibody are epitopes that are not normally bound at the same time by one monospecific antibody, such as, for example, a conventional antibody or an immunoglobulin single variable domain. The first antigen-binding domain and the second antigen-binding domain of a multispecific antibody may bind epitopes located within two distinct molecules (intermolecular binding). For example, a first antigen-binding domain of a multispecific antibody may bind to one epitope on one molecule, while a second antigen-binding domain of the multispecific antibody may bind to another epitope on a different molecule, thereby crosslinking the two molecules.

The antigen-binding domain of a multispecific antibody (such as a bispecific antibody) may comprise two VH/VL units, wherein a first VH/VL unit binds a first epitope and a second VH/VL unit binds a second epitope, wherein 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 (such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies, and triabodies, antibody fragments that have been covalently or non-covalently linked). A VH/VL unit further comprising at least part of the heavy chain variable region and/or at least part of the light chain variable region may also be referred to as an "arm" or "half antibody". The moiety may comprise a portion of the heavy chain variable region sufficient to permit intramolecular disulfide bond formation with the second moiety. In some embodiments, a moiety comprises a hole mutation or knob mutation, e.g., to allow heterodimerization with a second moiety or half-antibody comprising a complementary hole mutation or knob mutation. Section mutations and hole mutations are discussed below.

The multispecific antibodies provided herein can be bispecific antibodies. As used herein, the term "bispecific antibody" refers to a multispecific antibody comprising an antigen-binding domain capable of binding to two different epitopes on one molecule or capable of binding to an epitope on two different molecules. Bispecific antibodies may also be referred to herein as having "dual specificity" or being "dual specific". Exemplary bispecific antibodies can bind to both the molecule and any other antigen. One of the binding specificities may be for HER2 and the other for CD 3. See, for example, U.S. Pat. No.5,821,337. Bispecific antibodies can bind to two different epitopes of the same molecule. Bispecific antibodies can bind to two different epitopes on two different molecules. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing cancer-associated antigens. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.

Techniques for generating multispecific antibodies include, but are not limited to, recombinant co-expression of two pairs of immunoglobulin heavy and light chains with different specificities (see Milstein and Cuello, Nature305:537 (1983), WO 93/08829, and Traunker et al, EMBO J.10:3655 (1991)), and "node-in-pocket" engineering (see, e.g., U.S. Pat. No.5,731,168, WO2009/089004, US2009/0182127, US2011/0287009, Marvin and Zhu, actarmacol.sin. (2005)26 (6): 649-658, and Kormann (2005) Acta pharmacol.sin., 26: 1-9). As used herein, the term "knob-to-hole" or "KnH" technology refers to a technology for pairing two polypeptides together in vitro or in vivo by introducing a protuberance (knob) into one polypeptide and a cavity (hole) into the other polypeptide at the interface where the two polypeptides interact. For example, KnH has been introduced into the Fc: Fc binding interface, CL: CH1 interface or VH/VL interface of an antibody (see, e.g., US2011/0287009, US2007/0178552, WO 96/027011, WO 98/050431, Zhu et al (1997) Protein Science 6: 781-788, and WO 2012/106587). In some embodiments, KnH drives pairing together of two different heavy chains during the manufacture of multispecific antibodies. For example, multispecific antibodies having KnH in their Fc regions may further comprise a single variable domain linked to each Fc region, or further comprise different heavy chain variable domains paired with similar or different light chain variable domains. The KnH technique can also be used to pair together two different receptor ectodomains or any other polypeptide sequences comprising different target recognition sequences, including for example affinity antibodies (affibodies), peptibodies (peptibodies) and other Fc fusions.

As used herein, the term "knob mutation" refers to a mutation that introduces a protuberance into a polypeptide at the interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a hole mutation.

As used herein, the term "hole mutation" refers to a mutation that introduces a cavity (hole) into a polypeptide at the interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a knob mutation.

"protuberance" means that at least one amino acid side chain projects from the interface of the first polypeptide and can therefore be placed in a compensatory cavity adjacent to the interface (i.e., the interface of the second polypeptide), thereby stabilizing the heteromultimer and thereby, for example, facilitating heteromultimer formation beyond homomultimer formation. The protuberance may be present in the initial 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 the protuberance. To achieve this, a nucleic acid encoding at least one "initial" amino acid residue in the interface of the first polypeptide is replaced with a nucleic acid encoding at least one "import" amino acid residue having a larger side chain volume than the initial amino acid residue. There may be more than one initial and corresponding input residue. The side chain volumes of the various amino acid residues are shown, for example, in table 1 of US 2011/0287009. Mutations that introduce "bulges" may be referred to as "node mutations".

The import residue for bulge formation can be a naturally occurring amino acid residue selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Exemplary import residues are tryptophan or tyrosine. The initial residue used to form the bulge may have a smaller side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine or valine.

"cavity" refers to a recess of at least one amino acid side chain from an interface of a second polypeptide and thus accommodates a corresponding protrusion on an adjacent interface of a first polypeptide. The cavity may be present in the initial 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, a nucleic acid encoding at least one "initial" amino acid residue in the interface of the second polypeptide is replaced with DNA encoding at least one "import" amino acid residue having a smaller side chain volume than the initial amino acid residue. It will be appreciated that there may be more than one initial and corresponding input residue. The import residue for cavity formation may be a naturally occurring amino acid residue selected from the group consisting of alanine (a), serine (S), threonine (T) and valine (V). The import residue may be serine, alanine or threonine. The initial residues used to form the cavity have a large side chain volume, such as tyrosine, arginine, phenylalanine or tryptophan. Mutations introduced into "cavities" may be referred to as "hole mutations".

The protuberance is "placeable" in the cavity, which means that the spatial location of the protuberance and the cavity at the interface of the first polypeptide and the second polypeptide, respectively, and the dimensions of the protuberance and the cavity are such that the protuberance can be positioned in the cavity without significantly disrupting the normal association of the first and second polypeptides at the interface. Because protuberances such as Tyr, Phe, and Trp do not generally extend perpendicular to the axis of the interface and have a preferred conformation, the arrangement of the protuberances with corresponding cavities may in some cases rely on modeling of the protuberance/cavity pair based on three-dimensional structures, such as obtained by X-ray crystallography or Nuclear Magnetic Resonance (NMR). This can be accomplished using techniques that are widely accepted in the art.

An exemplary knob mutation in the IgG1 constant region is T366W (EU numbering). Exemplary hole mutations in the IgG1 constant region may comprise one or more mutations selected from T366S, L368A, and Y407V (EU numbering). One exemplary hole mutation in the IgG1 constant region may include T366S, L368A, and Y407V (EU numbering).

An exemplary knob mutation in the IgG4 constant region is T366W (EU numbering). An exemplary hole mutation in the constant region of IgG4 may comprise one or more mutations selected from T366S, L368A, and Y407V (EU numbering). One exemplary hole mutation in the IgG4 constant region comprises T366S, L368A, and Y407V (EU numbering).

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Comparison for the purpose of determining percent amino acid sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or megalign (dnastar) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. However, for purposes of this disclosure,% amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, inc and the source code has been submitted to the US Copyright Office (US Copyright Office, Washington d.c.,20559) along with the user document, where it is registered with US Copyright registration number TXU 510087. ALIGN-2 programs are publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from source code. All sequence comparison parameters were set by the ALIGN-2 program and were not changed.

In the case of employing ALIGN-2 to compare amino acid sequences, the% amino acid sequence identity of a given amino acid sequence a relative to (to), with (with), or against (against) a given amino acid sequence B (or may be stated as having or comprising a given amino acid sequence a relative to, with, or against a certain% amino acid sequence identity of a given amino acid sequence B) is calculated as follows:

fractional X/Y times 100

Wherein X is the number of amino acid residues scored as identical matches in the A and B alignments of the sequence alignment program by the program ALIGN-2, and wherein Y is the total number of amino acid residues in B. It will be appreciated that if the length of amino acid sequence a is not equal to the length of amino acid sequence B, then the% amino acid sequence identity of a relative to B will not equal the% amino acid sequence identity of B relative to a. Unless otherwise specifically indicated, all% amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

The term "variable region" or "variable domain" refers to a domain in an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The heavy and light chain variable domains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising 4 conserved Framework Regions (FR) and 3 hypervariable regions (HVRs). See, e.g., Kindt et al, Kuby Immunology, 6 th edition, w.h.freeman and co, page 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, libraries of complementary VL or VH domains can be screened using VH or VL domains, respectively, from antigen-binding antibodies to isolate antibodies that bind a particular antigen (Portolano et al (1993) J.Immunol.150: 880-628; Clarkson et al (1991) Nature352: 624-628).

"tumor associated antigens" (TAAs) are known in the art, as provided in the exemplary TAA list provided above, and can be prepared for use in generating human or humanized antibodies using methods and information well known in the art. In an attempt to find effective cellular targets for cancer diagnosis and treatment, researchers have sought to identify transmembrane or additional tumor-associated polypeptides that are specifically expressed on the surface of one or more specific types of cancer cells as compared to one or more normal non-cancerous cells. Typically, such tumor-associated polypeptides are more abundantly expressed on the surface of cancer cells than on the surface of non-cancerous cells. The identification of such tumor-associated cell surface antigen polypeptides has led to the ability to specifically target cancer cells for destruction via antibody-based therapies. Examples of TAAs include, but are not limited to, those described in U.S. patent nos. 8,679,767 and 8,541,178, which are expressly incorporated herein.

Recombinant methods and compositions can be used to generate antibody building blocks for ADCs useful in the methods of the present disclosure, e.g., as described in US4,816,567. Isolated nucleic acids encoding such antibodies described herein are provided. Such nucleic acids may encode an amino acid sequence comprising a VL of an antibody and/or an amino acid sequence comprising a VH (e.g., a light and/or heavy chain of an antibody). One or more vectors (e.g., expression vectors) comprising such nucleic acids are also provided. Host cells comprising such nucleic acids are also provided. The host cell may comprise (e.g., have been transformed with): (1) a vector comprising nucleic acids encoding an amino acid sequence comprising a VL of an antibody and an amino acid sequence comprising a VH of an antibody, or (2) a first vector comprising nucleic acids encoding an amino acid sequence comprising a VL of an antibody and a second vector comprising nucleic acids encoding an amino acid sequence comprising a VH of an antibody. The host cell may be eukaryotic, such as a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell). As such, a method of producing an antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expression of the antibody, as provided above, and optionally, recovering the antibody from the host cell (or host cell culture broth).

For recombinant production of antibodies, nucleic acids encoding the antibodies can be isolated and inserted into one or more vectors for further cloning and/or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of an antibody).

Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., US5,648,237; US5,789,199; U.S. Pat. No.5,840,523 (also Charlton, methods Molecular Biology, Vol.248 (B.K.C.Lo eds., Humana Press, Totowa, N.J.,2003), page 245-. After expression, the antibody can be isolated from the bacterial cell mass paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized" resulting in the production of antibodies with partially or fully human glycosylation patterns (Gerngross (2004) nat. Biotech.22: 1409-.

Host cells suitable for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified which can be used with insect cells, particularly for transfecting Spodoptera frugiperda (Spodoptera frugiperda) cells.

Plant cells may also be usedCultures as hosts (U.S. Pat. No.5,959,177; U.S. Pat. No.6,040,498; U.S. Pat. No.6,420,548; U.S. Pat. No.7,125,978; U.S. Pat. No.6,417,429, describing PLANTIBODIES useful for the production of antibodies in transgenic plants^TMA technique).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney lines (293 or 293 cells as described, for example, in Graham et al (1977) J.Gen Virol.36: 59); baby hamster kidney cells (BHK); mouse Sertoli (sertoli) cells (TM4 cells, as described, for example, in Mather (1980) biol. reprod.23: 243-; monkey kidney cells (CV 1); VERO cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); bovine murine (buffalo rat) hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TR1 cells, as described, for example, in Mather et al (1982) AnnalsN.Y.Acad.Sci.383: 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR^-CHO cells (Urlaub et al (1980) Proc. Natl. Acad. Sci. USA77: 4216); and myeloma cell lines such as Y0, NS0 and Sp 2/0. A review of certain mammalian host cell lines suitable for antibody production is provided, for example, by Yazaki and Wu, Methods in Molecular Biology, Vol.248 (edited by B.K.C.Lo, Humana Press, Totowa, N.J.), p.255-268 (2003).

The antibody component of the ADC may be identified, screened, or characterized for its physical/chemical properties and/or biological activity by a variety of assays known in the art. Antibodies can be tested for antigen binding activity, for example, by known methods such as ELISA or Western blot. Competition assays can also be used to identify antibodies that compete with another known antibody for binding to an antigen. A competing antibody can bind to the same epitope (e.g., a linear or conformational epitope) as the known antibody binds to. A detailed exemplary method for locating epitopes bound by antibodies is described in Morris (1996) "Epitope mapping protocols", Methods in Molecular Biology Vol.66(Humana Press, Totowa, N.J.).

Exemplary antibodies that form site-specific ADCs can include, but are not limited to, trastuzumab (trastuzumab), ocrelizumab (ocrelizumab), pertuzumab (pertuzumab), anti-PD 1, anti-PD-L1, anti-neuropilin (neuropilin) -1, anti-MUC 16, rituximab (rituximab), anti-mesothelin, anti-LY 6E, anti-STEAP 1, anti-FcRH 5, anti-CD 22, anti-B7H 4, anti-LGR 5, anti-CD 79B, and anti-Napi 2B.

The drug moiety forming the drug member of the ADC may be covalently attached to the antibody via a linker unit to form an antibody-drug conjugate to achieve a targeted therapeutic effect. An exemplary embodiment of an ADC compound comprises an antibody (Ab) that targets, e.g., a tumor cell, a cytotoxic or cytostatic drug module (D), and a linker module (L) that attaches the Ab to D. Attaching the antibody to D via one or more amino acid residues (such as lysine and cysteine) through a linker moiety (L); the composition has the formula: ab- (L-D)_pWherein p is 1 to about 20, or about 2 to about 5. The number of drug moieties that can be conjugated to an antibody molecule via a reactive linker moiety can be limited by the number of cysteine residues, including free cysteine residues present in the antibody or that can be introduced by the methods described herein, or native cysteines that form interchain disulfide bonds of the antibody.

Exemplary drug moieties include, but are not limited to, peptides (including therapeutic peptides comprising one or more non-natural amino acids, such as cyclic peptides, β peptides, stabilizing peptides, and cysteine-knot peptides), polyamides, maytansinoids, doramesins, auristatins, calicheamicin, Pyrrolobenzodiazepines (PBD), PNU-159682, anthracyclines, duocarmycins, vinca alkaloids, taxanes, single-ended sporins, CC1065, duocarmycin, camptothecin, einitastatin, rifamycin or a rifamycin analog, a cytotoxic or cytostatic derivative thereof, and radioactive isotope derivatives thereof including radioactive isotope derivatives thereof.

Fc region variants

One or more amino acid modifications can be introduced into the Fc region of the antibodies forming site-specific ADCs provided herein, thereby generating Fc region variants. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

The present invention encompasses antibody variants possessing some, but not all, effector functions, which make them desirable candidates for applications where the in vivo half-life of the antibody is important, while certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and/or in vivo cytotoxicity assays may be performed to confirm the reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays may be performed to ensure that the antibody lacks fcyr binding (and therefore potentially lacks ADCC activity), but retains FcRn binding ability. The major cells mediating ADCC, NK cells, express Fc γ RIII only, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of ravatch and Kinet, Annu. Rev. Immunol.9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of molecules of interest are described in U.S. Pat. No.5,500,362 (see, e.g., Hellstrom, I.et al, Proc. nat' l Acad. Sci. USA 83: 7059-; 5,821,337 (see Bruggemann, M. et al, J.Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be employed (see, e.g., ACT for flow cytometry)I^TMNon-radioactive cytotoxicity assays (Celltechnology, Inc. mountain View, CA; and CytoTox)Non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively/additionally, the ADCC activity of a molecule of interest can be assessed in vivo, for example in an animal model such as that disclosed in Clynes et al, Proc. nat' l Acad. Sci. USA 95: 652-. A C1q binding assay may also be performed to confirm that the antibody is unable to bind C1q, and therefore lacks CDC activity. See, e.g., WO2006/029879 and WO 2005/100402 for C1q and C3C binding ELISA. To assess complement activation, CDC assays can be performed (see, e.g., Gazzano-Santoro et al, J.Immunol. methods202:163 (1996); Cragg, M.S. et al, Blood 101: 1045-. FcRn binding and in vivo clearance/half-life assays can also be performed using methods known in the art (see, e.g., Petkova, s.b. et al, Int' l.immunol.18(12): 1759-.

One or more amino acid modifications may be introduced in the Fc portion of the antibody to increase IgG binding to the neonatal Fc receptor. The antibody may comprise the following three mutations according to EU numbering: M252Y, S254T, and T256E ("YTE mutations") (U.S. Pat. No.8,697,650; see also Dall' Acqua et al, Journal of Biological Chemistry281(33): 23514. sup. 23524 (2006). YTE mutations do not affect the ability of an antibody to bind to its cognate antigen.

YTE mutants may provide a means to modulate the ADCC activity of an antibody. YTEO mutants can provide a means to modulate the ADCC activity of humanized IgG antibodies against human antigens. See, e.g., U.S. patent nos. 8,697,650; see also Dall' Acqua et al, Journal of Biological Chemistry281(33): 23514-.

YTE mutants may allow for the simultaneous modulation of serum half-life, tissue distribution, and antibody activity (e.g. ADCC of IgG antibodies). See, e.g., U.S. patent nos. 8,697,650; see also Dall' Acqua et al, Journal of biological chemistry281(33) 23514-23524 (2006).

Antibodies with reduced effector function include those with substitutions in one or more of residues 238, 265, 269, 270, 297, 327 and 329 of the Fc region according to EU numbering (U.S. Pat. No.6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327 according to EU numbering, including so-called "DANA" Fc mutants having substitutions of residues 265 and 297 to alanine according to EU numbering (i.e., D265A and N297A according to EU numbering) (U.S. Pat. No.7,332,581). In certain embodiments, the Fc mutant comprises the following two amino acid substitutions: D265A and N297A. In certain embodiments, the Fc mutant consists of two amino acid substitutions: D265A and N297A.

The proline (P329) at position 329(EU numbering) of the wild type human Fc region may be replaced by glycine or arginine or an amino acid residue of a proline sandwich formed between tryptophan residues W87 and W110 of P329 and FcgRIII sufficiently large to disrupt Fc within the Fc/Fc γ receptor interface (Sondermann et al: Nature 406, 267-273(20July 2000)). In yet another embodiment, 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, said at least one further amino acid substitution is L234A and L235A of the human IgG1Fc region or S228P and L235E of the human IgG4Fc region, both according to EU numbering (US8,969,526, incorporated by reference in their entirety).

The polypeptide may comprise an Fc variant of a wild-type human IgG Fc region, wherein the polypeptide has the human IgG Fc region with P329 substituted with glycine, and wherein the Fc variant comprises at least two further amino acid substitutions, L234A and L235A of the human IgG1Fc region or S228P and L235E of the human IgG4Fc region, and wherein the residues are numbered according to the EU numbering (US8,969,526, which is incorporated by reference). Polypeptides comprising P329G, L234A and L235A (EU numbering) substitutions exhibit reduced affinity for human fcyriiia and fcyriia for down-regulating ADCC to at least 20% of the ADCC induced by a polypeptide comprising a wild-type human IgG Fc region and/or for down-regulating ADCP (U.S. patent No.8,969,526, incorporated by reference).

A polypeptide comprising an Fc variant of a wild-type human Fc polypeptide may comprise triple mutations: amino acid substitutions at position Pro329 according to EU numbering, the L234A and L235A mutations (P329/LALA) (US8,969,526, which is incorporated by reference). In exemplary embodiments, the polypeptide comprises the following amino acid substitutions: P329G, L234A, and L235A according to EU numbering.

Certain antibody variants with improved or reduced binding to FcR are described (see, e.g., U.S. Pat. No.6,737,056; WO 2004/056312, and Shields et al, J.biol. chem.9 (2): 6591-6604 (2001)).

An antibody variant may comprise an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and/or 334(EU numbering) of the Fc region.

Changes can be made to the Fc region that result in altered (i.e., improved or reduced) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in US6,194,551, WO99/51642, and Idusogie et al (2000) j.immunol.164: 4178-.

Antibodies with extended half-life and improved binding to the neonatal 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 US 2005/0014934. Those antibodies comprise an Fc region having one or more substitutions therein that improve the binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434 of the Fc region according to EU numbering, for example, substitution of residue 434 of the Fc region (US7,371,826). Also found in Duncan and Winter, Nature 322:738-40 (1988); US5,648,260; US5,624,821; and WO 94/29351, which concerns other examples of Fc region variants.

Cysteine engineered antibody variants

It may be desirable to create cysteine engineered antibodies, e.g. "Thiomab^TMAn antibody ", wherein one or more residues of the antibody are substituted with a cysteine residue. In particular embodiments, the substitute residues are present at accessible sites of the antibody. By replacing those residues with cysteine, the reactive thiol group is thus located at a accessible site of the antibody and can be used to conjugate the antibody to other moieties, such as a drug moiety or linker-drug intermediate, to create an immunoconjugate, as further described herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: v205 of the light chain (Kabat numbering); a140 for heavy chain (EU numbering); l174 for heavy chain (EU numbering); y373 of the heavy chain (EU numbering); k149(Kabat numbering) of the light chain; a118 of the heavy chain (EU numbering); and S400 of the heavy chain Fc region (EU numbering). In particular embodiments, the antibodies described herein comprise HC-a140C (EU numbering) cysteine substitutions. In particular embodiments, the antibodies described herein comprise an LC-K149C (Kabat numbering) cysteine substitution. In particular embodiments, the antibodies described herein comprise HC-a118C (EU numbering) cysteine substitutions. Cysteine engineered antibodies can be as described, for example, in U.S. patent nos. 7,521,541; 9,000,130.

The antibody may comprise one of the following heavy chain cysteine substitutions:

chain (HC/LC)	Residue of	EU mutation site #	Kabat mutation site #
				HC	A	118	114
HC	T	114	110
				HC	A	140	136
HC	L	174	170
				HC	L	179	175
HC	T	187	183
				HC	T	209	205
HC	V	262	258
				HC	G	371	367
HC	Y	373	369
				HC	E	382	378
HC	S	424	420
				HC	N	434	430
HC	Q	438	434

The antibody may comprise one of the following light chain cysteine substitutions:

chain (HC/LC)	Residue of	EU mutation site #	Kabat mutation site #
				LC	I	106	106
LC	R	108	108
				LC	R	142	142
LC	K	149	149
				LC	C	205	205

Joint

A "linker" (L) is a bifunctional or multifunctional moiety that can be used to link one or more drug moieties (D) to an antibody (Ab) to form an ADC of formula I. In some embodiments, ADCs may be prepared using linkers having reactive functionality for covalent attachment to drugs and antibodies. For example, in some embodiments, the cysteine thiol of the cysteine engineered antibody (Ab) may form a bond with a reactive functional group of a linker or drug-linker intermediate to generate an ADC.

The linker may have a functionality capable of reacting with free cysteines present on the antibody to form covalent disulfide bonds (see, e.g., Klussman et al (2004), Bioconjugate Chemistry15(4):765-773, page 766 conjugation methods, and the examples herein).

Exemplary spacer members include valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), and p-aminobenzyloxycarbonyl ("PAB"). A variety of linker components are known in the art.

Non-limiting illustrative such reactive functionalities include maleimides, haloacetamides, α -haloacetyl, activated esters such as succinimidyl esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, chlorinated acids, sulfonyl chlorides, isocyanates, and isothiocyanates see, for example, Klussman et al (2004) Bioconjugate Chemistry15(4) 765 773, page 766, and the examples herein.

The linker may have a functionality capable of reacting with electrophilic groups present on the antibody. Exemplary such electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups. The heteroatom of the reactive functionality of the linker is capable of reacting with an electrophilic group on an antibody and forming a covalent bond with an antibody unit. Non-limiting examples of such reactive functionalities include, but are not limited to, hydrazide (hydrazine), oxime (oxime), amino (amino), hydrazine (hydrazine), thiosemicarbazone (thiosemicarbazone), hydrazine carboxylate (hydrazine carboxylate), and arylhydrazide (arylhydrazide).

The linker may comprise 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-succinimido-4- (2-pyridylthio) pentanoate ("SPP"), and 4- (N-maleimidomethyl) cyclohexane-1 carboxylate ("MCC"). A variety of linker components are known in the art, some of which are described below.

The linker may be a "cleavable linker" to facilitate release of the drug. Non-limiting exemplary cleavable linkers include acid labile linkers (e.g., comprising a hydrazone), protease sensitive (e.g., peptidase sensitive) linkers, photolabile linkers, or disulfide-containing linkers (Chari et al (1992) Cancer Research52: 127-.

The linker may comprise one or more spacer units between the disulfide group and the drug moiety. One example includes a linker having the formula:

-A_a-W_w-Y_y-，

wherein A is a "stretcher unit" and a is an integer from 0 to 1; w is an "amino acid unit" and W is an integer from 0 to 12; y is a "spacer unit", and Y is 0,1, or 2; and Ab, D, and p are as defined above for formula I. Exemplary embodiments of such linkers are described in U.S. patent No.7,498,298, which is expressly incorporated herein by reference.

The linker component may comprise an "extender unit" that links the antibody to another linker component or drug moiety. Exemplary extender units are shown below (where the wavy line indicates the site of covalent attachment to an antibody, drug, or other linker component):

the linker may be a peptidomimetic linker such as those described in WO2015/095227, WO2015/095124 or WO2015/095223, which documents are hereby incorporated by reference.

Medication module

The site-specific ADC compounds of the invention comprise an antibody conjugated to one or more drug moieties, including the following:

maytansine and maytansinoids

In some embodiments, the ADC comprises an antibody conjugated to one or more maytansinoid molecules. Maytansinoids are derivatives of maytansine and are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was originally isolated from the east non-shrub maytansine (Maytenus serrata) (US 3896111). It was subsequently found that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (US 4151042). Synthetic maytansinoids are disclosed in, for example, US 4137230; US 4248870; US 4256746; US 4260608; US 4265814; US 4294757; US 4307016; US 4308268; US 4308269; US 4309428; US 4313946; US 4315929; US 4317821; US 4322348; US 4331598; US 4361650; US 4364866; US 4424219; US 4450254; US 4362663; and US 4371533.

Maytansinoid drug moieties are attractive drug moieties in antibody-drug conjugates because they: (i) relatively accessible for preparation by fermentation or chemical modification or derivatization of fermentation products; (ii) are readily derivatized with functional groups suitable for conjugation to antibodies via non-disulfide linkers; (iii) is stable in plasma; and (iv) effective against a variety of tumor cell lines.

Certain maytansinoid compounds suitable for use as maytansinoid drug moieties are known in the art and can be isolated from natural sources according to well known methods or produced using genetic engineering techniques (see, e.g., Yu et al (2002) Proc. Natl. Acad. Sci. U.S.A.99: 7968-. Maytansinoids can also be prepared synthetically according to known methods.

Exemplary maytansinoid drug moieties include, but are not limited to, those having modified aromatic rings, such as: c-19-dechlorination (U.S. Pat. No.4,256,746) (e.g., prepared by lithium aluminum hydride reduction of ansamycine P2); c-20-hydroxy (or C-20-demethyl) +/-C-19-dechlorine (U.S. Pat. Nos. 4,361,650 and 4,307,016) (prepared, for example, by demethylation using Streptomyces (Streptomyces) or Actinomyces (Actinomyces) or by dechlorination using LAH); and C-20-demethoxy, C-20-acyloxy (OCOR), +/-dechlorinated (U.S. Pat. No.4,294,757) (prepared, for example, by acylation using an acyl chloride) and those having modifications at other positions on the aromatic ring.

Exemplary maytansinoid drug moieties also include those having modifications such as: C-9-SH (US4,424,219) (e.g. by reacting maytansinol with H₂S or P₂S₅Reaction preparation); C-14-Alkoxymethyl (demethoxy/CH)₂OR) (US4,331,598); c-14-hydroxymethyl or acyloxymethyl (CH)₂OH or CH₂OAc) (US 4450254) (e.g. made by Nocardia); c-15-hydroxy/acyloxy (US 4364866) (prepared for example by transformation of maytansinol from Streptomyces); c-15-methoxy (US4,313,946 and US4,315,929) (e.g. isolated from Trewia nudlflora); C-18-N-demethylation (US4,362,663 and US4,322,348) (prepared, for example, by demethylation of maytansinol with Streptomyces); and 4, 5-deoxy (US4,371,533) (e.g. prepared by phthalide/LAH reduction of maytansinol).

Many positions on maytansinoid compounds are useful as attachment sites. For example, ester linkages can be formed by reaction with hydroxyl groups using conventional coupling techniques. The reaction can be carried out at the C-3 position having a hydroxyl group, the C-14 position modified with a hydroxymethyl group, the C-15 position modified with a hydroxyl group and the C-20 position having a hydroxyl group. The linkage is formed at the C-3 position of maytansinol or a maytansinol analogue.

Maytansinoid drug moieties include those having the following structure:

wherein the wavy line indicates the covalent attachment of the sulfur atom of the maytansinoid drug moiety to the linker of the ADC. Each R may independently be H or C₁-C₆An alkyl group. The alkylene chain attaching the amide group to the sulphur atom may be a methyl, ethyl or propyl group, i.e. m is 1,2 or 3(US 633410; US 5208020; Chari et al (1992) Cancer Res.52: 127-.

For the ADC of the present invention, attention is directed to all stereoisomers of the maytansinoid drug moiety, i.e., any combination of R and S configurations at chiral carbons (US 7276497; US 6913748; US 6441163; US 633410(RE 39151); US 5208020; Widdison et al (2006) J.Med.chem.49:4392-4408, which is incorporated by reference in its entirety). In some embodiments, the maytansinoid drug moiety has the following stereochemistry:

exemplary embodiments of maytansinoid drug moieties include, but are not limited to, DM 1; DM 3; and DM4, having the following structure:

wherein the wavy line indicates the covalent attachment of the sulfur atom of the drug to the linker (L) of the antibody-drug conjugate.

Immunoconjugates comprising maytansinoids, methods for their production, and therapeutic uses thereof are disclosed, for example, in US 5208020 and US 5416064; US 2005/0276812a 1; and european patent EP 0425235B 1, the disclosures of which are hereby expressly incorporated by reference. Also see Liu et al Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996); and Chari et al cancer Research52: 127-.

Antibody-maytansinoid conjugates can be prepared by chemically linking an antibody to a maytansinoid molecule without significantly impairing the biological activity of either the antibody or the maytansinoid molecule. See, for example, U.S. Pat. No.5,208,020 (the disclosure of which is hereby expressly incorporated by reference). ADCs conjugated with an average of 3-4 maytansinoid molecules per antibody molecule show efficacy in enhancing cytotoxicity to target cells without negatively impacting the function or solubility of the antibody. In some cases, even one molecule of toxin/antibody is expected to enhance cytotoxicity compared to the use of naked antibodies.

Exemplary linking groups for use in generating antibody-maytansinoid conjugates include, for example, those described herein and U.S. patent No. 5208020; european patent 0425235; chari et al, Cancer Research52: 127-; US 2005/0276812; and those disclosed in US 2005/016993, the disclosure of which is hereby expressly incorporated by reference.

Auristatin and dolastatin

The pharmaceutical module may include dolastatin, auristatin, and analogs and derivatives thereof (US 565483; US 5780588; US 5767237; US 6124431). Auristatin is a derivative of dolastatin-10, a marine mollusk compound. Although not intending to be bound by theory, dolastatin and auristatin have been shown to interfere with microtubule dynamics, GTP hydrolysis and nuclear and cell division (Woyke et al (2001) Antichronoicrob. Agents and Chemother.45(12):3580-3584) and to have anti-cancer (US 5663149) and anti-fungal activity (Pettit et al (1998) Antichronoicrob. Agents Chemother.42: 2961-2965). The dolastatin/auristatin drug moiety may be attached to the antibody via the N (amino) terminus or the C (carboxy) terminus of the peptide drug moiety (WO 02/088172; Doronina et al (2003) Nature Biotechnology 21(7): 778-.

Exemplary auristatin embodiments include N-terminally linked monomethylauristatin drug modules, which are disclosed in US 7498298 and US 7659241, the disclosures of which are expressly incorporated herein by reference in their entirety.

Formula D_EAn exemplary auristatin embodiment of (a) is MMAE, wherein the wavy line indicates the covalent attachment to the linker (L) of the antibody-drug conjugate:

formula D_FOne exemplary auristatin embodiment of (a) is MMAF, where the wavy line indicates the covalent attachment to the linker (L) of the antibody-drug conjugate:

other exemplary embodiments include monomethylvaline compounds having a phenylalanine carboxyl group modification at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007/008848) and monomethylvaline compounds having a phenylalanine side chain modification at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007/008603).

Typically, peptide-based drug moieties can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for example, according to liquid phase synthesis methods (see, e.g., E.And K.L ü bke, "the peptides", volume 1, pp 76-136,1965, Academic Press). Auristatin/dolastatin drug modules can be prepared according to the methods in the following documents: US 7498298; US 5635483; US 5780588; pettit et al (1989) J.am.chem.Soc.111: 5463-5465; pettit et al (1998) Anti-Cancer Drug Design13: 243-277; pettit, g.r., et al.synthesis,1996, 719-725; pettit et al (1996) J.chem.Soc.Perkin Trans.15: 859-863; and Doronina (2003) nat. Biotechnol.21(7): 778-.

Formula D_E(such as MMAE) and D_FAuristatin/dolastatin drug modules (such as MMAF), and drug-linker intermediates and derivatives thereof, such as MC-MMAF, MC-MMAE, MC-vc-PAB-MMAF, and MC-vc-PAB-MMAE can be used US 7498298; doronina et al (2006) Bioconjugate chem.17: 114-124; and Doronina et al (2003) nat.Biotech.21:778-784, and then conjugated to an antibody of interest.

Calicheamicin

The calicheamicin family of antibiotics and analogs thereof are capable of generating double-stranded DNA breaks at sub-picomolar concentrations (Hinman et al, (1993) Cancer Research 53: 3336-. Calicheamicin has an intracellular site of action, but in some cases does not readily cross the plasma membrane. Thus, in some embodiments, cellular uptake of these agents via antibody-mediated internalization may greatly enhance their cytotoxic effects. Non-limiting exemplary methods of preparing antibody-drug conjugates having a calicheamicin drug moiety are described in, for example, US5,712,374; US5,714,586; US5,739,116; US5,767,285; and WO 2017/068511.

The drug moiety conjugated to the antibody is a calicheamicin compound having the formula:

wherein X is Br or I; l is a linker; r is hydrogen, C_1-6Alkyl, or-C (═ O) C_1-6An alkyl group; and R is^aIs hydrogen or C_1-6An alkyl group.

Pyrrolobenzodiazepines

The ADC may comprise a Pyrrolobenzodiazepine (PBD) drug moiety. PDB dimers can recognize and bind to specific DNA sequences. The natural product ampamycin (an antrramycin), a PBD, was first reported in 1965 (Leimgruber et al, (1965) J.am.Chem.Soc.87: 5793-. Since then, some PBDs (both naturally occurring and analogues) were reported (Thurston et al (1994) chem. Rev.1994, 433-465), including dimers of tricyclic PBD scaffolds (US 6,884,799; US7,049,311; US7,067,511; US7,265,105; US7,511,032; US7,528,126; US7,557,099). Without intending to be bound by theory, it is believed that the dimeric structure imparts a suitable three-dimensional shape to achieve co-helicity (isochelicity) with the minor groove of type B DNA, resulting in a close fit at the binding site (Kohn, in Antibiotics III. Springer-Verlag, New York, pp.3-11 (1975); Hurley and New ham-VanDevanter (1986) Acc. chem. Res.19: 230-. Dimeric PBD compounds carrying a C2 aryl substituent 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.Letter19 (22): 6463-6466).

PBD compounds can be used as prodrugs by protecting them at the N10 position with a nitrogen protecting group which is removable in vivo (WO 00/12507; WO 2005/023814).

PBD dimers have been conjugated to antibodies and the resulting ADCs have been shown to have anti-cancer properties (US 2010/0203007). Non-limiting exemplary attachment sites on the PBD dimer include five-membered pyrrole rings, tethers between PBD units, and N10-C11 imine groups (WO 2009/016516; US 2009/304710; US 2010/047257; US 2009/036431; US 2011/0256157; WO 2011/130598).

The linker may be attached to one of the various sites of 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 tethering unit that connects the a ring (see structures C (i) and C (ii) below).

In some embodiments, an exemplary PBD dimer building block of an ADC has the structure of formula a-1:

wherein n is 0 or 1.

In some embodiments, an exemplary PBD dimer building block of an ADC has the structure of formula a-2:

wherein n is 0 or 1.

In some embodiments, an exemplary PBD dimer building block of an ADC has the structure of formula a-3:

wherein R is^EAnd R^E”Each independently selected from H or R^DWherein R is^DAs defined above; and is

Wherein n is 0 or 1.

In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, R^EAnd/or R^E”Is H. In some embodiments, R^EAnd R^E”Is H. In some embodiments, R^EAnd/or R^E”Is R^DWherein R is^DIs optionally substituted C_1-12A hydrocarbyl group. In some embodiments, R^EAnd/or R^E”Is R^DWherein R is^DIs methyl.

In some embodiments, an exemplary PBD dimer building block of an ADC has the structure of formula a-4:

wherein Ar is¹And Ar²Each independently is optionally substituted C_5-20An aryl group; wherein Ar is¹And Ar²May be the same or different; and is

Wherein n is 0 or 1.

An exemplary PBD dimer building block of an ADC has the structure of formula a-5:

wherein Ar is¹And Ar²Each independently is optionally substituted C_5-20An aryl group; wherein Ar is¹And Ar²May be the same or different; and is

Wherein n is 0 or 1.

Ar¹And Ar²Each of which may be independently selected from optionally substituted phenyl, furyl, thiophenyl and pyridyl. In some embodiments, Ar¹And Ar²Each independently is optionally substituted phenyl. In some embodiments, Ar¹And Ar²Each independently is optionally substituted thiophen-2-yl or thiophen-3-yl. In some embodiments, Ar¹And Ar²Each independently is optionally substituted quinolinyl or isoquinolinyl. The quinolinyl or isoquinolinyl group may be bound to the PBD core via any available ring position. For example, a quinolinyl group may be quinolin-2-yl, quinolin-3-yl, quinolin-4-yl, quinolin-5-yl, quinolin-6-yl, quinolin-7-yl and quinolin-8-yl. In some embodiments, the quinolinyl is selected from quinolin-3-yl and quinolin-6-yl. The isoquinolinyl group may be isoquinolin-1-yl, isoquinolineQuinolin-3-yl, isoquinolin-4-yl, isoquinolin-5-yl, isoquinolin-6-yl, isoquinolin-7-yl, and isoquinolin-8-yl. In some embodiments, the isoquinolinyl group is selected from isoquinolin-3-yl and isoquinolin-6-yl.

An exemplary PBD dimer building block of an ADC has the structure of formula a-6:

another non-limiting exemplary PBD dimer building block of an ADC has formula B:

and salts and solvates thereof, wherein:

wavy lines indicate the sites of covalent attachment to the linker;

wavy lines attached to OH represent S or R configurations;

R^V1and R^V2Independently selected from H, methyl, ethyl and phenyl (phenyl may be optionally substituted with fluoro, especially in the 4 position) and C_5-6A heterocyclic group; wherein R is^V1And R^V2May be the same or different; and is

n is 0 or 1.

R^V1And R^V2May be independently selected from H, phenyl, and 4-fluorophenyl.

Non-limiting exemplary PBD dimer building blocks of ADCs include tethered formulas c (i) and c (ii):

formulas C (I) and C (II) are shown as their N10-C11 imines. Exemplary PBD drug modules also include methanolamine and protected methanolamine forms, as shown in the table below:

wherein:

x is CH₂(N ═ 1 to 5), N, or O;

z and Z' are independently selected from OR and NR₂Wherein R is a primary, secondary or tertiary alkyl chain containing 1 to 5 carbon atoms; r₁，R’₁，R₂And R'₂Each independently selected from H, C₁-C₈Alkyl radical, C₂-C₈Alkenyl radical, C₂-C₈Alkynyl, C_5-20Aryl (including substituted aryl), C_5-20Heteroaryl radical, -NH₂-NHMe, -OH, and-SH, wherein, in some embodiments, alkyl, alkenyl, and alkynyl chains contain up to 5 carbon atoms;

R₃and R'₃Independently selected from H, OR, NHR, and NR₂Wherein R is a primary, secondary or tertiary alkyl chain containing 1 to 5 carbon atoms;

R₄and R'₄Independently selected from H, Me, and OMe;

R₅is selected from C₁-C₈Alkyl radical, C₂-C₈Alkenyl radical, C₂-C₈Alkynyl, C_5-20Aryl (including aryl substituted with halogen, nitro, cyano, hydrocarbyloxy, hydrocarbyl, heterocyclyl) and C_5-20Heteroaryl groups, wherein, in some embodiments, alkyl, alkenyl, and alkynyl chains contain up to 5 carbon atoms;

R₁₁is H, C₁-C₈A hydrocarbon group, or a protecting group (such as acetyl, trifluoroacetyl, tert-Butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ), 9-fluorenylmethylenoylcarbonyl (Fmoc), or a moiety comprising a self-sacrificial unit such as valine-citrulline-PAB);

R₁₂is H, C₁-C₈A hydrocarbyl group, or a protecting group;

wherein R is₁，R’₁，R₂，R’₂，R₅Or R is₁₂One hydrogen of one or-OCH between A rings₂CH₂(X)_nCH₂CH₂One hydrogen of the O-spacer is replaced with a bond to the linker of the ADC.

The ADC comprising the PBD dimer described herein can be generated by conjugating a linker-drug intermediate comprising a pyridine leaving group to the cysteine thiol of an antibody via a sulfur atom to form a disulfide linkage. Further, in some embodiments, ADCs comprising PBD dimers described herein may be generated by conjugation to a linker-drug intermediate comprising a thiopyridyl leaving group, wherein the pyridyl ring is substituted with one or more nitro groups. In some embodiments, the pyridine ring is substituted with-NO₂Monosubstituted. In some embodiments, -NO₂Monosubstitution is para with respect to disulfide. In some embodiments, the PBD dimer is linked via the N10 position. For example, a non-limiting exemplary PBD dimer-containing ADC can be generated by conjugating monomethylethylpyridyl disulfide, N10-linked PBD linker intermediate (shown below) to an antibody:

PBD dimers and ADCs comprising PBD dimers can be prepared according to methods known in the art. See, e.g., WO 2009/016516; US 2009/304710; US 2010/047257; US 2009/036431; US 2011/0256157; WO 2011/130598; WO 2013/055987.

Anthracene ring

The site-specific ADCs of the present disclosure may comprise an anthracycline. Anthracyclines are antibiotic compounds that exhibit cytotoxic activity. Without intending to be bound by theory, anthracyclines may operate to kill cells by a number of different mechanisms, including: 1) inserting a drug molecule into the DNA of the cell, thereby inhibiting DNA-dependent nucleic acid synthesis; 2) the generation Of free radicals from drugs, which then react with cellular macromolecules to cause damage to the cells, And/or 3) the interaction Of drug molecules with cell membranes (see, e.g., c. peterson et al, "Transport And Storage Of anti-multicycle In Experimental Systems And human leukamia" In anti-multicycle In Cancer Therapy; n.r. bachur, "free radial dam" id.at pp.97-102). Due to their cytotoxic potential, anthracyclines have been used to treat a wide variety of cancers such as leukemias, breast cancers, lung cancers, ovarian adenocarcinomas and sarcomas (see, e.g., P.H-Wiernik, Inantharcycline: Current Status and New Developments p 11).

Exemplary anthracyclines include doxorubicin, epirubicin, idarubicin, daunomycin, nemorubicin, and derivatives thereof. 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.Letters16: 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) Med.chem.chem.45: 4336-4343; EP 0328147; US6,630,579). The antibody-drug conjugate BR 96-doxorubicin reacts specifically with the tumor-associated antigen Lewis-Y and has been evaluated in phase I and II studies (Saleh et al (2000) J. Clin. Oncology 18: 2282-.

PNU-159682 is a potent metabolite (or derivative) of nemorubicin (Quinieri et al (2005) Clinical Cancer Research 11(4): 1608-1617). Nemorubicin is a semisynthetic analog of doxorubicin, having a 2-methoxymorpholine group on the glycosidic amino group, and has been under Clinical evaluation (Grandi et al (1990) Cancer treat. Rev.17: 133; Ripamonti et al (1992) Brit. J.cancer 65:703), including phase II/III trials for hepatocellular carcinoma (Sun et al (2003) Proceedings of the American Society for Clinical Oncology 22, Abs 1448; Quinieri (2003) Proceedings of the American Association of Cancer Research 44:1 Ed, Abs 4649; Pacciai et al (2006) journal. Clin. Clinocolgy 24: 16).

In some embodiments, the nemorubicin component of the nemorubicin-containing ADC is PNU-159682:

wherein the wavy line indicates attachment to the linker (L).

Anthracyclines, including PNU-159682, may be conjugated to antibodies via several attachment sites and various linkers, including those described herein (US 2011/0076287; WO 2009/099741; US 2010/0034837; WO 2010/009124).

Exemplary ADCs can be generated by conjugating a pyridyl disulfide PNU amide (shown below) to an antibody as follows:

to generate a disulfide-linked PNU-159682 antibody-drug conjugate:

PNU-159682, the linker of the Maleimide Acetal-Ab is acid labile, while PNU-159682-val-cit-PAB-Ab, PNU-159682-val-cit-PAB-spacer-Ab, and PNU-159682-val-cit-PAB-spacer (R)¹R²) The linker of the-Ab is protease cleavable.

1- (chloromethyl) -2, 3-dihydro-1H-benzo [ e ] indole (CBI) dimer drug moiety

In some embodiments, the ADC comprises 1- (chloromethyl) -2, 3-dihydro-1H-benzo [ e ] indole (CBI). DNA minor groove alkylating agents of the 5-amino-1- (chloromethyl) -1, 2-dihydro-3H-benzo [ e ] indole (amino CBI) class are potent cytotoxins (Atwell et al (1999) j. med. chem.,42:3400) and have been used as effector units for a wide variety 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 carbamates (Hay et al (2003) J.Med.Chem.46:2456) and the corresponding nitro CBI derivatives as hypoxia activated prodrugs (Tercel et al (2011) Angew.Chem., Int.Ed.,50: 2606-. CBI and pyrrolo [2,1-c ] [1,4] benzodiazepine (PBD) pharmacophores have been linked together by alkyl chains (Tercel et al (2003) J. Med. Chem46: 2132-.

Site-specific ADCs may comprise 1- (chloromethyl) -2, 3-dihydro-1H-benzo [ e ] indole (CBI) dimers (WO 2015/023355). The dimer may be a heterodimer, wherein one half of the dimer is a CBI module and the other half of the dimer is a PBD module.

An exemplary CBI dimer comprises the formula:

wherein

R¹Selected from H, P (O)₃H₂，C(O)NR^aR^bOr a bond to the linker (L); r²Selected from H, P (O)₃H₂，C(O)NR^aR^bOr a bond to the linker (L);

R^aand R^bIndependently selected from H and C optionally substituted with one or more F₁-C₆Alkyl, or R^aAnd R^bTo form a five or six membered heterocyclyl group;

t is selected from C₃-C₁₂Alkylene, Y, (C)₁-C₆Alkylene) -Y- (C)₁-C₆Alkylene), (C)₁-C₆Alkylene) -Y- (C)₁-C₆Alkylene) -Y- (C)₁-C₆Alkylene), (C)₂-C₆Alkenylene) -Y- (C)₂-C₆Alkenylene), and (C)₂-C₆Alkynylene) -Y- (C₂-C₆Alkynylene);

wherein Y is independently selected from O, S, NR¹Aryl, and heteroaryl;

wherein alkylene, alkenylene, aryl, and heteroaryl are independently and optionally substituted with F, OH, O (C)₁-C₆Alkyl), NH₂，NHCH₃，N(CH₃)₂，OP(O)₃H₂And C and₁-C₆alkyl substituted, wherein alkyl is optionally substituted with one or more F;

or alkylene, alkenylene, aryl, and heteroaryl are independently and optionally substituted with a bond to L;

d' is a drug moiety selected from:

wherein the wavy line represents the site of attachment to T; x¹And X²Independently selected from O and NR³Wherein R is³Selected from H and C optionally substituted with one or more F₁-C₆An alkyl group; r⁴Is H, CO₂R, or a bond to a linker (L), wherein R is C₁-C₆Alkyl or benzyl; and R is⁵Is H or C₁-C₆An alkyl group.

Amanitin and amanitin

Amanitin is a cyclic peptide consisting of 8 amino acids that can be isolated or synthetically prepared from the mushroom amanitin (amanitia pharioides.) amanitin specifically inhibits DNA-dependent RNA polymerase II of mammalian cells and thereby also inhibits transcription and protein biosynthesis in affected cells inhibition of transcription causes growth and proliferation arrest in cells, see, e.g., Moldenhauer et al jnci 104:1-13(2012), WO2010115629, WO2012041504, WO2012119787, WO2014043403, WO2014135282, and WO 2012012112119787, which are hereby incorporated by reference in their entirety.

Other drug modules

The drug moiety may further include geldanamycin (geldanamycin) (Mandler et al (2000) J. Nat. cancer Inst.92(19): 1573) 1581; Mandler et al (2000) Bioorganic & Med. chem. letters 10: 1025) 1028; Mandler et al (2002) Bioconjugate chem.13:786-791) and enzymatically active toxins and fragments thereof, including but not limited to diphtheria toxin A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa (Pseudomonas aeruginosa), ricin A chain (ricin Achain), abrin A chain (abrin A chain), magadirin A chain (ecoscin A chain), α -sarcin (alpha-saiercin), oleotoxin (Alfordii) A chain, oleanolicin, shikonin A protein (oleanolicin), trichothecin A chain (trichothecin A chain), or trichothecin (e.g. protein), such as trichothecin (I), and the like, and the inhibitory protein of phytoncicin (phytotoxin, such as echinomycin, trichothecin (phytotoxin I), a protein, e.g. a protein (trichothecin, e.g. a protein, a protein (trichothecin, a protein (trichothecin, a protein (trichothecin) and a protein, a protein (trichothecin, a protein (trichothecene) may also include a protein, a.

It is understood that where more than one nucleophilic group reacts with a drug-linker intermediate or with a linker reagent, the resulting product is a mixture of ADC compounds having a distribution of one or more drug moieties attached to an antibody.

Linker-drug intermediates may be prepared by reaction according to WO 2013/055987; WO 2015/023355; WO 2010/009124; the protocol of WO2015/095227 was coupled to a drug moiety and linker reagent and prepared by conjugation to proteins described herein, including cysteine engineered antibodies.

Methods for analyzing and quantifying antibodies and drug moieties in site-specific ADCs

ADCs are targeted anti-cancer therapeutics designed to reduce non-specific toxicity and increase efficacy relative to conventional small molecule and antibody cancer chemotherapy. They exploit the powerful targeting capabilities of monoclonal antibodies to specifically deliver highly potent, conjugated small molecule therapeutics to cancer cells. In order to assess such properties of these ADCs as efficacy, stability, homology, pharmacokinetics and toxicity, it is useful to accurately characterize and quantify antibody building blocks and drug modules from solutions, plasma, urine, and other biological samples via sample analytical analysis.

The present disclosure provides reproducible, accurate, and efficient analytical methods for quantifying and analyzing characteristics of antibodies and drug components of site-specific ADC therapeutic constructs. Figure 1b shows a cartoon of a workflow of one ADC sample assay of the present disclosure, including optional affinity capture of the ADC from the sample, site-specific enzymatic digestion (which may include cleavage and release of the drug from the ADC), and subsequent analysis of the drug and peptide fragments by chromatographic and/or mass spectrometry methods.

In these methods, the site-specific ADC construct is digested with one or more specific enzymes to form a digested ADC composition comprising at least one peptide fragment not linked to a drug moiety and at least one peptide fragment linked to a drug moiety. One or both of the drug and digested antibody building blocks/fragments are then analyzed by chromatography/mass spectrometry to determine characteristics of the ADC, which may include, but are not limited to, protein concentration of the ADC composition, total antibody concentration of the ADC, drug concentration and/or average DAR of the ADC, ADC metabolite or catabolite structure, and extinction coefficient of the ADC.

The protein building blocks of the ADC samples can be digested with proteolytic enzymes such as IdeS protease, IdeZ protease, IgdE protease, SpeB protease, gingipain, endoglycosidase, and combinations of these enzymes.

IdeS is an extracellular cysteine protease produced by streptococcus pyogenes (s.pyogenes) and commercially available from Promega (Madison, WI) and Genovis AB (Cambridge, MA). This enzyme, designated IdeS due to the immunoglobulin G degrading enzyme of streptococcus pyogenes, cleaves human IgG with high specificity below the hinge region, producing a homogeneous pool of F (ab')2 and Fc fragments. Thus, other human proteins (including immunoglobulins M, a, D and E) are not digested by IdeS. This enzyme cleaves IgG antibodies bound to surface structures of streptococci with high efficiency, thereby inhibiting phagocytic killing of streptococcus pyogenes, leading to the identification of this enzyme as a determinant and potential therapeutic target of bacterial virulence (von Pawel-Rammingen et al, EMBO J. (2002)21(7): 1607-15). The proteolytic cleavage sites of the IdeS enzyme are shown in the following table:

IgG class and subclass	IdeS cleavage site	SEQ ID No.
			Human IgG1	..CPAPELLG/GPSVF..	1
Human IgG2	..CPAPPVA/GPSVF..	2
			Human IgG3	..CPAPPVA/GPSVF..	2
Human IgG4	..CPAPPVA/GPSVF..	2
			Mouse IgG1	Without cutting
Mouse IgG2a	..CPAPPVA/GPSVF..	2
			Mouse IgG2b	Without cutting
Mouse IgG3	..CPAPPVA/GPSVF..	2
			Rat IgG2b	..CPAPPVA/GPSVF..	2
Kiwi fruit	..CPAPPVA/GPSVF..	2
			Rabbit	..CPAPPVA/GPSVF..	2

HTCPPCPAPELLGGPSVF SEQ ID NO.：1

HTCPPCPAPPVAGPSVF SEQ ID NO.：2

IdeZ protease (IgG-specific) is an antibody-specific protease cloned from Streptococcus equi (Streptococcus equi) subspecies zooepidemicus (zoepemicus), recognizes all human, sheep, monkey, and rabbit IgG subclasses, specifically cleaves a single recognition site below the hinge region, yielding a homogenous pool of F (AB')2 and Fc fragments, and is commercially available from NewEngland Biolabs (Ipswich, MA), Promega (Madison, WI), and Genovis AB (Cambridge, MA). IdeZ protease has significantly improved activity against the mouse IgG2a and IgG3 subclasses compared to IdeS protease.

IgdE is a protease in Streptococcus suis (Streptococcus suis) that targets porcine IgG alone. This enzyme, designated IgdE for Streptococcus suis immunoglobulin G degrading enzymes, is a cysteine protease different from Streptococcus immunoglobulin degrading proteases of the IdeS family and cleaves the hinge region of porcine IgG with high specificity (Spoerry et al, J Biol Chem, (2016)291(15): 7915-25).

SpeB is a cysteine protease isolated from Streptococcus pyogenes (Streptococcus pyogenes) that degrades IgA, IgM, IgE, and IgD and cleaves IgG antibodies in the hinge region after reduction, i.e., cleaves IgG molecules in a reduced state, e.g., in the presence of Dithiothreitol (DTT), β -mercaptoethanol, or L-cysteine (Persson et al, Infect. Immun. (2013)81(6): 2236-41).

Gingipain, Kgp (also known as Lys-gingipain), is a cysteine protease secreted by Porphyromonas gingivalis, which cleaves human IgG1 in the upper hinge region (between K223 and T224) fragment under mild reducing conditions to produce a homogeneous pool of Fab and Fc. One recombinant form of gingipain, Kgp, is commercially available from Genovis AB (Cambridge, MA).

Endoglycosidases represent a family of enzymes expressed by streptococcus pyogenes, capable of releasing terminal sialic acid residues, and in particular Asp279 of IgG, from glycoproteins such as immunoglobulins. EndoS is a specific endoglycosidase used for antibody deglycosylation. Additional endoglycosidases that may be useful in the methods of the present disclosure include one or more of EndoS2, EndoH, EndoA, EndoM, EndoF1, EndoF2, and EndoF 3. Endoglycosidases can be used in the assays of the present disclosure in combination with one or more proteases described above in the preparation of digested site-specific ADCs for chromatographic/spectroscopic analysis.

The proteolytic enzyme used to digest the site-specific ADC construct can be selected to generate unique peptide fragments for detection and quantification. One or more peptide fragments unique to the antibody of the ADC are detected and quantified, thereby eliminating non-specific proteins or other contaminants that may be present in the background or in the analytical sample applied to chromatography or spectroscopy that do not form part of the ADC.

In an exemplary embodiment, the ADC sample is not digested with trypsin, papain, pepsin, endoprotease LysC, endoprotease ArgC, Staphylococcus aureus V8, chymotrypsin, Asp-N, Asn-C, PNGaseF, endoprotease GluC, LysN, or any combination of these enzymes. Thus, in exemplary embodiments, the ADC sample does not contain detectable amounts of trypsin, papain, pepsin, endoprotease LysC, endoprotease ArgC, Staphylococcus aureus V8, chymotrypsin, Asp-N, Asn-C, PNGaseF, endoprotease GluC, or LysN during the digestion or analysis protocol.

Depending on the identity of the linker moiety of the ADC and the chemical treatment applied for reduction, denaturation, and/or digestion of the protein moiety of the sample, the drug moiety of the ADC may be cleaved from the antibody/peptide moiety of the ADC and thus may be detected and quantified as an unconjugated drug moiety in an LC-MS/MS analysis.

Alternatively or additionally, the drug moiety member of the ADC may remain attached to the antibody/peptide member of the ADC after reduction and denaturation of the ADC, and thus may be detected and quantified in the assay as a peptide-bound drug moiety.

The ADC-containing sample for analysis/quantification may be submitted for digestion (and optionally reduction and/or denaturation) without any preliminary sample cleaning or enrichment (i.e., "direct digestion" of the sample). Alternatively, or in addition, the sample containing the ADC may be enriched or concentrated for further analysis prior to digestion. Such concentration of low abundance peptides or drugs may include such enrichment techniques as size exclusion chromatography, dialysis, selective precipitation, differential centrifugation, filtration, gel electrophoresis, liquid chromatography, reverse phase chromatography, immunoprecipitation, SpinTrap columns including protein a and protein G, NHS and streptavidin iron or phosphorous or immobilized antibodies or lectins, paramagnetic beads, immuno-subtraction, fractionation, solid phase extraction, phosphopeptide enrichment, polyacrylamide gel electrophoresis, desalting, and the like.

ADC can be reduced by contacting with a composition comprising at least one reducing agent, such as Dithiothreitol (DTT), 2-mercaptoethanol, or tris (2-carboxyethyl) phosphine (TCEP). It is also possible to use a mixture comprising at least one denaturant, for example formamide, dimethylformamide, acetonitrile, SDS, urea, guanidine, sodium 3- ((1- (furan-2-yl) undecyloxy) carbonylamino) propane-1-sulfonate (ProteaseMax)^TM) And/or acid-labile surfactants such as those containing dioxolane (dioxolane) or dioxane (dioxane) functional groups, such as RapidGest^TMContacting a composition of-SF surfactants (as described in US7,229,539 and US8,580,533; incorporated herein by reference) denatures the ADC. ADCs may be simultaneously reduced and denatured by contact with a composition comprising at least one reducing agent and at least one denaturing agent. Such compositions may include additional solvents, buffers and/or pH modifiers such as acetonitrile, methanol, ethanol, HCl, ammonium bicarbonate, ammonium acetate, and/or formic acid, dephosphorylating agents, including phosphatases such as calf intestinal alkaline phosphatase, or lambda protein phosphatase.

The ADCs presented for analysis may also be present in solution or suspension, such as in a pharmaceutical composition formulated for administration to an animal or human, or in cell cultures or supernatants that may be present during the production steps of the ADCs, or in biological samples obtained from an animal or human. Thus, the ADC may be present in a matrix selected from the group consisting of buffer, whole blood, serum, plasma, cerebrospinal fluid, saliva, urine, lymph, bile, feces, sweat, vitreous humor, tear fluid, and tissue. Biological samples frequently presented for analysis of various safety, efficacy and pharmacokinetic/biodistribution parameters of ADCs include human, cynomolgus, rat, and mouse plasma and tissue samples, as well as biological samples from other non-human species.

When presented as part of such a biological sample, the ADC may be contacted with an affinity capture medium. Affinity capture is a widely used method for enriching/isolating intact proteins, identifying binding partners and protein complexes, or investigating post-translational modifications. The protein or protein complex can be separated by non-specific means (e.g., gel electrophoresis, protein A or G media, type 1 anti-neuronal nuclear autoantibodies (ANNA-1, also known as "anti-Hu"), or specific means (e.g., extracellular domain (ECD) antibodies, or anti-idiotypic antibodies). the ADC can then be eluted from the affinity capture media as a means of sample cleaning, followed by digestion (optionally including reduction and/or denaturation), and subsequent chromatographic/mass spectrometric analysis of the digests.

Alternatively/additionally, the ADC sample is analyzed with affinity capture by bead or resin supported protein a/G, followed by bead digestion (which may include proteolysis, deglycosylation, dephosphorylation, reduction, and/or denaturation), followed by elution of the enriched digested antibody sample from the affinity capture medium, and subsequent chromatography/mass spectrometry analysis. Methods for detecting and screening antibody-drug conjugates by immunoaffinity membrane (IAM) capture and mass spectrometry have been disclosed (US7,662,936), including bead-based affinity capture methods (US8,541,178).

The analytical sample (or at least a portion thereof) comprising one or both of the drug (or peptide-linker-drug) and the digestive antibody moiety of the site-specific ADC is then applied to a detection methodology that may include High Performance Liquid Chromatography (HPLC), reverse phase liquid chromatography (RP-LC), Mass Spectrometry (MS) or tandem mass spectrometry (MS/MS), both drug and antibody moieties of the ADC to detect and quantify.

Mass spectrometry can be used to establish the mass-to-charge ratio of at least one peptide fragment of the digested antibody, and/or the mass-to-charge ratio of the drug (or peptide-linker-drug) moiety of the ADC.

The molar extinction coefficient (or mass attenuation coefficient) is equal to the molar attenuation coefficient multiplied by the molar mass. The molar extinction coefficient of a protein at 280nm is largely dependent on the number of aromatic residues, particularly tryptophan, and can be predicted from the amino acid sequence. Thus, if the molar extinction coefficient is known, it can be used to determine the concentration of protein in solution.

Each publication or patent cited herein is incorporated by reference in its entirety.

The disclosure now generally described will be more readily understood by reference to the following examples, which are included merely for purposes of illustrating certain aspects of embodiments of the present disclosure. The examples are not intended to limit the disclosure, as one of ordinary skill in the art will recognize from the above teachings and examples that follow other techniques and methods can be satisfied by the claims and can be employed without departing from the scope of the claimed disclosure.

Examples

A material. Human lithium heparin plasma was purchased from bioremodelionivt (New York, u.s.a.). Streptavidin-coated Dyna beads M-280 was purchased from Invitrogen (CA, u.s.a.). IdeS, fabricotto, was purchased from Genovis corporation (Cambridge, MA). Other reagents include HBS-EP buffer containing 0.01M HEPES, pH7.4, 0.15M NaCl, 3mM EDTA, 0.005% polysorbate 20(GE Healthcare; Little Chalfount, U.K.) and peptide N-glycosidase F (PNGaseF; Prozyme; CA, U.S.A.). All TDCs and specific ADC capture reagents, such as ECD, were generated at Genentech (south san Francisco, CA, u.s.a.). The ECD was biotinylated with 10 molar equivalents of Sulfo-NHS-LC-Biotin (Pierce/Thermo Fisher Scientific, Rockford, IL, U.S. A.) for 60 minutes at room temperature in 10mM sodium phosphate/150 mM NaCl, pH 7.8. Excess unbound biotin was removed using a Zeba spin desalting column (Pierce/Thermo FisherScientific) following the manufacturer's protocol. The biotinylated ECD concentration was determined spectrophotometrically by measuring the absorbance at 280nm using GeneQuant 1300 (GEHealthcare).

Animal plasma samples. All animal studies were conducted in accordance with the National Institutes of health (National Institutes of health) guidelines for the Care and Use of laboratory animals (guidelines for the Care and Use of laboratory animals) and were approved by the institutional animal Care and Use Committee (institutional animal Care and Use Committee) of the Genentech company. For the PK study, female c.b-17SCID mice (charles river Laboratories) were administered a single dose of iv bolus injection of ADC and whole blood was collected from the animals via peripheral heart puncture. Blood samples were collected into tubes containing lithium heparin and allowed to stand on wet ice until centrifugation (within 15 minutes of collection). The collected plasma samples were stored at-70 ℃ until analysis. Plasma samples from Sprague-Dawley rats were obtained in a similar manner.

An apparatus. Affinity capture was performed on a KingFisher 96 magnetic particle processor (Thermo Electron) using a 2mL square top 96 deep well plate (Analytical Sales and Service, Pompton plates, NJ, u.s.a.). The eluate was transferred to a VWR Dynablock 96-well 0.5mL plate (VWR Scientific Products). In the coupling of SciexCapillary RPLC-MS was performed on a 5600 water nanoacquisition UPLC system (Cambridge, MA, u.s.a.) on a mass spectrometer (Redwood City, CA, USA).

Example 1: IdeS (generation 2) affinity capture LC-MS assay design and validation

Affinity capture LC-MS assays (Su, d.et al. (2016) anal. chem.,88(23): 11340-. To test and validate the multiplex assay of the present disclosure, IdeS protease (fig. 1A) removes the glycan-containing Fc region at specific sites, thereby reducing the size of the analyte and the heterogeneity of ADC catabolites. Bead elimination using IdeS is expected to rapidly generate F (ab')2 (about 100kDa) for final LC-MS analysis, instead of intact ADC (about 150 kDa). The reduced analyte size and rapid digestion provided by IdeS results in improved sensitivity and resolution, and minimal equivalent recovery of individual DAR species during the artificial drug modification or decomposition and enrichment process, compared to generation 1 affinity capture LC-MS used to test TDC for in vivo stability and PK assessment. This generation 2 affinity capture LC-MS showed surprising improvements for analysis of DAR and catabolite characterization of site-specific ADCs when tested against multiple TDCs with different antibodies, linker-drugs, and conjugation sites.

Fig. 1B provides a cartoon representation of the generation 2 LC-MS assay tested in this example, while fig. 1C shows a cartoon representation comparing embodiments of the generation 1 and generation 2 assays tested and compared in this example.

In vivo plasma samples were collected from mouse, rat and cynomolgus models to which TDC had been administered intravenously. All animal studies were conducted according to NIH experimental animal care and instructions for use. Plasma was purchased from biorelevationivt (New York, USA). All TDC and specific ADC capture reagents, such as the extracellular domain (ECD) and anti-human (Fab region) antibodies, were generated at Genentech (south san Francisco, CA, USA). ECD and anti-human (Fab region) antibodies were biotinylated with 10 molar equivalents of sulfoo-NHS-LC-biotin (Pierce/Thermo Fisher Scientific, Rockford, Ill., USA) for ECD or anti-human (Fab region) antibodies at 10mM sodium phosphate/150 mM NaCl, pH 7.8, for 60 minutes at room temperature. Using ZEBA^TMA rotating desalting column (Pierce/Thermo Fisher Scientific) followed the manufacturer's protocol to remove excess unbound biotin. By using GENEQUANT^TM1300(GE Healthcare) at 28The absorbance was measured at 0nm and the concentration of biotinylated ECD or anti-human (Fab region) antibody was determined spectrophotometrically.

Assay details for generation 1 affinity capture LC-MS have been previously described (Kaur et al, US 8541178; Xuet al, anal. biochem.2011,412(1): 56-66). For comparative testing of the 2 nd generation assay, 100 μ L of streptavidin paramagnetic beads (streptavidin-coated Dyna beads M-280; Invitrogen (CA, USA)) were added to a 96-deep well plate containing an excess of a specific biotinylated capture reagent, such as ECD, in HBS-EP buffer (300 μ L; 0.01M HEPES, pH7.4, 0.15M NaCl, 3mM EDTA, 0.005% P20(GE Healthcare; Little Chalfount, UK)) and incubated at Room Temperature (RT) for 1 hour with shaking. Plasma samples containing TDC were then added (maximum 2 μ g or 250 μ L, whichever is less) to ECD-immobilized beads to a total volume of 300-.

Affinity capture of biotinylated anti-human IgG F (ab')2 antibodies with a universal capture reagent is useful for capturing all the different humanized therapeutic antibody ADCs and is appropriate in studies with different therapeutic arms or in cases where no specific ECD is available. Results from the 2 nd generation LC-MS assay showed that the universal anti-human IgG F (ab')2 was similar to the specific ECD capture of the ADC analyte.

Affinity capture was performed on a KingFisher 96 magnetic particle processor (Thermo Electron) using 2-mL square top 96 deep well plates (Analytical Sales and Service, Pompton plants, NJ, USA). The eluate was transferred to a VWR Dynablock 96-well 0.5mL plate (VWR Scientific Products). Capillary RPLC-MS was performed on a Waters nanoACQUITY UPLC system (Cambridge, MA, USA) coupled to an AB Sciex 5600 triple time-of-flight (TOF) mass spectrometer (Redwood City, USA).

PNGaseF (PROZYME) described in relation to the 1st generation method^TM(ii) a CA) overnight digestion for comparison, affinity-captured TDC was treated with IdeS protease (FABRICATOR) in HBS-EP buffer (300. mu.L)^TMGenovis AB) (40 units) was digested at 37 ℃ for 1 hour. All shaking was carefully performed to ensure beads were in solution throughout the digestion protocolMedium to medium suspension is preferred. The newly generated F (ab')2 fragments were washed sequentially on the beads with HBS-EP buffer, water and 10% ACN, and then eluted by incubation at RT in 50 μ L of 30% acetonitrile containing 1% formic acid for 10 min. The subsequent F (ab')2 eluates were spun at 4000rpm for 10 minutes at RT in a Bruker centrifuge and then transferred to 96-well plates to remove residual beads with a magnet. The final elution was spun at 4000rpm for 10 minutes at RT before LC-MS to avoid injection of any residual beads. A5. mu.L aliquot eluted with F (ab')2 was submitted to LC-MS analysis.

Capillary LC-MS was performed on a tripleTOF 5600 mass spectrometer coupled to Waters NanoAcquisity. Preliminary desalting and preconcentration were performed on a PS-DVB single-root column (500- μm i.d.. times.5 cm, Thermo Fisher Scientific, Waltham, Mass.) using gradient conditions at 65 ℃ with mobile phases A, 0.1% Formic Acid (FA) and B, Acetonitrile (ACN) containing 0.1% FA at a flow rate of 15 μ L/min. The LC gradient was 0% B (0-4 min), 0-40% B (4-8 min), 40% B (8-11 min), 40-100% B (11-12.5 min), 100% B (12.5-13.5 min), 100-0% B (13.5-14.2 min), 0% B (14.2-15 min). The LC stream was diverted to waste for the first 6 minutes. The triple TOF 5600 was operated with the DuoSpray ion source with the following key settings: ion source temperature, 425.0 ℃; an ion source Gas (GS)1, 40; GS2, 35; curtain gas, 30; the ion ejection voltage floats, 5000V; declustering voltage, 250; collision energy, 20. Using Analyst^TMTF 1.6 mass spectra were acquired in intact protein mode. Using BIOANALYST^TM1.5.1 deconvolution is performed. Relative ratios of individual TDC DAR species are obtained based on peak areas in the deconvoluted mass spectra. The results calculated within ± 15% were considered to be not significantly different: average DAR ═ Σ (% peak area x number of conjugated drugs)/100.

For method development, direct LC-MS was tested and compared to both generation 1 and generation 2 affinity capture LC-MS. The total concentration of 100. mu.g/mL was used as DAR0: DAR2 ≈ 1:1 mixed naked antibody (DAR0) and TDC standard (DAR 2). For passage 1 and 2 affinity capture LC-MS, 20 μ L aliquots of TDC mixtures were spiked with 100 μ L human plasma to a final concentration of 20 μ g/mL, respectively. Another 10. mu.L aliquot of the TDC mixture was spiked with 50. mu.L of 30% ACN containing 1% FA and submitted directly to LC-MS analysis. A TDC mixture of known ratios allows for the determination of whether all individual DARs have similar LC-MS responses by DAR profiling of direct LC-MS. For example, if the relative DAR ratio by direct LC-MS is consistent with theoretical values, there is no significant shift in LC recovery and ionization efficiency for any individual DAR species. Agreement in DAR profiling by affinity capture LC-MS and direct LC-MS would suggest unbiased recovery of individual DAR species during the affinity enrichment process.

By injecting the same amount of starting TDC standard mixture, FIG. 1D shows a comparison between direct LC-MS (1D-1), generation 1 (1D-3) and generation 2 (1D-2) affinity capture LC-MS. Exemplary TDCs contain pyrrolobenzodiazepine dimer (PBD) as the cytotoxic drug payload. Similar retention times and charge envelopes (charge envelopes) suggest no significant difference in ionization efficiency of different TDC DAR species, allowing half-quantification using the relative ratio of individual DAR species (based on their peak areas in the deconvolution mass spectrum). The ADC catabolites contain glycation and/or other modifications. There was no significant difference in the relative ratio of DAR0 and DAR2 between direct LC-MS and generation 2 affinity capture LC-MS, indicating offset-free capture of individual DAR species (DAR0 and DAR 2). The 2 nd generation affinity capture method was further tested with a wide variety of TDC standards (DAR0 and DAR2) with different antibodies, conjugation sites in the Fab region, linkers (maleimide and disulfide), and toxins (DNA damaging agents, including anthracyclines, CBI dimers and PBD dimers, and tubulin conjugates). Similar ionization efficiencies and relative ratios of DAR0 and DAR2 were observed by the direct LC-MS approach, confirming that generation 2 affinity capture LC-MS is applicable to a wide variety of Fab site-specific antibody-drug conjugates.

PNGaseF digestion, passage 1, affinity Capture LC-MS assay method Using TDC (DAntibody drug conjugates) standard mixture comprising a cytotoxic drug monomethyl auristatin e (mmae) conjugated via a maleimido-hexanoyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB) linkerAmino acid engineered, anti-MUC 16 antibodies. This approach was later discovered to depend on the linker-drug and antibody showing different responses to individual DARs. The measured DAR0: DAR2 was compared to theoretical 1 with no significant difference from the calculated results, within ± 15%. The difference between measured DAR0: DAR2 from theoretical values for generation 1 and/or overnight digest of IdeS protease affinity capture LC-MS was significant, indicating that prolonged (e.g., overnight) bead digestion results in potentially biased recovery of the individual DAR during the affinity enrichment step. Shortened and optimized incubation times for ECD immobilization, ADC and ECD binding, and elimination steps on beads in generation 2 affinity capture LC-MS minimize the potential offset capture of different DARs and thus provide more accurate information on DAR profiling. Thus, the advantages of generation 2 affinity capture LC-MS include:

the MS intensity (maximum 230cps) of the 2 nd generation LC-MS is higher than that of the 1st generation analysis (maximum 48 cps).

The generation 2 affinity capture LC-MS allows detection of TDC down to about 20ng (0.2. mu.g/mL. times.100. mu.L).

The 2 nd generation assay resolved the adjacent MS peaks better.

More complete glycan removal was observed for the 2 nd generation capture LC-MS.

Fc removal by IdeS protease was completed in about 1 hour compared to deglycosylation by PNGaseF overnight digestion, greatly improving assay efficiency (1 day for generation 2 LC-MS versus 2 days for generation 1 LC-MS).

Affinity capture LC-MS was specifically designed to identify ADC catabolites, characterize DAR profiles, and thus understand ADC fate and PK behavior in the cycle. It is therefore important to preserve ADC integrity throughout the sample preparation process in order to accurately reflect in vivo biotransformation. However, using PNGaseF digestion, generation 1 affinity capture LC-MS assay, it was observed that ADCs containing unstable cytotoxic drugs undergo unintended changes after prolonged incubation, such as ex vivo payload metabolism (fig. 1F-1). Unstable TDC, TDC-L2 analysis by affinity capture LC-MS whole antibody assay (left) and F (ab')2 assay in rat plasma in vivo (fig. 1F-2). Artificial partial drug loss (-PD) from ex vivo payload metabolism was minimized by the IdeS digestion for one hour at 37 ℃ and affinity capture LC-MS F (ab')2 assay. TDC-L2 has a CBI dimer drug moiety and a maleimide linker. The production of such artificial ADC catabolites was minimized in generation 2 affinity capture LC-MS (fig. 1F-2), where IdeS digestion was completed within 1 hour. The shortened digestion time enables minimal unintended changes in ADC integrity and thus provides more accurate information about ADC biotransformation and PK behavior in vivo.

Figure 3A shows DAR profiling of TDC (PBD dimer drug, disulfide linker) standard mixtures (DAR0: DAR2 ═ 1:1) by direct LC-MS assay, affinity capture LC-MS F (ab')2 assay, and affinity capture LC-MS whole antibody assay, with standard deviations of 0.13, 0.09, and 0.14 for 3 replicates, respectively. LC-MS analysis was performed using affinity capture of human plasma (100 μ L) containing the spiked TDC standard mixture and injection of 5 μ L of eluate. Figure 3B shows the analysis of the TDC standard mixture (DAR0: DAR2 ═ 1:1) of PBD dimers (TDC1 and TDC4), anthracyclines (TDC2) and TDCs of CBI dimer (TDC5) drug modules with disulfide linkers covalently attached to cysteine engineered antibodies by direct LC-MS, affinity capture LC-MS F (ab ')2 assay with IdeS digestion for 1 hour, and DAR (drug-antibody ratio) profile of affinity capture LC-MS F (ab')2 assay with IdeS digestion overnight. LC-MS analysis was performed using affinity capture of human plasma (100 μ L) containing the spiked TDC standard mixture and injection of 5 μ L of eluate. By IdeS overnight digestion affinity capture LC-MS, the measured DAR0: DAR2 error of TDC2 was significant, indicating that prolonged on-bead incubation (e.g., overnight digestion) resulted in potentially biased recovery of different drug-loaded TDC2 species during sample preparation. For example, in affinity capture LC-MS F (ab')2 assays, the significantly shortened bead elimination time minimizes the potentially biased recovery of different ADC species and thus provides more accurate information on DAR assessment.

In the analysis of complex ADC catabolites, adjacent deconvolved MS peaks require near baseline separation to allow accurate assignment of ADC catabolite structures. FIG. 1E shows linker-drug deconjugation (-LD) by cleavage of the thiol-maleimide bond. Due to the loss of 42Da from the drug molecules, a variety of TDC catabolites were generated in vivo in mouse plasma. Their MS peaks were not resolved by PNGaseF digestion, generation 1 affinity capture LC-MS (fig. 1E-1), but by IdeS digestion, generation 2 affinity capture LC-MS gave near baseline resolution (fig. 1E-2), which enables reliable catabolite identification and more accurate DAR calculation. This precise information helps understand ADC efficacy and toxicity profiles as well as ADC drug metabolism, which in turn helps optimize new cytotoxic ADC designs, focusing on new types of antibody platforms, conjugation chemistries, linkers, and drugs. The multiple drugs and metabolic parameters and complex in vivo catabolic organisms that must be validated during drug development of ADCs pose challenges to bioanalytical analysis. For example, the discovery of generation 1 affinity capture LC-MS (an exploratory assay useful for DAR and catabolite characterization) is generally not suitable for next generation ADCs due to its limited sensitivity, resolution, efficiency and potentially offset response to certain DAR species. This PNGaseF digestion, the 2 nd generation assay, which accommodates low dose and unstable site-specific ADCs, became dominant in drug development. FIG. 1E shows characterization of complex TDC catabolites in mouse plasma in vivo by affinity capture LC-MS whole antibody assay (1E-1) versus affinity capture LC-MS F (ab')2 assay (1E-2). Partial drug loss by linker-drug deconjugation (-PD) significantly affected the potency of TDC-L1, resulting in a corresponding reduction in DAR.

The generation 2 affinity capture method employed in this comparative example utilizes IdeS protease for deglycosylation by removing most of the localized Fc fragment in glycans. The resulting F (ab')2 fragment of the linker-drug (about 100kDa) was retained by LC-MS analysis instead of the traditional intact ADC (about 150 kDa). Compared to deglycosylation by overnight digestion with PNGaseF, rapid removal of Fc and reduced F (ab')2 analyte size by IdeS digestion on beads resulted in improved assays with greater sensitivity, resolution, and efficiency, and minimal unintended changes in ADC profile and integrity during sample processing, as summarized in the following table:

table: variations and modifications to the 1st generation assay

The affinity capture LC-MS F (ab')2 assay was extended to analyze conventional ADCs where the drug was bound via interchain disulfide. Without IdeS protease digestion, LC split was required to elute the light chain (25kD) and heavy chain (50kD) fragments after overnight deglycosylation by PNGaseF, as the light chain fragment (smaller size) suppressed the ionization/MS signal of the heavy chain fragment (larger size). With IdeS protease digestion, the light and heavy chains are of similar size (about 23-29kDa) and can be eluted and analyzed simultaneously, with minimal MS bias against the heavy chain. The affinity capture LC-MSF (ab ')2 assay allows parallel comparison of ADC biotransformations between ADCs with the same drug conjugated to different antibodies when using a universal capture reagent, such as a biotinylated anti-human F (ab')2 antibody. For more accurate and detailed bioconversion and PK information, this comparative example demonstrates the potential of IdeS protease digestion, passage 2 assays to accommodate low dose, unstable, and complex site-specific ADCs, such as TDCs. Such information helps to optimize cytotoxic drug design, drive the development of appropriate PK bioanalytical strategies, and lead to the discovery of new ADC catabolites. The method is applicable to a variety of site-specific ADCs with conjugation sites in the Fab region, and to analysis of conventional ADCs conjugated via interchain disulfides.

Example 2: protein concentration determination

Accurate protein concentration determination is crucial for assessing the in vitro and in vivo efficacy, as well as toxicity, of protein-drug conjugates. The inventors have developed a method to determine protein concentration for ADCs containing small molecule payloads that contribute to the absorbance of the protein at 280nm, for example due to the presence of aromatic rings (particularly when the extinction coefficient of the small molecule at 280nm and/or its maximum absorbance is unknown). In this example, protein concentration was determined independently of the conjugated payload by proteolytic digestion with immunoglobulin degrading enzyme (IdeS) of streptococcus pyogenes and subsequent LC-MS analysis.

IdeS cleaves human IgG1 at a site below the hinge region with high specificity, generating F (ab')2 and Fc fragments (fig. 1A). These species can be separated chromatographically on reversed phase (FIG. 2A-1). The non-covalent interaction between the two arms of Fc is disrupted by the acidic and organic solvent concentrations of the mobile phase, resulting in an Fc/2 peak of about 25kD in size. In ADCs where the drug payload is conjugated to an interchain disulfide or site-specifically to F (ab ')2 or Fc region, the resulting drug-free antibody fragment (either of the Fc/2 or F (ab')2 fragment peaks) can be used to quantify the protein concentration of the sample. This method is useful for characterizing both traditional ADCs via interchain disulfide conjugation and THIOMABTM antibody drug conjugates (TDCs) with each antibody having two engineered cysteine residues for site-specific conjugation located in either the Fab or Fc regions. The antibody-drug conjugate was digested with IdeS and then injected onto reverse phase LC-MS and detected at absorbance of 280 nm. In the case of TDC conjugated on Fab and traditional ADC, the antibody fragment containing the drug is separated from the Fc/2 fragment by chromatography, allowing protein concentration quantification using Fc/2 fragment peak area. This value was interpolated using linear regression of a standard curve of an antibody standard digested with IdeS, where the starting concentration was plotted against the Fc/2 peak area (fig. 2A). In the case of TDC via engineered cysteine conjugation on Fc, the drug-containing Fc/2 fragment is separated from the F (ab ')2 fragment by chromatography, allowing protein concentration quantification using F (ab')2 fragment peak area. This value was interpolated using linear regression of a standard curve of antibody standards digested with IdeS, where the starting concentration was plotted against the F (ab')2 peak area (fig. 2A).

Standard curves (Fc/2 peak area versus concentration; FIG. 2B, top) over the range of 0.5-20mg/ml were generated using trastuzumab digested with IdeS protease (F (ab')2 peak area versus concentration; FIG. 2B, bottom). The protein concentration of TDC specifically conjugated at the upper site on f (ab) can be determined using the peak area of Fc/2 of TDC (top curve) and linear regression. This approach can also be used to characterize traditional ADCs conjugated on interchain disulfides, since the Fc/2 fragment in these conjugates is also drug-free. The protein concentration of TDC specifically conjugated at the upper site on Fc can be determined using the peak area of F (ab')2 of TDC (bottom curve) and linear regression.

Method of producing a composite material

Thiomab with 2 engineered cysteine residues per antibody^TMIncubated with 3-fold molar excess of thiol-reactive linker-drug for 2 hours at pH 7.5. Excess linker-drug was purified away by cation exchange and the conjugate was formulated in pH 5.5 buffer. Drug-to-antibody ratios (DAR) for TDCs were determined by LC-MS analysis using the abundance of deconvoluted masses with and without drug species (fig. 2A-2). All conjugates examined had a DAR of ≧ 1.7. The size range of the linker-drug payload is 700 and 1500 Da.

Mixing 30 units (30U/. mu.l) of IdeS (Fabrictor)^TMGenovis AB) to 10 μ l of antibody or antibody-drug conjugate at a concentration ranging from 0.52 to 20 mg/ml. The reaction mixture was adjusted to a final volume of 50. mu.l with PBS and the final reaction pH was about pH 6.5. Samples were incubated for 1 hour at 37 ℃ prior to LC-MS analysis. Samples were analyzed by reverse phase High Performance Liquid Chromatography (HPLC) using an HPLC system (Agilent 1260 definition) coupled with an electrospray ionization time-of-flight mass spectrometer (Agilent 6224 TOF-LC). Samples were injected in 10. mu.l volumes on PLRP-S1000A °,8 μm 50X 2.1mm column (Agilent) heated to 80 ℃. A gradient was generated using 0.05% trifluoroacetic acid (mobile phase A) and acetonitrile containing 0.05% trifluoroacetic acid (mobile phase B) at a flow rate of 0.5 ml/min. The column was held at 5% B for 0.7 min, followed by a gradient of 30% B to 40% B for 4.3 min. At 5 minutes, the concentration increased to 90% B, held for 1 minute. The column was then equilibrated in 5% B for an additional 2 minutes. Data were acquired and analyzed using AgilentMassHunter software.

Deconvolution mass spectrometry data was used to confirm that all antibodies and antibody drug conjugates had been digested completely and without intact antibodies.

Trastuzumab (HERCEPTIN) digested with IdeS of known concentration was used^TM(ii) a Anti Her2 human IgG1 antibody) generated a standard curve. Preparing a site-specific trastuzumab construct having:

1) linker-drug site-specifically conjugated at engineered cysteine K149C on f (ab) (conjugate a; fig. 2C);

2) linker-drug site-specifically conjugated at engineered cysteine S400C on Fc (conjugate B; fig. 2D); or

3) Linker-drug conjugated on interchain disulfide (conjugate C; fig. 2E).

Trastuzumab was serially diluted from 20mg/ml to 0.52mg/ml in 20mM histidine acetate pH 5.5, 240mM sucrose with a factor of 1.5. This buffer was chosen for dilution because it is the final formulated buffer for many ADCs and mimics the conditions of the experimental samples in the assay. Samples were diluted in triplicate for a total of ten concentrations.

Volumes of 10. mu.l of each dilution were added to 39. mu.l of phosphate buffered saline pH 7.2 and 1. mu.l of IdeS (30U/. mu.l) was added to a total volume of 50. mu.l. The final pH of these samples was in the optimal activity range for IdeS activity. The samples were incubated at 37 ℃ for 1 hour. The samples were then run on LC-MS sequentially or at increasing concentrations. A volume of 10. mu.l of antibody digest was injected onto the reverse phase column and eluted with a gradient to separate the Fc/2 and F (ab')2 peaks. Samples resulting in injection of >5 μ g antibody were followed by a blank run of the same LC-MS method without injection to ensure no sample was left to the next run.

The protein concentration of the antibody-drug conjugate was determined by proteolytic digestion with IdeS and subsequent LC-MS analysis. Standard curves were generated using trastuzumab digested with known concentrations of IdeS ranging from 0.52mg/ml to 20mg/ml (fig. 2C, 2D, 2E). Standards were digested and run in triplicate with minimal error. The starting concentration is plotted against the peak area,generating a linear regression (R) of the set of standards²0.9999) (fig. 2F). This linear regression equation was used to determine the protein concentration of unknown samples according to the following table:

1) for linker-drugs specifically conjugated at the f (ab) site (fig. 2C):

2) for linker-drug specific conjugation at the site on the Fc (FIG. 2D)

3) For linker-drug conjugation on interchain disulfide (FIG. 2E)

Protein concentrations of 81 TDCs were determined using this method and by BCA, a accepted colorimetric assay for protein concentration determination. Concentration values determined by each method were plotted against each other, showing strong correlation, verifying the accuracy and reproducibility of IdeS digestion method (fig. 2F). The concentration values determined by this method can then be used to calculate the extinction coefficient of a TDC or ADC at a280 absorbance using Beer-Lambert's law.

Figure 3A shows DAR profiling of TDC (PBD dimer drug, disulfide linker) standard mixtures (DAR0: DAR2 ═ 1:1) by direct LC-MS assay, IdeS digestion, affinity capture LC-MS F (ab')2 assay, and PNGaseF, affinity capture LC-MS whole antibody assay, with standard deviations of 0.13, 0.09, and 0.14 for 3 replicates, respectively. LC-MS analysis was performed using affinity capture of human plasma (100 μ L) containing the spiked TDC standard mixture and injection of 5 μ L of eluate.

Figure 3B shows TDC standard mixtures (DAR0: DAR2 ═ 1:1) with PBD dimers (TDC1 and TDC4), anthracyclines (TDC2) and TDCs of CBI dimer (TDC5) drug modules covalently attached with disulfide linkers to cysteine engineered antibodies were analyzed by direct LC-MS, IdeS digestion affinity capture LC-MS F (ab ')2 assay with IdeS digested for 1 hour, and DAR (drug-to-antibody ratio) profile of affinity capture LC-MS F (ab')2 assay with PNGaseF digested overnight (green). LC-MS analysis was performed using affinity capture of human plasma (100 μ L) containing the spiked TDC standard mixture and injection of 5 μ L of eluate. The error in measurement of DAR0: DAR2 by IdeS overnight digestion affinity capture of TDC2 of LC-MS was significant, indicating that prolonged on-bead incubation (e.g., overnight digestion) resulted in potentially biased recovery of different drug-loaded TDC2 species during sample preparation.

These methods provide a fast, robust, and reproducible assay for protein concentration determination of ADCs, regardless of the spectral characteristics of the payloads to which they are conjugated. Antibodies conjugated to other experimental payloads such as fluorophores can also be assayed using this method to determine their protein concentration, independent of fluorophore or absorbance. Protein concentrations of conventional ADCs can also be determined by this method, particularly for ADCs in which the payload is conjugated to interchain disulfide, since the Fc/2 region does not have these conjugation sites.

The assay of the invention involves a relatively rapid digestion step and continues to completion without over-digestion. Thus, this methodology has significant advantages over the use of conventional proteases such as pepsin, papain, and endopeptidase lysine C. For example, pepsin digestion of IgG1 occurs slower and preferably below pH 5. Papain digestion is performed at neutral pH and the site of protein cleavage is not specific and often causes multiple protein cleavage events. Limited LysC digestion may over digest the Fc region. IdeS does not risk over-digestion because it cleaves the IgG1 antibody at a specific site on the heavy chain below the hinge region. IdeS is stable in solution at 4 ℃ for up to one month. These features contribute to the robust protein concentration assay presented herein.

For routine concentration measurements, the method can be performed on HPLC without an online mass spectrometer. For example, trastuzumab standards are digested and analyzed along with test samples for quality control. Complete digestion can be confirmed in this assay because the intact antibody has a known retention time and can be detected without mass spectrometry. The peak area of absorbance at 280nm was then directly correlated with protein concentration without the need for MS analysis.

Although the foregoing invention has been described in some detail by way of illustration for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Sequence listing

<110> Gene Tak corporation (Genentech, Inc.)

Haofume Roche GmbH (F. Hoffmann-La Roche AG)

<120> bioanalytical method for characterization of site-specific antibody-drug conjugates

<130>P33200-WO

<141>2017-05-26

<150>US 62/342,825

<151>2016-05-27

<160>2

<210>1

<211>18

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>1

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

1 5 10 15

Ser Val Phe

<210>2

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>2

His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser

1 5 10 15

Val Phe

Claims (24)

1. A method of evaluating an Antibody Drug Conjugate (ADC), comprising:

(a) digesting an ADC comprising at least one drug moiety attached to an antibody at a recombinantly engineered site selected from the group consisting of a cysteine amino acid residue, a selenocysteine amino acid residue, a glutamine amino acid residue, a non-naturally occurring amino acid residue, and a glycomodified glycan residue with a protease that cleaves the ADC to form a digested ADC composition comprising at least one peptide fragment not attached to the at least one drug moiety and at least one peptide fragment attached to the at least one drug moiety; and are

(b) Analyzing the digested ADC composition by High Performance Liquid Chromatography (HPLC) and/or Mass Spectrometry (MS) to detect at least one peptide fragment not linked to the at least one drug moiety.

2. The method of claim 1, wherein the antibody is selected from the group consisting of an IgG antibody, an antibody fragment, a human or humanized antibody, a glycosylated or phosphorylated antibody, and a cysteine engineered antibody.

3. The method of claim 1 or 2, wherein the antibody portion of the ADC is an antibody that binds to one or more tumor associated antigens or cell surface receptors selected from (1) - (53):

(1) BMPR1B (bone morphogenetic protein receptor-type IB);

(2)E16(LAT1，SLC7A5)；

(3) STEAP1 (six transmembrane epithelial antigen of prostate);

(4)MUC16(0772P，CA125)；

(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiator, mesothelin);

(6) napi2B (Napi-3B, NPTIIb, SLC34a2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3B);

(7) sema5B (FLJ10372, KIAA1445, mm.42015, Sema5B, SEMAG, semaphorin 5B Hlog, Sema domain, seven thrombospondin repeats (type 1 and type 1), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B);

(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA2700050C12, RIKEN cDNA2700050C12 gene);

(9) ETBR (endothelin type B receptor);

(10) MSG783(RNF124, hypothetical protein FLJ 20315);

(11) STEAP2(HGNC _8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-associated gene 1, prostate cancer-associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein);

(12) TrpM4(BR22450, FLJ20041, TrpM4, TrpM4B, transient receptor potential cation channel, subfamily M, member 4);

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

(14) CD21(CR2 (complement receptor 2) or C3DR (C3 d/epstein-barr virus receptor) or Hs 73792);

(15) CD79B (CD79B, CD79 β (immunoglobulin-related β), B29);

(16) FcRH2(IFGP4, IRTA4, spa 1A (SH 2 domain containing phosphatase dockerin 1a), spa 1B, spa 1C);

(17)HER2；

(18)NCA；

(19)MDP；

(20)IL20Rα；

(21) curdlan;

(22)EphB2R；

(23)ASLG659；

(24)PSCA；

(25)GEDA；

(26) BAFF-R (B cell activating factor receptor, BLyS receptor 3, BR 3);

(27) CD22(B cell receptor CD22-B isoform);

(28) CD79a (CD79A, CD79 α, immunoglobulin related α);

(29) CXCR5 (burkitt's lymphoma receptor 1);

(30) HLA-DOB (β subunit of MHC class II molecules (Ia antigen));

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

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

(33) LY64 (lymphocyte antigen 64(RP105), a Leucine Rich Repeat (LRR) family of type I membrane proteins);

(34) FcRH1(Fc receptor-like protein 1);

(35) FcRH5(IRTA2, 2 associated with receptor translocation of the immunoglobulin superfamily);

(36) TENB2 (putative transmembrane proteoglycan);

(37) PMEL17(silver homolog; SILV; D12S 53E; PMEL 17; SI; SIL);

(38) TMEFF1 (transmembrane protein 1 with EGF-like and two follistatin-like domains; Tomoregulin-1);

(39) GDNF-Ra1(GDNF family receptor α 1; GFRA 1; GDNFR; GDNFRA; RETL 1; TRNR 1; RET 1L; GDNFR- α 1; GFR- α -1);

(40) ly6E (lymphocyte antigen 6 complex, locus E; Ly67, RIG-E, SCA-2, TSA-1);

(41) TMEM46(SHISA homolog 2 (Xenopus laevis); SHISA 2);

(42) ly6G6D (lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT 1);

(43) LGR5 (leucine rich repeat-containing G protein-coupled receptor 5; GPR49, GPR 67);

(44) RET (RET proto-oncogene; MEN 2A; HSCR 1; MEN 2B; MTC 1; PTC; CDHF 12; Hs.168114; RET 51; RET-ELE 1);

(45) LY6K (lymphocyte antigen 6 complex, locus K; LY 6K; HSJ 001348; FLJ 35226);

(46) GPR19(G protein-coupled receptor 19; Mm.4787);

(47) GPR54(KISS1 receptor; KISS 1R; GPR 54; HOT7T 175; AXOR 12);

(48) ASPHD 1(1 containing aspartate β -hydroxylase domain; LOC 253982);

(49) tyrosinase (TYR; OCAIA; OCA 1A; tyrosinase; SHEP 3);

(50) TMEM118 (Ring finger protein, transmembrane 2; RNFT 2; FLJ 14627);

(51) GPR172A (G protein-coupled receptor 172A; GPCR 41; FLJ 11856; D15Ertd747 e);

(52) CD 33; and

(53)CLL-1。

4. the method of any one of claims 1 to 3, wherein the drug moiety is linked to the antibody moiety of the ADC via a linker and is selected from the group consisting of peptides, polyamides, maytansinoids (maytansinoids), dolastatins (dolastatins), auristatins (auristatins), calicheamicins (calicheamicins), Pyrrolobenzodiazepines (PBD), PNU-159682, anthracyclines (anthracyclines), duocarmycins (duocarmycins), vinca alkaloids (vinca alkloids), taxanes (taxanes), trichothecenes (trichothecenes), CC1065, duocarmycins (duocarmycins), camptothecins (camptothecins), elenides (elinafifides), antibiotics, fluorophores, radioisotopes, and stereoisomers, isosteres, metabolites, analogs or derivatives thereof.

5. The method of any one of claims 1 to 4, wherein the protease is selected from the group consisting of: IdeS protease, IdeZ protease, IgdE protease, SpeB protease, gingipain (gingipain), endoglycosidase, and combinations thereof.

6. The method of any one of claims 1 to 5, wherein the digesting comprises incubating the ADC with the protease at a temperature of between about 20 ℃ and about 45 ℃ at a pH of between about pH 5 and about pH 9 for a period of time of between about 0.1 hour and about 48 hours.

7. The method of claim 6, wherein the digesting comprises incubating the ADC with the protease at a temperature of about 37 ℃ for a period of about 1 hour at a pH of about 7.

8. The method of any one of claims 1 to 7, wherein the analysis comprises at least one of RP-LC, RP-LC/MS and LC-MS/MS.

9. The method of any one of claims 1 to 8, wherein prior to the digesting step, the ADC is suspended in a matrix selected from the group consisting of buffer, whole blood, serum, plasma, cerebrospinal fluid, saliva, urine, lymph, bile, feces, sweat, vitreous fluid, tears, and tissue.

10. The method of any one of claims 1 to 9, wherein the ADC is suspended in whole blood, serum, plasma, or tissue of a mammal selected from the group consisting of human, cynomolgus monkey, rat, and mouse.

11. The method of any one of claims 1 to 10, wherein prior to the digestion step, the ADC is enriched by a technique selected from the group consisting of size exclusion chromatography, dialysis, selective precipitation, differential centrifugation, filtration, gel electrophoresis, liquid chromatography, reverse phase chromatography, immunoprecipitation, SpinTrap column comprising protein a and protein G, NHS and streptavidin iron or phosphorous or immobilized antibodies or lectins, paramagnetic beads, immuno-subtraction, fractionation, solid phase extraction, phosphopeptide enrichment, polyacrylamide gel electrophoresis, and desalting.

12. The method of any one of claims 1 to 11, wherein the ADC is bound to an affinity capture medium comprising at least one of a bead or resin supported protein a/G, a target antigen-paramagnetic bead capture medium, an anti-idiotypic antibody, an anti-Hu antibody, and an anti-drug antibody.

13. The method of claim 12, further comprising washing the ADC bound to the affinity capture medium to reduce non-antibody proteins in contact with the ADC.

14. The method of claim 12, further comprising dephosphorylating the ADC bound to the affinity capture medium.

15. The method of claim 12, wherein the digesting step occurs simultaneously with binding of the ADC to the affinity capture medium.

16. The method of claim 12, further comprising eluting the ADC from the affinity capture medium prior to the step of digesting the ADC.

17. The method of any one of claims 1 to 16, wherein the total antibody concentration of the ADC is calculated from an analysis of the digested ADC composition.

18. The method of any one of claims 1 to 17, wherein the antibody conjugated drug concentration of the ADC is calculated from an analysis of the digested ADC composition.

19. The method of any one of claims 1 to 18, wherein the average drug-to-antibody ratio (DAR) of the ADC is calculated from an analysis of the digested ADC composition.

20. The method of any one of claims 1 to 19, wherein metabolite (metabolate) or catabolite (catabolate) structures are determined from analysis of the digested ADC composition.

21. The method of any one of claims 1 to 20, wherein the protein concentration of the ADC is calculated from an analysis of the digested ADC composition.

22. The method of claim 21, wherein the protein concentration is correlated with peak area from RP-LC and/or MS analysis of at least one Fc fragment from the digested ADC.

23. The method of any one of claims 1 to 22, wherein the extinction coefficient of the ADC is calculated from an analysis of the digested ADC composition.

24. The method of any one of claims 1 to 23, wherein an average drug-to-antibody ratio (DAR) of the ADC, metabolite or catabolite structure is determined from the analysis of the digested ADC and the protein concentration is correlated with peak area from RP-LC and/or MS analysis of at least one Fc fragment of the digested ADC.