Methods and tools for conjugation to antibodies

Abstract

The present invention relates to an antibody comprising a tag that can be covalently conjugated to a primary amino group of a linker in the presence of microbial transglutaminase. Also provided by the present invention are methods for producing antibody-linker-conjugates by mixing an antibody with a linker and microbial transglutaminase. Also provided are kits of parts comprising antibody-linker conjugates and antibody-drug-conjugates producible according to the methods of the present invention, as well as buffers and microbial transglutaminase.

Description

[Technical field]

[0001] The present invention relates to an antibody comprising a tag that can be covalently conjugated to a primary amino group of a linker in the presence of microbial transglutaminase. Also provided by the present invention are methods for producing antibody-linker-conjugates by mixing an antibody with a linker and microbial transglutaminase. Also provided are kits of parts comprising antibody-linker conjugates and antibody-drug-conjugates producible according to the methods of the present invention, as well as buffers and microbial transglutaminase. [Background technology]

[0002] Antibody-drug conjugates or ADCs are a class of biopharmaceuticals designed as targeted therapies, for example to treat cancer (Hamilton GS (September 2015). “Antibody-drug conjugates for cancer therapy: The technological and regulatory challenges of developing drug-biologic hybrids”. Biologicals. 43(5): 318-32). Unlike chemotherapy, ADCs are intended to specifically treat target cells while minimizing drug exposure for healthy cells. A stable link between the antibody and the drug (e.g., cytotoxic (anticancer) agent) is a key aspect of ADCs. A stable ADC linker ensures that the cytotoxic payload is slightly degraded before reaching the target cells, e.g., tumor cells, improving safety and reducing dosage. Drugs linked to the antibody via the linker include therapeutic agents, such as cancer treatment agents for different solid tumor indications, including CRC, pancreatic cancer, gastric cancer, NSCLC, esophageal cancer and prostate cancer.

[0003] The production of antibody-drug conjugates (ADCs) involves a bioconjugation step that requires the attachment of APIs (active pharmaceutical ingredients), which are chemically synthesized small molecule drugs, to biotechnologically produced targeting moieties, such as antibodies. The attachment of APIs to monoclonal antibodies (mAbs) is preferably done in a strictly controlled manner with respect to the conjugation sites and the number of drugs attached (specific drug-to-antibody ratio DAR). One way to obtain such site-specific conjugates is by the use of microbial transglutaminase (mTG, Tgase). However, the conjugation of APIs with mTG typically requires prior modification of the mAb, for example, either by genetic engineering or by removal of native glycosylation. These modifications prevent the direct use of native mAbs and may lead to negative effects on certain properties of the final ADC product.

[0004] New technologies are needed to overcome such negative effects and allow more flexibility in ADC design. In view of the above, there remains a need for new and improved antibody-linker conjugation methods that provide higher drug-to-antibody ratios (DAR). Summary of the Invention

[0005] Transglutaminase from S. ladakanum is described in US 6660510 B2. Methods for conjugation of native mAbs using transglutaminase are mentioned in WO 2019 / 057772 and WO 2020 / 188061 A1. However, these publications do not contain sufficient information about ideal reaction buffer conditions and lack additional measures to maximize conjugation efficiency, including preferred conjugation tags that can be used for the antibodies provided by the present invention.

[0006] The present invention addresses this need and provides methods to achieve site-specific conjugation of two or more linkers (e.g., drug-linkers) to a fully glycosylated antibody. One targeted conjugation site is glutamine Q295 (Eu numbering) on ​​the heavy chain of the antibody, which is very close to the typical N-glycosylation site N297. Conjugation to this position 295 is facilitated by using certain transglutaminases, certain reaction conditions, specific mutations or insertions on the antibody sequence (e.g., glutamine-containing peptide tags), or a combination of the former. In some cases, conjugation is targeted not only to Q295 but also to additional positions simultaneously, producing ADCs with, for example, four or six drug-linkers attached. These additional positions are located either on peptide tags fused or integrated into the antibody. Some ADCs with drug-to-antibody ratios of two or more exhibit excellent pharmacological in vivo properties (PK).

[0007] Although the use of peptide tags fused to mAbs as MTG recognition motifs has been described, it was an unexpected finding disclosed herein that a particularly suitable combination of an improved reaction buffer system and an alternative peptide tag could achieve a particularly high DAR as shown in the examples provided herein.

[0008] These novel enzyme-based conjugation methods provide improved antibody-drug conjugates, so that various antibodies can be used in the methods of the present invention. The methods of the present invention can be used to provide, for example, immunoconjugates (also referred to herein as antibody-drug conjugates (ADCs)) that contain monoclonal antibodies against CEACAM5. These immunoconjugates have cytotoxic effects, killing tumor cells in vitro and inhibiting tumor growth in vivo. However, the application of the present invention is not limited to anti-CEACAM5 antibodies, rather, the present invention is applicable to the purpose of preparing antibody-linker conjugates or antibody-drug conjugates based on antibodies against any target. The present invention relates to the embodiments described in the claims and further described herein below.

[0009] Aspects of the invention relate to antibody modification and payload conjugation strategies that significantly reduce off-target cellular catabolism of such ADCs, thereby reducing the levels of released payload while improving efficacy through higher ADC exposure. These modifications thus provide drugs with improved therapeutic windows with reduced side effects and increased antitumor activity. As described herein, the exposure and half-life of ADCs in accordance with the invention may be improved by, for example, modifying the antibody heavy chain with YTE mutations and / or by using an innovative enzymatic conjugation method that uses a microbial transglutaminase (also referred to herein as "TGase" and "mTG") enzyme to attach the drug to the antibody.

[0010] Surprisingly, high exposure leading to low clearance values ​​in human predictive animal models was obtained by the above-mentioned approach. Such modifications improved the exposure of the ADC by a factor of about 10x compared to variants that did not contain these modifications. This resulted in a significant reduction in the released payload levels and a significant increase in the concentration of antitumor active ADC in circulation. The improved ADC resembled the PK profile of the original YTE antibody without the drug linker and showed an improved half-life over the ADCs of the prior art.

[0011] These results represent reduced toxicity and improved efficacy in clinical cancer therapy compared to prior art molecules. [Brief description of the drawings]

[0012] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1: Typical SEC chromatograms showing the purity of input mAb and final BDS for ADC7-M, ADC2-M, and ADC5-M. [Diagram 2] Figure 2: Typical RP-HPLC chromatograms illustrating the DAR determination of the final BDS for ADC7-M, ADC2-M and ADC5-M. [Figure 3-1] Figure 3: In vitro efficacy of ADC2-M, ADC5-M, and free payload against antigen-positive SK-CO-1 (Fig. 3a), SNU-16 (Fig. 3b), MKN-45 (Fig. 3c), and LS174T (Fig. 3d) cell lines compared to antigen-negative MDA-MB-231 (Fig. 3e) cell line. One representative experiment (mean ± SD of duplicate experiments) is shown. [Figure 3-2] Figure 3: In vitro efficacy of ADC2-M, ADC5-M, and free payload against antigen-positive SK-CO-1 (Fig. 3a), SNU-16 (Fig. 3b), MKN-45 (Fig. 3c), and LS174T (Fig. 3d) cell lines compared to antigen-negative MDA-MB-231 (Fig. 3e) cell line. One representative experiment (mean ± SD of duplicate experiments) is shown. [Figure 3-3] Figure 3: In vitro efficacy of ADC2-M, ADC5-M, and free payload against antigen-positive SK-CO-1 (Fig. 3a), SNU-16 (Fig. 3b), MKN-45 (Fig. 3c), and LS174T (Fig. 3d) cell lines compared to antigen-negative MDA-MB-231 (Fig. 3e) cell line. One representative experiment (mean ± SD of duplicate experiments) is shown. [Figure 4] Figure 4 Pharmacokinetic profiles in huFcRn Tg276 mice for ADC1, ADC1-M, ADC2-M, ADC6-M and ADC7-M (total antibody). [Diagram 5] FIG. 5 Tumor volume change following treatment with ADC1-M and ADC2-M versus vehicle control. [Figure 6] Figure 6. Potent bystander effect of ADC1-M, ADC2-M, ADC6-M and ADC7-M on antigen-negative MDA-MB-231 cells in co-culture with antigen-positive SK-CO-1 cells compared to ADC SAR DM4. One representative experiment (mean ± SD of duplicate experiments) is shown. [Figure 7] FIG. 7 Efficacy of ADC1-M and ADC3-M compared with ADC8 in the HPAF-II xenograft model. [Figure 8-1]Figure 8 (A) Scheme of small molecule drug / molecule conjugation to native fully glycosylated mAb. [Figure 8-2] FIG. 8 (B) Exemplary drug-linkers used as mTG substrates: Gly3-valine-citrulline-MMAE ("4M" = "Drug-Linker 4M") and Gly3-beta-glucuronide exatecan ("1M"). Also referred to herein as "Drug-Linker 1M" or "Drug-Linker Compound 1-M", "Compound DL1-M" or "DL1-M". [Figure 9] Figure 9 (A) Conjugation of drug-linker 4M to native mAb trastuzumab using MTG from S. mobaraensis. 5 mg / mL antibody, 20 molar equivalents of drug-linker, and various concentrations of mTG (purchased from Zedira, Germany) were mixed in 10% DMSO, 150 mM NaCl, 25 mM Tris-HCl, pH 8.0. The amount of enzyme was gradually increased from 0 U / ml to 75 U / ml. The reaction was incubated for 16 h and stopped with MTG blocker (Zedira), followed by DAR determination using RP-HPLC as described in section 11.3.5. A DAR of 2 could not be reached even at high enzyme concentrations when using this prior art buffer, 150 mM NaCl, 25 mM Tris-HCl, pH 8.0 (i.e., as described in Strop, et al.; Chemistry & Biology (2013). 20(2), 161-167 or Dickgiesser, et al.; Bioconjugate Chemistry 2020 31(4), 1070-1076) (. (B) mTG from S. ladakanum (mTG variant 1, i.e., MTG_1 according to SEQ ID NO: 45), and the same as (A) (150 mM NaCl, 25 mM Tris-HCl, pH 8.0) or Dickgiesser et al. al. Reactions were performed as described in 11.3.4, with increasing amounts of enzyme ranging from 2.5 to 20 U / ml. Although conjugation efficiency was higher with this mTG variant, a DAR of 2 was still not reached with this prior art buffer. [Figure 10]Figure 10. Scheme of the high-throughput HTRF (homogeneous time-resolved fluorescence) procedure. Cadaverine-biotin is conjugated to native trastuzumab using mTG (A). The conjugated antibody is then incubated with commercially available streptavidin-d2 and terbium-labeled anti-IgG antibody (B). Once d2 and terbium are in close proximity, the HTRF signal can be read (C). [Figure 11-1] Figure 11. (A) Effect of pH variation on transglutaminase-mediated bioconjugation in a wide range of buffer systems as indicated in the legend. [Figure 11-2] FIG. 11. (B) Effect of salt concentration (in mM NaCl) on transglutaminase conjugation efficiency for HEPES and Tris-HCl buffer systems. (C) Effect of buffer substance HEPES concentration on transglutaminase conjugation efficiency. [Figure 12] FIG. 12. (A) Conjugation of drug-linker 4M to native fully glycosylated mAb using S. ladakanum mTG in different buffer systems obtained from high-throughput screening. Buffers are as indicated, control buffer is 25 mM Tris-HCl, 150 mM NaCl, pH 8.0. Improved buffer with 24 mM HEPES at pH 7.5 performed best. (B) Comparison of conjugation between improved buffer and prior art (150 mM NaCl, 25 mM Tris-HCl, pH 8.0) buffer with increasing mTG. While efficient conjugation of drug-linker 4M in improved buffer can be achieved with only 10 U / ml mTG, achieving DAR=2 using S. ladakanum mTG in prior art buffer typically requires much more mTG enzyme, i.e., about 70 U / ml. [Figure 13] Figure 13. HIC-HPLC analysis of a monoclonal antibody containing the amino acid sequence "TLQS" on the light chain of the antibody. The corresponding ADC is shown. [Figure 14-1] Figure 14. Mass spectrometry analysis of a monoclonal antibody containing the amino acid sequence "TLQS" on the antibody light chain and the corresponding ADC. [Figure 14-2]Figure 14. Mass spectrometry analysis of a monoclonal antibody containing the amino acid sequence "TLQS" on the antibody light chain and the corresponding ADC. [Figure 15-1] Figure 15. HIC-HPLC analysis of tag fusion antibodies mAb_tag2, mAb_tag3 and mAb_tag4 and the corresponding ADCs. [Figure 15-2] Figure 15. HIC-HPLC analysis of tag fusion antibodies mAb_tag2, mAb_tag3 and mAb_tag4 and the corresponding ADCs. [Figure 16] Figure 16. Scheme of conjugation of mAb with a glutamine tag fused to the light chain C-terminus using native mAb conjugation technology resulting in an ADC with DAR=4. [Figure 17-1] Figure 17. Reduced RP-HPLC showing efficient conjugation to both the light chain (with peptide tag) and the heavy chain (native glutamine). (A) Conjugation of drug-linker 4M to tag fusion antibody mAb_tag5 resulting in DAR3.9. [Figure 17-2] FIG. 17 (B) Conjugation of drug-linker 1M to tag-fusion antibody mAb_tag5, resulting in DAR3.9. [Figure 17-3] Figure 17 (C) Conjugation of drug-linker 1M to mAb7-M' (top) resulting in ADC7-M' bottom, DAR 3.9. [Figure 18] Figure 18. pH dependence of transglutaminase-mediated bioconjugation in a wide range of buffer systems. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Detailed Description of the Invention definition As used herein, "CEACAM5" refers to "carcinoembryonic antigen-related cell adhesion molecule 5", also known as "CD66e" (cluster of differentiation 66e). CEACAM5 is a glycoprotein involved in cell adhesion. CEACAM5 is highly expressed on the surface of, for example, colon cancer, gastric cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, prostate cancer and other solid tumors.

[0014] A "domain" or "region" is any region of a protein that is generally defined on the basis of sequence homology and often relates to a particular structural or functional entity.

[0015] A sequence that "encodes" an expression product such as a polypeptide, protein, or enzyme, or a "coding sequence" is a nucleotide sequence that, when expressed, results in the production of that polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes the amino acid sequence of that polypeptide, protein, or enzyme. A protein coding sequence includes a start codon (usually ATG) and a stop codon.

[0016] The reference of a particular protein (e.g., an antibody) as used herein can include polypeptides having a native amino acid sequence, as well as mutants and modified forms, regardless of their origin or mode of preparation. A protein having a native amino acid sequence is a protein having the same amino acid sequence as that obtained from nature. Such native sequence proteins can be isolated from nature or prepared using standard recombinant and / or synthetic methods. Native sequence proteins specifically encompass naturally occurring truncated or soluble forms, naturally occurring mutant forms (e.g., alternatively spliced ​​forms), naturally occurring allelic variants, and forms that include post-translational modifications. Native sequence proteins include proteins that carry post-translational modifications, such as glycosylation or phosphorylation, or other modifications of some amino acid residues.

[0017] The term "gene" refers to a DNA sequence that codes for or corresponds to a specific sequence of amino acids, including all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, which determine the conditions under which the gene is expressed. Some genes that are not structural genes are transcribed from DNA into RNA but are not translated into amino acid sequences. Other genes function as regulators of structural genes or as regulators of DNA transcription. In particular, the term gene contemplates genomic sequences that code for proteins, i.e., sequences that include regulatory elements, promoters, introns and exon sequences.

[0018] As used herein, a sequence that is "at least 85% identical" to a reference sequence is a sequence that has 85% or more, for example 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the full length of the reference sequence.Accordingly, the percentage of "sequence identity" is determined by comparing two such sequences over their full length by global pairwise alignment using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443 (1970)), for example, by using the program Needle (EMBOSS) with BLOSUM62 matrix and the following parameters: gap open=10, gap extension=0.5, end gap penalty=not applicable, end gap open=10, end gap extension=0.5 (these are standard settings).

[0019] A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain with similar chemical properties (e.g., charge, size, or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of a protein. Examples of groups of amino acids with side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Conservative amino acid substitutions can also be defined based on amino acid size.

[0020] An "antibody" (also referred to as an "immunoglobulin") is, for example, a natural or conventional type of antibody in which two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda (I) and kappa (k). There are five main heavy chain classes (or isotypes) that determine the functional activity aspects of the antibody molecule: IgM, IgD, IgG, IgA and IgE. Each antibody chain contains distinct sequence domains (or regions). The light chain of a typical IgG antibody contains two regions, the variable region (VL) and the constant region (CL). The heavy chain of a typical IgG antibody contains four regions, the variable region (VH) and the constant region (CH), and the remaining three constant domains (CH1, CH2 and CH3). The variable regions of both the light and heavy chains determine the binding and specificity to the antigen. The constant regions of the light and heavy chains can confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement binding, and binding to Fc receptors (FcRs). The Fv fragment is the N-terminal portion of the Fab fragment of an antibody and consists of the variable portions of one light chain and one heavy chain.

[0021] The specificity of an antibody lies in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site is mainly made up of residues that are from the so-called hypervariable or complementarity determining regions (CDRs). Complementarity determining regions (CDRs) therefore refer to amino acid sequences that together define the binding affinity and specificity of the Fv region of an antibody. The light (L) and heavy (H) chains of an antibody have three CDRs, designated CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. Thus, the antigen binding site of a conventional antibody encompasses six CDRs, including a set of CDRs from each of the heavy and light chain variable regions.

[0022] "Framework region" (FR) refers to the amino acid sequence inserted between the CDRs, i.e., the portion of the immunoglobulin light and heavy chain variable regions that is relatively conserved among different immunoglobulins of a single species. The light and heavy chains of an immunoglobulin each have four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L, and FR1-H, FR2-H, FR3-H, FR4-H, respectively. As used herein, a "human framework region" is a framework region that is substantially (about 85% or more, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identical to the framework region of a naturally occurring human antibody.

[0023] In the context of the present invention, the definition of CDR / FR in an immunoglobulin light or heavy chain is determined based on the definition of IMGT (Lefranc et al. Dev. Comp. Immunol., 2003, 27(1):55-77; www.imgt.org).

[0024] As used herein, the term "antibody" includes conventional antibodies and fragments thereof, as well as single domain antibodies and fragments thereof, such as the variable heavy chains of single domain antibodies. The term "antibody" as used herein also includes chimeric, humanized, bispecific or multispecific antibodies, as well as other types of engineered antibodies. The term "antibody" includes monoclonal antibodies.

[0025] The term "monoclonal antibody" or "mAb" as used herein refers to an antibody molecule of a single amino acid sequence directed against a specific antigen and should not be construed as requiring production of the antibody by any particular method. A monoclonal antibody can be produced, for example, by a single clone of a B cell or hybridoma, but is produced by methods including recombinant, e.g., genetic or protein engineering.

[0026] The term "chimeric antibody" in its broadest sense refers to an engineered antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies. In some embodiments, chimeric antibodies contain the VH and VL of an antibody derived from a non-human animal, together with the CH and CL of another antibody, which in some embodiments is a human antibody. The non-human animal may be any animal, such as mouse, rat, hamster, rabbit, etc. Chimeric antibodies also refer to multispecific antibodies that have specificity for at least two different antigens.

[0027] The term "humanized antibody" refers to an antibody that is wholly or partially of non-human origin and has been modified, for example, by replacing certain amino acids in the framework regions of VH and VL to avoid or minimize immune responses in humans. The constant regions of a humanized antibody are typically human CH and CL regions.

[0028] A "fragment" of an antibody (e.g., of a conventional antibody) comprises a portion of an intact antibody, such as an IgG, in particular the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, and bispecific and multispecific antibodies formed from antibody fragments. A fragment of a conventional antibody can also be a heavy chain antibody or a single domain antibody, such as a VHH.

[0029] The term "Fab" refers to an antibody fragment having a molecular weight of about 50,000 Da and antigen-binding activity, in which about half of the N-terminal heavy chain and the entire light chain are linked via disulfide bonds. It is usually obtained from the fragments by treating IgG with the protease papain.

[0030] The term "F(ab')2" refers to an antibody fragment with a molecular weight of about 100,000 Da and antigen-binding activity, which is slightly larger than two identical Fab fragments linked via disulfide bonds in the hinge region. It is usually obtained from among the fragments by treating IgG with the protease pepsin.

[0031] The term "Fab" refers to an antibody fragment having a molecular weight of about 50,000 Da and antigen-binding activity, which is obtained by cleaving the disulfide bond in the hinge region of F(ab')2.

[0032] Single chain Fvs ("scFvs") are covalently linked VH::VL heterodimers, which are usually expressed from gene fusions that include genes encoding VH and VL linked by a peptide-encoding linker. The human scFv fragments of the invention include CDRs that are held in the proper configuration, for example by using recombinant gene technology. Bivalent and multivalent antibody fragments form spontaneously by association of monovalent scFvs, or are produced by coupling monovalent scFvs by a peptide linker, such as bivalent sc(Fv)2. A "dsFv" is a VH::VL heterodimer stabilized by a disulfide bond. "(dsFv)2" refers to two dsFvs linked by a peptide linker.

[0033] The term "bispecific antibody" or "BsAb" refers to an antibody that contains two different antigen-binding sites. Thus, BsAbs are capable of binding, for example, to two different antigens simultaneously. Genetic engineering is frequently used to design, modify, and produce antibodies or antibody derivatives with a desired set of binding properties and effector functions, as described, for example, in EP 2 050 764 A1.

[0034] The term "multispecific antibody" refers to an antibody that contains two or more different antigen-binding sites.

[0035] The term "diabody" refers to small antibody fragments with two antigen-binding sites, which contain a heavy chain variable domain (VH) connected to a light chain variable domain (VL) on the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites.

[0036] The term "hybridoma" refers to cells obtained by subjecting B cells prepared by immunizing a non-human mammal with an antigen to cell fusion with myeloma cells derived from a mouse or the like, which produce the desired monoclonal antibody having antigen specificity.

[0037] "Purified" or "isolated," when referring to a polypeptide (e.g., an antibody) or a nucleotide sequence, means that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. As used herein, the term "purified" means that at least 75%, 85%, 95%, 96%, 97%, or 98% by weight of the same type of biological macromolecules are present. An "isolated" nucleic acid molecule encoding a particular polypeptide refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode the polypeptide of interest. However, the molecule may include some additional bases or moieties that do not adversely affect the basic properties of the composition.

[0038] As used herein, the term "subject" refers to a mammal, such as a rodent, cat, dog, primate, or human. In embodiments of the invention, the subject (or patient) is a human.

[0039] As used herein, the term "about" in relation to a numerical value means that the value can vary within + / - 10% of said value.

[0040] Unless otherwise specified, the concentrations of DMSO used herein are given as % (v / v).

[0041] antibody Any antibody may be used in the conjugation method of the invention. Preferred antibodies include monoclonal antibodies, which contain an amino acid sequence selected from the group consisting of GGTLQSPP (SEQ ID NO: 74), TLQSG (SEQ ID NO: 77), TLQSPP (SEQ ID NO: 78), GGTLQSG (SEQ ID NO: 79) and TLQSA (SEQ ID NO: 80), more preferably the amino acid sequence TLQSPP (SEQ ID NO: 78) or GGTLQSPP (SEQ ID NO: 74) (most preferably the amino acid sequence GGTLQSPP) in at least one, preferably both, of its light chain constant region (CL) and / or in at least one, preferably both, of its heavy chain constant region (CH), and / or contain a glutamine at position 295 (EU numbering). It has been found that these amino acid sequences, even when incorporated into the antibody light chain constant region (CL) and / or heavy chain constant region (CH), are suitable to serve as substrates for microbial transglutaminase (also herein "mTG" or "MTG"). Therefore, these amino acid sequences are also referred to herein as "mTG tags". When incorporated into the CL and / or CH chains of an antibody (preferably at the C-terminus of said chains), the primary amino groups of a linker (preferably a drug linker, but the linker may also include other payloads other than a drug, such as a detectable label or a secondary antibody), or a primary amino group of a therapeutic agent, can react with the glutamine of these aforementioned amino acid sequences of the antibody in the presence of the mTG enzyme.

[0042] [ka]

[0043] In the above reaction scheme, the acyl acceptor is the linker (preferably a drug-linker) or the therapeutic agent, which contains the primary amino group.

[0044] The present inventors have previously generated, screened and selected specific anti-CEACAM5 antibodies, which surprisingly showed a combination of several properties that make them ideally suited for use in cancer therapy, especially as part of an immunoconjugate (antibody-drug conjugate). These antibodies can also be used in the conjugation method of the present invention. Some of these antibodies have been used as examples of the present invention, for example, in conjugation by the method of the present invention. However, as mentioned, the application of the present invention is not limited to anti-CEACAM5 antibodies, but rather, the present invention is applicable for the purpose of preparing antibody-linker conjugates or antibody-drug conjugates based on antibodies against any target. Preferred antibodies that can be used in the methods of the invention include isolated antibodies (e.g., those that bind to human CEACAM5 protein), The isolated antibody comprises: (i) at least one light chain constant region (CL) comprising a sequence selected from the group consisting of GGTLQSPP, TLQSG, TLQSPP and TLQSA (preferably GGTLQSPP), preferably comprising this sequence at its C-terminus; and / or (ii) at least one heavy chain constant region (CH) comprising one or more of the following amino acid substitutions: (a) L234A and L235A (LALA mutations); (b) L234A and L235A and P329G (LALA-PG mutation); (c) L235A and G237A (LAGA mutations); (d) M252Y and S254T and T256E (YTE mutations); (e)K222R: Eu numbering is used for the amino acid substitutions. Further preferred antibodies that may be used in the methods of the present invention include isolated antibodies that bind to human CEACAM5 protein, wherein the isolated antibody comprises: (i) at least one light chain constant region (CL) comprising a sequence selected from the group consisting of GGTLQSPP, TLQSG, TLQSPP and TLQSA (preferably GGTLQSPP), preferably comprising this sequence at its C-terminus; And (ii) at least one heavy chain constant region (CH) comprising one or more of the following amino acid substitutions: (a) L234A and L235A (LALA mutations); (b) L234A and L235A and P329G (LALA-PG mutation); (c) L235A and G237A (LAGA mutations); (d) M252Y and S254T and T256E (YTE mutations); (e) K222R; Eu numbering is used for the amino acid substitutions. The isolated antibody comprises a CDR1-H consisting of the amino acid sequence of SEQ ID NO: 3, a CDR2-H consisting of the amino acid sequence of SEQ ID NO: 4, a CDR3-H consisting of the amino acid sequence of SEQ ID NO: 5, a CDR1-L consisting of the amino acid sequence of SEQ ID NO: 6, a CDR2-L consisting of the amino acid sequence of SEQ ID NO: 7, and a CDR3-L consisting of the amino acid sequence of SEQ ID NO: 8.

[0045] Preferably, an isolated antibody that may be used in the method of the invention comprises framework regions FR1, FR2, FR3, FR4, FR5, FR6, FR7, and FR8 with the structures FR1-CDR1-H-FR2-CDR2-H-FR3-CDR3-H-FR4 and FR5-CDR1-L-FR6-CDR2-L-FR7-CDR3-L-FR8, where FR1 consists of SEQ ID NO: 54, FR2 consists of SEQ ID NO: 55, FR3 consists of SEQ ID NO: 56, FR4 consists of SEQ ID NO: 57, FR5 consists of SEQ ID NO: 58, FR6 consists of SEQ ID NO: 59, FR7 consists of SEQ ID NO: 60 and FR8 consists of SEQ ID NO: 61. The invention also provides the conjugation methods described herein using an isolated antibody (e.g., one that binds to a human CEACAM5 protein), wherein the isolated antibody comprises: (i) at least one heavy chain constant region (CH) comprising one or more of the following amino acid substitutions: (a) L234A and L235A (LALA mutations); (b) L234A and L235A and P329G (LALA-PG mutation); (c) L235A and G237A (LAGA mutations); (d) M252Y and S254T and T256E (YTE mutations); (e) K222R; And (ii) at least one light chain constant region (CL) comprising the sequence GGTLQSPP, preferably at its C-terminus. Eu numbering is used for the amino acid substitution. The Eu numbering system is well known (see Edelman et al., Proc. Natl. Acad. Sci. USA 1969, 63, 78-85 and Kabat, EA et al., National Institutes of Health (US) Office of the Director. Sequences of Proteins of Immunological Interest, 5th ed.; DIANE Publishing: Collingdale, PA, USA, 1991), and the position of the indicated amino acid substitution follows this numbering system. Amino acid substitution is specified using one-letter amino acid code. Also, GGTLQSPP can be included multiple times in the light chain constant region (CL), and alternatively or additionally, can also be included in the heavy chain constant region (CH). Preferably, GGTLQSPP is included once per light chain constant region (CL) in both light chain constant regions (CL) of the antibody of the present invention.

[0046] In one embodiment of the isolated antibody that may be used in the conjugation method of the invention, both heavy chain constant regions (CH) comprise one or more of said amino acid substitutions (a) to (e) and / or both light chain constant regions comprise said sequence GGTLQSPP. Preferred combinations of CL and CH chain modifications are outlined in Table 4 below, which shows the modification combinations for antibodies mAb1-M, mAb2-M, mAb3-M, mAb6-M and mAb7-M. Preferably, the antibody of the invention comprises any of the following heavy chain constant region (CH) and light chain constant region (CL) modifications: (a) the C H contains the amino acid substitutions L234A, L235A (LALA mutation) and M252Y, S254T, and T256E (YTE mutation); or (b) the CH comprises the amino acid substitutions L234A, L235A (LALA mutation) and M252Y, S254T and T256E (YTE mutation), and a light chain constant region (CL) comprising (preferably at the C-terminus) the sequence GGTLQSPP; or (c) the CH comprises the amino acid substitutions L234A, L235A (LALA mutation), and M252Y, S254T and T256E (YTE mutation), and K222R, and a light chain constant region (CL) comprising (preferably at the C-terminus) the sequence GGTLQSPP; or (d) C-H contains the amino acid substitutions L234A, L235A (LALA mutation); or (e) CH comprises the amino acid substitutions L234A, L235A (LALA mutation), and a light chain constant region (CL) comprising (preferably at the C-terminus) the sequence GGTLQSPP.

[0047] Preferably, both CL and both CH regions of an antibody of the invention contain the modifications outlined in (a) to (e) above.

[0048] Any combination of the above and below aspects herein forms part of the present invention.

[0049] In some embodiments of the conjugation methods of the invention, the antibody used in the methods of the invention is a conventional antibody, such as a conventional monoclonal antibody, or an antibody fragment, a bispecific or multispecific antibody.

[0050] In some embodiments, the antibody comprises or consists of an IgG or a fragment thereof.

[0051] In some embodiments, the antibody used in the conjugation method of the present invention is, for example, a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody. A myriad of methods for humanizing antibody sequences are known in the art; see, for example, the review by Almagro & Fransson (2008) Front Biosci 13:1619-1633. One commonly used method is CDR grafting or antibody reshaping, which involves grafting the CDR sequences of a donor antibody, typically a murine antibody, onto the framework scaffold of a human antibody of different specificity. Since CDR grafting reduces the binding specificity and affinity, and therefore the biological activity, of the CDR-grafted non-human antibody, back mutations are introduced at selected positions of the CDR-grafted antibody to retain the binding specificity and affinity of the parent antibody. Identification of possible back mutation positions can be performed using information available from the literature and antibody databases. Amino acid residues that are candidates for back mutations are typically those located on the surface of the antibody molecule, and buried or low surface-exposed residues will usually not be altered. An alternative humanization technique to CDR grafting and backmutation is resurfacing, in which non-surface exposed residues of non-human origin are retained and surface residues are changed to human residues. Another alternative technique is known as "guided selection" (Jespers et al. (1994) Biotechnology 12, 899), which can be used to derive fully human antibodies from murine antibodies that retain the epitope and binding specificity of the parent antibody.

[0052] For chimeric antibodies, humanization typically involves altering the framework regions of the variable region sequences.

[0053] Although amino acid residues that are part of the CDRs are typically not changed in connection with humanization, in certain cases it may be desirable to change individual CDR amino acid residues, for example to remove glycosylation sites, deamidation sites, or unwanted cysteine ​​residues. N-linked glycosylation occurs by attachment of an oligosaccharide chain to an asparagine residue in the tripeptide sequence Asn-X-Ser or Asn-X-Thr, where X is any amino acid except Pro. Removal of an N-glycosylation site is achieved, for example, by mutating either the Asn or Ser / Thr residue to a different residue by conservative substitution. Deamidation of asparagine and glutamine residues may occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation when present primarily in the sequence Asn-Gly, and to a lesser extent in other dipeptide sequences such as Asn-Ala. Thus, when such a deamidation site, e.g. Asn-Gly, is present in a CDR sequence, it is desirable to remove the site, typically by conservative substitution to remove one of the involved residues. Substitutions on the CDR sequences to remove one of the involved residues are also intended to be encompassed by the invention.

[0054] In a humanized antibody or fragment thereof, the heavy and light chain variable domains comprise human receptor framework regions. The humanized antibody further comprises human heavy and light chain constant domains, where present.

[0055] In some embodiments, the antibody used in the conjugation methods of the invention is an antibody fragment (e.g., a humanized antibody fragment) selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, and diabody.

[0056] In some embodiments, the antibody is a bispecific or multispecific antibody formed from antibody fragments, at least one antibody fragment being a fragment of an antibody according to the invention. Multispecific antibodies are multivalent protein complexes, e.g. as described in EP 2 050 764 A1 or US 2005 / 0003403 A1.

[0057] Antibodies useful in the conjugation methods of the invention may be produced by any technique known in the art. Antibodies according to the invention may, for example, be used in isolated (e.g., purified) form or may be contained in a vector, such as a membrane or lipid vesicle (e.g., liposome).

[0058] When an antibody that is not an IgG antibody is used in the conjugation methods of the invention, it is preferred that the antibody comprises an amino acid sequence selected from the group consisting of GGTLQSPP, TLQSG, TLQSPP, GGTLQSG and TLQSA.

[0059] Nucleic Acids and Host Cells A further aspect of the present disclosure relates to an isolated nucleic acid comprising or consisting of a nucleic acid sequence encoding an antibody of the invention as defined above.

[0060] Typically, the nucleic acid is a DNA or RNA molecule, which is comprised on any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.

[0061] The terms "vector," "cloning vector," and "expression vector" refer to a vehicle that introduces DNA or RNA sequences (e.g., foreign genes) into a host cell to transform the host and promote expression (e.g., transcription and translation) of the introduced sequences.

[0062] Thus, a further aspect of the present disclosure relates to a vector comprising a nucleic acid as defined above.

[0063] Such vectors may contain regulatory elements, such as promoters, enhancers, terminators, etc., to induce or direct expression of the polypeptide upon administration to a subject. Examples of promoters and enhancers used in expression vectors for animal cells include the SV40 early promoter and enhancer (Mizukami T. et al. 1987), Moloney murine leukemia virus LTR promoter and enhancer (Kuwana Y et al. 1987), immunoglobulin H chain promoter (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983), etc.

[0064] Any expression vector for animal cells can be used as long as the gene encoding the human antibody C region can be inserted and expressed. Examples of suitable vectors include pAGE107 (Miyaji H et al. 1990), pAGE103 (Mizukami T et al. 1987), pHSG274 (Brady G et al. 1984), pKCR (O'Hare K et al. 1981), pSG1 beta d2-4- (Miyaji H et al. 1990), etc.

[0065] Other examples of plasmids include replicative plasmids that contain an origin of replication, or integrative plasmids, such as pUC, pcDNA, pBR, and the like.

[0066] Other examples of viral vectors include adenovirus, retrovirus, herpes virus and AAV vectors. Such recombinant viruses are produced by techniques known in the art, for example by transfecting packaging cells or by transient transfection with helper plasmid or virus. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses can be found, for example, in WO 95 / 14785, WO 96 / 22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94 / 19478.

[0067] A further object of the present disclosure relates to host cells transfected, infected or transformed with the nucleic acids and / or vectors.

[0068] The term "transformation" refers to the introduction of a "foreign" (i.e., exogenous) gene, DNA or RNA sequence into a host cell, where the host cell expresses the introduced gene or sequence to produce a desired substance, typically a protein or enzyme, encoded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA has been "transformed."

[0069] The nucleic acids are used to produce the antibodies of the invention in a suitable expression system. The term "expression system" means a host cell and a compatible vector under suitable conditions for the expression of a protein encoded by, for example, foreign DNA carried by the vector and introduced into the host cell.

[0070] Common expression systems include E. coli host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli, Kluyveromyces or Saccharomyces yeast, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (e.g., produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, neuronal cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells lacking the dihydrofolate reductase gene (hereinafter referred to as the "DHFR gene") (Urlaub G et al; 1980), rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cells"), etc. In some embodiments, YB2 / 0 cells are used because the ADCC activity of chimeric or humanized antibodies is enhanced when expressed in YB2 / 0 cells.

[0071] For the expression of humanized antibody, the expression vector is either a type in which the gene encoding the antibody heavy chain and the gene encoding the antibody light chain are present on separate vectors, or a type in which both genes are present on the same vector (tandem type). In terms of the ease of constructing the humanized antibody expression vector, the ease of introducing it into animal cells, and the balance of the expression levels between the antibody H chain and L chain in animal cells, the humanized antibody expression vector is a tandem type (Shitara K et al. J Immunol Methods. 1994 Jan 3; 167 (1-2): 271-8). Examples of tandem type humanized antibody expression vectors include pKANTEX93 (WO 97 / 10354), pEE18, etc.

[0072] The present disclosure also relates to a method for producing a recombinant host cell expressing an antibody according to the invention, said method comprising the steps of: (i) introducing into a competent host cell, in vitro or ex vivo, the above-mentioned recombinant nucleic acid or vector, (ii) culturing in vitro or ex vivo the obtained recombinant host cell, and (iii) optionally selecting cells that express and / or secrete said antibody.

[0073] Such recombinant host cells can be used for the production of the antibodies of the invention.

[0074] Methods for Producing Antibodies Antibodies that can be used for conjugation with a linker or drug linker using the methods of the present invention can be obtained or produced by any technique known in the art, such as, without limitation, chemical, biological, genetic or enzymatic techniques, alone or in combination.

[0075] Knowing the amino acid sequence of the desired antibody, one of skill in the art can readily produce said antibody or immunoglobulin chains using standard techniques for the production of polypeptides. For example, they can be synthesized using a commercially available peptide synthesizer (such as those produced by Applied Biosystems, Foster City, California) using the well-known solid-phase method, following the manufacturer's instructions. Alternatively, the antibodies and immunoglobulin chains of the invention can be produced by recombinant DNA techniques, as is well known in the art. For example, these polypeptides (e.g., antibodies) are obtained as DNA expression products following incorporation of a DNA sequence encoding the desired polypeptide onto an expression vector and introduction of such vector into a suitable eukaryotic or prokaryotic host that will express the desired polypeptide, from which they can then be isolated using well-known techniques.

[0076] The present invention further relates to a method for producing an antibody of the present invention, the method comprising the steps of: (i) culturing the transformed host cell; (ii) expressing the antibody; and (iii) recovering the expressed antibody.

[0077] The antibodies of the invention can be suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0078] In some embodiments, the humanized chimeric antibodies of the present invention can be produced by obtaining nucleic acid sequences encoding the humanized VL and VH regions as described above, constructing a human chimeric antibody expression vector by inserting them into an expression vector for animal cells having genes encoding a human antibody CH and a human antibody CL, and expressing the coding sequences by introducing the expression vector into animal cells.

[0079] The CH domain of the human chimeric antibody may be any region belonging to a human immunoglobulin heavy chain. For example, an IgG class domain is suitable, and any one of the subclasses belonging to the IgG class, such as IgG1, IgG2, IgG3, and IgG4, may be used. The CL domain of the human chimeric antibody may be any region belonging to a human immunoglobulin light chain, and may be any of the kappa class or lambda class.

[0080] Methods for producing humanized or chimeric antibodies involve conventional recombinant DNA and gene transfection techniques and are well known in the art (see, for example, Morrison SL. et al. (1984) and patent documents US 5,202,238; and US 5,204,244).

[0081] Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (see, for example, Riechmann L. et al. 1988; Neuberger MS. et al. 1985). Antibodies can be humanized using various techniques known in the art, such as the techniques disclosed in application WO2009 / 032661, CDR grafting (EP 239,400; PCT Publication WO91 / 09967; U.S. Patent Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska MA. et al. al. (1994), and chain shuffling (U.S. Patent No. 5,565,332). General recombinant DNA techniques for the preparation of such antibodies are also known (see European Patent Application EP 125023 and International Patent Application WO 96 / 02576).

[0082] The Fab of the present invention can be obtained by treating an antibody (e.g., IgG) of the present invention with a protease such as papain. Alternatively, the Fab can be produced by inserting DNA sequences encoding both chains of the Fab of the antibody into a vector for prokaryotic or eukaryotic expression and introducing the vector into a prokaryotic or eukaryotic cell (as appropriate) to express the Fab.

[0083] The F(ab')2 of the present invention can be obtained by treating the antibody (e.g., IgG) of the present invention with the protease pepsin. F(ab')2 can also be produced by linking the Fab' described below via a thioether bond or disulfide bond.

[0084] The Fab' of the present invention can be obtained by treating the F(ab')2 of the present invention with a reducing agent such as dithiothreitol. Alternatively, the Fab' can be produced by inserting a DNA sequence encoding the Fab' chain of the antibody into a vector for prokaryotic expression or a vector for eukaryotic expression, and introducing the vector into a prokaryotic or eukaryotic cell (as appropriate) to effect its expression.

[0085] The scFv of the present invention can be produced by taking the sequences of the CDRs or VH and VL domains as previously described for the antibodies of the present invention, constructing DNA encoding the scFv fragment, inserting the DNA into a prokaryotic or eukaryotic expression vector, and then introducing the expression vector into a prokaryotic or eukaryotic cell (as appropriate) to express the scFv. To generate humanized scFv fragments, a well-known technique called CDR grafting is used. This involves selecting complementarity determining regions (CDRs) according to the present invention and grafting them onto a human scFv fragment framework of known three-dimensional structure (see, for example, WO98 / 45322; WO 87 / 02671; US5,859,205; US5,585,089; US4,816,567; EP0173494).

[0086] Modification of the antibodies of the present invention Amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, modifications may be desired to improve the binding affinity and / or other biological properties of the antibody.

[0087] Modifications and changes can be made to the structure of the antibodies of the invention and the DNA sequences encoding them, resulting in functional antibodies or polypeptides possessing desired properties.

[0088] When making changes to the amino acid sequence of a polypeptide, the hydropathic index of the amino acids is taken into consideration. The importance of the hydropathic amino acid index to the interactive biological function of a protein is generally understood in the art. It is accepted that the relative hydropathic properties of amino acids contribute to the secondary structure of the resulting protein, which in turn determines the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); aspartic acid (-3.5); lysine (-3.9); and arginine (-4.5).

[0089] Further aspects of the invention also encompass function-conservative variants of the polypeptides of the invention.

[0090] For example, certain amino acids can be substituted by other amino acids in a protein structure without appreciable loss of activity. Because the interactive capacity and properties of a protein determine its biological functional activity, certain amino acid substitutions can be made in a protein sequence and, of course, in its coding DNA sequence, while still obtaining a protein with similar properties. It is therefore contemplated that various modifications can be made in the antibody sequences of the present invention, or in the corresponding DNA sequences encoding said polypeptides, without appreciable loss of their biological activity.

[0091] It is known in the art that certain amino acids can be substituted by other amino acids with similar hydropathic index or score to result in proteins with similar biological activity, i.e., biologically functional equivalent proteins. It is also possible to use well-established techniques such as alanine scanning approach to identify all amino acids that can be substituted in the antibody or polypeptide of the present invention without significant loss of binding to the antigen. Such residues are considered neutral, since they are not involved in antigen binding or in maintaining the structure of the antibody. One or more of these neutral positions can be substituted by alanine or another amino acid without changing the main properties of the antibody or polypeptide of the present invention.

[0092] Neutral positions can be seen as positions where any amino acid substitution can be incorporated. In fact, in the principle of alanine scanning, alanine is chosen because this residue has no particular structural or chemical characteristics. It is generally accepted that if a particular amino acid can be substituted with alanine without changing the properties of the protein, it is likely that many, if not all, other amino acid substitutions will also be neutral. In the opposite case, where alanine is the wild-type amino acid, if a particular substitution can be shown as neutral, it is likely that other substitutions will also be neutral.

[0093] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions taking into account any of the foregoing characteristics are well known to those of skill in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0094] It may also be desirable to modify the antibody of the present invention with respect to effector function, for example, to enhance the antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody, or to alter, for example, binding to Fc receptors. This is accomplished by introducing one or more amino acid substitutions into the Fc region of the antibody. Alternatively, or in addition, a cysteine ​​residue(s) is / are introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus produced has improved internalization capacity and / or increased complement-mediated cell killing and / or antibody-dependent cellular cytotoxicity (ADCC) (Caron PC. et al. 1992; and Shopes B. 1992). In some embodiments, the antibody of the present invention is an antibody with a modified amino acid sequence that reduces or eliminates binding to most Fcγ receptors. This may reduce uptake and toxicity in normal cells and tissues expressing such receptors, such as macrophages, hepatic sinusoidal cells, etc. An example of such an antibody is one that contains a substitution of two leucine (L) residues at positions 234 and 235 to alanine (A) (i.e., LALA). This double substitution has been demonstrated to reduce Fc binding to FcγR and, consequently, also reduces ADCC and reduces complement binding / activation. Another example of such an antibody is one that contains the substitution P329G in addition to the LALA double substitution (i.e., PG-LALA; see, e.g., Schlothauer et al., Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions, Protein Engineering, Design and Selection, Volume 29, Issue 10, October 2016, p. 457-466).In some embodiments, an antibody of the present invention is therefore an antibody that (i) contains, for example, a LALA or PG-LALA set of substitutions and (ii) has an amino acid sequence that is otherwise identical to the amino acid sequence of one of the antibodies of the present invention described herein above with reference to the respective SEQ ID NOs.

[0095] Another type of amino acid modification of the antibodies of the invention is useful for altering the original glycosylation pattern of the antibody, i.e., by deleting one or more carbohydrate moieties found on the antibody and / or adding one or more glycosylation sites that are not present on the antibody. The presence of either of the tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid other than proline, creates a potential glycosylation site. Addition or deletion of glycosylation sites of the antibody can conveniently be accomplished by altering the amino acid sequence such that it contains one or more of the above-mentioned tripeptide sequences (for N-linked glycosylation sites).

[0096] Another type of modification involves the removal of sequences identified in silico or experimentally that potentially lead to degradation products or heterogeneity of antibody preparations. As an example, deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation when present primarily in the sequence Asn-Gly, and to a lesser extent in other dipeptide sequences such as Asn-Ala. Thus, when such deamidation sites, particularly Asn-Gly, are present in antibodies or polypeptides, it is considered to remove the site, typically by conservative substitution to remove one of the involved residues. Such substitutions on sequences to remove one or more of the involved residues are also intended to be encompassed by the present invention.

[0097] Another type of covalent modification involves chemically or enzymatically coupling glycosides to the antibody. These procedures are advantageous in that they do not require production of the antibody in a host cell that has glycosylation capabilities for N- or O-linked glycosylation. Depending on the mode of coupling used, the sugar(s) are attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as on cysteine, (d) free hydroxyl groups, such as on serine, threonine, or hydroxyproline, (e) aromatic residues, such as on phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. For example, such methods are described in WO87 / 05330.

[0098] Removal of carbohydrate moieties present on antibodies is accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the antibody to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), but leaves the antibody intact. Chemical deglycosylation is described in Sojahr H. et al. (1987) and Edge, A. S. et al. (1981). Enzymatic cleavage of carbohydrate moieties on antibodies can be accomplished by the use of various endo- and exoglycosidases as described in Thotakura, N. R. et al. (1987).

[0099] Another type of covalent modification of the antibody involves linking it to one of a variety of nonproteinaceous polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, e.g., by the methods described in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192, or 4,179,337.

[0100] Other amino acid sequence modifications known in the art may also be applied to the antibodies of the invention.

[0101] immunoconjugate The present invention provides immunoconjugates, also referred to herein as antibody-drug conjugates, or more simply as conjugates.All these terms used herein have the same meaning and can be interchanged.Suitable methods for preparing immunoconjugates are known in the art.The immunoconjugates of the present invention are prepared by in vitro methods, for example as described herein.Preferably, they can be prepared by the method according to the present invention.

[0102] The present invention provides an immunoconjugate comprising an antibody of the present invention covalently linked via a linker to at least one growth inhibitory agent or at least one other agent, including a detectable label or a therapeutic agent. The therapeutic agent is preferably a cytotoxic drug.

[0103] The term "growth inhibitory agent" (also referred to as "anti-proliferative agent") refers to a molecule or compound or composition that inhibits the growth of cells, such as tumor cells, in vitro and / or in vivo.

[0104] In some embodiments, the growth inhibitory agent is a cytotoxic drug (also referred to as a cytotoxic agent). In some embodiments, the growth inhibitory agent is a radioactive moiety.

[0105] The term "cytotoxic drug" as used herein refers to a substance that directly or indirectly inhibits or interferes with the function of a cell and / or causes the destruction of a cell. The term "cytotoxic drug" includes, for example, chemotherapeutic drugs, enzymes, antibiotics, toxins, such as small molecule toxins or enzymatically active toxins, toxoids, vincas, taxanes, maytansinoids or maytansinoid analogs, tomaymycin or pyrrolobenzodiazepine derivatives, cryptophycin derivatives, leptomycin derivatives, auristatins or dolastatins analogs, prodrugs, topoisomerase I inhibitors, topoisomerase II inhibitors, DNA alkylating agents, antitubulin agents, CC-1065 and CC-1065 analogs.

[0106] Topoisomerase I inhibitors are molecules or compounds that inhibit the human enzyme topoisomerase I, which is responsible for changing DNA topology by catalyzing the temporary interruption and reconnection of single strands of DNA.Topoisomerase I inhibitors are highly toxic, for example, to dividing mammalian cells.Examples of suitable topoisomerase I inhibitors include camptothecin (CPT) and its analogs, such as topotecan, irinotecan, ciratecan, cositecan, exatecan, lutotecan, gimatecan, belotecan and rubitecan.

[0107] In some embodiments, the immunoconjugate of the present invention comprises the cytotoxic drug exatecan as a growth inhibitory agent. Exatecan has the chemical name (1S,9S)-1-amino-9-ethyl-5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H-benzo(de)pyrano(3',4':6,7)indolizino(1,2-b)quinoline-10,13-dione. Exatecan is represented by the following structural formula (I): [ka]

[0108] In further embodiments of the present invention, other CPT analogs and other cytotoxic drugs are used, for example as listed above. Examples of some cytotoxic drugs and methods of conjugation are further described in application WO2008 / 010101, which is incorporated by reference.

[0109] The term "radioactive moiety" means At 211 , Bi 212 , Er 169 , I 131 , I 125 , Y 90 , In 111 , P 32 , Re 186 , Re 188 , Sm 153 , Sr 89 Radioisotopes generally emit mainly beta radiation. In some embodiments, the radioisotope is an alpha-emitting isotope, such as thorium-227, which emits alpha radiation. The immunoconjugate can be prepared, for example, as described in application WO2004 / 091668.

[0110] In the immunoconjugates of the present invention, the antibodies of the present invention are covalently linked to at least one growth inhibitory drug via a linker. As used herein, "linker" refers to a chemical moiety that includes a covalent bond and / or any chain of atoms that covalently attaches a growth inhibitory drug to an antibody. Linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups. Conjugation of the antibodies of the invention with cytotoxic drugs or other growth inhibitory agents can be accomplished using a variety of bifunctional protein coupling agents, including, for example, but not limited to, N-succinimidyl pyridyldithiobutyrate (SPDB), butanoic acid 4-[(5-nitro-2-pyridinyl)dithio]-2,5-dioxo-1-pyrrolidinyl ester (nitro-SPDB), 4-(pyridin-2-yldisulfanyl)-2-sulfo-butyric acid (sulfo-SPDB), N-succinimidyl(2-pyridyldithio)propionate (SPDP), succinimidyl(N-maleimidomethyl) Cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)-hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al. (1987). Carbon-labeled 1-isothiocyanatobenzylmethyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotides to antibodies (WO 94 / 11026).

[0111] In an embodiment of the present invention, the linker is a "cleavable linker". This facilitates the release of cytotoxic drugs or other growth-inhibitory agents in or near cells, such as tumor cells. In some embodiments, the linker is a linker that can be cleaved in the endosome of mammalian cells. For example, an acid-labile linker, a peptidase-sensitive linker, an esterase-labile linker, a photolabile linker, or a disulfide-containing linker is used (see, for example, US Patent No. 5,208,020).

[0112] The following nomenclature is also used herein when referring to structural formulas representing immunoconjugates, where a combination of a growth inhibitory drug and a linker is also referred to as a [(linker)-(growth inhibitory drug)] moiety, e.g., a combination of an exatecan molecule and a linker is also referred to as a [(linker)-(exatecan)] moiety.

[0113] In some particular embodiments of the invention, the linker is a linker cleavable by the human enzyme glucuronidase. Thus, for example, the immunoconjugate prepared by the methods of the invention has the following formula (IIA) which includes a linker cleavable by glucuronidase: [ka] wherein the antibody is an antibody of the invention (preferably a monoclonal antibody), and n is the number of [(linker)-(growth inhibitory agent)] moieties covalently linked to the antibody. In embodiments using Formula IIA above, n is preferably between 3 and 4, and most preferably between 3.5 and 4.0 (i.e., about 4).

[0114] The number n is also referred to as the "drug-to-antibody ratio" (or "DAR"). This number n should always be understood as the average number for any given (preparation of) immunoconjugate.

[0115] In formula (IIA) above, the chemical structure between the antibody and the growth inhibitory drug is a linker. One of these linkers is also contained in formula (VIIIA), which is further illustrated below.

[0116] In any one of the embodiments having a glucuronidase-cleavable linker, as described above, the growth inhibitory drug is, for example, exetecan.

[0117] Thus, in some embodiments, the methods of the invention provide an immunoconjugate comprising an antibody according to the invention covalently linked via a linker to exatecan, the conjugate having the following formula (IVA): [ka] where n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody. The number n (also referred to as DAR) is, for example, between 1 and 10. In embodiments using formula IVA above, n is preferably between 3 and 4, most preferably between 3.5 and 4.0 (i.e., about 4).

[0118] Thus, in some embodiments, the methods of the invention provide an immunoconjugate comprising an antibody according to the invention covalently linked to exatecan via a linker, the conjugate having the following formula (VIA): [ka] where n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody. The number n (also referred to as DAR) can be, for example, between 1 and 10. In embodiments using formula VIA above, n is preferably between 3 and 5, more preferably between 3.5 and 4.5, and most preferably 4.

[0119] In preparing any of the above-mentioned immunoconjugates using the methods of the present invention, antibodies against any of the targets can be used.

[0120] Thus, in some embodiments, the methods of the present invention provide an immunoconjugate comprising an antibody according to the present invention covalently linked to exatecan via a linker, the conjugate having the following formula (VIIIA): [ka] where n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody. The number n (also referred to as DAR) can be, for example, between 1 and 10. The number n (also referred to as DAR) is, for example, between 1 and 10.

[0121] In other embodiments of the invention, the linker may be a "non-cleavable linker" (e.g., an SMCC linker). Release of the growth inhibitory drug from the antibody may occur by lysosomal degradation of the antibody.

[0122] In another embodiment of the invention, the immunoconjugate is a fusion protein comprising an antibody of the invention and a cytotoxic or growth inhibitory polypeptide (as a growth inhibitory agent). Such fusion proteins are produced by recombinant techniques or peptide synthesis, methods well known in the art. The coding DNA molecule contains respective regions encoding the two parts of the conjugate (the antibody and the cytotoxic or growth inhibitory polypeptide, respectively), either adjacent to each other or separated by a region encoding a linker peptide.

[0123] The antibodies of the invention may also be used in directed enzyme prodrug therapy, such as antibody-directed enzyme prodrug therapy, by conjugating the antibody to a prodrug-activating enzyme that converts a prodrug (e.g., a peptidyl chemotherapy drug, see WO 81 / 01145) into an active, cytotoxic drug (see, e.g., WO 88 / 07378 and U.S. Pat. No. 4,975,278). The enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug to convert it into its more active, cytotoxic form. Enzymes that are useful in this context include, but are not limited to: alkaline phosphatases, which are useful for converting phosphate-containing prodrugs to free drugs; arylsulfatases, which are useful for converting sulfate-containing prodrugs to free drugs; cytosine deaminases, which are useful for converting the non-toxic fluorocytosine to the anticancer drug 5-fluorouracil; proteases such as Serratia protease, thermolysin, subtilisin, carboxypeptidase, and cathepsins (e.g., cathepsins B and L), which are useful for converting peptide-containing prodrugs to free drugs. D-alanylcarboxypeptidases, useful for converting prodrugs containing D-amino acid substituents; carbohydrate cleaving enzymes such as O-galactosidase and neuraminidase, useful for converting glycosylated prodrugs to the free drug; P-lactamases, useful for converting P-lactam derivatized drugs to the free drug; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting drugs derivatized at their amine nitrogen with phenoxyacetyl or phenylacetyl groups, respectively, to the free drug. Enzymes can be covalently attached to the antibody by techniques well known in the art, for example, the use of linkers discussed above.

[0124] Suitable methods for preparing immunoconjugates are well known in the art (see, for example, Hermanson GT, Bioconjugate Techniques, Third Edition, 2013, Academic Press). For example, methods for conjugating cytotoxic drugs to antibodies via linkers that are covalently attached to cysteine ​​residues of the interchain disulfide bridges of the antibody are well known.

[0125] Generally, the immunoconjugates are obtained by a process, which for example comprises the following steps: (i) preparing a compound comprising a linker and a growth inhibitory drug (e.g., a cytotoxic drug), also referred to herein as a "drug-linker compound"; (ii) contacting an optionally buffered aqueous solution of the antibody with a solution of the Drug-Linker Compound: (iii) optionally then separating the conjugate formed in (ii) from unreacted antibody and / or drug-linker compound.

[0126] Aqueous solutions of antibodies can be buffered with buffers such as histidine, potassium phosphate, acetate, citrate, or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes buffer). The buffer is selected depending on the nature of the antibody. The drug-linker compound can be dissolved in an organic polar solvent such as dimethylsulfoxide (DMSO) or dimethylacetamide (DMA).

[0127] For conjugation to cysteine ​​residues of the antibody, the antibody is subjected to reduction (e.g. using TCEP) prior to step (ii). Suitable reducing conditions for reducing only interchain disulfide bonds are known in the art.

[0128] The reaction temperature for conjugation is usually between 20° C. and 40° C. The reaction time can vary, typically from 1 to 24 hours. The reaction between the antibody and the drug-linker compound can be monitored by refractometry and / or size exclusion chromatography (SEC) with a UV detector. If the conjugate yield is too low, the reaction time can be extended.

[0129] A number of different chromatographic methods can be used by those skilled in the art to carry out the separation of step (iii). The conjugate can be purified, for example, by SEC, adsorption chromatography (e.g., ion exchange chromatography IEC), hydrophobic interaction chromatography (HIC), affinity chromatography, mixed support chromatography such as hydroxyapatite chromatography, or high performance liquid chromatography (HPLC) such as reverse phase HPLC. Purification by dialysis or filtration or diafiltration can also be used.

[0130] After steps (ii) and / or (iii), the conjugate-containing solution may be subjected to an additional step (iv) of purification, for example by chromatography, ultrafiltration and / or diafiltration. Such additional steps of purification, for example by chromatography, ultrafiltration and / or diafiltration, may also be performed on the antibody-containing solution after the reduction reaction, in cases where reduction is performed prior to conjugation.

[0131] The conjugate is recovered in aqueous solution at the end of such process. Drug-to-antibody ratio (DAR) is a number that can vary depending on the nature of the antibody and drug-linker compound used, as well as the experimental conditions used for conjugation (e.g., ratio (drug-linker compound) / (antibody), reaction time, nature of solvent and co-solvent if any). Thus, contact between antibody and drug-linker compound can lead to a mixture containing several conjugates that differ from each other by different drug-to-antibody ratios. Thus, the DAR determined is an average value.

[0132] An exemplary method that can be used to determine the DAR isD The method consists of spectrophotometrically measuring the ratio of the absorbance of a solution of the purified conjugate at λ and 280 nm, a wavelength commonly used to measure protein concentrations, such as antibody concentrations. D is selected to allow the drug to be distinguished from the antibody, i.e., as would be readily known to one skilled in the art, and λ D is the wavelength at which the drug has high absorbance, and λ D is far enough away from 280 nm to avoid substantial overlap of the drug and antibody absorbance peaks. For example, λ D may be selected as 370 nm for exatecan (or camptothecin or other camptothecin analogues), or 252 nm for maytansinoid molecules.

[0133] The method for calculating DAR is derived from, for example, Antony S. Dimitrov (ed), LLC, 2009, Therapeutic Antibodies and Protocols, vol 525, 445, Springer Science. λD ) and 280 nm (A 280 The absorbance of the conjugate in is measured at the monomer peak in a size exclusion chromatography (SEC) analysis (calculating the "DAR(SEC)" parameter) or using a classical spectrophotometer instrument (calculating the "DAR(UV)" parameter). The absorbance can be expressed as: A λD =(CD×ε DλD )+(C A ×ε AλD ) A 280 =(CD×ε D280 )+(C A ×ε A280 ) During the ceremony: C D and C A are the concentrations of drug and antibody in solution, respectively, ε DλD and ε D280 are λ Dand the molar extinction coefficient of the drug at 280 nm, ε AλD and εA 280 are λ D and the molar extinction coefficient of the antibody at 280 nm.

[0134] Solving these two equations with two unknowns leads to the following equation: C D =[(ε A280 ×A λD )-(ε AλD ×A 280 )] / [(ε DλD ×ε A280 )-(ε AλD ×ε D280 )] C A =[A 280 -(C D ×ε D280 )] / ε A280

[0135] The average DAR is then calculated from the ratio of drug concentration to antibody: DAR=C D / C A .

[0136] An alternative method for preparing an immunoconjugate is next described, which is the method of the present invention. This method can be used in particular for antibodies that comprise, in at least one of their light chain constant regions (CL) and / or in at least one of their heavy chain constant regions (CH), an amino acid sequence selected from the group consisting of GGTLQSPP, TLQSG, TLQSPP and TLQSA. Thus, a further aspect of the present invention relates to a method for producing an antibody-linker conjugate, the method comprising the steps of: (1) providing an antibody comprising in at least one, preferably both, of its light chain constant regions (CL) and / or in at least one, preferably both, of its heavy chain constant regions (CH), an amino acid sequence selected from the group consisting of GGTLQSPP, TLQSG, TLQSPP and TLQSA, more preferably the amino acid sequence TLQSPP or GGTLQSPP (most preferably the sequence GGTLQSPP): (2) Mixing at least the following components in a reaction buffer: (a) the antibody provided in step (1); (b) a microbial transglutaminase, preferably a transglutaminase comprising the following amino acid sequence: MGSGSGSGTGEEKRSYAETHRLTADDVDDINALNESAPAASSAGPSFRAP DSDERVTPPAEPLDRMPDPYRPSYGRAETIVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFAFFDEDKYKNELKNGRPRSGETRAEFEGRVAKDSFDEAKGFQRARDVASVMNKALENAHDEGAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKDRNGGNHDPSKMKAVIYSKHFWSGQDRSGSSDKRKYGDPEAFRPDRGTGLVDMSRDRNIPRSPTSPGESFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWSDGYSDFDRGAYVVTFVPKSWNTAPDKVTQGWP (SEQ ID NO: 44); or FRAP DSDERVTPPAEPLDRMPDPYRPSYGRAETIVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFAFFDEDKYKNELKNGRPRSGETRAEFEGRVAKDSFDEAKGFQRARDVASVMNKALENAHDEGAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKDRNGGNHDPSKMKAVIYSKHFWSGQDRSGSSDKRKYGDPEAFRPDRGTGLVDMSRDRNIPRSPTSPGESFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWSDGYSDFDRGAYVVTFVPKSWNTAPDKVTQGWPLE (SEQ ID NO: 73); or FRAPDSDERVTPPAEPLDRMPDPYRPSYGRAETIVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFAFFDEDKYKNELKNGRPRSGETRAEFEGRVAKDSFDEAKGFQRARDVASVMNKALENAHDEGAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKDRNGGNHDPSKMKAVIYSKHFWSGQDRSGSSDKRKYGDPEAFRPDRGTGLVDMSRDRNIPRSPTSPGESFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWSDGYSDFDRGAYVVTFVPKSWNTAPDKVTQGWP (SEQ ID NO: 45); or DSDERVTPPAEPLDRMPDPYRPSYGRAETIVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFAFFDEDKYKNELKNGRPRSGETRAEFEGRVAKDSFDEAKGFQRARDVASVMNKALENAHDEGAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKDRNGGNHDPSKMKAVIYSKHFWSGQDRSGSSDKRKYGDPEAFRPDRGTGLVDMSRDRNIPRSPTSPGESFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWSDGYSDFDRGAYVVTFVPKSWNTAPDKVTQGWPLE (SEQ ID NO: 48); or DSDERVTPPAEPLDRMPDPYRPSYGRAETIVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFAFFDEDKYKNELKNGRPRSGETRAEFEGRVAKDSFDEAKGFQRARDVASVMNKALENAHDEGAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKDRNGGNHDPSKMKAVIYSKHFWSGQDRSGSSDKRKYGDPEAFRPDRGTGLVDMSRDRNIPRSPTSPGESFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWSDGYSDFDRGAYVVTFVPKSWNTAPDKVTQGWP (SEQ ID NO: 49); and (c) a linker comprising an H2N moiety capable of reacting with the antibody from step (1) in the presence of said transglutaminase, wherein the linker is preferably a drug-linker, and wherein said linker is covalently attached to the drug: (3) separating the antibody-linker conjugates produced in step (2) from unreacted linker and said transglutaminase, preferably by subjecting said mixture from step (2) to size exclusion chromatography.

[0137] In one embodiment, the transglutaminase is ATGGGTAGCGGTAGCGGTTCAGGCACCGGTGAAGAAAAACGTAGCTATGCAGAAACCCATCGTCTGACCGCAGATGATGTTGATGATATTAATGCACTGAATGAAAGCGCACCGGCAGCAAGCAGCGCAGGTCCGAGCTTTCGTGCACCG

[0138] In a preferred embodiment of the method, the reaction buffer is 7% DMSO, 24 mM HEPES, pH 7.0. In the methods of the present invention, the linker has the formula [ka] is a linker having the formula: wherein R is the remainder of the linker, optionally including a drug, the drug being preferably exatecan. In a more preferred embodiment of the method of the present invention, the linker is NH2-GGG-beta-glucuronide.

[0139] In an exemplary embodiment of the method of the present invention, in step (2), the mixture comprises the following drug-linker: [ka] Includes.

[0140] In a further embodiment of the method of the present invention, the mixture of step (2) comprises 5 molar equivalents of linker or drug-linker, respectively, per conjugation site, the conjugation site being the sequence GGTLQSPP, TLQSG, TLQSPP or TLQSA contained in the light chain constant region (CL) and / or the heavy chain constant region (CH) of said antibody.

[0141] A further aspect of the invention relates to antibody-linker conjugates producible according to the methods of the invention.

[0142] Exemplary methods of the invention for preparing immunoconjugates are described in the Examples. Drug-Linker Compounds

[0143] The present disclosure also provides compounds comprising a linker and a growth inhibitory drug (e.g., a cytotoxic drug), also referred to herein as "drug-linker compounds." For example, the present disclosure provides compounds of the following formula (XA): [ka] or a physiologically acceptable salt thereof. Compounds according to formula XA are also referred to herein as "Drug-Linker Compound 1-M," "Compound DL1-M," or "DL1-M."

[0144] These Drug-Linker compounds are used to prepare immunoconjugates using the methods of the invention described herein above and below.

[0145] The disclosed exemplary drug-linker compounds (eg, those depicted above as (XA)) are prepared by chemical synthesis, for example, as further described in the Examples below.

[0146] Pharmaceutical Compositions The antibodies or immunoconjugates of the invention are combined with pharma- ceutically acceptable carriers, diluents and / or excipients, optionally sustained release matrices of classes including, but not limited to, biodegradable polymers, non-biodegradable polymers, lipids or sugars, to form a pharmaceutical composition.

[0147] Accordingly, another aspect of the present invention relates to a pharmaceutical composition comprising an antibody or immunoconjugate of the invention and a pharma- ceutically acceptable carrier, diluent and / or excipient.

[0148] Therapeutic Methods and Uses The present invention also provides an immunoconjugate (obtainable by the method of the present invention) or a pharmaceutical composition of the present invention comprising such an immunoconjugate, for use as a medicament. For example, the present invention provides an immunoconjugate or pharmaceutical composition of the present invention for use in treating cancer. The present invention further provides a method of treating cancer, comprising administering an immunoconjugate or pharmaceutical composition of the present invention to a subject in need thereof.

[0149] The antibodies disclosed herein, as well as any other antibodies, can be conjugated (linked) to a growth inhibitory drug or another drug using the methods of the invention, whereby the antibody will be useful for targeting the drug (e.g., a growth inhibitory drug) to a target cell (e.g., a cancerous cell) that expresses or overexpresses the antigen (preferably a cell surface antigen) to which the antibody binds.

[0150] In some embodiments, antibodies that may be used in the methods of the present invention are antibodies with altered amino acid sequences that reduce or eliminate binding to most Fcγ receptors, which may reduce uptake and toxicity in normal cells and tissues that express such receptors, e.g., macrophages, hepatic sinusoidal cells, etc.

[0151] One aspect of the present invention relates to a method of treating cancer comprising administering a therapeutically effective amount of an immunoconjugate or pharmaceutical composition of the present invention to a subject in need thereof.

[0152] The efficacy of treatment with an antibody or immunoconjugate or pharmaceutical composition according to the invention can be readily analyzed in vivo, e.g. in a mouse model of cancer, by measuring, e.g., the change in tumor volume, % tumor regression, partial regression or complete regression between treated and control groups.

[0153] kit The present invention provides in a further aspect a kit of parts comprising a reaction buffer as defined in the context of the method of the present invention, the kit comprising a microbial transglutaminase from S. ladakanum, preferably a transglutaminase comprising the amino acid sequence as set out in SEQ ID NO: 44, 47, 48, 49 or 45, preferably comprising the amino acid sequence of SEQ ID NO: 45.

[0154] In one embodiment of the kit of parts of the invention, the kit further comprises a linker having a primary amino group, the linker being a substrate for microbial transglutaminase, the linker preferably further being covalently attached to the payload. A preferred linker has the formula: [ka] where R is the remainder of the linker and may also optionally include a drug or a detectable label. Examples of detectable labels include radioactive and fluorescent groups.

[0155] In one embodiment of the kit of parts of the invention, the kit further comprises an instruction manual with instructions for using the reaction buffer and microbial transglutaminase of the kit to conjugate a linker to an antibody, preferably a monoclonal antibody, via a primary amino group.

[0156] Conjugation Methods of the Invention As shown in the examples, the present inventors have developed a surprisingly improved method for efficiently covalently conjugating antibodies to primary amines of a linker using a transglutaminase enzyme-based system.

[0157] Thus, the present invention provides in a further aspect a method for producing an antibody-linker conjugate, the method comprising the steps of: (1) providing an antibody optionally comprising in at least one, preferably both, of its light chain constant regions (CL) and / or in at least one, preferably both, of its heavy chain constant regions (CH), an amino acid sequence selected from the group consisting of GGTLQSPP, TLQSG, TLQSPP, GGTLQSG and TLQSA, more preferably the amino acid sequence TLQSPP or GGTLQSPP (most preferably the amino acid sequence GGTLQSPP); (2) Mixing at least the following components in a reaction buffer: (a) the antibody provided in step (1); (b) a microbial transglutaminase, preferably a transglutaminase comprising the amino acid sequence set forth in SEQ ID NO: 44, 47, 48, 49, 45, 73, 81, 82, 83 or 84, preferably comprising the amino acid sequence of SEQ ID NO: 45; and (c) a linker or therapeutic agent comprising an H2N moiety capable of reacting with the antibody from step (1) in the presence of said transglutaminase, said linker preferably being a drug-linker, said linker being covalently attached to the drug; (3) separating the antibody-linker conjugates produced in step (2) from unreacted linker and said transglutaminase, preferably by subjecting said mixture from step (2) to size exclusion chromatography.

[0158] In method step (1), the antibody optionally comprises an amino acid sequence selected from the group consisting of GGTLQSPP, TLQSG, TLQSPP, GGTLQSG and TLQSA. Such sequences are useful because they contain a glutamine that can react with the linker or the therapeutic agent in method step (2)(c). However, the conjugation reaction can also be performed using a native antibody, for example as shown in FIG. 12. Thus, if the antibody does not contain an amino acid sequence as described above, it is preferred that an antibody containing a glutamine at position 295 (Eu numbering) or a glutamine at a different position that can react with the linker or the therapeutic agent in method step 2(c) in the presence of the reaction buffer and the microbial transglutaminase is used in the method of the invention.

[0159] In method step (2)(c), the H2N portion of the linker or therapeutic agent reacts with a glutamine of the antibody, which can be, for example, a glutamine at position 295 (Eu numbering) of the heavy chain of the antibody and / or a glutamine on an amino acid sequence of the antibody, the amino acid sequence being selected from the group consisting of GGTLQSPP, TLQSG, TLQSPP, GGTLQSG and TLQSA. Preferably, the reaction buffer is a reaction buffer that is between 0% and 7% DMSO, between 20 mM and 30 mM HEPES (preferably 24 mM HEPES), and a pH between 6.8 and 7.4 (preferably pH 7); or a reaction buffer containing between 3% and 10% DMSO, between 5 mM and 100 mM HEPES, less than 150 mM NaCl, and having a pH between 6 and 8; or The reaction buffer comprises between 3% and 10% DMSO, between 5 mM and 60 mM HEPES, less than 10 mM NaCl (preferably no NaCl), and has a pH between 6 and 7.5.

[0160] In a further embodiment of the method of the present invention, the linker has the formula: [ka] where R is the remainder of the linker, which may also include a therapeutic agent or a detectable label.

[0161] In a further embodiment of the method of the present invention, in step (2) of the method, the mixture comprises the following drug-linker: [ka] Includes.

[0162] In a further embodiment of the method of the present invention, the mixture of step (2) comprises 5 molar equivalents of linker or drug-linker, respectively, per conjugation site, the conjugation site being the sequence GGTLQSPP, TLQSG, TLQSPP, GGTLQSG or TLQSA contained in the light chain constant region (CL) and / or the heavy chain constant region (CH) of said antibody, and / or the conjugation site is a glutamine at position 295 of the heavy chain of the antibody, and the antibody is a monoclonal antibody, wherein Eu numbering is used to position said glutamine at position 295.

[0163] In a further embodiment of the method of the present invention, the antibody is a monoclonal antibody, preferably an anti-CEACAM5 antibody as defined in any of (a), (b), or (c) below: (a) the antibody comprises: (i) at least one light chain constant region (CL) comprising a sequence selected from the group consisting of GGTLQSPP, TLQSG, TLQSPP, GGTLQSG and TLQSA, preferably comprising this sequence at the C-terminus of the light chain constant region; and (ii) at least one heavy chain constant region (CH) comprising one or more of the following amino acid substitutions: (a) L234A and L235A (LALA mutations); (b) L234A and L235A and P329G (LALA-PG mutation); (c) L235A and G237A (LAGA mutations); (d) M252Y and S254T and T256E (YTE mutation); (e) K222R; wherein Eu numbering is used for said amino acid substitutions; wherein the isolated antibody comprises a CDR1-H consisting of the amino acid sequence of SEQ ID NO:3, a CDR2-H consisting of the amino acid sequence of SEQ ID NO:4, a CDR3-H consisting of the amino acid sequence of SEQ ID NO:5, a CDR1-L consisting of the amino acid sequence of SEQ ID NO:6, a CDR2-L consisting of the amino acid sequence of SEQ ID NO:7, and a CDR3-L consisting of the amino acid sequence of SEQ ID NO:8: (b) the heavy chain constant region (CH) and the light chain constant region (CL) of the antibody have any of the following sequence combinations: (1) both CHs comprise a sequence of SEQ ID NO:31 and both CLs comprise a sequence of SEQ ID NO:12; or (2) both CHs comprise a sequence of SEQ ID NO:31 and both CLs comprise a sequence of SEQ ID NO:33; or (3) both CHs comprise a sequence of SEQ ID NO:32 and both CLs comprise a sequence of SEQ ID NO:33; or (4) both CHs comprise a sequence of SEQ ID NO:50 and both CLs comprise a sequence of SEQ ID NO:12; or (5) both CHs comprise a sequence of SEQ ID NO:50 and both CLs comprise a sequence of SEQ ID NO:33; or (6) one CH comprises the sequence of SEQ ID NO:31 and one CL comprises the sequence of SEQ ID NO:12; or (7) one CH comprises the sequence of SEQ ID NO: 31 and one CL comprises the sequence of SEQ ID NO: 33; or (8) one CH comprises the sequence of SEQ ID NO: 32 and one CL comprises the sequence of SEQ ID NO: 33; or (9) one CH comprises the sequence of SEQ ID NO:50 and one CL comprises the sequence of SEQ ID NO:12; or (10) One CH comprises the sequence of SEQ ID NO:50 and one CL comprises the sequence of SEQ ID NO:33. (c) the antibody comprises: two identical heavy chains (HC) comprising the amino acid sequence of SEQ ID NO:34 and two identical light chains (LC) comprising the amino acid sequence of SEQ ID NO:14; or two identical heavy chains (HC) comprising the amino acid sequence of SEQ ID NO: 34 and two identical light chains (LC) comprising the amino acid sequence of SEQ ID NO: 36; or two identical heavy chains (HC) comprising the amino acid sequence of SEQ ID NO: 35 and two identical light chains (LC) comprising the amino acid sequence of SEQ ID NO: 36; or two identical heavy chains (HC) comprising the amino acid sequence of SEQ ID NO:51 and two identical light chains (LC) comprising the amino acid sequence of SEQ ID NO:14; or Two identical heavy chains (HC) comprising the amino acid sequence of SEQ ID NO:51 and two identical light chains (LC) comprising the amino acid sequence of SEQ ID NO:36. and / or The drug is a growth inhibitory agent as defined in (d), (e), or (f) below: (d) The growth inhibitory agent is a cytotoxic drug or a radioactive moiety. (e) the growth inhibitory agent is selected from the group consisting of a chemotherapeutic agent, an enzyme, an antibiotic, a toxin, e.g., a small molecule toxin or an enzymatically active toxin, a toxoid, a vinca, a taxane, a maytansinoid or a maytansinoid analog, a tomaymycin or a pyrrolobenzodiazepine derivative, a cryptophycin derivative, a leptomycin derivative, an auristatin or a dolastatin analog, a prodrug, a topoisomerase I inhibitor, a topoisomerase II inhibitor, a DNA alkylating agent, an antitubulin agent, CC-1065, and a CC-1065 analog; (f) The growth inhibitory agent is exatecan.

[0164] The present invention further provides an antibody-linker conjugate producible according to any of claims 1 to 7, wherein the linker is a linker or a drug linker as defined below: [ka] where n is the number of [(linker)-(growth inhibitory agent)] moieties covalently linked to the antibody; or [ka] where n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody; where n is preferably between 1 and 10, more preferably 4.

[0165] In a further embodiment of the antibody-linker conjugate of the invention, the linker is a drug linker and the drug is a growth inhibitory drug, preferably exatecan.

[0166] In a further embodiment of the method of the present invention, in step (2), the mixture comprises the following drug-linker: [ka] Includes.

[0167] In a further embodiment of the method of the invention, a glutamine of an amino acid sequence selected from the group consisting of GGTLQSPP, TLQSG, TLQSPP, GGTLQSG and TLQSA of the antibody reacts in step (2) of the method in the presence of the microbial transglutaminase to form a covalent bond with the H2N portion of the linker.

[0168] In a further embodiment of the method of the present invention, the antibody is a monoclonal antibody.

[0169] In a further embodiment of the method of the present invention, the microbial transglutaminase is a microbial transglutaminase from S. ladakanum.

[0170] In a further embodiment of the method of the present invention, the reaction mixture obtained in step (2) is incubated at a temperature between 20° C. and 38° C. until the antibody-linker conjugates form and can be separated in method step (3).

[0171] In further embodiments of the method of the invention, the reaction buffer comprises one or more of the following buffers adjusted to a pH between pH 7 and pH 8.8:In further embodiments of the method of the invention, the reaction buffer comprises Tris-HCl adjusted to a pH between 7.7 and 8.5:or comprises Bicine and / or Bicine / Tris adjusted to a pH between 7.8 and 8.8:or comprises Tricine adjusted to a pH between 7.8 and 8.0.

[0172] In a further embodiment of the method of the present invention, the reaction buffer comprises HEPES in an amount between 5 mM and 100 mM.

[0173] In a further embodiment of the method of the invention, the reaction buffer is a reaction buffer comprising between 3% and 10% DMSO, between 5 mM and 60 mM HEPES, less than 100 mM NaCl (preferably no NaCl) and having a pH between 6 and 7.5; The antibody further comprises the sequences GGTLQSPP, TLQSG, TLQSPP, GGTLQSG, and TLQSA in both light chain constant regions (CL) and / or both heavy chain constant regions (CH); and the linker has the formula: [ka] where R is the remainder of the linker, optionally also including the drug.

[0174] In a further embodiment of the method of the present invention, said reaction buffer comprises less than 150 mM NaCl, preferably less than 100 mM NaCl, more preferably less than 75 mM NaCl, even more preferably 5 mM NaCl or less, and most preferably said reaction buffer does not contain NaCl.

[0175] In a further embodiment of the method of the invention, the total amount of all buffers contained in said reaction buffer is lower than 160 mM, preferably lower than 155 mM.

[0176] In a further embodiment of the method of the present invention, the total amount of all buffering agents contained in said reaction buffer is less than 70 mM.

[0177] In a further embodiment of the method of the invention, the total amount of all buffers contained in said reaction buffer is lower than 155 mM, the reaction buffer contains less than 10 mM NaCl and the total amount of microbial transglutaminase is between 2.5 and 12 U / ml.

[0178] In a further embodiment of the method of the invention, the total amount of all buffers contained in said reaction buffer is lower than 70 mM, the reaction buffer contains less than 50 mM NaCl, the total amount of microbial transglutaminase is between 2.5 and 12 U / ml, and the pH of the reaction buffer is between pH 5.9 and pH 8.1. In a further embodiment of the method of the invention, the antibody is trastuzumab, rituximab or labetuzumab; or said antibody comprises the amino acid Q295 in its heavy chain constant region (CH), where Eu numbering is used to define the position of said glutamine at position 295; or The antibody comprises amino acid Q295 in its heavy chain constant region (CH), where Eu numbering is used to define the position of the glutamine at position 295; the antibody is an IgG monoclonal antibody.

[0179] In a further embodiment of the method of the invention, the reaction buffer comprises at least one buffer selected from the group consisting of imidazole, HEPES, bicine, Tris-HCl, MOPS / BisTrisPropane and MOPS / HEPES Sodium, the buffer concentration being selected in the range of 10-160 mM, preferably the buffer is HEPES at a concentration between 10 and 30 mM, more preferably at a concentration of about 24 mM.

[0180] In a further embodiment of the method of the present invention, said reaction buffer has a pH between 6.5 and 8.7, more preferably between 6.8 and 8.2, most preferably a pH of about 7, or said reaction buffer has a pH between about 7 and 8.5, and the reaction buffer comprises less than 10 mM NaCl and between 20 mM and 120 mM of at least one of the following buffers: HEPES, Tris-HCl, MOPS / NaHEPES, bicine, bicine / tris, imidazole and tricine.

[0181] In a further embodiment of the method of the invention, said reaction buffer comprises between 4% and 7% DMSO.

[0182] In a further embodiment of the method of the invention, the reaction buffer comprises said transglutaminase, said transglutaminase comprising the amino acid sequence of SEQ ID NO: 45, and said microbial transglutaminase is included in the mixture of step (2) of the method at a concentration between 5 U / mL and 25 U / mL, preferably at a concentration between 10 U / mL and 20 U / mL.

[0183] In a further embodiment of the method of the present invention, said reaction buffer does not contain sodium chloride and further comprises about 24 mM HEPES, said reaction buffer has a pH of about 7.5, and a microbial transglutaminase concentration of about 12.5 U / mL.

[0184] In a further embodiment of the method of the present invention, the reaction buffer does not contain sodium chloride and further comprises about 24 mM HEPES, the reaction buffer has a pH of about 7 and contains about 4% DMSO, and the concentration of microbial transglutaminase in the mixture of step (2) of the method is about 20 U / mL.

[0185] In a further embodiment of the method of the present invention, the reaction buffer does not contain sodium chloride and further comprises about 24 mM HEPES, the reaction buffer has a pH of about 7 and contains about 7% DMSO, and the concentration of microbial transglutaminase in the mixture of step (2) of the method is about 20 U / mL.

[0186] In a further embodiment of the method of the invention, said antibody comprises the amino acid sequence TLQSG, GGTLQSPP or GGTLQSG at the C-terminus of its light chain constant region (CL) and the amino acid Q295 in its heavy chain constant region (CH), wherein Eu numbering is used to position said glutamine at position 295.

[0187] In a further embodiment of the method of the invention, the drug-antibody ratio (DAR) of the antibody-linker conjugate produced in step (2) is between 3 and 4.5, preferably 3.8 to 4.1.

[0188] In a further embodiment of the method of the present invention, the drug-antibody ratio (DAR) of the antibody-linker conjugate produced in step (2) is between 1.9 and 2.

[0189] In a further embodiment of the method of the invention, said antibody comprises the amino acid Q295 in its heavy chain constant region (CH), wherein Eu numbering is used to define the position of said glutamine at position 295, and said antibody is an IgG monoclonal antibody.

[0190] In a further embodiment of the method of the present invention, the mTG enzyme is from S. ladakanum, said antibody is a monoclonal antibody comprising a glutamine at position 295, the reaction buffer comprises DMSO and further comprises HEPES in an amount between about 20 mM and 30 mM, the reaction buffer comprises less than 100 mM NaCl, and the pH of the reaction buffer is between about pH 7 and pH 7.5, wherein Eu numbering is used to define the position of said glutamine at position 295.

[0191] In a further embodiment of the method of the present invention, the mTG enzyme is from S. ladakanum, said antibody is a monoclonal antibody comprising a glutamine at position 295, the reaction buffer comprises DMSO and further comprises HEPES in an amount between about 20 mM and 30 mM, the reaction buffer comprises less than 100 mM NaCl, the pH of the reaction buffer is between about pH 7 and pH 7.5, wherein Eu numbering is used to define the position of said glutamine at position 295, and the C-terminus of the light chain of the antibody comprises the amino acid sequence GGTLQSPP.

[0192] In a further embodiment of the methods of the present invention, the linker has the structure (A) or (B) shown below, where R is a therapeutic agent or another payload: [ka]

[0193] In a further embodiment of the method of the present invention, the antibody comprises one or more amino acid sequences selected from the group consisting of GGTLQSPP, TLQSG, TLQSPP, GGTLQSG and TLQSA, which amino acid sequence is located at the C-terminus of the antibody light chain and / or at the C-terminus of the antibody heavy chain.

[0194] In a further embodiment of the method of the invention, a molar excess of drug linker is used per conjugation site of the antibody in step (2) of the method, preferably at least 4 molar equivalents of drug-linker per conjugation site.

[0195] In a further embodiment of the antibody-drug conjugate of the invention, the immunoconjugate has the formula (IVA) and n is between 1 and 10, preferably about 4.

[0196] In a further aspect, the present invention provides an antibody comprising an amino acid sequence selected from the group consisting of GGTLQSPP, GGTLQSG and TLQSA at the C-terminus of the antibody heavy chain and / or an amino acid sequence selected from the group consisting of GGTLQSPP, GGTLQSG and TLQSA at the C-terminus of the antibody light chain.

[0197] A brief description of the sequence Amino acid sequence: SEQ ID NO: 1 Human CEACAM5 protein sequence according to GenBank accession number AAA51967.1 SEQ ID NO:2 Macaca fascicularis CEACAM5 protein sequence (NCBI reference sequence XP_005589491.1) SEQ ID NO: 3 CDR1-H of mAb1 SEQ ID NO: 4 CDR2-H of mAb1 SEQ ID NO: 5 CDR3-H of mAb1 SEQ ID NO: 6 CDR1-L of mAb1 SEQ ID NO: 7 CDR2-L of mAb1 SEQ ID NO: 8 CDR3-L of mAb1 SEQ ID NO: 9 VH of mAb1 SEQ ID NO: 10 VL of mAb1 SEQ ID NO: 11 CH of mAb1 SEQ ID NO: 12 CL of mAb1, mAb1-M and mAb6-M SEQ ID NO: 13 HC of mAb1 SEQ ID NO: 14 LC of mAb1, mAb1-M and mAb6-M

[0198] Nucleic acid sequence: SEQ ID NO: 15 DNA sequence encoding the HC of mAb1 SEQ ID NO: 16 DNA sequence encoding the LC of mAb1

[0199] Amino acid sequence: SEQ ID NO: 17 HC of antibody hu8G4 SEQ ID NO: 18 LC of antibody hu8G4 SEQ ID NO: 19 Optimized antibody variant 1 HC SEQ ID NO: 20 LC of optimized antibody variant 1 SEQ ID NO: 21 LC of optimized antibody variant 2 SEQ ID NO: 22 LC of optimized antibody variant 4 SEQ ID NO: 23 LC of optimized antibody variant 5 SEQ ID NO: 24 Optimized antibody variant 6 HC SEQ ID NO: 25 HC of huMab2-3 (allotype) SEQ ID NO: 26 LC of huMab2-3 SEQ ID NO:27 hmn-14 HC SEQ ID NO:28 LC of hmn-14 SEQ ID NO:29 rb8G4HC SEQ ID NO: 30 LC of rb8G4 SEQ ID NO: 31 CH(CH-LALA-YTE) of mAb1-M & mAb2-M SEQ ID NO: 32 CH of mAb3-M (CH-K222R-LALA-YTE) SEQ ID NO: 33 CL (CL-tag) of mAb2-M, mAb3-M and mAb7-M SEQ ID NO: 34 HC of mAb1-M and mAb2-M (HC-LALA-YTE) SEQ ID NO: 35 HC of mAb3-M (HC-K222R-LALA-YTE) SEQ ID NO: 36 LC (LC-tag) of mAb2-M, mAb3-M and mAb7-M SEQ ID NO: 40 HC of mAb5-M (anti-CD20-HC) SEQ ID NO: 41 LC of mAb5-M (anti-CD20-LC) SEQ ID NO: 44 Transglutaminase SEQ ID NO: 45 Transglutaminase (activated, version 1) SEQ ID NO: 47 Transglutaminase (activated, from S. mobaraensis) SEQ ID NO: 48 Transglutaminase (activated, version 3) SEQ ID NO: 49 Transglutaminase (activated, version 4) SEQ ID NO: 50 CH(CH-LALA) of mAb6-M and mAb7-M SEQ ID NO: 51 HC of mAb6-M and mAb7-M (HC-LALA) SEQ ID NO: 54 Framework region FR1 SEQ ID NO: 55 Framework region FR2 SEQ ID NO: 56 Framework region FR3 SEQ ID NO: 57 Framework region FR4 SEQ ID NO: 58 Framework region FR5 SEQ ID NO: 59 Framework region FR6 SEQ ID NO: 60 Framework region FR7 SEQ ID NO: 61 Framework region FR8

[0200] Nucleic acid sequence: SEQ ID NO: 37 DNA sequence encoding the HC (HC-LALA-YTE) of mAb1-M and mAb2-M SEQ ID NO: 38 DNA sequence encoding HC (LC-K222R-LALA-YTE) of mAb3-M SEQ ID NO: 39 DNA sequence encoding the LC (LC-tag) of mAb2-M, mAb3-M and mAb7-M SEQ ID NO: 42 DNA sequence encoding the HC of mAb5-M (anti-CD20-HC) SEQ ID NO: 43 DNA sequence encoding the LC of mAb5-M (anti-CD20-LC) SEQ ID NO: 46 DNA sequence encoding transglutaminase SEQ ID NO: 52 DNA sequence encoding the HC (HC-LALA) of mAb6-M and mAb7-M SEQ ID NO: 53 DNA sequence encoding the LC of mAb1-M and mAb6-M

[0201] Amino acid sequence: SEQ ID NO: 62 Labetuzumab heavy chain SEQ ID NO:63 Labetuzumab light chain SEQ ID NO: 64 Rituximab HC SEQ ID NO: 65 Rituximab LC SEQ ID NO:66 mAb_tag2 _LC SEQ ID NO: 67 mAb_tag2_HC (HC-tag) SEQ ID NO: 68 mAb_tag3_LC (LC-tag1) SEQ ID NO: 69 mAb_tag4_LC (LC-tag2) SEQ ID NO: 70 mAb_tag3&4_HC(HC) SEQ ID NO: 71 mAb_tag5_LC (LC-tag) SEQ ID NO: 72 mAb 7-M_HC(ceacam-HC) SEQ ID NO: 73 Transglutaminase (activated, version 2, from S. ladakanum) SEQ ID NO: 74 mTG tag GTLQSPP SEQ ID NO: 77 mTG tag TLQSG SEQ ID NO: 78 mTG tag TLQSPP SEQ ID NO: 79 mTG tag GGTLQSG SEQ ID NO: 80 mTG tag TLQSA SEQ ID NO: 81 MTG_5 (from S. mobaraensis) SEQ ID NO: 82 MTG_6 (from S. mobaraensis) SEQ ID NO: 83 MTG_7 (from S. mobaraensis) SEQ ID NO: 84 MTG_8 (from S. mobaraensis)

[0202] Example 1: Synthesis of Drug-Linker Compounds with Glucuronide-Based Linkers: Drug-Linker Compound 1-M (DL1-M) [ka]

[0203] Synthetic route to compound 9).

[0204] [ka] Synthetic Route to Compound 11 (also referred to herein as Drug-Linker Compound 1-M (DL1-M)

[0205] Protocol for the chemical preparation of compound 9 Step 1: Compound 1

[0206] [ka]

[0207] To a stirred solution of (2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-bromo-tetrahydro-pyran-2-carboxylic acid methyl ester (8.30 g; 20.90 mmol; 1.00 eq.) and 4-hydroxy-3-nitro-benzaldehyde (5.24 g; 31.35 mmol; 1.50 eq.) in acetonitrile (83.00 ml; 10.00 V) was added silver(I) oxide (9.69 g; 41.80 mmol; 2.00 eq.). The reaction mixture was stirred at RT for 16 h. The reaction mixture was filtered through Celite. The filtrate was concentrated under vacuum to give a solid. The solid was dissolved in EtOAc and washed with 10% aqueous NaHCO3 to remove excess 4-hydroxy-3-nitro-benzaldehyde. The organic layer was concentrated under vacuum to give compound 1 as a sand-colored solid. Yield: 9.0g Yield: 89.1%

[0208] Analysis data : NMR: 1 H-NMR (400 MHz, DMSO-d6): 9.98 (s,1H), 8.46 (s, 1H),8.25-8.21 (m, 1H),7.64 (d, J = 11.60Hz, 1H), 5.94 (d, J= 10.00 Hz,1H),5.51-5.44 (m, 1H),5.20-5.09 (m, 2H),4.80 (d, J = 13.20 Hz, 1H), 3.64 (s,3H), 2.09 (s, 9H).

[0209] Step 2: Compound 2 [ka]

[0210] To a stirred solution of compound 1 (9.00 g; 18.62 mmol; 1.00 eq.) in propan-2-ol (33.00 ml; 3.67 V) and CHCl3 (167.00 ml; 18.56 V), silica gel 60-120 (3.60 g; 112.09 mmol; 6.02 eq.) was added followed by sodium borohydride (1.80 g; 46.55 mmol; 2.50 eq.). The reaction mixture was stirred at RT for 1 h. After completion, the reaction mixture was quenched with cold H2O and filtered through Celite. The filtrate was extracted with dichloromethane and dried over Na2SO4. The solvent was concentrated to give compound 2 as an off-white powder. Yield: 8.70g Yield: 92.4%. Analysis data LCMS: Column: ATLANTIS dC18 (50 x 4.6 mm) 5 μm, mobile phase A: 0.1% HCOOH, in H2O: ACN (95:5), B: ACN RT(min): 2.05, M+H: 503.2, purity: 96.6%

[0211] Step 3: Compound 3 [ka]

[0212] To a stirred solution of compound 2 (8.70 g; 17.21 mmol; 1.00 eq.) in ethyl acetate (100.00 ml; 11.49 V) and THF (100.00 ml; 11.49 V) was added palladium on carbon (10% w / w) (2.50 g; 2.35 mmol; 0.14 eq.). The reaction mixture was stirred under hydrogen atmosphere at RT for 3 h. After completion, the reaction mixture was filtered through Celite. The solvent was concentrated under vacuum to give compound 3 as an off-white solid. Yield: 8.5g Yield: 100%

[0213] Analytical data: LCMS: Column: ATLANTIS dC18 (50 x 4.6 mm) 5 μm, mobile phase A: 0.1% HCOOH, in H2O: ACN (95:5), B: ACN RT(min): 1.73, M+H: 456.10, purity: 95.1%

[0214] Step 4: Compound 4 [ka]

[0215] To a stirred solution of compound 3 (10.00 g; 20.89 mmol; 1.00 eq.) and (9H-fluoren-9-ylmethoxycarbonylamino)-acetic acid (7.60 g; 25.06 mmol; 1.20 eq.) in DCM (250.00 ml; 25.00 V), 2-ethoxy-2H-quinoline-1-carboxylic acid ethyl ester (15.65 g; 62.6 mmol; 3.00 eq.) was added at 0° C. The reaction mixture was stirred at RT for 16 h. After completion, the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (56% EtOAc:Petroleum Ether) to obtain the compound with 80% purity. The compound was further purified by washing with 30% EtOAc and Pet Ether to obtain compound 4 as a white solid. Yield: 8.5g Yield: 50.7%

[0216] Analytical data: LCMS: Column: ATLANTIS dC18 (50 x 4.6 mm) 5 μm, mobile phase A: 0.1% HCOOH, in H2O, ACN (95:5), B: ACN RT(min):3.03, M+H:735.2, Purity:81.9%

[0217] Step 5: Compound 5 [ka]

[0218] To a stirred solution of compound 4 (2.00 g; 2.49 mmol; 1.00 eq.) in THF (40.00 ml; 20.00 V) at 0° C. was added carbonic acid bis-(4-nitro-phenyl) ester (3.06 g; 9.97 mmol; 4.00 eq.) and DIPEA (4.40 ml; 24.92 mmol; 10.00 eq.). The reaction mixture was stirred at RT for 12 h. After completion of the reaction, the reaction mixture was concentrated under vacuum. The crude product was purified by column chromatography using silica gel (230-400) and Pet ether / ethyl acetate as eluent to give compound 5 as a pale yellow solid. Yield: 2.0g Yield: 84.6%

[0219] Analytical data: LCMS: Column: X-Bridge C8 (50X4.6) mm, 3.5 μm; Mobile phase: A: 0.1% TFA in Milli-Q water; B: ACN RT(min):3.24;M+H:900.20, Purity:94.9%

[0220] Step 6: Compound 6 [ka]

[0221] Compound 5 (1,369 g; 1,00 eq.) was dissolved in N,N-dimethylformamide (15,00 ml) and exatecan mesylate (679,7 mg; 1,00 eq.), 4-methylmorpholine (0,422 ml; 3,00 eq.) and 1-hydroxybenzotriazole (172,8 mg; 1,00 eq.) were added for synthesis. The reaction mixture was stirred overnight at room temperature. After the stirring time, the reaction suspension turned into a brown solution. The reaction was monitored by LC-MS, which showed complete conversion of the starting material. The reaction mixture was purified by RP flash chromatography. The product-containing fractions were combined, concentrated under vacuum and lyophilized overnight to give compound 6 as a yellow solid. Yield: 1.59g Yield: 87.5%

[0222] Analytical data: LCMS: Column: Chromolis HR RP-18e (50-4,6 mm); Mobile phase A: 0.05% HCOOH in HO; B: 0.04% HCOOH and 1% HO in ACN; T: 40 °C; Flow rate: 3,3 ml / min; MS: 100-2000, amu positive; 1% → 100% B: 0 → 2,0 min; 100% B: 2,0 → 2,5 min RT(min):1.95;M+H:1196.40, Purity:84.4%.

[0223] Step 7: Compound 7 [ka]

[0224] Compound 6 (1,586 g; 1,00 eq.) was dissolved in tetrahydrofuran (50,00 ml) and a solution of LiOH (0.1 M) (containing lithium hydroxide hydrate (281,77 mg; 6,00 eq.) in water (67,100 ml)) was added dropwise at 0° C. The pH value was checked during the addition. The pH should not exceed 10. The addition of the solution of LiOH was complete after 1.5 h. The reaction was monitored by LC-MS and showed complete conversion of the starting material. The reaction was quenched with a citric acid solution and the pH was adjusted to 5. The reaction mixture was concentrated under reduced pressure. The crude product was purified by preparative HPLC. The product-containing fractions were combined and lyophilized to give compound 7 as a dark yellow solid. Yield: 728mg Yield: 54.8%

[0225] Analytical data: LCMS: Column: Chromolis HR RP-18e (50-4,6 mm); Mobile phase A: 0.05% HCOOH in HO; B: 0.04% HCOOH and 1% HO in ACN; T: 40 °C; Flow rate: 3,3 ml / min; MS: 100-2000, amu positive; 1% → 100% B: 0 → 2,0 min; 100% B: 2,0 → 2,5 min

[0226] RT(min):1.68;M+H:1056.30, Purity:98.5%.

[0227] Step 8: Compound 8 [ka]

[0228] Compound 7 (728,000 mg; 1,00 eq.) was dissolved in N,N-dimethylformamide (20,00 ml). Piperidine (136,513 μl; 2,00 eq.) was added and the solution was stirred at RT for a total of 4 h. The reaction was monitored by LC-MS, which showed complete conversion of the starting material. The reaction mixture was concentrated under reduced pressure and the crude product was purified by RP flash chromatography. The product-containing fractions were combined, the solvent was partially removed and it was lyophilized overnight to provide compound 8 as a yellow solid. Yield: 706mg Yield: 100%

[0229] Analytical data: LCMS: Column: Chromolis HR RP-18e (50-4,6 mm); Mobile phase A: 0.05% HCOOH in HO; B: 0.04% HCOOH and 1% HO in ACN; T: 40 °C; Flow rate: 3,3 ml / min; MS: 100-2000, amu positive; 1% → 100% B: 0 → 2,0 min; 100% B: 2,0 → 2,5 min RT(min):1.22;M+H:834.30, Purity:97.6%.

[0230] Step 9: Compound 9 [ka]

[0231] To a solution of compound 8 (854 mg; 1,00 eq.) in dimethylformamide (30,00 ml) was added N-ethyldiisopropylamine (149,234 μl; 1,00 eq.) and 3-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-propionic acid 2,5-dioxo-pyrrolidin-1-yl ester (233,61 mg; 1,00 eq.). The reaction mixture was stirred at RT for 3 h. The reaction was monitored by LC-MS, which showed complete conversion of the starting material. The reaction mixture was concentrated under reduced pressure and the crude product was purified by RP flash chromatography. The product-containing fractions were combined, concentrated and lyophilized to give the desired product in 91% purity. This material was again purified by RP chromatography to give compound 9 as a yellow solid. Yield: 580mg Yield: 60.1%

[0232] Analytical data: LCMS: Column: Chromolis HR RP-18e (50-4,6 mm); Mobile phase A: 0.05% HCOOH in HO; B: 0.04% HCOOH and 1% HO in ACN; T: 40 °C; Flow rate: 3,3 ml / min; MS: 100-2000, amu positive; 1% → 100% B: 0 → 2,0 min; 100% B: 2,0 → 2,5 min RT(min): 1.38; M+H: 985.30, purity: 90% (the other 10% of the isomer can be removed by HPLC)

[0233] 1H NMR (500 MHz, DMSO-d6) δ 13.10 - 12.44 (m, 1H), 9.08 (s, 1H), 8.32 (t, J = 5.8 Hz, 1H), 8.16 (s, 1H), 8.02 (d, J = 8.8 Hz, 1H), 7.76 (d, J = 10.9 Hz, 1H), 7.31 (s, 1H), 7.15 - 7.09 (m, 2H), 6.98 (s, 2H), 5.48 - 5.38 (m, 2H), 5.32 - 5.22 (m, 3H), 5.11 - 5.01 (m, 2H), 4.87 (d, J = 7.6 Hz, 1H), 3.92 - 3.88 (m, 1H), 3.89 - 3.84 (m, 2H), 3.65 - 3.61 (m, 2H), 3.46 - 3.41 (m, 1H), 3.42 - 3.37 (m, 1H), 3.38 - 3.31 (m, 1H), 3.28 - 3.20 (m, 1H), 3.15 - 3.07 (m, 1H), 2.48 - 2.44 (m, 2H), 2.38 (s, 3H), 2.24 - 2.13 (m, 2H), 1.94 - 1.80 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H).

[0234] Compound 11 chemical preparation method ステップ1:Compound 10

change

[0235] To a solution of compound 8 (289.00 mg; 1.00 eq) in dimethylformamide (10.00 ml) was added N-ethyldiisopropylamino (0.104 ml, 2.00 eq) and 2,5-dioxopyrrolidin-1-yl(tert-butoxycarbonyl)glycylglycinate (105.70 mg; 1.00 eq). The reaction mixture was stirred at room temperature for 1 hour and the reaction was reflected by LC-MS. After completion, the solvent was removed under vacuum and the crude product was purified by preparative HPLC through a Sunfire column. The product-containing fractions were combined and lyophilized to provide the trifluoroacetate salt of compound 10 as a yellow solid.

[0236] Yield: 162.00 mg, 0.14 mmol. Yield: 44.8%

[0237] Analytical data: LCMS: Column: Chromolis HR RP-18e (50-4,6 mm); Mobile phase A: 0.05% HCOOH in HO; B: 0.04% HCOOH and 1% HO in ACN; T: 40 °C; Flow rate: 3,3 ml / min; MS: 100-2000, amu positive; 1% → 100% B: 0 → 2,0 min; 100% B: 2,0 → 2,5 min RT(min):1.41;M+H:1048.1, Purity:97.9%

[0238] Step 2: Compound 11 [ka]

[0239] To a suspension of compound 10 (162.00 mg; 1.00 eq) in dichloromethane (5.00 ml) was added 4.0 M hydrogen chloride solution in 1,4-dioxane (682.43 μl, 20.00 eq), which was stirred at room temperature for 3.5 h and the reaction was monitored by LC-MS. After completion, the reaction mixture was concentrated under vacuum and the crude product was purified by preparative HPLC. The product-containing fractions were combined and lyophilized to give the trifluoroacetate salt of compound 11 as a yellow solid.

[0240] Yield: 113 mg; 0.11 mmol. Yield: 77.0%

[0241] Analytical data: LCMS: Column: Chromolis HR RP-18e (50-4,6 mm); Mobile phase A: 0.05% HCOOH in HO; B: 0.04% HCOOH and 1% HO in ACN; T: 40 °C; Flow rate: 3,3 ml / min; MS: 100-2000, amu positive; 1% → 100% B: 0 → 2,0 min; 100% B: 2,0 → 2,5 min RT(min):1.22;M+H:948.0, M+2H:474.8;Purity:98.8%

[0242] 1H NMR (700 MHz, DMSO-d6) δ 12.93 - 12.79 (m, 1H), 9.14 (s, 1H), 8.61 (t, J = 5.8 Hz, 1H), 8.41 (t, J = 5.9 Hz, 1H), 8.14 (s, 1H), 8.05 (d, J = 8.8 Hz, 1H), 8.03 - 7.93 (m, 3H), 7.79 (d, J = 10.8 Hz, 1H), 7.31 (s, 1H), 7.13 (s, 2H), 6.58 - 6.47 (m, 1H), 5.80 - 5.61 (m, 1H), 5.48 - 5.40 (m, 2H), 5.31 - 5.24 (m, 3H), 5.08 (d, J = 12.2 Hz, 1H), 5.03 (d, J = 12.3 Hz, 1H), 4.91 (d, J = 7.7 Hz, 1H), 3.96 (d, J = 5.8 Hz, 2H), 3.95 - 3.86 (m, 3H), 3.62 (q, J = 5.8 Hz, 2H), 3.43 (t, J = 9.3 Hz, 1H), 3.41 (t, J = 8.5 Hz, 1H), 3.35 (t, J = 9.0 Hz, 1H), 3.27 - 3.20 (m, 1H), 3.15 - 3.09 (m, 1H), 2.38 (s, 3H), 2.23 - 2.13 (m, 2H), 1.93 - 1.81 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H).

[0243] Example 2: Expression and purification of exemplary engineered antibodies and transglutaminase The following describes the expression and purification of exemplary modified antibodies. These exemplary modified antibodies can be used to produce the respective antibody-drug conjugates using the method of the present invention. The characteristics of modified antibodies mAb1-M, mAb2-M, mAb3-M, mAb4-M, mAb5-M, mAb6-M and mAb7-M are summarized in Table 4 below. [Table 4]

[0244] DNA sequences encoding antibodies mAb1-M, mAb2-M, mAb3-M, mAb5-M, mAb6-M and mAb7-M were synthesized at GeneArt (Life Technologies) and cloned into pTT5 plasmid for recombinant expression. The resulting plasmids were used for transient transfection and recombinant protein expression in shake flasks. ExpiCHO expression system (Gibco™, Thermo Fisher Scientific Inc.) was used. Seven days after transfection, the supernatant was harvested and the expressed antibodies were purified. A standard stepwise process was used, including Protein A affinity chromatography (HiTrap MabSelect SuRe column, Cytiva) and size exclusion chromatography (HiLoad Superdex 200pg column, Cytiva).

[0245] mAb4-M / rituximab (F. Hoffmann-La Roche Ltd., Basel, Switzerland) was purchased of pharmacy grade quality.

[0246] Expression and purification of transglutaminase The DNA sequence (SEQ ID NO: 46) encoding the transglutaminase enzyme mTG was synthesized by GeneArt (Life Technologies) and cloned into pET30a plasmid for recombinant expression. The Escherichia coli BL21(DE3) strain transformed with the produced plasmid was cultured overnight in a shake flask at 28°C and 130 rpm (50 mm shaker diameter) in lysogen broth medium supplemented with 5 g / l glucose, 10 ml / 100 ml of 10x phosphate buffered saline and 30 mg / l kanamycin. This culture was used to inoculate a fermenter containing 9.5 l of growth medium (50 g / l yeast extract, 10 g / l peptone, 0.5 g / l MgSO4x7H2O, and 2 ml of 50% Desmophen (antifoam by Rhein Chemie Rheinau) at an optical density of 0.00002. The fermenter was run overnight (16 h) at 28 °C, 800 U / min rotation, 5 Nl / min aeration, and pH 7.0-7.4. At OD5, the culture was induced with 0.1 mM IPTG until an OD of ~30 was reached (5-6 h). In case of foaming or a drop in oxygen concentration below 2 mg / ml, more Desmophen was added or the rotation was increased to 1000 rpm, respectively. The cell mass was harvested by continuous flow-through centrifugation.

[0247] 50 g of cell pellet was resuspended in 250 ml 50 mM Na-Acetate, 1 mM DTT, 5 mM MgCl2, 25 U / ml Benzonase (Millipore) pH 5.5 and disrupted using a French press. The supernatant was clarified by centrifugation and filtration (0.8 / 0.2 μm pore size), the pH was adjusted to 5.5, and loaded onto a Fractogel® SO3-(M) 85 ml column (Millipore) and eluted with a 20 CV linear gradient from 0 to 1 M NaCl. Fractions with purified protein of the expected size were identified by SDS-PAGE, pooled and dialyzed overnight at 4° C. against 50 mM Tris / HCl, 300 mM NaCl, pH 8.0. The transglutaminase was then proteolytically processed at 37°C for 60 min at a final concentration of 2mg / ml using 0.5U / ml Dispase® I (Sigma Aldrich) and 2mM CaCl2, followed by the addition of 5mM EDTA. The reaction mix was dialyzed overnight at 4°C against 50mM sodium phosphate buffer pH 6.0, loaded onto a Fractogel® SO3-column (Millipore) and eluted with a 20CV linear gradient from 0-1M NaCl. Fractions with efficiently cleaved and purified protein were identified by SDS-PAGE, pooled, concentrated and purified using a HiLoad Superdex 75pg size-exclusion column (Cytiva) with 24 HEPES, pH 7, 100mM NaCl as the running buffer. Transglutaminase-containing fractions were pooled, concentrated to >20 mg / ml, flash frozen in liquid nitrogen, and stored at −80° C. Enzyme activity was determined using the ZediXclusive Microbial Transglutaminase Assay (Zedira).

[0248] Example 3: Exemplary ADC preparation, conjugation and characterization of ADC7-M, ADC2-M, ADC5-M and ADC3-M 3.1. Antibody preparation and conjugation Monoclonal antibodies (mAbs) were stored at -80°C. Prior to conjugation, mAbs were thawed at RT and buffer exchanged into 24 mM HEPES, pH 7.0 using a HiTrap desalting column in combination with an Akta liquid chromatography (LC) system (Cytiva). Microbial transglutaminase (mTG) was used to link drug linkers (DLs) to antibodies. The reaction setup was as follows: 5 mg / ml mAb, 5 molar equivalents of DL1-M per conjugation site, 20 U / ml mTG, 7% DMSO, 24 mM HEPES, pH 7.0. The reaction was carried out at 37°C for 18 h. ADCs were separated from DLs and mTG by size exclusion chromatography (SEC). Prior to SEC purification, the NaCl concentration of the samples was adjusted to 100 mM using a 5 M NaCl stock solution. SEC was performed using a HiLoad Superdex 200 26 / 60 Increase column coupled to an Akta LC system (Cytiva) at a flow rate of 2.5 ml / min and 10 mM histidine, 100 mM NaCl, pH 5.5 as the running buffer. The fractionated samples containing the ADC material were pooled and concentrated to 8 mg / ml using an Amicon Ultra 15 50K centrifuge (Millipore), followed by a final buffer exchange to 10 mM histidine, 40 mM NaCl, 6% trehalose, 0.05% TWEEN, pH 5.5 using a HiTrap desalting column coupled to an Akta LC system (Cytiva). The final bulk drug substance (BDS) was filtered through a 0.22 μm sterile filter unit (Millipore) and flash frozen in liquid nitrogen until further use.

[0249] 3.2 Quality attributes ADC7-M [Table A]

[0250] ADC2-M [Table B]

[0251] ADC3-M [Table C]

[0252] ADC5-M [Table D]

[0253] 3.3. Methods: Characterization of drug substances Monomer content and purity were assessed by size-exclusion chromatography on a TOSOH TSKgel column. Injection volumes ranged from 1 to 5 μl (up to 10 μg protein). Wavelength 214nm. Column: Tosoh TSKgel 7.8mm x 300mm, 5μm. Mobile phase 50 mM sodium phosphate monobasic, 0.4 M sodium perchlorate, pH 6.3. Injection volume 1-5 μL. Column temperature 25°C. Flow rate 0.5mL / min A typical SEC chromatogram showing the purity of the input mAb and the final BDS is shown in FIG.

[0254] Reverse Phase HPLC (RP-HPLC) Method RP-HPLC method parameters. Wavelength 214nm. Column: PLRP-S 1000 Å (50 × 2.1 mm, 8 μm) column, Agilent. Mobile phase A Lichrosolv H2O+0.1%TFA. Mobile phase B 100%AcN+0.1%TFA. Gradient 30-45%B(7.5min) Injection volume 10 μL. Column temperature: 65°C. Flow rate 1.0mL / min

[0255] Sample preparation: 40 μl of sample (2.2 mg / ml) was mixed with 4 μl of 0.5 M TCEP and incubated at RT for 5 min. Then, 40 μl of 0.5 M iodoacetamide was added and incubated at 37° C. for 15 min. 10 μl of this reaction was injected.

[0256] A typical RP-HPLC chromatogram illustrating the DAR determination of the final BDS is shown in FIG. Free Drug Method Wavelength 254nm. Column Phenomenex Gemini, C18, 2 x 150mm, 3μm (P / N 00F-4439-B0). Mobile phase A 0.1% formic acid in water. Mobile phase B 0.1% formic acid in acetonitrile. gradient [Table E] Injection volume 10.00 μL. Column temperature: 50°C. Flow rate 0.75mL / min. Sample preparation: Protein dropwise: 100 μL drug substance + 250 μL cold MeOH + 50 μL 3M MgCl2. Spin at 20,000 rpm for 10 min.

[0257] Standard preparation: Mix 20 μL 20 mM DL (MSC2702209A in DMSO) + 20 μL DMSO + 40 μL MeOH + 20 μL 200 mM NAC in diafiltration buffer. Incubate overnight to obtain 4 mM DL-NAC. Dilute 4 mM DL-NAC with MeOH to obtain 4 μM DL-NAC standard.

[0258] Endotoxin characterization Endotoxin was determined by a kinetic chromogenic LAL assay using the Endosafe PTS endotoxin system (Charles River). Buffers and antibodies were diluted 10-fold with LAL reagent water. ADC was diluted 10-fold with LAL reagent water. All samples were analyzed on 0.01-1 EU / mL cartridges. EU / mL values ​​were converted to EU / mg by dividing by ADC[P]mg / mL.

[0259] Example 4: ADC2-M specifically kills cancer cells in vitro with high efficacy Human cancer cell lines were used to evaluate the efficacy of ADC2-M in killing cancer cells. ADC2-M demonstrated subnanomolar and subnanomolar to single-digit nanomolar in vitro efficacy against different CEACAM5-positive cell lines, respectively (Table 3). In contrast, the effect of ADC2-M was minor against the CEACAM5-negative cell line MDA-MB-231 (Table 3). As shown in the exemplary dose-response curves, ADC2-M was highly effective against the CEACAM5-positive cell lines SK-CO-1, SNU-16, MKN-45 and LS174T (Figures 3a-d).

[0260] In contrast, ADC2-M had only a minor effect on the viability of antigen-negative MDA-MB-231 cells (FIG. 3e). An isotype control ADC utilizing the same linker payload as ADC2-M demonstrated much lower efficacy against the CEACAM5-positive cell lines tested (Figure 3).

[0261] In conclusion, ADC2-M specifically kills CEACAM5-expressing human cancer cell lines in vitro with high efficacy. [Table 3]

[0262] Table 3. Efficacy of ADC2-M and free payload against multiple human cell lines. Maximum efficacy compared to untreated control at highest tested compound concentration is indicated in brackets. Efficacy cutoff reported as -50% efficacy, otherwise "n / a". CEACAM5 expression is shown for each cell line.

[0263] Methods – Viability Assay: The cytotoxic effect of ADCs on cancer cell lines was measured by cell viability assay. The day before treatment, cells were seeded in 96-well plates in a volume of 90 μL. Test compounds (ADCs or free payload) were formulated in cell culture medium at 10x the starting concentration. Test compounds were serially diluted (1:4) and 10 μL of each dilution was added to cells in triplicate. Plates were cultured at 37° C. in a CO2 incubator for 6 days. For cell viability measurements, Cell Titer-Glo® reagent (Promega™ Corp, Madison, WI) was added to each well and plates were processed according to the manufacturer's instructions. Luminescence signals were measured using a Varioskan plate reader (Thermo Fisher). Luminescence readings were converted to % viability relative to untreated cells. Data were fitted with nonlinear regression analysis using log(inhibitor) vs. response, variable slope, 4-parameter fit equation using Genedata Screener or GraphPad Prism. Data are presented as % relative cell viability versus compound molar concentration, and error bars indicate standard deviation (SD) of duplicate or triplicate. Geometric mean IC50 values ​​from multiple experiments were calculated.

[0264] ADC2-M was also compared to ADC SAR DM4 in terms of its cytotoxic effect on antigen-positive SK-CO-1 and antigen-negative MDA-MB-231 cell lines. ADC2-M showed similar efficacy to ADC SAR DM4 on SK-CO-1 cancer cells (Figure 3a compared to Figure 26a). Non-specific effects on antigen-negative MDA-MB-231 were higher for ADC SAR DM4 compared to ADC2-M (Figure 3e compared to Figure 26b).

[0265] ADC2-M was also generated utilizing an antibody backbone lacking the YTE mutation, and this ADC (ADC7-M) showed comparable results to the respective ADC (ADC2-M) that contained the YTE mutation.

[0266] Example 5: Pharmacokinetic study of ADC Pharmacokinetic studies in human FcRn transgenic (276 hemizygous model) mice were performed after a single intravenous dose of 3 mg / kg ADC1, ADC1-M, ADC6-M, ADC7-M, ADC2-M. Samples were taken from 6 animals per treatment group at 1, 24, 48, 72, 144, 168, 240, 336, and 504 h post-dose for ADC1-M, ADC6-M, ADC7-M, and ADC2-M, and from 9 animals per treatment group at 0.08 (approximately 5 min), 4, 24, 48, 72, 168, 240, 336, and 504 h post-dose for ADC1. Data were pooled for PK analysis. See Figure 4 for plasma concentrations per treatment. Back-extrapolation of plasma concentrations at time 0 (C0) yielded concentrations slightly lower than the theoretical maximum for ADC1-M, ADC7-M, and ADC2-M, and close to the theoretical maximum for ADC6-M and ADC1 (assuming dilution by a plasma volume of 40 mL / kg). Plasma concentrations declined with a biphasic profile. A sharp decrease in plasma concentrations was observed up to 24 h, followed by a slower decrease until the last sampling time (504 h) in all molecular groups, with the exception of animals treated with ADC6-M, which were quantifiable up to 336 h.

[0267] The longest terminal half-life (t1 / 2) value was calculated for ADC2-M with Lala-YTE variant with DAR=4 (190h), followed by ADC7-M with Lala variant with DAR=4 (122h), ADC1-M with Lala-YTE variant and DAR=8 (64.5h), and ADC6-M with Lala variant with DAR=8 (33.8h), and finally ADC1 with IgG1.4 with DAR=8 (29.6h). The % of extrapolated AUCinf was lower than 20%, allowing reliable calculation of AUC0-inf and derived parameters (Cl, Vz and Vss). In transgenic mice, the highest AUC0-inf and lowest Cl values ​​were calculated for ADC2-M (Lala-YTE variant, DAR=4), followed by ADC7-M, ADC6-M, ADC1-M, and ADC1. The AUC0-inf and Cl values ​​ranged from 1.36 million to 10.2 million h*ng / mL and 0.293 to 1.16 mL / h / kg, respectively. No obvious differences in volume of distribution (Vss) were observed with Vss ranging from 49.8 to 113 mL / kg.

[0268] In conclusion, after a single iv dose of 3 mg / kg in transgenic mice, the best PK profile was exhibited by ADC2-M with the highest plasma exposure, lowest Cl and longest t1 / 2. The PK profile was improved by moving from IgG1.4 to the Lala variant, and the strongest effect was observed by further including the YTE variant, reducing the DAR from 8 to 4.

[0269] Table 5: PK parameters of exemplary ADCs following iv administration of 3 mg / kg. Antibody-drug conjugates (ADCs) and drug-to-antibody ratios (DARs) are also indicated. [Table 5]

[0270] Example 6: Efficacy of ADC1-M and ADC2-M in pancreatic cell line derived tumor models The efficacy of ADC1-M and ADC2-M was evaluated in the human pancreatic cell line-derived xenograft model BxPC3 (ATCC, CRL-1687). 6 BxPC3 cells were injected subcutaneously into the right flank of 6- to 8-week-old immunodeficient female mice (Hsd: Athymic Nude-Foxn1nu, Envigo). 3 When the tumor reached an average volume of 1000 mg / kg, 10 mice / group were treated intravenously once with (saline solution) or ADC1-M (5 mg / kg; day 0), or with ADC2-M (5 mg / kg or 10 mg / kg; day 0). The length (L) and width (W) of the tumor were measured with calipers, and the tumor volume was calculated using L x (W^2) / 2.

[0271] Single treatment with ADC1-M at a dose of 5 mg / kg and ADC2-M at a dose of 5 mg / kg or 10 mg / kg led to significant antitumor effects. Both ADCs have comparable efficacy in this model (Figure 5). Treatment had no significant effect on body weight (data not shown).

[0272] Example 7: Preparation of ADC Labetuzumab Govitecan Analog ADC8 7.1 Antibodies For purposes of further comparative experiments, additional analogs of the ADC labetuzumab govitecan were prepared based on a monoclonal antibody having the following sequence: Heavy Chain: DIQLTQSPSS LSASVGDRVT ITCKASQDVG TSVAWYQQKP GKAPKLLIYW TSTRHTGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCQQ YSLYRSFGQG TKVEIKRTVA APSVFIFPPS DEQLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL SKADYEKHKV YACEVTHQGL SSPVTKSFNR GECEVQLVES GGGVVQPGRS LRLSCSASGF DFTTYWMSWV RQAPGKGLEW IGEIHPDSST INYAPSLKDR FTISRDNAKN TLFLQMDSLR PEDTGVYFCA SLYFGFPWFA YWGQGTPVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKRV EPKSCDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK (SEQ ID NO: 62) Light Chain: DIQLTQSPSS LSASVGDRVT ITCKASQDVG TSVAWYQQKP GKAPKLLIYW TSTRHTGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCQQ YSLYRSFGQG TKVEIKRTVA APSVFIFPPS DEQLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC (SEQ ID NO: 63)

[0273] 7.2 Drug-Linker Compounds As a drug-linker molecule to be conjugated to the above-mentioned antibody, the following structure: [ka] The molecule represented by the formula:

[0274] This drug-linker molecule was purchased from SyntaBio LLC, 10239 Flanders Ct, San Diego, CA 92121. Lot No. S041070422.

[0275] 7.3 Conjugation Monoclonal antibodies (mAbs) were thawed at 2-8°C for up to 3 days prior to conjugation and further stored at 2-8°C in PBS, pH 6.8 until use. On the day of conjugation, the pH of the mAb solution was adjusted to pH 8.5, final concentration of 5% (v / v) by addition of 0.5 M Tris, 0.025 M EDTA. After pH adjustment, the mAbs were reduced using 10 molar equivalents of TCEP by incubation at 20°C for 120 min. The mAb solution was then diluted 1:1 with 20 mM histidine, 80 mM NaCl, pH 5.5, the DMSO concentration was adjusted to 10% (v / v), and the reaction was initiated by adding 16 molar equivalents of the drug-linker described above. The reaction was incubated at 20°C for 60 min and finally quenched by addition of 100 mM NAC (n-acetyl-cysteine). The conjugated mAb (i.e., ADC) was then processed by preparative size-exclusion chromatography. 7.4 Preparative Size-Exclusion Chromatography, Desalting and Filtration

[0276] The reaction mixture was purified using preparative size-exclusion chromatography. A GE HiLoad 26 / 60 Superdex S200 column was connected to an Akta Avant 25 system (GE Healthcare) and equilibrated with 20 mM histidine, 80 mM NaCl, pH 5.5 according to the manufacturer's instructions. The reaction mixture was then injected and run through the column at a flow rate of 5 ml / min using 20 mM histidine, 80 mM NaCl, pH 5.5 as the running buffer. ADC-containing fractions were determined by UV light absorption at 280 nm, pooled, and concentrated. The ADC material was concentrated using a Vivaspin VS2022 device (Sartorius UK Ltd.) according to the manufacturer's instructions. The concentrated ADC material was transferred to formulation buffer (10 mM histidine, 100 mM NaCl, 3% trehalose, 0.05% (w / v) PS20, pH 5.5). A HiPrep 26 / 10 desalting column (GE Healthcare) was used at a flow rate of 10 ml / min on an Akta Avant 25 system (GE Healthcare) according to the manufacturer's instructions. The final ADC material was filtered using a 0.2 μm filter (0.2 μm PES filter, Merck Millipore), aliquoted, and subsequently flash frozen in liquid nitrogen. The final concentration of the ADC material (drug substance) was 7.7 mg / ml. The material was kept at -80°C until further use. The ADC resulting from this work is referred to herein as "ADC8". This ADC is an analogue of labetuzumab govitecan.

[0277] 7.5 Further Characterization of the ADC8 Drug Substance The ADC8 drug substance obtained above was further analyzed (a) by size exclusion chromatography (SEC), which showed a monomeric purity of 99.3%, (b) by reversed phase HPLC (RP-HPLC), which showed a DAR of 7.7, and (c) by a RP HPLC based free drug method, which showed residual free drug levels below 0.02% (by molar ratio).

[0278] Example 8: ADC1-M, ADC2-M, ADC6-M and ADC7-M kill cancer cells with higher specificity than ADC SAR DM4 (analogue of tusamitamab ravtansine) and ADC8 (analogue of labetuzumab govitecan) Using the same method described in Example 21, human cancer cell lines were used to compare the effects of ADC1-M, ADC2-M, ADC6-M and ADC7-M, as well as the ADC SARs DM4 and ADC8, on cancer cells with CEACAM5 expression versus their effects on cancer cells lacking CEACAM5 expression. The fold reduction in IC50, defined as the specificity index, was calculated by dividing the IC50 on CEACAM5-negative MDA-MB-231 cells by the IC50 on each CEACAM5-positive cell line (see Table 6). The higher the value of the specificity index, the more specific the ADC tested. ADC1-M, ADC2-M, ADC6-M and ADC7-M showed much lower IC50 in CEACAM5-positive SK-CO-1, SNU-16 and MKN-45 cells than in CEACAM5-negative MDA-MB-231 cells. These had specificity indices ranging from 116 to 874. The specificity indexes of ADC2-M and ADC7-M may be underestimated due to their lack of effect on MDA-MB-231 cells in the tested concentration range (as shown for ADC2-M in Table 3 and Figure 3e). For this reason, the specificity index was calculated only for ADC2-M and ADC7-M, setting the IC50 at the highest tested concentration of 100 nM. In contrast, ADC SAR DM4 and ADC8 showed lower specificity indices ranging from 26 to 73 and 1.8 to 4.8, respectively. In conclusion, a more selective killing of CEACAM5-positive cancer cells by ADC1-M, ADC2-M, ADC6-M and ADC7-M was shown in vitro compared to ADC SAR DM4 and ADC8.

[0279] [Table 6]

[0280] Table 6. Increased relative efficacy of ADCs in CEACAM5-positive cell lines compared to the CEACAM5-negative MDA-MB-231 cell line (specificity index). The respective specificity index of each ADC was calculated by dividing the IC50 in MDA-MB-231 by the IC50 in each of the three CEACAM5-positive cell lines (theoretical index of 1 for MDA-MB-231 is reported for illustrative purposes only). Calculations were based on the geometric mean IC50 of two to four individual experiments.

[0281] Example 9: ADC1-M, ADC2-M, ADC6-M, ADC7-M mediate stronger bystander effects than ADC SAR DM4 against antigen-negative cells in co-culture with antigen-positive cells Using the same method as in Example 10, the efficacy of ADC1-M, ADC2-M, ADC6-M, ADC7-M and ADC SAR DM4 in mediating bystander effects on antigen-negative cells in close proximity to antigen-positive cells was evaluated in a bystander analysis. ADC1-M, ADC2-M, ADC6-M and ADC7-M showed a strong bystander effect on CEACAM5-negative MDA-MB-231 cells in the presence of CEACAM5-positive SK-CO-1 (Figure 6). Co-culture experiments were performed at an ADC concentration of 1 nM, which causes maximum inhibition of CEACAM5-positive SK-CO-1 cell viability with ADC1 (Figure 26A), but has no effect on CEACAM5-negative MDA-MB-231 cells alone (Figure 26B). Consistent with these findings, non-specific effects of ADC1-M, ADC2-M, ADC6-M, ADC7-M and ADC SAR DM4 were not observed in MDA-MB-231 only controls in the bystander analysis setting.

[0282] In conclusion, ADC1-M, ADC2-M, ADC6-M, and ADC7-M mediate strong bystander effects against antigen-negative cancer cells in coculture with antigen-positive cells. These findings support the efficacy of effectively targeting tumors with heterogeneous target expression.

[0283] Compared with the ADC SAR DM4, ADC1-M, ADC2-M, ADC6-M and ADC7-M mediated a much stronger bystander effect on antigen-negative cells in coculture with antigen-positive cells (Figure 6).

[0284] For all ADCs tested, the magnitude of the bystander effect was increased by increasing the number of antigen-positive cells added to a fixed number of antigen-negative cells. Without being bound by theory, this is a result of more ADC being processed by a greater number of antigen-positive cells, releasing more free payload, which is responsible for the bystander effect on the antigen-negative cells.

[0285] No non-specific effects of the tested ADCs were observed in bystander analyses in MDA-MB-231 cells alone for all tested ADCs at 1E-9M test concentrations.

[0286] Example 10: Efficacy of ADC1-M and ADC3-M compared to ADC8 The efficacy of ADC1-M and ADC3-M compared to ADC8 was evaluated in a human pancreatic adenocarcinoma cell line-derived xenograft model, HPAF-II (ATCC, CRL-1997). Six- to eight-week-old immunodeficient female mice (Hsd: Athymic Nude-Foxn1nu, Envigo) were inoculated with 5 × 10 6 HPAF-II cells were injected subcutaneously into the right flank. 3 When the tumor reached an average volume of 1000 mg / kg, 10 mice / group were treated once intravenously with ADC1-M (1 mg / kg or 6 mg / kg) or ADC3-M (1 mg / kg or 6 mg / kg) or ADC8 (1 mg / kg or 6 mg / kg). The length (L) and width (W) of the tumor were measured with calipers, and the tumor volume was calculated using the formula L x W^2 / 2.

[0287] Single treatment with ADC1-M or ADC3-M at a dose of 1 or 6 mg / kg led to significant antitumor effects. The effect is dose-dependent, since a single treatment with 1 mg / kg led to only a mild and temporary antitumor effect, while higher doses showed much stronger antitumor effects. In contrast, single treatment with the same dose of ADC8 did not show significant antitumor effects at either dose (Figure 7). All treatments had no significant effect on body weight (data not shown).

[0288] Example 11: Development of improved conjugation reaction conditions 11.1 mTG variants Site-specific conjugation to position Q295 of the native mAb by commonly used mTG conjugation techniques (see also figure legend in Figure 9) is not very efficient and does not produce a homogenous ADC with DAR=2 (see Figure 9A).

[0289] Results using prior art buffer systems were ineffective, even when alternative mTG mutants from, for example, S. ladakanum were used (see FIG. 9B).

[0290] 11.2 Buffer composition, excipients and pH As mentioned, mTG from S. ladakanum shows better conjugation efficiency at position Q295 compared to mTG from S. mobaraensis, but the conjugation efficiency using the published reaction conditions is still not optimal. Therefore, a high-throughput (HT) assay was developed that is capable of efficiently screening improved reaction conditions, e.g., for buffer composition, excipients, pH, etc. The assay (Figure 10) consists of a first conjugation step, where a biotin-containing mTG-compatible substrate is conjugated with mTG under various reaction conditions. The efficiency of conjugation is then determined by a homogeneous time-resolved fluorescence (HTRF) assay, which allows the assay to be performed in a high-throughput manner.

[0291] More than 10,000 different reaction conditions were screened to identify reaction parameters that increased the conjugation efficiency. Procedures for the conjugation reaction and evaluation of conjugation efficiency are described in Sections 11.3.1 to 11.3.3. Figures 11(A), (B) and (C) show the effect of pH (A), salt (B) and buffer concentration (C) on mTG conjugation efficiency.

[0292] A pH of about 5.9 to 8.7 was found to be beneficial for the conjugation reaction (Figure 11(A)). Furthermore, reducing the sodium chloride concentration was found to improve the conjugation efficiency (Figure 11(B)). For the concentrations of buffering substances exemplified in the experiments using HEPES, lower concentrations of buffering agents were also found to be beneficial.

[0293] The discovery that the most suitable reaction buffers are those that do not contain sodium chloride and contain low concentrations of buffering agents was surprising because physiological concentrations of salt are typically used in such reactions.

[0294] It was also unexpectedly shown that buffer type and pH play a role when calculated in terms of normalized conjugation efficiency, as shown in the table below (excerpt from FIG. 11A): [Table F]

[0295] As shown above, too acidic a pH or too much sodium chloride resulted in poorer conjugation efficiency. Furthermore, the following types of buffers resulted in high conjugation efficiency at pH between 7 and 8.5: HEPES, Tris-HCl, MOPS / NaHEPES, bicine, bicine / tris, imidazole, and tricine.

[0296] Then, the conjugation of the drug-linker 4M was evaluated using a suitable buffer selected from the high-throughput screening results. The procedure for the conjugation reaction and the evaluation of the conjugation efficiency were as follows: 11.3.4 and 11.3.5The results are described in Section 12. The results confirm highly improved conjugation efficiency with all selected buffers compared to previously used buffers (see Figures 12A and 12B). Two very good performing buffers included a buffer containing 24 mM HEPES at a pH of 7.5 (see Figure 12A) and a buffer containing 24 mM Tris-HCl at a pH of 8. These buffers provided a much higher DAR than reactions in the prior art buffer (25 mM Tris-HCl, 150 mM NaCl, pH 8.0).

[0297] The improved reaction conditions reduced the amount of mTG enzyme required to effectively conjugate the linker to the antibody compared to when the reaction was carried out in prior art buffer systems.

[0298] 11.3 Overview of the method Methods for expression and purification of transglutaminase are described in Example 2 above.

[0299] 11.3.1 Conjugation Reaction Methods for High Throughput Bioconjugation Screening 2 μL of 5× concentrated buffer plate (shown in Table 7) (Molecular Dimensions) was mixed with 6 μL of pre-diluted additive (shown in Table 8) (Jena Bioscience). Herceptin (Trastuzumab, Genentech / Roche) and N-(biotinyl)cadaverine (B002, Zedira) were pre-mixed and 1 μL of the mixture was added to the buffer dilution, resulting in a final 5 mg / mL of Herceptin and 687.6 μM of N-(biotinyl)cadaverine in the reaction mixture. 1 μL of enzyme "MTG_1" (mTG enzyme according to SEQ ID NO: 45) was added to result in a final 12.5 U / mL. After 16 hours of incubation at 37° C., the reaction was stopped by adding transglutaminase inhibitor C102 (Zedira) to a final of 0.1 mM. The transglutaminase reaction was transferred to a high throughput screening system and evaluated with an HTRF readout.

[0300] Screening controls: As a negative control, inhibitors were added prior to the addition of MTG_1 in one well per plate to measure background during HTRF reading.

[0301] Table 7. Customized buffers used in screening reaction conditions. All buffers were 5x concentrated: [Table 7-1] [Table 7-2]

[0302] Table 8. Pre-diluted additive concentrations for screening: [Table 8]

[0303] 11.3.2 HTRF Procedure Streptavidin-d2 (SA-d2, 610SADLA, Cisbio) and pAb anti-human IgG-Tb cryptate (IgG-Tb, 61HFCTAA, Cisbio) were diluted with 9 μL terbium buffer (61DB10RDF) to a final CSA-d2 = 1500 ng / mL and CIgG-Tb = 150 ng / mL on an HTRF 96-well low-volume white plate (66PL96100). The transglutaminase conjugation reaction was diluted with PBS, pH 7.4 (Gibco) to obtain a 10 nM antibody stock solution and 1 μL of the dilution was added to the HTRF reagent mix. It was pipetted carefully to avoid air bubbles in the wells. To avoid evaporation, the plate was sealed with adhesive strips and incubated for 4 h at room temperature. Background fluorescence was detected by two blank measurements with PBS as the sample volume. Detection of the TR-FRET signal was performed with an EnVision 2104 multiplate reader using excitation filter UV2 (320 nm) and emission filters APC (665 nm) and Europium (615 nm). Data processing was performed with EnVision Workstation version 1.12.

[0304] 11.3.3 Data Reduction Step 1: For each well, a ratio is calculated as shown in equation Eq. 1 below: This calculation minimizes well-to-well variability because the donor background noise is normalized for each well.

[0305]

number

[0306] Step 2: The result of the calculation, given in equation Eq. 2 below, normalizes the measurements for each well such that plate-to-plate variability is minimized.

[0307]

number

[0308] 11.3.4 mTG-Mediated Drug-Linker Conjugation and Titration Experiments Typically, Herceptin / Trastuzumab was conjugated with the linker-payload Gly3-VC-MMAE 1 (G3-MMAE, Moradec LLC). Increasing concentrations of MTG_1 were used in different buffer systems with otherwise constant reaction conditions. DAR was determined using reverse phase chromatography as described in 11.3.5.

[0309] The following reaction conditions were used: 5 μL of 5x concentrated buffer was mixed with 15 μL of deionized water. 1.04 μL of 120 mg / mL Herceptin was added for a final yield of 5 mg / mL, then 1.46 μL of 10 mM Gly3-VC-MMAE was added for a final concentration of 583.3 μM. MTG_1 was diluted to 200, 150, 100, 50 and 25 U / mL in water. 2.5 μL of the pre-dilution of MTG_1 was added to the previously prepared reagent mixture. The conjugation reaction was carried out for 16 h at 37 °C with shaking at 600 rpm. The reaction was stopped by adding 2.5 μL of transglutaminase inhibitor B102 (Zedira).

[0310] 11.3.5 Drug-Antibody Ratio (DAR) Analysis Reverse-phase HPLC was performed using a PLRP-S 1000 Å (50 × 2.1 mm, 8 μm) column (Agilent) run at 1.0 mL / min and 65 °C. 5–7.5 μg of antibody or ADC was injected and chromatographed using a 7.5 min linear gradient of 30–45% solvent B / A (solvent A: Lichrosolv HO + 0.1% TFA; solvent B: 100% AcN + 0.1% TFA) run with absorbance monitored at 214 nm.

[0311] MTG Layout As used herein, the mTG enzymes having the following names have the respective amino acid sequences shown below: MTG_Proenzyme (=SEQ ID NO: 44) MTG_1 (activated type) (=SEQ ID NO: 45) MTG_2 (activated type) (=SEQ ID NO: 73) MTG_3 (activated type) (=SEQ ID NO: 48) MTG_4 (activated type) (=SEQ ID NO: 49)

[0312] The following mTG enzymes from S. mobaraensis may also be used in the methods of the present invention: MTG_5 (from S. mobaraensis) (=SEQ ID NO: 81): DSDDRV TPPAEPLDRM PDPYRPSYGR AETVVNNYIR KWQQVYSHRD GRKQQMTEEQ REWLSYGCVG VTWVNSGQYP TNRLAFASFD EDRFKNELKN GRPRSGETRA EFEGRVAKES FDEEKGFQRA REVASVMNRA LENAHDESAY LDNLKKELAN GNDALRNEDA RSPFYSALRN TPSFKERNGG NHDPSRMKAV IYSKHFWSGQ DRSSSADKRK YGDPDAFRPA PGTGLVDMSR DRNIPRSPTS PGEGFVNFDY GWFGAQTEAD ADKTVWTHGN HYHAPNGSLG AMHVYESKFR NWSEGYSDFD RGAYVITFIP KSWNTAPDKV KQGWP

[0313] MTG_6 (from S. mobaraensis) (=SEQ ID NO: 82): DSDDRV TPPAEPLDRM PDPYRPSYGR AETVVNNYIR KWQQVYSHRD GRKQQMTEEQ REWLSYGCVG VTWVNSGQYP TNRLAFASFD EDRFKNELKN GRPRSGETRA EFEGRVAKES FDEEKGFQRA REVASVMNRA LENAHDESAY LDNLKKELAN GNDALRNEDA RSPFYSALRN TPSFKERNGG NHDPSRMKAV IYSKHFWSGQ DRSSSADKRK YGDPDAFRPA PGTGLVDMSR DRNIPRSPTS PGEGFVNFDY GWFGAQTEAD ADKTVWTHGN HYHAPNGSLG AMHVYESKFR NWSEGYSDFD RGAYVITFIP KSWNTAPDKV KQGWPLE

[0314] MTG_7 (from S. mobaraensis) (=SEQ ID NO: 83): FRAPDSDDRV TPPAEPLDRM PDPYRPSYGR AETVVNNYIR KWQQVYSHRD GRKQQMTEEQ REWLSYGCVG VTWVNSGQYP TNRLAFASFD EDRFKNELKN GRPRSGETRA EFEGRVAKES FDEEKGFQRA REVASVMNRA LENAHDESAY LDNLKKELAN GNDALRNEDA RSPFYSALRN TPSFKERNGG NHDPSRMKAV IYSKHFWSGQ DRSSSADKRK YGDPDAFRPA PGTGLVDMSR DRNIPRSPTS PGEGFVNFDY GWFGAQTEAD ADKTVWTHGN HYHAPNGSLG AMHVYESKFR NWSEGYSDFD RGAYVITFIP KSWNTAPDKV KQGWP

[0315] MTG_8 (from S. mobaraensis) (=SEQ ID NO: 84): FRAPDSDDRV TPPAEPLDRM PDPYRPSYGR AETVVNNYIR KWQQVYSHRD GRKQQMTEEQ REWLSYGCVG VTWVNSGQYP TNRLAFASFD EDRFKNELKN GRPRSGETRA EFEGRVAKES FDEEKGFQRA REVASVMNRA LENAHDESAY LDNLKKELAN GNDALRNEDA RSPFYSALRN TPSFKERNGG NHDPSRMKAV IYSKHFWSGQ DRSSSADKRK YGDPDAFRPA PGTGLVDMSR DRNIPRSPTS PGEGFVNFDY GWFGAQTEAD ADKTVWTHGN HYHAPNGSLG AMHVYESKFR NWSEGYSDFD RGAYVITFIP KSWNTAPDKV KQGWPLE

[0316] Example 12: mTG Recognition Tag To achieve even higher DARs by the mTG conjugation method described above (i.e., previously, two drugs were conjugated at position Q295 (Eu numbering) of both heavy chains of the mAb), certain peptide motifs were genetically fused to the mAb of interest to allow further conjugation at these motifs. Several examples of such peptide tags have been described in the prior art. However, unexpectedly, new and functional additional peptide sequences (e.g., GGTLQSPP and TLQS) that can be used in a similar manner were identified. Conjugation of antibodies fused with such peptide motifs using mTG from A. ladakanum resulted in ADCs with a DAR of about 4 (see FIG. 17).

[0317] 12.1 Identification and evaluation of MTG recognition tags Conjugation of mAb_tag1 antibody The antibody containing the amino acid sequence "TLQS" on the light chain of the antibody was recombinantly expressed using a pTT5-based expression vector and a HEK293 expression system (Gibco™, Thermo Fisher Scientific Inc.), and then purified using Protein A chromatography. The antibody was conjugated with the drug-linker 4M using microbial transglutaminase (T001, Zedira GmbH).

[0318] Conjugation was performed using 34.4 μM mAb, 344 μM G3-MMAE and 6 U / ml MTG (purchased from Zedira, Germany), 25 mM, in Tris-HCl, pH 8.0, 150 mM sodium chloride, and incubated at 37° C. for 16 h. ADC was separated from G3-MMAE 4M and possible high molecular weight species (HMWS) by size exclusion chromatography (SEC). SEC was performed using a Superdex 200 increase 10 / 300 column with PBS, pH 7.0 as running buffer. Fractionated samples containing ADC material were pooled and concentrated to >1 mg / ml using an Amicon Ultra 15 50K centrifuge (Millipore).

[0319] The ADC was analyzed using hydrophobic interaction chromatography (HIC) and mass spectrometry (MS), resulting in a drug-to-antibody ratio (DAR) of 1.7 (see Figure 13).

[0320] The conjugated and unconjugated antibodies were digested by chymotrypsin digestion using the Smart Digest kit (Thermo Fisher Scientific) and subsequently analyzed by MALDI-MS using a Bruker Ultraflex MALDI TOF / TOF mass spectrometer with an α-cyano-4 hydroxycinnamic acid matrix. Comparison of the detected masses revealed a peak at 877.368 Da that was present in the mAb but not in the ADC (see FIG. 14). This mass is consistent with the theoretical mass of the light chain peptide QSGVPSRF (876.968 Da). Comparison of the detected masses revealed a peak at 2154.126 Da that was present in the ADC but not in the mAb (see FIG. 14). This mass is close to the theoretical mass (2154.968 Da) of the light chain peptide QSGVPSRF conjugated with one Gly3-VC-MMAE peptide. These results confirm that the Gly3-VC-MMAE linker-payload can be successfully conjugated to the antibody light chain peptide QSGVPSRF.

[0321] Conjugation of mAb_tag2, mAb_tag3 and mAb_tag4 antibodies The peptide identified above is located on the light chain of the antibody as part of the sequence YTSTLQSGVPS. Its core sequence TLQSG was fused to the sequence of a trastuzumab-derived HER2-binding antibody, replacing lysine 447 of the heavy chain (mAb_tag2). The antibody was recombinantly expressed using the Expi293 expression system (Gibco™) using a pTT5-based plasmid and purified using protein A and size-exclusion chromatography. Mass spectrometry revealed partial truncation of the C-terminal QSG peptide. The same procedure was used to produce further antibodies with the sequence GGTLQSG (mAb_tag3) and LC-GGTLQSPP (mAb_tag4) fused to the light chain C-terminus. Mass spectrometry did not reveal truncation of the peptide tag in mAb_tag4.

[0322] antibody mAb_tag2, mAb_tag3 and mAb_tag4 was conjugated and purified as described above using mTG enzyme from S. mobaraensis for antibodies containing the "TLQS" peptide. Drug-to-antibody ratios (DAR) were assessed using HIC and MS, resulting in DAR values ​​of 1.9, 1.7 and 1.9 for mAb_tag2, _tag3 and _tag4, respectively (see FIG. 15).

[0323] 12.2 Conjugation of mAbs with recognition tags (mAb_tag5 and mAb7-M) using techniques for native mAb conjugation Antibodies containing glutamine-containing recognition tags were conjugated using native mAb conjugation with mTG from S. ladakanum in combination with reaction conditions determined to be suitable for simultaneous conjugation of four drug-linkers per antibody.

[0324] The tag sequence GGTLQSPP was fused to the light chain C-terminal sequence of the CD20-specific mAb, mAb_tag5, derived from rituximab, and the ceacam-5-specific mAb7-M. Antibodies were recombinantly expressed using the Expi293 Expression System (Gibco™) using pTT5-based plasmids and purified using Protein A and size-exclusion chromatography. Drug-linker 4M or 1M was conjugated to the antibody followed by SEC purification and DAR determination using RP-HPLC as described above.

[0325] For this, the mAb was buffer exchanged into 24 mM HEPES, pH 7.0 using a HiTrap desalting column in combination with an Akta liquid chromatography (LC) system (Ctiva). The conjugation mixture was set up as follows: 5 mg / ml mAb, 5 molar equivalents of drug-linker 4M or 1M per conjugation site, 20 U / ml MTG_1, 4% DMSO, 24 mM HEPES, pH 7.0. The reaction was carried out for 18 h at 37°C. The ADC was separated from the drug-linker and mTG by size-exclusion chromatography (SEC). Prior to SEC purification, the NaCl concentration of the sample was adjusted to 100 mM using a 5 M NaCl stock solution. SEC was performed using a HiLoad Superdex 200 10 / 30 increase column with 10 mM histidine, 100 mM NaCl, 3% trehalose, pH 7.0 as the running buffer. Fractionated samples containing ADC material were pooled and concentrated to >1 mg / ml using an Amicon Ultra 15 50K centrifuge (Millipore). Drug-to-antibody ratio and purity were assessed using reversed-phase (RP) chromatography.

[0326] Efficient conjugation to both the light and heavy chains resulting in a drug-to-antibody ratio of 3.9 was observed in all examined cases (see FIG. 17). We demonstrated successful conjugation of the drug-linker to Q295 of the heavy chain, plus the naturally occurring glycan at N297, plus an additional drug-linker attachment to the glutamine tag GGTLQSPP fused to the LC.

[0327] Antibody sequences used in conjugation experiments: >mAb_tag2_LC(Trastuzumab) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 66) >mAb_tag2_HC (Trastuzumab-HC-tag) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEAYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG TLQSG (SEQ ID NO:67) >mAb_tag3_LC(Trastuzumab-LC-tag1) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC GGTLQSG (SEQ ID NO:68) >mAb_tag4_LC (Trastuzumab-LC-tag2) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC GGTLQSPP (SEQ ID NO:69) >mAb_tag3&4_HC (Trastuzumab HC) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDRTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEAYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 70) >mAb_tag5_HC(~RituximabHC=SEQ ID NO:40) QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAG TTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 40) >mAb_tag5_LC tag (Rituximab-LC-tag) QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC GGTLQSPP (SEQ ID NO:71) >mAb7-M_HC(ceacam-HC) EVQLQESGPGLVKPSQTLSLTCTVSDGSVSRGGYYLTWIRQHPGKGLEWIGYIYYSGSTYFNPSLRSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARGIAVAPFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTC PPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 72) >mAb7-M_LC (ceacam LC tag = SEQ ID NO: 36) EIVLTQSPATLSVSPGERATLSCRTSQSVRSNLAWYQQKPGQAPRLLIYAASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYTNWPFTFGGPGTKVDIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC GGTLQSPP (SEQ ID NO:36)

Claims

1. 1. A method for producing an antibody-linker-conjugate, comprising the steps of: (1) providing an antibody, optionally comprising in at least one, preferably both, of its light chain constant regions (CL) and / or in at least one, preferably both, of its heavy chain constant regions (CH), an amino acid sequence selected from the group consisting of GGTLQSPP, TLQSG, TLQSPP, GGTLQSG, and TLQSA, more preferably the amino acid sequence TLQSPP or GGTLQSPP (most preferably the amino acid sequence GGTLQSPP); (2) Mixing at least the following components in a reaction buffer: (a) the antibody provided in step (1); (b) a microbial transglutaminase, preferably comprising an amino acid sequence as defined in SEQ ID NO: 44, 47, 48, 49, 45, 73, 81, 82, 83, or 84, preferably comprising the amino acid sequence of SEQ ID NO: 45; and (c) an H capable of reacting with the antibody from step (1) in the presence of the transglutaminase; 2 a linker or therapeutic agent comprising an N moiety, wherein the linker is preferably a drug-linker, wherein the linker in said drug-linker is covalently attached to the drug; (3) separating the antibody-linker-conjugate produced in step (2) from unreacted linker and the transglutaminase, preferably by subjecting the mixture from step (2) to size exclusion chromatography. The method comprising:

2. The reaction buffer is A reaction buffer that is between 0% and 7% DMSO, between 20 mM and 30 mM HEPES (preferably 24 mM HEPES), and a pH between 6.8 and 7.4 (preferably pH 7); or A reaction buffer comprising between 1% and 10% DMSO, between 5 mM and 100 mM HEPES, and less than 150 mM NaCl, and having a pH between 6 and 8; or A reaction buffer comprising between 1% and 10% DMSO, between 5 mM and 60 mM HEPES, and less than 10 mM NaCl (preferably no NaCl), and having a pH between 6 and 7.5; or A reaction buffer comprising between 5 mM and 100 mM HEPES and less than 150 mM NaCl, and having a pH between 6 and 8; or A reaction buffer containing between 5 mM and 60 mM HEPES and less than 10 mM NaCl (preferably no NaCl) and having a pH between 6 and 7.

5. The method of claim 1, wherein

3. the antibody, which is a monoclonal antibody, comprises a glutamine at position 295 of the heavy chain of the antibody, and Eu numbering is used to define the position of the glutamine at position 295, and / or the antibody comprises an amino acid sequence selected from the group consisting of GGTLQSPP, GGTLQSG, and TLQSA, at the C-terminus of the heavy and / or light chain of the antibody; and The reaction mixture contained less than 10 mM NaCl, and the reaction buffer was the following buffer adjusted to a pH between pH 7 and pH 8.8: Bicine, Bicine / Tris, Tris-HCl, HEPES, and / or Tricine The method of claim 1 or 2, comprising one or more of the following:

4. The linker has the formula: 【Chemistry 1】 is a linker having the formula: The method of any one of claims 1 to 3, wherein R is the remainder of the linker, which may also include a therapeutic agent or a detectable label.

5. In step (2), the mixture is a drug-linker 【Chemistry 2】 5. The method of claim 4, comprising:

6. 6. The method of any one of claims 1 to 5, wherein the mixture in step (2) comprises a molecular excess of linker or drug-linker or therapeutic agent, preferably at least 2, more preferably at least 5 molar equivalents of linker or drug-linker or therapeutic agent, respectively, per conjugation site, wherein the conjugation site is the sequence GGTLQSPP, TLQSG, TLQSPP, GGTLQSG, or TLQSA contained in the light chain constant region (CL) and / or heavy chain constant region (CH) of the antibody; and / or wherein the conjugation site is a glutamine at position 295 of the heavy chain of the antibody, wherein the antibody is a monoclonal antibody, and wherein Eu numbering is used to define the position of the glutamine at position 295.

7. the antibody is a monoclonal antibody, preferably an anti-CEACAM5 antibody; and / or The drug is a growth inhibitory drug as defined in (a), (b), or (c) as specified below: (a) the growth inhibitory agent is a cytotoxic drug or a radioactive moiety; (b) the growth inhibitory agent is selected from the group consisting of a chemotherapeutic agent, an enzyme, an antibiotic, a toxin, e.g., a small molecule toxin or an enzymatically active toxin, a toxoid, a vinca, a taxane, a maytansinoid or a maytansinoid analog, a tomaymycin or a pyrrolobenzodiazepine derivative, a cryptophycin derivative, a leptomycin derivative, an auristatin or a dolastatin analog, a prodrug, a topoisomerase I inhibitor, a topoisomerase II inhibitor, a DNA alkylating agent, an antitubulin agent, CC-1065, and a CC-1065 analog; (c) The method of any one of claims 1 to 5, wherein the growth inhibitory agent is exatecan.

8. The linker is preferably a linker or drug linker as defined below: 【Chemistry 3】 where n is the number of [(linker)-(growth inhibitory agent)] moieties covalently linked to the antibody; or 【Chemistry 4】 An antibody-linker conjugate producible according to the method of any one of claims 1 to 7, wherein n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody; and wherein n is preferably between 1 and 10, more preferably 4.

9. The antibody-linker conjugate of claim 8, wherein the linker is a drug-linker.

10. In step (2), the mixture is a drug-linker: 【Chemistry 5】 5. The method of claim 4, comprising:

11. In step (2) of the method, a glutamine of an amino acid sequence selected from the group consisting of GGTLQSPP, TLQSG, TLQSPP, GGTLQSG, and TLQSA of the antibody is reacted in the presence of the microbial transglutaminase to form the H of the linker. 2 The method of any one of claims 1 to 10, wherein a covalent bond is formed with the N moiety.

12. The method according to any one of claims 1 to 11, wherein the antibody is a monoclonal antibody.

13. The method according to any one of claims 1 to 12, wherein the microbial transglutaminase is a microbial transglutaminase from S. ladakanum or from S. mobaraensis.

14. The method according to any one of claims 1 to 13, wherein the reaction mixture obtained in step (2) is incubated at a temperature between 20°C and 38°C until the antibody-linker-conjugates are formed and can be separated in method step (3).

15. The reaction buffer is the following buffer adjusted to a pH between pH 7 and pH 8.8: Bicine, Bicine / Tris, Tris-HCl, HEPES, and / or Tricine The method according to any one of claims 1 to 14, comprising one or more of the following:

16. The reaction buffer comprises Tris-HCl adjusted to a pH between 7.7 and 8.5; or containing bicine and / or bicine / tris adjusted to a pH between 7.8 and 8.8; or 16. The method of any one of claims 1 to 15, comprising tricine adjusted to a pH between 7.8 and 8.

0.

17. The method of any one of claims 1 to 16, wherein the reaction buffer comprises HEPES in an amount between 5 mM and 100 mM.

18. The reaction buffer is a reaction buffer containing between 0% and 10% DMSO, between 5 mM and 60 mM HEPES, and less than 100 mM NaCl (preferably no NaCl), and having a pH between 6 and 7.5; the antibody further comprises the sequences GGTLQSPP, TLQSG, TLQSPP, GGTLQSG, and TLQSA in both light chain constant regions (CL) and / or both heavy chain constant regions (CH); and The linker has the formula: 【Chemistry 6】 is a linker having the formula:

18. The method of any one of claims 1 to 17, wherein R is the remainder of a linker and may also optionally include a drug.

19. 19. The method according to any one of claims 1 to 18, wherein the reaction buffer comprises less than 150 mM NaCl, preferably less than 100 mM NaCl, more preferably less than 75 mM NaCl, even more preferably 5 mM NaCl or less, and most preferably the reaction buffer does not contain any NaCl.

20. The method according to any one of claims 1 to 19, wherein the total amount of all buffering agents contained in the reaction buffer is lower than 160 mM, preferably lower than 155 mM.

21. The method according to any one of claims 1 to 20, wherein the total amount of all buffering agents contained in the reaction buffer is less than 70 mM.

22. 22. The method of any one of claims 1 to 21, wherein the total amount of all buffering agents contained in the reaction buffer is lower than 155 mM; and the reaction buffer contains less than 10 mM NaCl and the total amount of microbial transglutaminase is between 2.5 U / ml and 12 U / ml.

23. 23. The method of any one of claims 1 to 22, wherein the total amount of all buffering agents contained in the reaction buffer is less than 70 mM; and the reaction buffer contains less than 50 mM NaCl; the total amount of microbial transglutaminase is between 2.5 U / ml and 12 U / ml, and the pH of the reaction buffer is between pH 5.9 and pH 8.

1.

24. the antibody is trastuzumab, rituximab, or labetuzumab; or the antibody comprises the amino acid Q295 in its heavy chain constant region (CH), where Eu numbering is used to define the position of the glutamine at position 295; or 24. The method of any one of claims 1 to 23, wherein the antibody comprises amino acid Q295 in its heavy chain constant region (CH), where Eu numbering is used to define the position of the glutamine at position 295; and wherein the antibody is an IgG monoclonal antibody.

25. 25. The method according to any one of claims 1 to 24, wherein the reaction buffer comprises at least one buffering agent selected from the group consisting of imidazole, HEPES, bicine, Tris-HCl, MOPS / Bis-Tris propane, and MOPS / HEPES sodium, and the buffering agent concentration is selected within the range of 10 to 160 mM, preferably the buffering agent is HEPES at a concentration between 10 and 30 mM, more preferably at a concentration of 24 mM.

26. 26. The method of any one of claims 1 to 25, wherein the reaction buffer has a pH between 6.5 and 8.7, more preferably a pH of 6.8-8.2, most preferably a pH of about 7; or wherein the reaction buffer has a pH between about 7 and 8.5, and wherein the reaction buffer comprises at least one of the following buffering agents: HEPES, Tris-HCl, MOPS / NaHEPES, bicine, bicine / tris, imidazole, and tricine, between 20 mM and 120 mM, and less than 10 mM NaCl.

27. 27. The method according to any one of claims 1 to 26, wherein the reaction buffer comprises between about 0.5% and 8% DMSO, preferably between about 4% and 7% DMSO, most preferably up to 5% DMSO.

28. 28. The method according to any one of claims 1 to 27, wherein the reaction buffer comprises the transglutaminase, wherein the transglutaminase comprises the amino acid sequence of SEQ ID NO: 45, and wherein the microbial transglutaminase is included in the mixture in step (2) of the method at a concentration of between 5 U / mL and 25 U / mL, preferably at a concentration of between 10 U / mL and 20 U / mL.

29. 29. The method of any one of claims 1 to 28, wherein the reaction buffer does not contain any sodium chloride and further comprises about 24 mM HEPES, the reaction buffer has a pH of about 7.5, and the microbial transglutaminase concentration is about 12.5 U / mL.

30. 30. The method of any one of claims 1 to 29, wherein the reaction buffer does not contain any sodium chloride and further comprises about 24 mM HEPES, the reaction buffer has a pH of about 7 and comprises about 4% DMSO, and the concentration of microbial transglutaminase in the mixture in step (2) of the method is about 20 U / mL.

31. 31. The method of any one of claims 1 to 30, wherein the reaction buffer does not contain any sodium chloride and further comprises about 24 mM HEPES, the reaction buffer has a pH of about 7 and contains about 7% DMSO, and the concentration of microbial transglutaminase in the mixture in step (2) of the method is about 20 U / mL.

32. 32. The method of any one of claims 1 to 31, wherein the antibody comprises the amino acid sequence TLQSG, GGTLQSPP, or GGTLQSG at the C-terminus of its light chain constant region (CL) and the amino acid Q295 in its heavy chain constant region (CH), wherein Eu numbering is used to define the position of the glutamine at position 295.

33. 33. The method of any one of claims 1 to 32, wherein the drug-antibody ratio (DAR) of the antibody-linker-conjugate produced in step (2) is between 3 and 4.5, preferably 3.8 to 4.

1.

34. 33. The method of any one of claims 1 to 32, wherein the drug-antibody ratio (DAR) of the antibody-linker-conjugate produced in step (2) is between about 1.9 and 2.

35. The method of claim 34, wherein the antibody comprises amino acid Q295 in its heavy chain constant region (CH), where Eu numbering is used to define the position of the glutamine at position 295; and the antibody is an IgG monoclonal antibody.

36. 35. The method of claim 34, wherein the mTG enzyme is from S. ladakanum; the antibody is a monoclonal antibody that contains a glutamine at position 295 and the reaction buffer contains HEPES in an amount of between about 20-30 mM; the reaction buffer does not contain any NaCl and the pH of the reaction buffer is between about pH 7 and pH 7.5; and Eu numbering is used to define the position of the glutamine at position 295.

37. 35. The method of claim 34, wherein the mTG enzyme is from S. ladakanum; the antibody is a monoclonal antibody comprising a glutamine at position 295 and the reaction buffer comprises HEPES in an amount of between about 20-30 mM; the reaction buffer does not comprise any NaCl and the pH of the reaction buffer is between about pH 7 and pH 7.5; and Eu numbering is used to define the position of the glutamine at position 295; and the C-terminus of the light chain of the antibody comprises the amino acid sequence GGTLQSPP.

38. The method of any one of claims 1 to 37, wherein the linker has the structure (A) or (B) shown below, where R is a therapeutic agent or another payload: 【Chemistry 7】 【Chemistry 8】

39. 39. The method of any one of claims 1 to 38, wherein the antibody comprises one or more amino acid sequences selected from the group consisting of GGTLQSPP, TLQSG, TLQSPP, GGTLQSG, and TLQSA; wherein said amino acid sequences are located at the C-terminus of the antibody light chain and / or at the C-terminus of the antibody heavy chain.

40. 40. The method of any one of claims 1 to 39, wherein in step (2), a molar excess of drug-linker per conjugation site of the antibody is used, preferably at least 4 molar equivalents of drug-linker per conjugation site.

41. 41. An antibody-drug-conjugate or antibody-linker conjugate producible by the method of any one of claims 1 to 40.

42. 42. A kit of parts comprising a buffer as defined in any one of claims 1 to 41 and a microbial transglutaminase from S. ladakanum, preferably comprising an amino acid sequence as defined in SEQ ID NO: 44, 47, 48, 49 or 45, preferably comprising the amino acid sequence of SEQ ID NO:

45.

43. 43. The kit of parts according to claim 42, further comprising a linker having a primary amino group, wherein the linker is a substrate for microbial transglutaminase, and wherein the linker is preferably further covalently attached to the payload.

44. 44. The kit of parts of claim 42 or 43, further comprising an instruction manual instructing how to use the buffer and microbial transglutaminase of the kit to conjugate a linker having a primary amino group to an antibody.

45. An antibody comprising an amino acid sequence selected from the group consisting of GGTLQSPP, GGTLQSG, and TLQSA at the C-terminus of its heavy chain, and / or an amino acid sequence selected from the group consisting of GGTLQSPP, GGTLQSG, and TLQSA at the C-terminus of its light chain.

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