Methods and tools for conjugation to antibodies

Abstract

The present invention relates to antibodies comprising a tag that can be covalently conjugated to a primary amino group of a linker in the presence of a microbial transglutaminase. The invention also provides a method of producing an antibody-linker-conjugate by mixing an antibody with a linker and a microbial transglutaminase. Also provided are antibody-linker conjugates and antibody-drug-conjugates that can be produced according to the methods of the invention, as well as kits comprising a buffer and a microbial transglutaminase.

Description

Technical Field

[0001] The present invention relates to antibodies comprising a tag that can be covalently conjugated to the primary amino group of a linker in the presence of a microbial transglutaminase. The present invention also provides a method for producing an antibody-linker-conjugate by mixing an antibody with a linker and a microbial transglutaminase. Antibody-linker conjugates and antibody-drug conjugates that can be produced according to the method of the present invention and a kit comprising a buffer and a microbial transglutaminase are also provided. Background Art

[0002] Antibody-drug conjugates or ADCs are a class of biopharmaceutical drugs designed as targeted therapies, such as for the treatment of cancer (Hamilton GS (September 2015). "Antibody-drug conjugates for cancer the rapy: The technological and regulatory challenges of developing drug-biologic hybrids". Biologicals. 43 (5): 318–32). Unlike chemotherapy, ADCs are expected to specifically treat target cells while minimizing drug exposure to healthy cells. Stable connection of antibodies and drugs (e.g., cytotoxic (anticancer) agents) is an important aspect of ADCs. Stable ADC linkers ensure that less cytotoxic payloads fall off before reaching target cells, such as tumor cells, improving safety and limiting doses. Drugs connected to antibodies via linkers include therapeutic agents, such as therapeutic agents for the treatment of cancer, such as therapeutic agents for different solid tumor indications, including, for example, CRC, pancreatic cancer, gastric cancer, NSCLC, esophageal cancer, and prostate cancer.

[0003] The generation of antibody-drug conjugates (ADCs) involves a bioconjugation step, which requires the attachment of chemically synthesized small molecule drugs, i.e., APIs (active pharmaceutical ingredients), to biotechnologically produced targeting moieties, such as antibodies. The attachment of APIs to monoclonal antibodies (mAbs) is preferably completed in a strictly controlled manner with respect to the number of attached drugs (specific drug-antibody ratio, DA R) ​​and conjugation sites. One way to obtain such site-specific conjugates is via the use of microbial transglutaminase (mTG, Tgase). However, the conjugation of APIs using mTG typically requires prior modification of mAbs, such as via genetic engineering or removal of native glycosylation. These modifications prevent the direct use of native mAbs and may result in negative effects on certain features of the final ADC product.

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

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

[0006] The present invention addresses this need and provides a method for achieving site-specific conjugation of two or more linkers (e.g., drug-linkers) to fully glycosylated antibodies. A targeted conjugation site is glutamine Q295 (EU numbering) in the heavy chain of the antibody, which is very close to the typical N-glycosylation site N297. By using a certain transglutaminase, certain reaction conditions, specific mutations or insertions in the antibody sequence (such as peptide tags containing glutamine) or a combination of the former, conjugation to this position 295 is promoted. In some cases, conjugation not only targets Q295, but also simultaneously targets other positions to produce ADCs with, for example, 4 or 6 drug-linkers attached. These other positions are located in peptide tags fused to the antibody or integrated into peptide tags within the antibody. Some of the ADCs with a drug-antibody ratio of 2 or higher show exquisite pharmacological in vivo characteristics (PK).

[0007] While the use of peptide tags fused to mAbs as MTG recognition motifs has been described, the unexpected discovery disclosed herein is that particularly suitable combinations of improved reaction buffer systems and alternative peptide tags can achieve particularly high DARs, as shown in the examples provided herein.

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

[0009] Aspects of the present invention relate to antibody modification and payload conjugation strategies that significantly reduce off-target cellular catabolism of such ADCs, thereby reducing the level of released payload while improving efficacy driven by higher ADC exposure. Thus, these modifications will provide drugs with improved therapeutic windows through reduced side effects and increased anti-tumor activity.

[0010] As described herein, the exposure and half-life of ADCs according to the present invention will be improved, for example, by modifying the antibody heavy chain with a YTE mutation and / or using an innovative enzyme conjugation method that uses microbial transglutaminase (also referred to herein as "TGase" and "mTG").

[0011] Surprisingly, high exposures that cause low clearance values ​​in human prediction animal models were obtained by the methods as above. Compared with variants that do not contain these modifications, with such modifications, the exposure of the ADC was improved by about 10 times, which therefore caused a significant reduction in the level of released effective load and a significant increase in the concentration of anti-tumor active ADC in the circulation. The improved ADC has similar PK characteristics to the original YTE antibody that does not contain a drug linker and exhibits an improved half-life relative to the prior art ADC.

[0012] These results will reduce the toxicity and increase the efficacy of clinical cancer therapies compared to prior art molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : Typical SEC chromatograms showing the purity of input mAbs and final BDS for ADC7-M, ADC2-M, and ADC5-M.

[0014] Figure 2 : Typical RP-HPLC chromatograms illustrating DAR determination of final BDS of ADC7-M, ADC2-M and ADC5-M.

[0015] Figure 3 : With antigen-negative MDA-MB-231 ( Figure 3 e) Cell lines compared to ADC2-M, ADC5-M and free payload against antigen-positive SK-CO-1 ( Figure 3 a) SNU-16( Figure 3 b) MKN-45( Figure 3 c) and LS174T( Figure 3 d) In vitro potency of cell lines. One representative experiment is shown, mean ± SD of duplicates.

[0016] Figure 4: Pharmacokinetic characteristics of ADC1, ADC1-M, ADC2-M, ADC6-M and ADC7-M in huFcRn Tg276 mice (total antibody).

[0017] Figure 5 : Changes in tumor volume after treatment with ADC1-M and ADC2-M compared to vehicle control.

[0018] Figure 6 : More potent bystander effects of ADC1-M, ADC2-M, ADC6-M and ADC7-M on antigen-negative MDA-MB-231 cells co-cultured with antigen-positive SK-CO-1 cells compared to ADC SAR DM4. One representative experiment is shown, mean ± SD of duplicates.

[0019] Figure 7 : Efficacy of ADC1-M and ADC3-M compared with ADC8 in the HPAF-II xenograft model.

[0020] Figure 8: (A) Scheme of conjugation of small molecule drugs / molecules to native fully glycosylated mAbs. (B) Exemplary drug-linkers used as mTG substrates: Gly3-valine-citrulline-MMAE ("4M" = "drug-linker 4M") and Gly3-β-glucuronide exatecan ("1M", also referred to herein as "drug-linker 1M" or "drug-linker compound 1-M", "compound DL1-M" or "DL1-M".).

[0021] Fig. 9:(A) Drug-linker 4M was conjugated to native mAb trastuzumab using MTG from S. mobaraensis. 5 mg / mL antibody, 20 molar equivalents of drug-linker and different mTG (purchased from Zedira, Germany) concentrations were mixed in 150 mM NaCl, 25 mM Tris-HCl, pH 8.0 with 10% DMSO. The amount of enzyme was titrated from 0 to 75 U / ml. The reaction was incubated for 16 h, stopped with M TG blocker (Zedira), followed by DAR determination using RP-HPLC, as described in Section 11.3.5. When using this prior art buffer (i.e. described in Strop et al.; Chemistry & Biology (2013). 20(2), 161–167 or Dickgiesser et al.; Bioconjugate Chemistry 2020 31(4), 1070-1076): 150 mM NaCl, 25 mM Tris-HCl, pH 8.0, it was not possible to achieve a DAR of 2 even at high enzyme concentrations. (B) Drug-linker 4M was conjugated to native mAb using mTG from Streptotrichum ladakhii (mTG variant 1, i.e. MTG_1 according to SEQ ID NO: 45) and the same buffer as in (A) or disclosed by Dickgiesser et al. (2020) (150 mM NaCl, 25 mM Tris-HCl, pH 8.0). The reaction was performed as described in 11.3.4, and the amount of enzyme was titrated in the range of 2.5-20 U / ml. The conjugation efficiency was higher with this mTG variant, but a DAR of 2 was still not reached with this prior art buffer.

[0022] Fig.10 : Scheme of 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 anti-IgG antibody labeled with terbium (B). Once d2 and terbium are in close proximity, the HTRF signal can be read (C).

[0023] Figure 11: (A) Effect of pH variation on transglutaminase-mediated bioconjugation in a range of buffer systems as shown in the legend; (B) Effect of salt concentration (mM NaCl) on transglutaminase conjugation efficiency for HEPES and Tris-HCl buffer systems; (C) Effect of buffer HEPES concentration on transglutaminase conjugation efficiency.

[0024] Figure 12: (A) Drug-linker 4M was conjugated to native fully glycosylated mAbs using Ladakh Streptotrichum mTG in different buffer systems obtained from high-throughput screening. Buffers were as shown, and the control buffer was 25mM Tris-HCl, 150mM NaCl, pH 8.0. The modified buffer containing 24mM HEPES at pH 7.5 performed best. (B) Conjugation of modified buffer and prior art (150mM NaCl, 25mM Tris-HCl pH 8.0) buffer was compared after titration of mTG. Effective conjugation of drug-linker 4M can be achieved at only 10U / ml mTG in the modified buffer, while achieving DAR=2 using Ladakh Streptotrichum mTG in the prior art buffer typically requires much more mTG enzyme, i.e., about 70U / ml.

[0025] Fig.13 : HIC-HPLC analysis of a monoclonal antibody containing the amino acid sequence "TLQS" within the antibody light chain. The corresponding ADC is shown.

[0026] Figure 14: Mass spectrometry analysis of a monoclonal antibody comprising the amino acid sequence "TLQS" within the antibody light chain and the corresponding ADC.

[0027] Figure 15: HIC-HPLC analysis of tag fusion antibodies mAb_Tag2, mAb_Tag3 and mAb_Tag4 and the corresponding ADCs.

[0028] Fig.16 : Conjugation scheme of a mAb with a glutamine tag fused to the C-terminus of the light chain using native mAb conjugation technology, resulting in an ADC with DAR=4.

[0029] Figure 17: Reduction RP-HPLC showing efficient conjugation to both the light chain (equipped with a peptide tag) and the heavy chain (native glutamine). (A) Conjugation of drug-linker 4M to tag-fused antibody mAb_tag5 resulted in a DAR of 3.9. (B) Conjugation of drug-linker 1M to tag-fused antibody mAb_tag5 with a DAR of 3.9. (C) Conjugation of drug-linker 1M to mAb7-M' (top) resulted in ADC7-M' (bottom; DAR of 3.9).

[0030] Fig.18 : pH dependence of transglutaminase-mediated bioconjugation in a range of buffer systems. DETAILED DESCRIPTION

[0031] definition

[0032] As used herein, "CEACAM5" designates "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 particularly highly expressed on the surface of, for example, colorectal cancer, gastric cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, prostate cancer and other solid tumors.

[0033] A "domain" or "region" can be any area of ​​a protein, which is generally defined based on sequence homology and often involves a specific structural or functional entity.

[0034] A "coding sequence" or a sequence "encoding" an expression product such as a polypeptide, protein or enzyme is a nucleotide sequence that, when expressed, causes the production of the polypeptide, protein or enzyme, i.e., the nucleotide sequence encodes the amino acid sequence of the polypeptide, protein or enzyme. The coding sequence of a protein may include a start codon (usually ATG) and a stop codon.

[0035] As used herein, reference to a particular protein (e.g., an antibody) may include polypeptides having a native amino acid sequence as well as variants and modified forms, regardless of their source or mode of preparation. A protein having a native amino acid sequence is a protein having an amino acid sequence identical to an amino acid sequence obtained from nature. Such native sequence proteins may be isolated from nature or may be prepared using standard recombinant and / or synthetic methods. Native sequence proteins specifically encompass naturally occurring truncated or soluble forms, naturally occurring variant forms (e.g., alternatively spliced ​​forms), naturally occurring allelic variants, and forms including post-translational modifications. Native sequence proteins include proteins carrying post-translational modifications such as glycosylation or phosphorylation or other modifications of some amino acid residues.

[0036] The term "gene" means a DNA sequence encoding or corresponding to a specific amino acid sequence, which includes all or part of one or more proteins or enzymes, and may or may not include a regulatory DNA sequence, such as a promoter sequence, which determines conditions such as gene expression. Some genes that are not structural genes can be transcribed into RNA from DNA, but are not translated into amino acid sequences. Other genes can act as regulatory genes of structural genes or as regulatory genes of DNA transcription. In particular, the term gene can mean a genomic sequence encoding a protein, i.e. a sequence comprising regulatory sequences, promoters, introns and exon sequences.

[0037] Herein, a sequence that is "at least 85% identical" to a reference sequence is a sequence that has 85% or more sequence identity to the full length of the reference sequence, for example, a sequence that has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. Thus, the percentage of "sequence identity" can be determined by comparing two such sequences over their full length using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 443 (1970)), for example using the program Needle (EMBOSS) with the BLOSUM62 matrix and the following parameters: start gap (gap open) = 10, gap extension (gap extend) = 0.5, end gap (endgap) penalty = false, start end gap (endg ap open) = 10, end gap extension (endgap extend) = 0.5 (which is the standard setting), by global pairwise alignment, comparing two such sequences over their full length.

[0038] A "conservative amino acid substitution" is one in which an amino acid residue is substituted with another amino acid residue having a side chain with similar chemical properties (e.g., charge, size, or hydrophilicity). In general, conservative amino acid substitutions will not substantially change the functional properties of the protein. Examples of amino acid groups having side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxy 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 substituents can also be defined based on amino acid size.

[0039] "Antibodies" (also referred to as "immunoglobulins") may, for example, be natural or conventional types of antibodies 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, λ (I) and κ (κ). There are five major heavy chain classes (or isotypes) that determine aspects of the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each antibody chain contains different sequence domains (or regions). The light chain of a typical IgG antibody comprises two regions, a variable region (VL) and a constant region (CL). The heavy chain of a typical IgG antibody comprises four regions, a variable region (VH) and a constant region (CH), the latter consisting of 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, complementary binding, and binding to Fc receptors (FcR). 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.

[0040] The specificity of an antibody lies in the structural complementarity of the antibody binding site and the antigenic determinant. The antibody binding site is composed of residues mainly from the so-called hypervariable or complementary determining region (CDR). Therefore, the complementary determining region (CDR) refers to the amino acid sequence that defines the binding affinity and specificity of the Fv region of the antibody together. The light (L) chain and heavy (H) chain of an antibody each have three CDRs, which are respectively named as CDR1-L, CDR2-L, CDR3-L and C DR1-H, CDR2-H, CDR3-H. Therefore, the antigen binding site of a conventional antibody comprises six CDRs, which comprise CDR sets from each heavy chain variable region and light chain variable region.

[0041] "Framework region" (FR) refers to the amino acid sequence inserted into CDR, i.e., those parts of the light chain variable region and heavy chain variable region of immunoglobulin that are relatively conserved in different immunoglobulins in a single species. The light chain and heavy chain of immunoglobulin each have four FRs, which are named FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively. As used herein, "human framework region" is a framework region that is substantially identical to the framework region of a naturally occurring human antibody (about 85% or more, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%).

[0042] In the context of the present invention, the CDR / FR definition in an immunoglobulin light or heavy chain is determined based on the IMGT definition (Lefranc et al., Dev. Comp. Immunol., 2003, 27(1):55-77; www.imgt.org).

[0043] As used herein, the term "antibody" includes conventional antibodies and fragments thereof, and single domain antibodies and fragments thereof, such as the variable heavy chain of a single domain antibody; as used herein, the term "antibody" also includes chimeric, humanized, bispecific or multispecific antibodies, and other types of engineered antibodies. The term "antibody" includes monoclonal antibodies.

[0044] The term "monoclonal antibody" or "mAb" as used herein refers to an antibody molecule that is directed against a single amino acid sequence of a specific antigen, and should not be construed as requiring the antibody to be produced by any particular method. Monoclonal antibodies can be produced, for example, by a single clone of a B cell or hybridoma, but can also be recombinant, for example, produced by methods involving genetic or protein engineering.

[0045] The term "chimeric antibody" 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 its broadest sense. In one embodiment, a chimeric antibody comprises VH and VL of an antibody derived from a non-human animal associated with CH and CL of another antibody, which in some embodiments is a human antibody. As non-human animals, any animal such as mice, rats, hamsters, rabbits, etc. can be used. Chimeric antibodies can also refer to multispecific antibodies that are specific for at least two different antigens.

[0046] The term "humanized antibody" refers to an antibody that is wholly or partially of non-human origin and is modified to replace, for example, certain amino acids in the framework regions of VH and VL in order to avoid or minimize immune responses in humans. The constant regions of humanized antibodies are typically human CH and CL regions.

[0047] "Fragments" of antibodies (e.g., conventional antibodies) include a portion of an intact antibody such as IgG, in particular the antigen binding region 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 by antibody fragments. Fragments of conventional antibodies may also be single domain antibodies, such as heavy chain antibodies or VHH.

[0048] The term "Fab" means an antibody fragment having a molecular weight of about 50,000 Da and antigen-binding activity, in which about half of the N-terminal side of the heavy chain and all the light chains are bound together by a disulfide bond. It is usually obtained in the fragment by treating IgG with a protease such as papain.

[0049] The term "F(ab')2" refers to an antibody fragment having a molecular weight of about 100,000 Da and antigen binding activity, which is slightly larger than two identical Fab fragments bound via a disulfide bond in the hinge region. It is usually obtained in the fragment by treating IgG with a protease such as pepsin.

[0050] 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 of the hinge region of F(ab')2.

[0051] Single-chain Fv ("scFv") is a covalently linked VH::VL heterodimer, which is usually expressed from a gene fusion comprising VH and VL encoding genes connected by a peptide encoding linker. The human scFv fragments of the present invention contain CDRs maintained in an appropriate conformation, for example, by using genetic recombination techniques. Divalent and multivalent antibody fragments can be formed spontaneously by the association of monovalent scFvs, or can be generated by coupling monovalent scFvs by peptide linkers, such as divalent sc(Fv)2. "dsFv" is a VH::VL heterodimer stabilized by a disulfide bond. "(dsFv)2" means two dsFvs coupled by a peptide linker.

[0052] The term "bispecific antibody" or "BsAb" refers to an antibody comprising two different antigen binding sites. Therefore, BsAb can, for example, simultaneously bind to two different antigens. Genetic engineering is used with increasing frequency to design, modify and produce antibodies or antibody derivatives with desired binding properties and effector function sets, as described in, for example, EP 2 050 764A1.

[0053] The term "multispecific antibody" refers to an antibody that contains two or more different antigen binding sites.

[0054] The term "diabody" refers to a small antibody fragment with two antigen-binding sites, which comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing of the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0055] The term "hybridoma" means a cell obtained by subjecting B cells prepared by immunizing a non-human mammal with an antigen to cell fusion with a myeloma cell derived from a mouse or the like, which produces a desired monoclonal antibody having antigen specificity.

[0056] "Purified" or "isolated" when referring to a polypeptide (e.g., an antibody) or nucleotide sequence means that the molecule is present in the substantial absence of other biological macromolecules of the same type. As used herein, the term "purified" means the presence of at least 75%, 85%, 95%, 96%, 97% or 98% by weight of biological macromolecules of the same type. 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 subject polypeptide; however, the molecule may contain some additional bases or moieties that do not adversely affect the basic characteristics of the composition.

[0057] As used herein, the term "subject" refers to a mammal, such as a rodent, a feline, a canine, a primate, or a human. In an embodiment of the present invention, the subject (or patient) is a human.

[0058] As used herein, the term "about" in connection with a numerical value means that the value may vary from + / - 10% to the stated value.

[0059] As used herein, concentrations of DMSO are given as % (v / v) unless otherwise indicated.

[0060] Antibody

[0061] Any antibody can be used in the conjugation method of the present invention. Preferred antibodies include monoclonal antibodies, which comprise an amino acid sequence selected from GGTLQSPP (SEQ ID NO: 74), TLQSG (SEQ ID NO: 77), TLQ SPP (SEQ ID NO: 78), GGTLQSG (SEQ ID NO: 79) and TLQSA (SEQ ID NO: 80) in at least one of its light chain constant regions (CL) and preferably in both of its light chain constant regions (CL) and / or in at least one of its heavy chain constant regions (CH) and preferably in both of its heavy chain constant regions (CH), and more preferably comprise the amino acid sequence TLQSPP (SEQ ID NO: 78) or GGTLQS PP (SEQ ID NO: 74) (most preferably comprise the amino acid sequence GGTLQSPP); and / or comprise glutamine at position 295 (EU numbering). These amino acid sequences were found to be suitable for use as substrates for microbial transglutaminases (also referred to herein as "mTG" or "MTG"), even when incorporated into the constant regions of the light chain (CL) and / or the heavy chain (CH) of antibodies. These amino acid sequences are therefore also named "mTG tags" herein. When included in the CL and / or CH chains of an antibody (preferably at the C-terminus of the chain), the primary amino group of a linker (preferably a drug linker; but the linker may also include other payloads other than the drug, such as a detectable label or a secondary antibody) or the primary amino group of a therapeutic agent may react with the glutamine of these aforementioned amino acid sequences of the antibody in the presence of the mTG enzyme:

[0062]

[0063] In the reaction schemes shown above, the acyl acceptor is the linker (preferably a drug-linker) or the therapeutic agent comprising the primary amino group.

[0064] The inventors have previously generated, screened and selected specific anti-CEACAM5 antibodies that surprisingly exhibit a combination of several features that make them ideally suited for cancer therapy, particularly as part of an immunoconjugate (antibody-drug conjugate). These antibodies can also be used in the conjugation methods of the present invention. Some of these antibodies are used as examples of the present invention, for example, for conjugation by the methods of the present invention. However, as mentioned, the application of the present invention is not limited to anti-CEACAM5 antibodies; rather, the present invention can be used for the purpose of preparing antibody-linker conjugates or antibody-drug conjugates based on antibodies against any target.

[0065] Preferred antibodies that can be used in the methods of the invention include an isolated antibody (e.g., one that binds to human CEACAM 5 protein);

[0066] and wherein the isolated antibody comprises

[0067] (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) and preferably comprising this sequence at the C-terminus of said light chain constant region;

[0068] and / or

[0069] (ii) at least one heavy chain constant region (CH) comprising one or more of the following amino acid substitutions:

[0070] (a) L234A and L235A (LALA mutation);

[0071] (b) L234A and L235A and P329G (LALA-PG mutation);

[0072] (c) L235A and G237A (LAGA mutations);

[0073] (d) M252Y, S254T, and T256E (YTE mutation);

[0074] (e) K222R;

[0075] and wherein EU numbering is used for the amino acid substitutions.

[0076] Additional preferred antibodies that can be used in the methods of the invention include isolated antibodies that bind to human CEACAM5 protein, and wherein the isolated antibody comprises

[0077] (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) and preferably comprising this sequence at the C-terminus of said light chain constant region;

[0078] and

[0079] (ii) at least one heavy chain constant region (CH) comprising one or more of the following amino acid substitutions:

[0080] (a) L234A and L235A (LALA mutation);

[0081] (b) L234A and L235A and P329G (LALA-PG mutation);

[0082] (c) L235A and G237A (LAGA mutations);

[0083] (d) M252Y, S254T, and T256E (YTE mutation);

[0084] (e) K222R;

[0085] and wherein EU numbering is used for said amino acid substitutions;

[0086] and wherein the isolated antibody comprises CDR1-H consisting of the amino acid sequence of SEQ ID NO:3, CDR2-H consisting of the amino acid sequence of SEQ ID NO:4, CDR3-H consisting of the amino acid sequence of SEQ ID NO:5, CDR1-L consisting of the amino acid sequence of SEQ ID NO:6, CDR2-L consisting of the amino acid sequence of SEQ ID NO:7, and CDR3-L consisting of the amino acid sequence of SEQ ID NO:8.

[0087] Preferably, the isolated antibody that can be used in the method of the present invention comprises framework regions FR1, FR2, FR3, FR4, FR5, FR6, FR7 and FR8 having the structures FR1-CDR1-H–FR2–CDR2-H–FR3–CDR3-H–FR4 and FR5-CDR1-L–FR6–CDR2-L–FR7–CDR3-L–FR8; wherein 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.

[0088] The present invention also provides a conjugation method as described herein using an isolated antibody (e.g., an isolated one that binds to human CEACAM5 protein), wherein the isolated antibody comprises

[0089] (i) at least one heavy chain constant region (CH) comprising one or more of the following amino acid substitutions:

[0090] (a) L234A and L235A (LALA mutation);

[0091] (b) L234A and L235A and P329G (LALA-PG mutation);

[0092] (c) L235A and G237A (LAGA mutations);

[0093] (d) M252Y, S254T, and T256E (YTE mutation);

[0094] (e) K222R;

[0095] and

[0096] (ii) at least one light chain constant region (CL) comprising the sequence GGTLQSPP and preferably comprising this sequence at the C-terminus of said light chain constant region;

[0097] and wherein 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 edition; DIANE Publishing: Collingdale, PA, USA, 1991), and the positions of the amino acid substitutions shown follow this numbering system. Amino acid substitutions are specified using single-letter amino acid codes. GGTLQSPP may be included several times in the light chain constant region (CL) and may alternatively or additionally be included in the heavy chain constant region (CH). Preferably, GGTLQSPP is included once in each of the two light chain constant regions (CL) of the antibodies of the invention.

[0098] In an embodiment of an isolated antibody that can be used in the conjugation method of the present invention, both heavy chain constant regions (CH) comprise one or more of the amino acid substitutions (a) to (e) and / or both light chain constant regions comprise the sequence GGTLQSPP. Preferred combinations of modifications of the CL and CH chains are summarized in Table 4 below, which indicates the modification combinations of antibodies mAb1-M, mAb2-M, mAb3-M, mAb6-M and mAb7-M. Preferably, the antibodies of the present invention comprise any of the following heavy chain constant region (CH) and light chain constant region (CL) modifications:

[0099] (a) CH comprises amino acid substitutions L234A, L235A (LALA mutation) and M252Y, S254T and T256E (YTE mutation); or

[0100] (b) 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 the sequence GGTLQSPP (preferably at the C-terminus); or

[0101] (c) 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 the sequence GGTLQSPP (preferably at the C-terminus); or

[0102] (d) CH comprises amino acid substitutions L234A, L235A (LALA mutation); or

[0103] (e) CH comprises amino acid substitutions L234A, L235A (LALA mutation); and a light chain constant region (CL) comprising the sequence GGTLQSPP (preferably at the C-terminus).

[0104] Preferably, both CL regions and both CH regions of the antibody of the present invention comprise the modifications outlined in (a) to (e) above.

[0105] Any combination of the embodiments described herein above and below forms part of the present invention.

[0106] In some embodiments of the conjugation method of the present invention, the antibody used in the method of the present invention is a conventional antibody, such as a conventional monoclonal antibody, or an antibody fragment, a bispecific or multispecific antibody.

[0107] In some embodiments, the antibody comprises or consists of IgG or a fragment thereof.

[0108] In some embodiments, the antibody used in the conjugation method of the present invention can be, for example, a mouse antibody, a chimeric antibody, a humanized antibody, or a human antibody. Numerous methods for humanization of antibody sequences are known in the art; see, for example, those reviewed by Almagro & Fransson (2008) Front Biosci. 13: 1619-1633. A commonly used method is CDR transplantation, or antibody remodeling, which involves transplanting the CDR sequence of a donor antibody, usually a mouse antibody, into a framework scaffold of a human antibody with different specificity. Since CDR transplantation may reduce the binding specificity and affinity of the non-human antibody to which the CDR is transplanted and thus reduce the biological activity, a back mutation can be introduced at the selected position of the CDR-transplanted antibody to retain the binding specificity and affinity of the parent antibody. The identification of the position of the possible back mutation can be performed using information available in the literature and in the antibody database. The 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 exposure residues are generally not changed. The alternative humanization technique for CDR grafting and back mutation is surface remodeling (resurfacing), wherein the non-surface exposed residues of non-human origin are retained, and the surface residues are changed to adult residues. Another alternative technique is called "guided selection (guided selection)" (Jespers et al., (1994) Biotechnology 12,899) and can be used to derive a fully human antibody that preserves the epitope and binding characteristics of the parent antibody from a mouse antibody.

[0109] For chimeric antibodies, humanization typically involves modification of the framework regions of the variable region sequences.

[0110] The amino acid residues that are part of the CDR will typically not be changed in association with humanization, although in some cases it may be desirable to change individual CDR amino acid residues, such as to remove glycosylation sites, deamidation sites or undesirable cysteine ​​residues. N-linked glycosylation occurs by attaching an oligosaccharide chain to an asparagine residue in a tripeptide sequence Asn-X-Ser or Asn-X-Thr, where X can be any amino acid except Pro. Removal of N-glycosylation sites can be achieved by mutating Asn or Ser / Thr residues to different residues, such as by conservative substitution. Deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation, primarily when present in the sequence Asn-Gly, and to a lesser extent in other dipeptide sequences such as Asn-Ala. When such deamidation sites, such as Asn-Gly, are present in a CDR sequence, it may therefore be desirable to remove the site, typically by conservative substitution to remove one of the residues involved. The invention is also intended to encompass substitutions within the CDR sequences to remove one of the implicated residues.

[0111] In a humanized antibody or fragment thereof, the variable domains of the heavy and light chains may comprise human acceptor framework regions. A humanized antibody may further comprise human constant heavy and light chain domains (where present).

[0112] In some embodiments, the antibody used in the conjugation method of the present invention can be an antibody fragment (e.g., a humanized antibody fragment) selected from Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, and diabodies,

[0113] In some embodiments, the antibody may be 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, as described, for example, in EP 2 050 764 A1 or US 2005 / 0003403 A1.

[0114] Antibodies that can be used in the conjugation method of the present invention can be produced by any technique known in the art.Antibodies according to the present invention can be used, for example, in an isolated (eg, purified) form or contained in a carrier such as a membrane or lipid vesicle (eg, liposome).

[0115] If an antibody other than an IgG antibody is used in the conjugation method of the present invention, it is preferred that the antibody comprises an amino acid sequence selected from the group consisting of GGTLQSPP, TLQSG, TLQSPP, GGTLQSG and TLQSA.

[0116] Nucleic acids and host cells

[0117] Additional aspects of the present disclosure relate to an isolated nucleic acid comprising or consisting of a nucleic acid sequence encoding an antibody of the invention as defined above.

[0118] Typically, the nucleic acid is a DNA or RNA molecule, which may be contained in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.

[0119] The terms "vector", "cloning vector" and "expression vector" mean a vehicle by which DNA or RNA sequences (eg, foreign genes) can be introduced into host cells in order to transform the host and promote the expression (eg, transcription and translation) of the introduced sequences.

[0120] Therefore, additional aspects of the disclosure relate to vectors comprising a nucleic acid as defined above.

[0121] Such vectors may include regulatory elements, such as promoters, enhancers, terminators, etc., to cause or direct the expression of the polypeptide after administration to a subject. Examples of promoters and enhancers used in expression vectors for animal cells include early promoters and enhancers of SV40 (Mizukami T. et al. 1987), LTR promoters and enhancers of Moloney murine leukemia virus (Kuwana Y et al., 1987), promoters of immunoglobulin H chains (Mason JO et al., 1985) and enhancers (Gillies SD et al., 1983), etc.

[0122] Any expression vector for animal cells can be used as long as the gene encoding human antibody C region can be inserted and expressed. Examples of suitable vectors include pAGE107 (Miyajih et al., 1990), pAGE103 (Mizukami T et al., 1987), pHSG274 (Brady G et al., 1984), pKCR (O'Hare K et al., 1981), pSG1βd2-4- (Miyajih et al., 1990) and the like.

[0123] Other examples of plasmids include replicating plasmids or integrating plasmids comprising an origin of replication, such as pUC, pcDNA, pBR, and the like.

[0124] Other examples of viral vectors include adenovirus, retrovirus, herpes virus and AAV vector. Such recombinant viruses can be produced by techniques known in the art, such as by transfection of packaging cells or by transient transfection with helper plasmids or viruses. Viral packaging cell typical examples 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, US5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94 / 19478.

[0125] A further object of the present disclosure relates to host cells transfected, infected or transformed by nucleic acids and / or vectors.

[0126] The term "transformation" means the introduction of a "foreign" (i.e., external) gene, DNA or RNA sequence into a host cell so that the host cell will express 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 the introduced DNA or RNA has been "transformed."

[0127] The nucleic acid can be 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 example, for expressing a protein encoded by foreign DNA carried by the vector and introduced into the host cell.

[0128] 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, but are not limited to, prokaryotic cells (e.g., bacteria) and eukaryotic cells (e.g., 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, neurons, adipocytes, etc.). Examples also include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells in which the dihydrofolate reductase gene (hereinafter referred to as "DHFR gene") is deficient (Urlaubg 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 these cells.

[0129] For the expression of humanized antibodies, expression vectors may belong to the type in which the gene encoding the heavy chain of the antibody and the gene encoding the light chain of the antibody are present on a separate vector, or belong to the type in which both genes are present on the same vector (tandem type). With regard to the ease of constructing the humanized antibody expression vector, the ease of introducing into the animal cell, and the balance of the expression level of the antibody H chain and L chain in the animal cell, the humanized antibody expression vector belongs to the tandem type (Shitara K et al., J Immunol Methods. January 3, 1994; 167 (1-2): 271-8). Examples of tandem type humanized antibody expression vectors include pKANTEX93 (WO 97 / 10354), pEE18, etc.

[0130] The present disclosure also relates to a method for producing a recombinant host cell expressing an antibody according to the present invention, the method comprising the steps consisting of: (i) introducing a recombinant nucleic acid or vector as described above into a competent host cell in vitro or ex vivo, (ii) culturing the obtained recombinant host cell in vitro or ex vivo and (iii) optionally selecting cells that express and / or secrete the antibody.

[0131] Such recombinant host cells can be used to produce the antibodies of the invention.

[0132] Methods of producing antibodies

[0133] Antibodies useful for conjugation to linkers or drug linkers using the methods of the invention can be obtained or produced by any technique known in the art, such as, but not limited to, any chemical, biological, genetic or enzymatic technique, alone or in combination.

[0134] Knowing the amino acid sequence of the desired antibody, those skilled in the art can easily produce the antibody or immunoglobulin chain using standard techniques for producing polypeptides. For example, they can be synthesized using a commercially available peptide synthesis device (such as that manufactured by Applied Biosystems, Foster City, California) and following the manufacturer's instructions using a well-known solid phase method. Alternatively, the antibodies and immunoglobulin chains of the present invention can be produced by recombinant DNA technology, as is well known in the art. For example, after the DNA sequence encoding the desired polypeptide is incorporated into an expression vector and such vectors are introduced into a suitable eukaryotic or prokaryotic host that will express the desired polypeptide, these polypeptides (such as antibodies) can be obtained as DNA expression products, and they can be separated from the eukaryotic or prokaryotic host later using well-known techniques.

[0135] The present invention further relates to a method of producing an antibody of the present invention, said method comprising the steps consisting of: (i) culturing the transformed host cell; (ii) expressing the antibody; and (iii) recovering the expressed antibody.

[0136] 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, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0137] In some embodiments, the humanized chimeric antibodies of the present invention can be produced by obtaining nucleic acid sequences encoding humanized VL and VH regions as previously described, constructing a human chimeric antibody expression vector for animal cells having genes encoding human antibody CH and human antibody CL, and expressing the coding sequence by introducing the expression vector into animal cells.

[0138] As the CH domain of the human chimeric antibody, any region belonging to the heavy chain of human immunoglobulin can be used, for example, those belonging to the IgG class are suitable, and any of the subclasses belonging to the IgG class, such as IgG1, IgG2, IgG3 and IgG4, can be used. In addition, as the CL of the human chimeric antibody, any region belonging to the light chain of human immunoglobulin can be used, and those belonging to the κ class or the λ class can be used.

[0139] Methods for producing humanized or chimeric antibodies may involve conventional recombinant DNA and gene transfection techniques, which are well known in the art (see, for example, Morrison SL. et al. (1984) and patent documents US Pat. No. 5,202,238; and US Pat. No. 5,204,244).

[0140] Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (see, e.g., Riechmann L. et al. 1988; Neuberger MS. et al. 1985). Antibodies can be humanized using various techniques known in the art, including, e.g., techniques disclosed in application WO2009 / 032661, CDR-grafting (EP 239,400; PCT publication WO91 / 09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or surface remodeling (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska MA. et al. (1994)) and chain shuffling (U.S. Pat. No. 5,565,332). General recombinant DNA techniques for preparing such antibodies are also known (see European Patent Application EP125023 and International Patent Application WO 96 / 02576).

[0141] Fab of the present invention can be obtained by treating the antibody of the present invention (e.g., IgG) with a protease such as papain. In addition, Fab can be produced by inserting the DNA sequence encoding the two chains of the Fab of the antibody into a vector for prokaryotic expression or for eukaryotic expression, and introducing the vector into a prokaryotic or eukaryotic cell (depending on the situation) to express Fab.

[0142] The F(ab')2 of the present invention can be obtained by treating the antibody of the present invention (eg, IgG) with a protease such as pepsin. In addition, F(ab')2 can be produced by bonding Fab' described below via a thioether bond or a disulfide bond.

[0143] 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. In addition, Fab' can be produced by inserting a DNA sequence encoding the Fab' chain of an antibody into a vector for prokaryotic expression or a vector for eukaryotic expression, and introducing the vector into a prokaryotic or eukaryotic cell (as the case may be) for expression thereof.

[0144] The scFv of the present invention can be produced by obtaining the sequence of the CDR or VH and VL domains as previously described for the antibody of the present invention, then constructing a 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 the case may be) to express the scFv. In order to generate a humanized scFv fragment, a well-known technique called CDR transplantation can be used, which involves selecting the complementary determining regions (CDRs) according to the present invention and transplanting them onto a human scFv fragment framework having a known three-dimensional structure (see, e.g., WO98 / 45322; WO 87 / 02671; US5,859,205; US5,585,089; US4,816,567; EP0173494).

[0145] Modification of the Antibodies of the Present Invention

[0146] Amino acid sequence modifications of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody.

[0147] Modifications and changes may be made in the structure of the antibodies of the invention and in the DNA sequences encoding them and still obtain a functional antibody or polypeptide possessing the desired characteristics.

[0148] When making changes to the amino acid sequence of a polypeptide, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index to the interactive biological function of a protein is generally understood in the art. It is generally believed that the relative hydropathic character of amino acids contributes to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules such as enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. The amino acids are assigned a hydropathic index based on their hydrophobicity and charge characteristics, which 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); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0149] Additional aspects of the invention also encompass function-conservative variants of the polypeptides of the invention.

[0150] For example, certain amino acids in a protein structure may be substituted with other amino acids without appreciable loss of activity. Since the ability and properties of a protein to interact define its biological functional activity, certain amino acid substitutions may be made in a protein sequence, and certainly in its encoding DNA sequence, while nevertheless obtaining a protein with similar properties. Thus, it is contemplated that various changes may be made in the antibody sequences of the present invention or the corresponding DNA sequences encoding the polypeptides without appreciable loss of their biological activity.

[0151] It is known in the art that certain amino acids can be replaced by other amino acids with similar hydropathic indexes or scores, and still obtain proteins with similar biological activity, i.e., still obtain proteins that are equivalent in biological function. It is also possible to use well-established techniques, such as alanine-scanning methods, to identify all amino acids that can be replaced without significant loss of antigen binding in the antibody or polypeptide of the present invention. Such residues can be characterized as neutral because they do not involve antigen binding or do not involve the structure of the antibody. One or more of these neutral positions can be replaced by alanine or by another amino acid without changing the main features of the antibody or polypeptide of the present invention.

[0152] Neutral positions can be considered as positions where any amino acid substitution can be incorporated. In fact, in the alanine scanning principle, alanine is selected because it does not carry specific structural or chemical features for this residue. It is generally recognized that if alanine can replace a specific amino acid without changing the properties of the protein, many other (if not all) amino acid substitutions may also be neutral. In the opposite case where alanine is the wild-type amino acid, if a specific substitution can be shown to be neutral, then other substitutions will also likely be neutral.

[0153] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions taking into account any of the foregoing characteristics are well known to those skilled in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0154] It may also be desirable to modify the antibodies of the invention with respect to effector functions, for example, to enhance the antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody, or, for example, to alter binding to Fc receptors. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. Alternatively or additionally, cysteine ​​residues can be introduced into the Fc region, thereby allowing the formation of inter-bond disulfide bonds in this region. The homodimeric antibodies thus generated can have improved internalization capacity and / or increased complement-mediated cell killing and / or antibody-dependent cellular toxicity (ADCC) (Caron PC. et al. 1992; and Shopes B. 1992). In some embodiments, the antibodies of the invention can be antibodies with modified amino acid sequences that cause reduced or eliminated binding to most Fcγ receptors, which can reduce uptake and toxicity in normal cells and tissues expressing such receptors, such as macrophages, liver sinusoidal cells, etc. An example of such an antibody is one comprising a substitution of two leucine (L) residues at positions 234 and 235 to alanine (A) (i.e., LALA); this double substitution has been shown to reduce the binding of Fc to FcγR and thus reduce ADCC and reduce complement binding / activation. Another example of such an antibody is one comprising a substitution P329G (i.e., PG-LALA; see, e.g., Schlothauer et al., Novelhu man IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions, Protein Engineering, Design and Selection, Vol. 29, No. 10, October 2016, pp. 457–466) in addition to the LA LA double substitution. In some embodiments, the antibody of the present invention may therefore be an antibody having an amino acid sequence that (i) contains, for example, a LALA or PG-L ALA substitution set and (ii) is otherwise consistent with the amino acid sequence of one of the antibodies of the present invention described herein above with reference to the respective SEQ ID NOs.

[0155] Another type of amino acid modification of the antibodies of the present invention can be used to change the original glycosylation pattern of the antibody, i.e., by deleting one or more carbohydrate moieties found in the antibody, and / or adding one or more glycosylation sites not present in the antibody. The presence of any one of the tripeptide sequences asparagine-X-serine and asparagine-X-threonine creates a potential glycosylation site, wherein X is any amino acid except proline. By changing the amino acid sequence so that it contains one or more of the tripeptide sequences described above, the addition or deletion of glycosylation sites of the antibody can be conveniently accomplished (for N-linked glycosylation sites).

[0156] Another type of modification relates to removing the sequence identified by computer or experiment, because potentially causing degradation products or heterogeneity of antibody preparations. As an example, the deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly prone to deamidation, mainly when present in the sequence Asn-Gly, and at other dipeptide sequences such as Asn-Ala, the degree is lower. When such deamidation sites, particularly Asn-Gly, are present in antibodies or polypeptides, it is therefore considered to remove the site, typically by conservative substitution to remove one of the residues involved. The present invention is also intended to encompass such substitutions in the sequence to remove one or more of the residues involved.

[0157] Another type of covalent modification involves chemical or enzymatic coupling of glycosides to antibodies. These procedures are advantageous in that they do not require the production of antibodies with glycosylation capabilities for N-linked glycosylation or O-linked glycosylation in host cells. Depending on the coupling mode used, sugars may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyls such as those of serine, threonine or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine or tryptophan, or (f) amide groups of glutamine. For example, such methods are described in WO87 / 05330.

[0158] Removal of carbohydrate moieties present on antibodies can be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the antibody to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment causes the cleavage of most or all sugars except the linked sugar (N-acetylglucosamine or N-acetylgalactosamine) while keeping the antibody intact. Chemical deglycosylation is described by Sojahrh. et al. (1987) and Edge, AS. et al. (1981). Enzymatic cleavage of carbohydrate moieties on antibodies can be achieved by using various endo- and exo-glycosidases as described by Thotakura, NR. et al. (1987).

[0159] Another type of covalent modification of the antibody involves linking the antibody to one of various nonproteinaceous polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, for example, in the manner shown in US Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

[0160] Other amino acid sequence modifications known in the art may also be applied to the antibodies of the invention.

[0161] Immunoconjugates

[0162] The present invention provides immunoconjugates, also referred to herein as antibody-drug conjugates, or more simply conjugates. As used herein, all of these terms have the same meaning and are interchangeable. Suitable methods for preparing immunoconjugates are known in the art. The immunoconjugates of the present invention can be prepared by in vitro methods, for example as described herein; preferably they can be prepared by methods according to the present invention.

[0163] The present invention provides an immunoconjugate comprising an antibody of the present invention covalently linked to at least one growth inhibitory agent or to at least one other agent via a linker. The other agent includes a detectable label or a therapeutic agent. The therapeutic agent is preferably a cytotoxic drug.

[0164] The term "growth inhibitory agent" (also known as an "antiproliferative 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.

[0165] 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.

[0166] As used herein, the term "cytotoxic drug" refers to a substance that directly or indirectly inhibits or prevents the function of a cell and / or causes destruction of a cell. The term "cytotoxic drug" includes, for example, chemotherapeutic agents, enzymes, antibiotics, toxins such as small molecule toxins or enzyme-active toxins, toxoids, vinca alkaloids (vincas), taxanes, maytansines or maytansine analogs, tomamycin or pyrrolobenzodiazepines. Derivatives, Nostoc cyclic peptide derivatives, leptomycin derivatives, auristatin or dolastatin analogs, prodrugs, topoisomerase I inhibitors, topoisomerase II inhibitors, DNA alkylating agents, anti-tubulin agents, CC-1065 and CC-1065 analogs.

[0167] Topoisomerase I inhibitors are molecules or compounds that inhibit the human enzyme topoisomerase I, which is involved in changing the topological structure of DNA by catalyzing the transient breakage and rejoining of single strands of DNA. Topoisomerase I inhibitors are highly toxic to dividing cells, such as dividing cells of mammals. Examples of suitable topoisomerase I inhibitors include camptothecin (CPT) and its analogs, such as topotecan, irinotecan, silatecan, cositecan, exatecan, lortotecan, gimatecan, belotecan and rubitecan.

[0168] In some embodiments, the immunoconjugates of the present invention include the cytotoxic drug exatecan as a growth inhibitor. 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):

[0169]

[0170] In further embodiments of the invention, other CPT analogs and other cytotoxic drugs may be used, for example as listed above. Some examples of cytotoxic drugs and conjugation methods are further given in the application WO2008 / 010101, incorporated by reference.

[0171] The term "radioactive moiety" refers to a chemical entity (e.g., a molecule, compound, or composition) that comprises or consists of a radioactive isotope useful for treating cancer, such as At 211 、Bi 212 , Er 169 ,I 131 ,I 125 , Y 90 、In 111 , P 32 、Re 186 、Re 188 、Sm 153 , Sr 89 or a radioactive isotope of Lu. Such radioactive isotopes generally emit mainly beta radiation. In some embodiments, the radioactive isotope is an alpha-emitter isotope, such as thorium 227, which emits alpha radiation. The immunoconjugates can be prepared, for example, as described in application WO2004 / 091668.

[0172] In the immunoconjugates of the present invention, the antibodies of the present invention are covalently linked to at least one growth inhibitor via a linker. As used herein, "linker" means a chemical moiety comprising a covalent bond and / or any atomic chain that covalently attaches a growth inhibitor 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. The conjugation of the antibodies of the present invention to cytotoxic drugs or other growth inhibitors can be carried out, for example, using various bifunctional protein coupling agents, including but not limited to N-succinimidyl pyridyl dithiobutyrate (SPDB), butyric acid 4-[(5-nitro-2-pyridyl) disulfide]-2,5-dioxo-1-pyrrolidinyl ester (nitro-SPDB), 4-(pyridin-2-yldisulfanyl)-2-sulfo-butyric acid (4-(Pyridin-2-yldisulfanyl)-2-sulfo-butyric acid) acid) (sulfo-SPDB), N-succinimidyl (2-pyridyldithio) propionate (SPDP), succinimidyl (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl diimidoadipate HCL), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis (p-azidobenzoyl)-hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., 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 Vitette et al. (1987). Carbon-labeled 1-isothiocyanatobenzylmethyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelator for conjugating radionucleotides to antibodies (WO 94 / 11026).

[0173] In embodiments of the invention, the linker can be a "cleavable linker" that can facilitate the release of a cytotoxic drug or other growth inhibitory agent inside or near a cell such as a tumor cell. In some embodiments, the linker is a cleavable linker in the endosome of a mammalian cell. For example, an acid-labile linker, a peptidase-sensitive linker, an esterase-labile linker, a photolabile linker, or a linker containing a disulfide (see, e.g., U.S. Pat. No. 5,208,020) can be used.

[0174] When referring to the structural formula of the immunoconjugate, the following nomenclature is also used herein: the growth inhibitory agent and the linker together are also referred to as the [(linker)-(growth inhibitory agent)] portion; for example, the exatecan molecule and the linker together are also referred to as the [(linker)-(exetatecan)] portion.

[0175] In some specific embodiments of the invention, the linker is a linker that can be cleaved by the human enzyme glucuronidase. For example, the immunoconjugate prepared by the method of the invention can therefore have the following formula (IIA), which includes a linker that can be cleaved by glucuronidase, and the formula (IIA) is:

[0176]

[0177] wherein the antibody is an antibody of the invention (preferably a monoclonal antibody), and wherein n is the number of [(linker)-(growth inhibitor)] moieties covalently attached to the antibody. In embodiments using Formula IIA above, n is preferably 3 to 4 and most preferably 3.5 to 4.0 (i.e., about 4).

[0178] The number n is also called the "drug to antibody ratio" (or "DAR"); this number n is always to be understood as the average number for any given (preparation of) immunoconjugate.

[0179] In formula (IIA) above, the chemical structures of the antibody and the growth inhibitory agent are linkers. One of these linkers is also included in (VIIIA) further depicted below.

[0180] In any of the embodiments having a linker cleavable by a glucuronidase as described above, the growth inhibitor can be, for example, exatecan.

[0181] 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, wherein the conjugate has the following formula (IVA):

[0182]

[0183] Wherein n is the number of [(linker)-(exatecan)] moieties covalently attached to the antibody. The number n (also referred to as DAR) can be, for example, from 1 to 10; in embodiments using Formula IVA above, n is preferably from 3 to 4 and most preferably from 3.5 to 4.0 (ie, about 4).

[0184] 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, wherein the conjugate has the following formula (VIA):

[0185]

[0186] Wherein n is the number of [(linker)-(exatecan)] moieties covalently attached to the antibody. The number n (also referred to as DAR) can be, for example, 1 to 10; in embodiments using Formula VIA above, n is preferably 3 to 5 and more preferably 3.5 to 4.5 and most preferably 4.

[0187] In preparing any of the immunoconjugates described above using the methods of the invention, antibodies directed against any target may be used.

[0188] 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, wherein the conjugate has the following formula (VIIIA):

[0189]

[0190] wherein n is the number of [(linker)—(exatecan)] moieties covalently attached to the antibody. The number n (also referred to as DAR) can be, for example, 1 to 10.

[0191] In other embodiments of the invention, the linker may be a "non-cleavable linker" (eg, an SMCC linker). Release of the growth inhibitory agent from the antibody may occur upon lysosomal degradation of the antibody.

[0192] In other embodiments of the invention, the immunoconjugate may be a fusion protein comprising an antibody of the invention and a cytotoxic or growth inhibitory polypeptide (as a growth inhibitor); such fusion proteins may be prepared by recombinant techniques or by peptide synthesis (i.e., methods well known in the art). The molecule encoding the DNA may comprise regions encoding the two parts of the conjugate (the antibody and the cytotoxic or growth inhibitory polypeptide, respectively), which are adjacent to each other or separated by a region encoding a linker peptide.

[0193] The antibodies of the invention can also be used to direct enzyme-catalyzed prodrug therapy, for example, by conjugating the antibody to a prodrug-activating enzyme that converts a prodrug (e.g., a peptide-based chemotherapeutic agent, see WO81 / 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 in ADEPT can include any enzyme that can act on a prodrug in a manner that converts the prodrug into its more active, cytotoxic form. Enzymes that can be used in this context include, but are not limited to, alkaline phosphatases, which can be used to convert phosphate-containing prodrugs into free drugs; arylsulfatases, which can be used to convert sulfate-containing prodrugs into free drugs; cytosine deaminases, which can be used to convert non-toxic fluorocytosine into the anticancer drug 5-fluorouracil; proteases, such as Serratia protease, thermolysin, subtilisin, carboxypeptidases, and cathepsins (e.g., cathepsins B and L), which can be used to convert peptide-containing prodrugs into free drugs. ; D-alanyl carboxypeptidase, which can be used to convert prodrugs containing D-amino acid substituents; carbohydrate lyases, such as O-galactosidase and neuraminidase, which can be used to convert glycosylated prodrugs into free drugs; β-lactamase, which can be used to convert β-lactam derived drugs into free drugs; and penicillin amidase, such as penicillin V amidase or penicillin G amidase, which can be used to convert phenoxyacetyl or phenylacetyl derived drugs at their amine nitrogens into free drugs. Enzymes can be covalently bound to antibodies by techniques well known in the art, such as using joints as discussed above.

[0194] Suitable methods for preparing immunoconjugates are well known in the art (see, e.g., Hermansong. T., Bioconjugate Techniques, 3rd 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 antibodies are well known.

[0195] In general, the immunoconjugate can be obtained, for example, by a method comprising the following steps:

[0196] (i) preparing a compound comprising a linker and a growth inhibitory agent (e.g., a cytotoxic drug), which is also referred to herein as a "drug-linker compound";

[0197] (ii) contacting an optionally buffered aqueous solution of the antibody with a solution of the drug-linker compound;

[0198] (iii) The conjugate formed in (ii) is then optionally separated from unreacted antibody and / or drug-linker compound.

[0199] The aqueous solution of the antibody can be buffered with a buffer such as histidine, potassium phosphate, acetate, citrate or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes buffer). The buffer can be selected depending on the properties of the antibody. The drug-linker compound can be dissolved in, for example, an organic polar solvent such as dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA).

[0200] For conjugation to cysteine ​​residues of the antibody, the antibody is subjected to reduction (eg using TCEP) prior to step (ii). Suitable reducing conditions that reduce only interchain disulfide bonds are known in the art.

[0201] The reaction temperature of the conjugation is generally 20°C to 40°C. The reaction time may vary and is typically 1 to 24 hours. The reaction of the antibody and drug-linker compound may be monitored by size exclusion chromatography (SEC) with a refractometer and / or UV detector. If the conjugate yield is too low, the reaction time may be extended.

[0202] A variety of different chromatographic methods are available to the skilled person for separation in step (iii): the conjugate may 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 may be used.

[0203] After step (ii) and / or (iii), the solution containing the conjugate may be subjected to an additional purification step (iv), for example by chromatography, ultrafiltration and / or diafiltration. In the case where reduction is performed prior to conjugation, such additional purification steps, for example by chromatography, ultrafiltration and / or diafiltration, may be performed with the solution containing the antibody after the reduction reaction.

[0204] At the end of such a process the conjugate is recovered in aqueous solution. The drug-antibody ratio (DAR) is a number that can vary with the nature of the antibody used and the nature of the drug-linker compound, together with the experimental conditions used for conjugation, such as the (drug-linker compound) / (antibody) ratio, reaction time, solvent, and co-solvent (if present). Thus, contacting of the antibody and the drug-linker compound may result in a mixture containing several conjugates that differ from one another due to different drug-antibody ratios. Thus, the measured DAR is an average value.

[0205] An exemplary method that can be used to determine DAR is to measure spectrophotometrically the concentration of a solution of a purified conjugate at λ D The wavelength λ is the ratio of the absorbance at λ to that at 280 nm. 280 nm is a wavelength commonly used to measure protein concentrations, such as the wavelength used to measure antibody concentrations. D In order to allow differentiation of the drug from the antibody, i.e., as the skilled person will readily appreciate, D is the wavelength at which the drug has high absorbance, and λ D far enough away from 280 nm to avoid substantial overlap of the absorbance peaks of the drug and antibody. D It may be chosen to be 370 nm for exatecan (or for camptothecin or other camptothecin analogs), or 252 nm for maytansine molecules.

[0206] The method of DAR calculation can be derived from, for example, Antony S. Dimitrov (ed), LLC, 2009, Therapeutic Antibodies and Protocols, Vol. 525, 445, Springer Science: Conjugates at λ D The absorbance at λD ) and the absorbance at 280 nm (A 280 ) is measured on the monomer peak of a size exclusion chromatography (SEC) analysis (allowing the calculation of the "DAR(SEC)" parameter), or using a classical spectrophotometer setup (allowing the calculation of the "DAR(UV)" parameter). The absorbance can be expressed as follows:

[0207] A λD＝ (C D Xε DλD )+(C A Xε AλD )

[0208] A 280 =(C D Xε D280 )+(C A Xε A280 )

[0209] in:

[0210] ·C D and C A are the concentrations of drug solution and antibody solution, respectively

[0211] ·ε DλD and ε D280 are the drugs in λ D Molar extinction coefficient at 280 nm

[0212] ·εAλD and ε A280 The antibodies are D The molar extinction coefficients at 280 nm and 300 nm are given in Table 1.

[0213] Solving these two equations with two unknowns yields the following equation:

[0214] C D =[(ε A280 XA λD )-(ε AλD XA 280 )] / [(ε DλD Xε A280 )-(ε AλD Xε D280 )]

[0215] C A =[A 280 -(C D Xε D280 )] / ε A280

[0216] Then, the average DAR was calculated based on the ratio of drug concentration to antibody concentration: DAR = C D / C A .

[0217] An alternative method for preparing an immunoconjugate is described below and is a method of the invention. This method can be used in particular for an antibody comprising an amino acid sequence selected from the group consisting of GGTLQSPP, TLQSG, TLQSPP and TLQSA in at least one of its light chain constant regions (CL) and / or in at least one of its heavy chain constant regions (CH). Thus, a further aspect of the invention relates to a method for producing an antibody-linker-conjugate comprising the following steps:

[0218] (1) providing an antibody, which comprises an amino acid sequence selected from the group consisting of GGTLQSPP, TLQSG, TLQSPP and TLQSA and more preferably comprises the amino acid sequence TLQSPP or GGTLQSPP (most preferably comprises the sequence GGTL QSPP) in at least one of its light chain constant regions (CL) and preferably in both of its light chain constant regions (CL) and / or in at least one of its heavy chain constant regions (CH) and preferably in both of its heavy chain constant regions (CH);

[0219] (2) Mix together at least the following components in a reaction buffer:

[0220] (a) the antibody provided in step (1);

[0221] (b) Microbial transglutaminase, preferably transglutaminase comprising the following amino acid sequence: MG SGSGSG TGEEKRSYAETHRLTADDVDDINALNESAPAASSAGPSFRAP DSDERVTPPAEPLDRMPDPYRPSYGRAETIVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFAFFDEDKYKNELKNGRPRSGETRAEFEGRVAKDSFDEAKGFQRARDVASVMNKALENAHDEGAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKDRNGGNHDPSKMKAVIYSKHFWSGQDRSGSSDKRKYGDPEAFRPDRGTGLVDMSRDRNIPRSPTSPGESFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWSDGYSDFDRGAYVVTFVPKSWNTAPDKVTQGWP (SEQ ID NO:44); or FRAP DS DERVTPPAEPLDRMPDPYRPSYGRAETIVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFAFFDEDKYKNELKNGRPRSGETRAEFEGRVAKDSFDEAKGFQRARDVASVMNKALENAHDEGAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKDRNGGNHDPSKMKAVIYSKHFWSGQDRSGSSDKRKYGDPEAFRPDRGTGLVDMSRDRNIPRSPTSPGESFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWSDGYSDFDRGAYVVTFVPKSWNTAPDKVTQGWPLE (SEQ ID NO:73); or FRAPDSDERVTPPAEPLDRMPDPYRPSYGRAETIVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFAFFDEDKYKNELKNGRPRSGETRAEFEGRVAKDSFDEAKGFQRARDVASVMNKALENAHDEGAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKDRNGGNHDPSKMKAVIYSKHFWSGQDRSGSSDKRKYGDPEAFRPDRGTGLVDMSRDRNIPRSPTSPGESFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWSDGYSDFDRGAYVVTFVPKSWNTAPDKVTQGWP(SEQ ID NO:45); or DSDE RVTPPAEPLDRMPDPYRPSYGRAETIVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFAFFDEDKYKNELKNGRPRSGETRAEFEGRVAKDSFDEAKGFQRARDVASVMNKALENAHDEGAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKDRNGGNHDPSKMKAVIYSKHFWSGQDRSGSSDKRKYGDPEAFRPDRGTGLVDMSRDRNIPRSPTSPGESFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWSDGYSDFDRGAYVVTFVPKSWNTAPDKVTQGWPLE(SEQ ID NO:48);or DSDER VTPPAELDRMPDPYRPSYGRAETIVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFAFFDEDKYKNELKNGRPRSGETRAEFEGRVAKDSFDEAKGFQRARDVASVMNKALENAHDEGAYLDNLKKEL ANGNDALRNEDARSPFYSALRNTPSFKDRNGGNHDPSKMKAVIYSKHFWSGQDRSGSSDKRKYGDPEAFRPDRGTGLVDMSRDRNIPRSPTSPGESFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWSDGYSD FDRGAYVVTFVPKSWNTAPDKVTQGWP (SEQ ID NO:49); and;

[0222] (c) a linker comprising an H2N-moiety capable of reacting with the antibody from step (1) in the presence of said transglutaminase, and wherein the linker is preferably a drug-linker, wherein said linker is covalently attached to the drug;

[0223] (3) separating the antibody-linker-conjugate produced in step (2) from unreacted linker and from the transglutaminase, preferably by subjecting the mixture from step (2) to size exclusion chromatography.

[0224] In one embodiment, the transglutaminase is encoded by the following polynucleotide: ATGGGTAGCGG TAGCGGTT CAGGCACCGGTGAAGAAAAACGTAGCTATGCAGAAACCCCATCGTCTGACCGCAGATGATGTTGATGATATTAATGC ACTGAATGAAAGCGCACCGGCAGCAAGCAGCGCAGGTCCGAGCTTTCGTGCACCGGATAGTGATGAACGTGTTACCCCTCCGGCAGAACCGCTGGATCGTATGCCGGATCCGTATCGTCCGAGCTATGGTCGTGCCGAAACCATTGTTAATAACTATATTCGTAAGTGGCAGCAGGTTTATAGCCATCGTGATGGTCGTAAACAGCAGATGACCGAAGAACAGCGTGAATGGCTGAGTTATGGTTGTGTTGGTGTTACCTGGGTTAATAGCGGTCAGTATCCGACCAATCGTCTGGCATTTGCATTTTTCGATGAGGACAAATACAAGAACGAGCTGAAAAATGGTCGTCCGCGTAGCGGTGAAACCCGTGCAGAATTTGAAGGTCGTGTTGCAAAAGATAGCTTTGATGAAGCAAAAGGTTTTCAGCGTGCACGTGATGTTGCAAGCGTTATGAATAAAGCACTGGAAAATGCACATGATGAAGGTGCCTATCTGGACAACCTGAAAAAAGAACTGGCAAATGGTAATGATGCCCTGCGTAATGAAGATGCACGTAGCCCGTTTTATAGCGCACTGCGTAATACCCCGAGCTTTAAAGATCGTAATGGTGGTAATCATGACCCGAGCAAAATGAAAGCAGTGATCTATAGCAAACACTTTTGGAGCGGTCAGGATCGTAGTGGTAGCAGCGATAAACGTAAATATGGTGATCCGGAAGCATTTCGTCCGGATCGTGGCACCGGTCTGGTTGATATGAGCCGTGATCGTAATATTCCGCGTAGTCCGACCAGTCCGGGTGAAAGCTTTGTTAATTTTGATTATGGTTGGTTTGGCGCACAGACCGAAGCAGATGCAGATAAAACCGTTTGGACCCATGGCAATCATTATCATGCACCGAATGGTAGCCTGGGTGCAATGCATGTTTATGAAAGCAAATTTCGCAATTGGAGCGACGGCTATAGCGATTTTGATCGTGGTGCCTATGTTGTTACCTTTGTTCCGAAAAGCTGGAATACCGCTCCGGATAAAGTTACCCAAGGTTGGCCGTAA(SEQID NO:46).

[0225] In a preferred embodiment of the method, the reaction buffer is 7% DMSO, 24 mM HEP ES, pH 7.0.

[0226] In the method of the present invention, the linker is a linker having the following formula:

[0227]

[0228] wherein R is the remainder of the linker and may optionally further comprise a drug,

[0229] Thereby, the drug is preferably exatecan. In a more preferred embodiment of the method of the present invention, the linker is NH2-GGG-β-glucuronide.

[0230] In an exemplary embodiment of the method of the present invention, in step (2) the mixture comprises the following drug-linker:

[0231]

[0232] In a further embodiment of the method of the present invention, the mixture in step (2) comprises 5 molar equivalents of a linker or a drug-linker / conjugation site, respectively, wherein the conjugation site is the sequence GGTLQSPP, TLQSG, TLQSPP or TLQSA contained in the light chain constant region (CL) and / or in the heavy chain constant region (CH) of the antibody.

[0233] A further aspect of the invention relates to antibody-linker conjugates producible according to the methods of the invention.

[0234] Exemplary methods of the invention for preparing immunoconjugates are described in the Examples.

[0235] Drug-Linker Compounds

[0236] The present disclosure also provides compounds comprising a linker and a growth inhibitor (e.g., a cytotoxic drug), which are also referred to herein as "drug-linker compounds." For example, the present disclosure provides a compound of the following formula (XA) or a physiologically acceptable salt thereof, wherein the formula (XA) is:

[0237]

[0238] The compound according to Formula XA is also referred to herein as "Drug-Linker Compound 1-M," "Compound DL 1-M," or "DL1-M."

[0239] These drug-linker compounds can be used to prepare immunoconjugates using the methods of the invention as described herein above and below.

[0240] Disclosed exemplary drug-linker compounds (eg, those depicted above as (XA)) can be prepared by chemical synthesis, for example, as described further in the Examples below.

[0241] Pharmaceutical composition

[0242] The antibodies or immunoconjugates of the present invention can be combined with pharmaceutically acceptable carriers, diluents and / or excipients and optionally with sustained release matrices to form pharmaceutical compositions, the sustained release matrices including but not limited to biodegradable polymers, non-biodegradable polymers, lipids or sugars.

[0243] Therefore, another aspect of the present invention relates to a pharmaceutical composition comprising an antibody or immunoconjugate of the present invention and a pharmaceutically acceptable carrier, diluent and / or excipient.

[0244] Treatments and uses

[0245] 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 for treating cancer comprising administering an immunoconjugate or pharmaceutical composition of the present invention to a subject in need thereof.

[0246] The antibodies disclosed herein and any other antibodies can be conjugated (linked) to a growth inhibitory agent or another agent using the methods of the invention. The antibodies will thus be useful for targeting the drug (e.g., growth inhibitory agent) to a target cell (e.g., cancer cell) that expresses or overexpresses the antigen (preferably a cell surface antigen) to which the antibody binds.

[0247] In some embodiments, the antibodies that can be used in the methods of the present invention can be antibodies with modified amino acid sequences that result in reduced or eliminated binding to most Fcγ receptors, which can reduce uptake and toxicity in normal cells and tissues expressing such receptors, such as macrophages, liver sinusoidal cells, etc.

[0248] Aspects of the invention relate to methods of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of an immunoconjugate or pharmaceutical composition of the invention.

[0249] The efficacy of treatment with an antibody or immunoconjugate or pharmaceutical composition according to the invention can be readily determined in vivo, for example in a mouse model of cancer, and by measuring, for example, changes in tumor volume, % tumor regression, partial regression or complete regression between treatment and control groups.

[0250] Reagent test kit

[0251] In a further aspect, the present invention provides a kit comprising a reaction buffer as defined herein in the context of the methods of the invention, and wherein the kit comprises a microbial transglutaminase from Streptococcus ladakii, preferably a transglutaminase comprising an amino acid sequence as defined in SEQ ID NO: 44, 47, 48, 49 or 45 and preferably a transglutaminase comprising the amino acid sequence of SEQ ID NO 45.

[0252] In one embodiment of the kit of parts of the invention, the kit further comprises a linker having a primary amino group, wherein the linker is a substrate for a microbial transglutaminase and wherein the linker is preferably further covalently attached to a payload. A preferred linker has the following formula:

[0253]

[0254] Wherein R is the remainder of the linker and may optionally further comprise a drug or a detectable label. Examples of detectable labels include radioactive groups and fluorescent groups.

[0255] In one embodiment of the kit of parts of the present invention, the kit further comprises an instruction manual with instructions on how to use the reaction buffer and microbial transglutaminase of the kit to conjugate a linker having a primary amino group to an antibody, preferably to a monoclonal antibody.

[0256] Conjugation method of the present invention

[0257] As shown in the Examples, the inventors have developed a surprisingly improved method for efficiently covalently conjugating antibodies to primary amines of a linker using a transglutaminase-based system.

[0258] Thus, in a further aspect, the invention provides a method for producing an antibody-linker-conjugate comprising the following steps:

[0259] (1) providing an antibody, which optionally comprises an amino acid sequence selected from the group consisting of GGTLQSPP, TLQSG, TLQSPP, GGTLQSG and TLQSA in at least one of its light chain constant regions (CL) and preferably in both of its light chain constant regions (CL) and / or in at least one of its heavy chain constant regions (CH) and preferably in both of its heavy chain constant regions (CH), and more preferably comprises the amino acid sequence TLQSPP or GGTLQSPP (most preferably comprises the amino acid sequence GGTLQSPP);

[0260] (2) Mix together at least the following components in a reaction buffer:

[0261] (a) the antibody provided in step (1);

[0262] (b) a microbial transglutaminase, preferably comprising SEQ ID NO: 44, 47, 48, 49, 4

[0263] 5, 73, 81, 82, 83 or 84 and preferably a transglutaminase comprising the amino acid sequence of SEQ ID NO 45; and

[0264] (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, and wherein said linker is preferably a drug-linker, wherein said linker is covalently attached to a drug;

[0265] (3) separating the antibody-linker-conjugate produced in step (2) from unreacted linker and from the transglutaminase, preferably by subjecting the mixture from step (2) to size exclusion chromatography.

[0266] In method step (1), the antibody optionally comprises an amino acid sequence selected from the group consisting of GGTLQSPP, TLQSG, TLQSPP, G GTLQSG and TLQSA. Such a sequence is useful because it comprises a glutamine that reacts with the linker or the therapeutic agent in method step (2)(c). However, the conjugation reaction can also be carried out using a natural antibody as shown, for example, in FIG12 . Therefore, if the antibody does not comprise such an amino acid sequence as shown above, it is preferred to use an antibody in the method of the present invention that comprises a glutamine at position 295 (EU numbering) or a glutamine at a different position that is capable of reacting with the linker or the therapeutic agent in method step 2(c) in the presence of the reaction buffer and the microbial transglutaminase.

[0267] In method step (2)(c), the H2N-part of the linker or the H2N-part of the therapeutic agent reacts with the glutamine of the antibody. The glutamine of the antibody can, for example, be the glutamine at position 295 (EU numbering) of the antibody heavy chain and / or can be the glutamine in the amino acid sequence of the antibody, wherein the amino acid sequence is selected from the group consisting of GGTLQSPP, TLQSG, TLQSPP, GGTLQSG and TLQSA.

[0268] Preferably, the reaction buffer

[0269] A reaction buffer of 0% to 7% DMSO, 20 mM to 30 mM HEPES (preferably 24 mM HEPES) and a pH of 6.8 to 7.4 (preferably pH 7); or

[0270] is a reaction buffer comprising 3% to 10% DMSO, 5 mM to 100 mM HEPES and containing less than 150 mM Na Cl and having a pH of 6 to 8; or

[0271] A reaction buffer comprising 3% to 10% DMSO, 5 mM to 60 mM HEPES and less than 10 mM NaCl (preferably no NaCl) and having a pH of 6 to 7.5.

[0272] In further embodiments of the methods of the invention, the linker is a linker having the formula:

[0273]

[0274] wherein R is the remainder of the linker, which may also include a therapeutic agent or a detectable label.

[0275] In another embodiment of the method of the present invention, in step (2) of the method, the mixture comprises the following drug-linker:

[0276]

[0277] In a further embodiment of the method of the invention, the mixture in step (2) comprises 5 molar equivalents of a linker or a drug-linker / conjugation site, respectively, wherein the conjugation site is the sequence GGTLQSPP, TLQSG, TLQSPP, GGTLQ SG or TLQSA contained in the light chain constant region (CL) and / or in the heavy chain constant region (CH) of said antibody; and / or wherein the conjugation site is the glutamine at position 295 of the antibody heavy chain, wherein the antibody is a monoclonal antibody and wherein EU numbering is used to define the position of the glutamine at position 295.

[0278] In further embodiments of the method of the invention, the antibody is a monoclonal antibody and is preferably an anti-CEACAM5 antibody as defined in (a), (b) or (c), said (a), (b) or (c) being as indicated below:

[0279] (a) Antibodies comprising

[0280] (i) at least one light chain constant region (CL) comprising a sequence selected from the group consisting of GGTLQSPP, TLQSG, TLQSPP, GGTLQSG and TLQSA, and preferably comprising this sequence at the C-terminus of said light chain constant region; and

[0281] (ii) at least one heavy chain constant region (CH) comprising one or more of the following amino acid substitutions:

[0282] (a) L234A and L235A (LALA mutation);

[0283] (b) L234A and L235A and P329G (LALA-PG mutation);

[0284] (c) L235A and G237A (LAGA mutations);

[0285] (d) M252Y, S254T, and T256E (YTE mutation);

[0286] (e) K222R;

[0287] and wherein EU numbering is used for said amino acid substitutions;

[0288] and 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;

[0289] (b) wherein the heavy chain constant region (CH) and light chain constant region (CL) of the antibody have any one of the following sequence combinations:

[0290] (1) both CHs contain the sequence of SEQ ID NO:31 and both CLs contain the sequence of SEQ ID NO:12; or

[0291] (2) both CHs contain the sequence of SEQ ID NO: 31 and both CLs contain the sequence of SEQ ID NO: 33; or

[0292] (3) both CHs contain the sequence of SEQ ID NO: 32 and both CLs contain the sequence of SEQ ID NO: 33; or

[0293] (4) both CHs contain the sequence of SEQ ID NO: 50 and both CLs contain the sequence of SEQ ID NO: 12; or

[0294] (5) both CHs contain the sequence of SEQ ID NO: 50 and both CLs contain the sequence of SEQ ID NO: 33; or

[0295] (6) a CH comprising a sequence of SEQ ID NO:31 and a CL comprising a sequence of SEQ ID NO:12; or

[0296] (7) a CH comprising a sequence of SEQ ID NO: 31 and a CL comprising a sequence of SEQ ID NO: 33; or

[0297] (8) a CH comprising a sequence of SEQ ID NO: 32 and a CL comprising a sequence of SEQ ID NO: 33; or

[0298] (9) a CH comprising a sequence of SEQ ID NO: 50 and a CL comprising a sequence of SEQ ID NO: 12; or

[0299] (10) A CH comprises a sequence of SEQ ID NO:50 and a CL comprises a sequence of SEQ ID NO:33.

[0300] (c) Antibodies are composed of

[0301] 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

[0302] 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

[0303] 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

[0304] 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

[0305] 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.

[0306] and / or

[0307] wherein the drug is a growth inhibitory agent as defined in (d), (e) or (f), said (d), (e) or (f) being as specified below:

[0308] (d) The growth inhibitory agent is a cytotoxic drug or a radioactive moiety.

[0309] (e) a growth inhibitory agent selected from a chemotherapeutic agent, an enzyme, an antibiotic, a toxin such as a small molecule toxin or an enzyme-active toxin, a toxoid, a vinca alkaloid, a taxane, a maytansine or a maytansine analog, tomamycin or a pyrrolobenzodiazepine derivatives, Nostoc cyclic peptide derivatives, leptomycin derivatives, auristatin or dolastatin analogs, prodrugs, topoisomerase I inhibitors, topoisomerase II inhibitors, DNA alkylating agents, anti-tubulin agents, CC-1065 and CC-1065 analogs;

[0310] (f) The growth inhibitor is exatecan.

[0311] The present invention further provides an antibody-linker conjugate which can be produced according to any one of claims 1 to 7, wherein the linker is a linker or a drug linker as defined below:

[0312]

[0313] wherein n is the number of [(linker)-(growth inhibitory agent)] moieties covalently attached to the antibody; or

[0314]

[0315] wherein n is the number of [(linker)—(exetatecan)] moieties covalently attached to the antibody; and wherein n is preferably from 1 to 10 and more preferably 4.

[0316] In further embodiments of the antibody-linker conjugates of the invention, the linker is a drug linker, and wherein the drug is a growth inhibitory agent and preferably exatecan.

[0317] In a further embodiment of the method of the present invention, in step (2) the mixture comprises the following drug-linker:

[0318]

[0319] In a further embodiment of the method of the present invention, the glutamine of the amino acid sequence of the antibody selected from the group consisting of GGTLQSPP, TLQSG, TLQSPP, GGTLQSG and TLQSA reacts in step (2) of the method in the presence of the microbial transglutaminase to form a covalent bond with the H2N-part of the linker.

[0320] In further embodiments of the methods of the invention, the antibody is a monoclonal antibody.

[0321] In a further embodiment of the method of the invention, the microbial transglutaminase is a microbial transglutaminase from Streptoverticillus ladakhii.

[0322] In a further embodiment of the method of the invention, the reaction mixture obtained in step (2) is incubated at a temperature of 20° C. to 38° C. until the antibody-linker-conjugate is formed and can be separated in method step (3).

[0323] In further embodiments of the methods of the invention, the reaction buffer comprises one or more of the following buffers adjusted to a pH of from 7 to pH 8.8:

[0324] BICINE, BICINE / Tris, Tris-HCl, HEPES, and / or Tricine.

[0325] In a further embodiment of the method of the present invention, wherein the reaction buffer comprises Tris-HCl adjusted to pH 7.7 to pH 8.5; or

[0326] comprising BICINE and / or BICINE / Tris adjusted to pH 7.8 to pH 8.8; or

[0327] Contains Tricine adjusted to pH 7.8 to pH 8.0.

[0328] In further embodiments of the methods of the invention, the reaction buffer comprises HEPES in an amount of 5 mM to 100 mM.

[0329] In a further embodiment of the method of the invention, the reaction buffer is a reaction buffer comprising 3% to 10% DMSO, 5 mM to 60 mM HEPES and comprising less than 100 mM NaCl (preferably no NaCl) and having a pH of 6 to 7.5;

[0330] The antibody further comprises GGTLQSPP, TLQSG, TLQSPP, GGTLQSG and TLQSA sequences in both light chain constant regions (CL) and / or in both heavy chain constant regions (CH); and the linker is a linker having the following formula:

[0331]

[0332] wherein R is the remainder of the linker and may optionally further comprise a drug.

[0333] In further embodiments of the method of the invention, 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, most preferably wherein the reaction buffer does not comprise NaCl.

[0334] In a further embodiment of the method of the invention the total amount of all buffering agents comprised in said reaction buffer is below 160 mM and preferably below 155 mM.

[0335] In a further embodiment of the method of the invention the total amount of all buffering agents comprised in said reaction buffer is lower than 70 mM.

[0336] In further embodiments of the method of the invention, the total amount of all buffers comprised in the reaction buffer is less than 155 mM; and wherein the reaction buffer comprises less than 10 mM NaCl and wherein the total amount of microbial transglutaminase is 2.5 U / ml to 12 U / ml.

[0337] In further embodiments of the method of the present invention, the total amount of all buffering agents contained in the reaction buffer is less than 70 mM; and wherein the reaction buffer comprises less than 50 mM NaCl; wherein the total amount of microbial transglutaminase is 2.5 U / ml to 12 U / ml and wherein the pH of the reaction buffer is pH 5.9 to pH 8.1.

[0338] In further embodiments of the methods of the invention, the antibody is trastuzumab, rituximab or labetuzumab; or

[0339] wherein the antibody comprises 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; or

[0340] wherein the antibody comprises 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; and wherein the antibody is an IgG monoclonal antibody.

[0341] In another embodiment of the method of the present invention, the reaction buffer comprises at least one buffer selected from imidazole, HEPES, BICINE, Tris-HCl, MOPS / Bis-Tris propane and MOPS / sodium HEPES, and wherein the buffer concentration is selected in the range of 10-160 mM, preferably wherein the buffer is HEPES at a concentration of 10 mM to 30 mM, more preferably HEPES at a concentration of about 24 mM.

[0342] In further embodiments of the method of the invention, the reaction buffer has a pH of 6.5 to 8.7, more preferably a pH of 6.8 to 8.2, most preferably a pH of about 7; or wherein the reaction buffer has a pH of about 7 to 8.5 and wherein the reaction buffer comprises less than 10 mM NaCl and 20 mM to 120 mM of at least one of the following buffers: HEPES, Tris-HCl, MOPS / NaHEPES, BICINE, BICINE / Tris, imidazole and Tricine.

[0343] In a further embodiment of the method of the invention, the reaction buffer comprises 4% to 7% DMSO.

[0344] In a further embodiment of the method of the present invention, the reaction buffer comprises the transglutaminase, wherein the transglutaminase comprises the amino acid sequence of SEQ ID NO: 45, and wherein in step (2) of the method the microbial transglutaminase is contained in the mixture at a concentration of 5 U / ml to 25 U / ml and preferably at a concentration of 10 U / ml to 20 U / ml.

[0345] In further embodiments of the methods of the invention, the reaction buffer does not comprise sodium chloride and further comprises about 24 mM HEPES, wherein the reaction buffer has a pH of about 7.5 and wherein the microbial transglutaminase concentration is about 12.5 U / ml.

[0346] In another embodiment of the method of the present invention, the reaction buffer does not contain sodium chloride and further comprises about 24 mM HEPES, wherein the reaction buffer has a pH of about 7 and comprises about 4% DMSO, wherein the concentration of microbial transglutaminase in the mixture in step (2) of the method is about 20 U / ml.

[0347] In another embodiment of the method of the present invention, the reaction buffer does not contain sodium chloride and further comprises about 24 mM HEPES, wherein the reaction buffer has a pH of about 7 and comprises about 7% DMSO, wherein the concentration of microbial transglutaminase in the mixture in step (2) of the method is about 20 U / ml.

[0348] In further embodiments of the methods of the invention, the antibody comprises the amino acid sequence TLQSG, GGTLQSPP or GGTLQSG at the C-terminus of its light chain constant region (CL) and 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.

[0349] In a further embodiment of the method of the invention, the antibody-linker-conjugate produced in step (2) has a drug-antibody ratio (DAR) of 3 to 4.5, preferably 3.8 to 4.1.

[0350] In a further embodiment of the method of the invention, the antibody-linker-conjugate produced in step (2) has a drug-antibody ratio (DAR) of 1.9 to 2.

[0351] In further embodiments of the methods of the invention, the antibody comprises 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; and wherein the antibody is an IgG monoclonal antibody.

[0352] In another embodiment of the method of the present invention, the mTG enzyme is from Streptococcus ladakii; wherein the antibody is a monoclonal antibody comprising glutamine at position 295, and wherein the reaction buffer comprises DM SO and further comprises HEPES in an amount of about 20 mM to 30 mM; wherein the reaction buffer comprises less than 100 mM NaCl, and wherein the pH of the reaction buffer is about pH 7 to pH 7.5; and wherein EU numbering is used to define the position of the glutamine at position 295.

[0353] In another embodiment of the method of the present invention, the mTG enzyme is from Streptococcus ladakii; wherein the antibody is a monoclonal antibody comprising glutamine at position 295, and wherein the reaction buffer comprises DM SO and further comprises HEPES in an amount of about 20 mM to 30 mM; wherein the reaction buffer comprises less than 100 mM NaCl, and wherein the pH of the reaction buffer is about pH 7 to pH 7.5; and wherein EU numbering is used to define the position of the glutamine at position 295; and wherein the C-terminus of the light chain of the antibody comprises the amino acid sequence GGTLQSPP.

[0354] In additional embodiments of the methods of the invention, the linker has structure (A) or (B) shown below, wherein R is a therapeutic agent or another payload:

[0355]

[0356]

[0357] In another embodiment of the method of the present invention, the antibody comprises one or more amino acid sequences selected from GGTLQSP P, TLQSG, TLQSPP, GGTLQSG and TLQSA; wherein this amino acid sequence is located at the C-terminus of the light chain of the antibody and / or at the C-terminus of the heavy chain of the antibody.

[0358] In further embodiments of the method of the invention, a molar excess of drug-linker / antibody conjugation site (size) is used in step (2) of the method and preferably at least 4 molar equivalents of drug-linker / conjugation site are used.

[0359] In further embodiments of the antibody-drug-conjugates of the invention, the immunoconjugate has formula (IV A), and n is 1 to 10 and preferably about 4.

[0360] In a further aspect, the present invention provides an antibody comprising an amino acid sequence selected from GGTLQSPP, GGTLQSG and TLQSA at the C-terminus of the heavy chain of the antibody and / or an amino acid sequence selected from GGTLQSPP, GGTLQSG and TLQSA at the C-terminus of the light chain of the antibody.

[0361] Sequence Description

[0362] Amino Acid Sequence:

[0363]

[0364]

[0365] Nucleotide sequence:

[0366] SEQ ID NO:15 DNA sequence encoding the HC of mAb1 SEQ ID NO:16 DNA sequence encoding the LC of mAb1 Amino acid sequence:

[0367]

[0368]

[0369] Nucleotide sequence:

[0370] Amino Acid Sequence:

[0371]

[0372]

[0373] Example

[0374] Example 1: Synthesis of Drug-Linker Compounds with Glucuronide Linkers: Drug-Linker Compounds 1-M(DL1-M)

[0375]

[0376] The synthetic route to compound 9).

[0377]

[0378] Synthetic route to Compound 11, also referred to herein as Drug-Linker Compound 1-M (DL1-M).

[0379] Chemical preparation scheme of compound 9

[0380] Step 1: Compound 1

[0381]

[0382] 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 equiv) and 4-hydroxy-3-nitro-benzaldehyde (5.24 g; 31.35 mmol; 1.50 equiv) in acetonitrile (83.00 ml; 10.00 V) was added silver (I) oxide (9.69 g; 41.80 mmol; 2.00 equiv). The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was filtered through celite. The filtrate was concentrated in vacuo to give a solid. The solid was dissolved in EtOAc and washed with a 10% aqueous solution of NaHCO3 to remove excess 4-hydroxy-3-nitro-benzaldehyde. The organic layer was concentrated in vacuo to give compound 1 as a sandy solid.

[0383] Yield: 9.0g

[0384] Percent yield: 89.1%

[0385] Analyze the data:

[0386] NMR: 1H-NMR (400MHz, 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=10.00Hz,1H),5.51-5.44(m,1H),5.20-5.09(m,2H),4.80(d,J=13.20Hz,1H),3.64(s,3H),2.09(s,9H).

[0387] Step 2: Compound 2

[0388]

[0389] To a stirred solution of compound 1 (9.00 g; 18.62 mmol; 1.00 equiv) in propan-2-ol (33.00 ml; 3.67 V) and CHCl (167.00 ml; 18.56 V) was added silica gel 60-120 (3.60 g; 112.09 mmol; 6.02 equiv) followed by sodium borohydride (1.80 g; 46.55 mmol; 2.50 equiv). The reaction mixture was stirred at room temperature for 1 h. Upon completion, the reaction mixture was quenched with cooled 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.

[0390] Yield: 8.70g

[0391] Percent yield: 92.4%

[0392] Analyze the data

[0393] LCMS: Column: ATLANTIS dC18 (50x4.6mm) 5μm; Mobile phase A: 0.1% HCOOH in H2O:ACN (95:5); B: ACN

[0394] Room temperature (min): 2.05; M+H: 503.2, purity: 96.6%

[0395] Step 3: Compound 3

[0396]

[0397] To a stirred solution of compound 2 (8.70 g; 17.21 mmol; 1.00 equiv) 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 equiv). The reaction mixture was stirred at room temperature under hydrogen atmosphere for 3 h. Upon completion, the reaction mixture was filtered off through celite. The solvent was concentrated in vacuo to afford compound 3 as an off-white solid.

[0398] Yield: 8.5g

[0399] Percent yield: 100%

[0400] Analyze the data:

[0401] LCMS: Column: ATLANTIS dC18 (50x4.6mm) 5μm; Mobile phase A: 0.1% HCOOH in H2O:ACN (95:5); B: ACN

[0402] Room temperature (min): 1.73; M+H: 456.10, purity: 95.1%

[0403] Step 4: Compound 4

[0404]

[0405] To compound 3 (10.00g; 20.89mmol; 1.00 equivalent) and (9H-fluorene-9-ylmethoxycarbonylamino)-acetic acid (7.60g; 25.06mmol; 1.20 equivalent) in DCM (250.00ml; 25.00V) stirring solution was added 2-ethoxy-2H-quinoline-1-carboxylic acid ethyl ester (15.65g; 62.66mmol; 3.00 equivalent) at 0°C. The reaction mixture was stirred at room temperature for 16h. After completion, the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (56% EtOAc: petroleum ether) to obtain a compound with 80% purity. The compound was further purified by washing with 30% EtOAc and petroleum ether to obtain compound 4 as a white solid.

[0406] Yield: 8.5g

[0407] Percent yield: 50.7%

[0408] Analyze the data:

[0409] LCMS: Column: ATLANTIS dC18 (50x4.6mm) 5μm; Mobile phase A: 0.1% HCOOH in H2O:ACN (95:5); B: ACN

[0410] Room temperature (min): 3.03; M+H: 735.2, purity: 81.9%

[0411] Step 5: Compound 5

[0412]

[0413] Carbonic acid bis-(4-nitro-phenyl) ester (3.06 g; 9.97 mmol; 4.00 equivalent) and DIP EA (4.40 ml; 24.92 mmol; 10.00 equivalent) were added to a stirred solution of compound 4 (2.00 g; 2.49 mmol; 1.00 equivalent) in THF (40.00 ml; 20.00 V) at 0 ° C. The reaction mixture was stirred at room temperature for 12 h. After the reaction was complete, the reaction mixture was concentrated in vacuo. The crude product was purified by column chromatography using silica gel (230-400) and petroleum ether / ethyl acetate as eluent to provide compound 5 as a light yellow solid.

[0414] Yield: 2.0g

[0415] Percent yield: 84.6%

[0416] Analyze the data:

[0417] LCMS: Column: X-Bridge C8 (50X4.6) mm, 3.5 μm; Mobile phase: A: 0.1% TFA in MilliQ water; B: ACN

[0418] Room temperature (min): 3.24; M+H: 900.20, purity: 94.9%

[0419] Step 6: Compound 6

[0420]

[0421] Compound 5 (1,369 g; 1,00 equivalent) was dissolved in N,N-dimethylformamide (15,00 ml), exatecan mesylate (679,7 mg; 1,00 equivalent), 4-methylmorpholine (0,422 ml; 3,00 equivalent) and 1-hydroxybenzotriazole (172,8 mg; 1,00 equivalent) for synthesis were added. The reaction mixture was stirred at room temperature overnight. After the stirring time, the reaction suspension became a brown solution. The reaction was monitored by LC-MS showing complete conversion of the starting material. The reaction mixture was purified via reverse phase flash chromatography. The product containing fractions were combined, concentrated in vacuo and freeze-dried overnight to provide compound 6 as a yellow solid.

[0422] Yield: 1.59 g

[0423] Percent yield: 87.5%

[0424] Analyze the data:

[0425] LCMS: Column: Chromolith HR RP-18e (50-4,6 mm); Mobile phase A: 0.05% HCOOH in H2O; B: 0.04% HCOOH and 1% H2O 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

[0426] Room temperature (min): 1.95; M+H: 1196.40, purity: 84.4%.

[0427] Step 7: Compound 7

[0428]

[0429] Compound 6 (1,586 g; 1,00 equiv) was dissolved in tetrahydrofuran (50,00 ml) at 0°C and a solution (0.1 M) of LiOH (containing lithium hydroxide hydrate (281,77 mg; 6,00 equiv) in water (67,100 ml)) was added dropwise. The pH value was checked during the addition. The pH should not exceed 10. The addition of the solution of LiOH was completed after 1.5 hours. The reaction was monitored by LC-MS which showed complete conversion of the starting material. The reaction was quenched with 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 freeze-dried to provide compound 7 as a dark yellow solid.

[0430] Yield: 728 mg

[0431] Percent yield: 54.8%

[0432] Analyze the data:

[0433] LCMS: Column: Chromolith HR RP-18e (50-4,6 mm); Mobile phase A: 0.05% HCOOH in H2O; B: 0.04% HCOOH and 1% H2O 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

[0434] Room temperature (min): 1.68; M+H: 1056.30, purity: 98.5%.

[0435] Step 8: Compound 8

[0436]

[0437] Compound 7 (728,000 mg; 1,00 equiv) was dissolved in N,N-dimethylformamide (20,00 ml). Piperidine (136,513 μl; 2,00 equiv) was added and the solution was stirred at room temperature for a total of 4 hours. 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 reverse phase flash chromatography. The product containing fractions were combined, the solvent was partially removed and it was freeze-dried overnight to provide compound 8 as a yellow solid.

[0438] Yield: 706 mg

[0439] Percent yield: 100%

[0440] Analyze the data:

[0441] LCMS: Column: Chromolith HR RP-18e (50-4,6 mm); Mobile phase A: 0.05% HCOOH in H2O; B: 0.04% HCOOH and 1% H2O 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

[0442] Room temperature (min): 1.22; M+H: 834.30, purity: 97.6%.

[0443] Step 9: Compound 9

[0444]

[0445] To a solution of compound 8 (854 mg; 1,00 equiv) in dimethylformamide (30,00 ml) was added N-ethyldiisopropylamine (149,234 μl; 1,00 equiv) 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 equiv). The reaction mixture was stirred at room temperature for 3 hours. 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 passed through reverse phase flash chromatography. The product containing fractions were combined, concentrated and freeze-dried to produce the desired product with a purity of 91%. The material was purified again by RP chromatography to produce compound 9 as a yellow solid.

[0446] Yield: 580 mg

[0447] Percent yield: 60.1%

[0448] Analyze the data:

[0449] LCMS: Column: Chromolith HR RP-18e (50-4,6 mm); Mobile phase A: 0.05% HCOOH in H2O; B: 0.04% HCOOH and 1% H2O 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

[0450] Room temperature (min): 1.38; M+H: 985.30, purity: 90% (another 10% of isomers can be removed by HPLC)

[0451] 1H NMR (500MHz, DMSO-d6) δ13.10–12.44(m,1H),9.08(s,1H),8.32(t,J=5.8Hz,1H),8.16(s,1H),8.02(d,J=8.8Hz,1H),7.76(d,J= 10.9Hz,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 .6Hz,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=7.3Hz,3H).

[0452] Chemical preparation scheme of compound 11

[0453] Step 1: Compound 10

[0454]

[0455] To a solution of compound 8 (289.00 mg; 1.00 equivalent) in dimethylformamide (10.00 ml), N-ethyldiisopropylamine (0.104 ml, 2.00 equivalent) and 2,5-dioxopyrrolidin-1-yl (tert-butoxycarbonyl) glycyl glycine salt (105.70 mg; 1.00 equivalent) were added. The reaction mixture was stirred at room temperature for 1 hour, and the reaction was monitored (mirror) by LC-MS. After completion, the solvent was removed in vacuo, and the crude product was purified by preparative HPLC through Sunfire columns. The fractions containing the product were combined and freeze-dried to provide compound 10 trifluoroacetate salts as yellow solids.

[0456] Yield: 162.00 mg, 0.14 mmol.

[0457] Percent yield: 44.8%

[0458] Analyze the data:

[0459] LCMS: Column: Chromolith HR RP-18e (50-4,6 mm); Mobile phase A: 0.05% HCOOH in H2O; B: 0.04% HCOOH and 1% H2O 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

[0460] Room temperature (min): 1.41; M+H: 1048.1, purity: 97.9%

[0461] Step 2: Compound 11

[0462]

[0463] To a suspension of compound 10 (162.00 mg; 1.00 equiv.) in dichloromethane (5.00 ml) was added 4.0 M hydrogen chloride in 1,4-dichloromethane. The mixture was stirred at room temperature for 3 hours at 4 °C for 1 h. The reaction mixture was concentrated in vacuo and the crude product was purified by preparative HPLC. The fractions containing the product were combined and freeze-dried to provide a trifluoroacetic acid salt of compound 11 as a yellow solid.

[0464] Yield: 113 mg; 0.11 mmol.

[0465] Percent yield: 77.0%

[0466] Analyze the data:

[0467] LCMS: Column: Chromolith HR RP-18e (50-4,6 mm); Mobile phase A: 0.05% HCOOH in H2O; B: 0.04% HCOOH and 1% H2O 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

[0468] Room temperature (min): 1.22; M+H: 948.0, M+2H: 474.8; Purity: 98.8%

[0469] 1H NMR(700MHz,DMSO-d6)δ12.93–12.79(m,1H),9.14(s,1H),8.61(t,J=5.8H z,1H),8.41(t,J=5.9Hz,1H),8.14(s,1H),8.05(d,J=8.8Hz,1H),8.03–7.9 3(m,3H),7.79(d,J=10.8Hz,1H),7.31(s,1H),7.13(s,2H),6.58–6.47(m,1 H),5.80–5.61(m,1H),5.48–5.40(m,2H),5.31–5.24(m,3H),5.08(d,J=12. 2Hz,1H),5.03(d,J=12.3Hz,1H),4.91(d,J=7.7Hz,1H),3.96(d,J=5.8Hz,2 H),3.95–3.86(m,3H),3.62(q,J=5.8Hz,2H),3.43(t,J=9.3Hz,1H),3.41(t ,J=8.5Hz,1H),3.35(t,J=9.0Hz,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=7.3Hz,3H).

[0470] Example 2: Expression and purification of exemplary modified antibodies and expression and purification of transglutaminase

[0471] Hereinafter, the expression and purification of exemplary modified antibodies will be described. These exemplary modified antibodies can be used to produce respective antibody-drug-conjugates using the methods 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:

[0472]

[0473]

[0474] Table 4

[0475] The DNA sequence of coding antibody mAb1-M, mAb2-M, mAb3-M, mAb5-M, mAb6-M and mAb7-M is synthesized and cloned on pTT5 plasmid, for recombinant expression at GeneArt (Life Technologies). Using ExpiCHO expression system (GibcoTM, Thermo Fisher Scientific Co., Ltd.), the plasmid produced is used for transient transfection and recombinant protein expression in shaking flask. Seven days after transfection, harvest supernatant and the antibody expressed is purified using standard step-by-step method, and the standard step-by-step method comprises protein A affinity chromatography (HiTrap MabSelect SuRe post, Cytiva) and size exclusion chromatography (HiLoad Superdex200pg post, Cytiva).

[0476] mAb4-M / rituximab (F. Hoffmann-La Roche GmbH, Basel, Switzerland) was purchased in pharmaceutical grade quality.

[0477] Expression and purification of transglutaminase

[0478] The DNA sequence encoding transglutaminase mTG (Seq ID 46) was synthesized and cloned into pET30a plasmid for recombinant expression in GeneArt (Life Technologies). E. coli BL21 (DE3) strain transformed with the generated plasmid was cultured overnight in shake flasks in lysogenic broth medium supplemented with 5 g / l glucose, 10 ml / 100 ml 10x phosphate buffered saline and 30 mg / l kanamycin at 28°C and 130 rpm (50 mm throw). This culture was used to inoculate a fermenter containing 9.5 liters of growth medium (50 g / l yeast extract, 10 g / l peptone, 0.5 g / l MgSO4 x 7H2O and 2 ml 50% Desmophen (antifoam from Rhein Chemie Rheinau) to an optical density of 0.00002. The fermenter was run overnight (16 h) at 28°C and 800 U / min rotation speed, 5 Nl / min aeration and pH 7.0-7.4. At OD 5, the culture was induced with 0.1 mM IPTG until an OD of ~30 was reached (5-6 hours). In the event of foam formation or the oxygen concentration dropped below 2 mg / ml, more Desmophen was added or the rotation speed was increased to 1000 rpm, respectively. The cell pellet was harvested by continuous flow-by centrifugation.

[0479] 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), pH adjusted to 5.5 and loaded onto S O3-(M) 85 ml column (Millipore), followed by 20 CV of linear gradient elution from 0-1 M NaCl. Fractions with the 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. Then, 0.5 U / ml I (Sigma Aldrich) and 2 mM CaCl2 were used to proteolyze transglutaminase at a final concentration of 2 mg / ml at 37°C for 60 min, followed by the addition of 5 mM EDTA. The reaction mixture was dialyzed against 50 mM sodium phosphate buffer pH 6.0 at 4°C overnight and loaded into SO3-column (Millipore) and eluted with a linear gradient of 0-1M NaCl in 20CV. The fractions with effective cleavage and purified protein were identified by SDS-PAGE, merged, concentrated and purified using HiLoad Superdex 75pg size exclusion column (Cyti va) with 24HEPES pH 7, 100mM NaCl as running buffer. The transglutaminase containing fractions were merged, concentrated to>20mg / ml, quickly frozen in liquid nitrogen and stored at -80°C. Enzyme activity was determined using ZediXclusive microbial transglutaminase assay (Zedira).

[0480] Example 3: Exemplary ADC preparation, conjugation and characterization of ADC7-M, ADC2-M, ADC5-M and ADC3-M

[0481] 3.1. Antibody preparation and conjugation

[0482] Monoclonal antibodies (mAbs) were stored at -80°C. Prior to conjugation, mAbs were thawed at room temperature and purified using HiTrap desalting columns. The buffer was exchanged to 24 mM HEPES, pH 7.0 using a liquid chromatography (LC) system (Cytiva). To couple the drug linker (DL) to the antibody, microbial transglutaminase (mTG) was used. The reaction was set up 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. The ADC was separated from DL and mTG via 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. A HiLoad Superdex 200 26 / 60 Increase column was used in combination with a 4% HPLC-MS / MS column. SEC was performed using a Cytiva 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. Fractions containing ADC material were pooled and concentrated to 8 mg / ml using an Amicon Ultra 15 50K centrifuge (Millipore) and subsequently purified using a HiTr ap desalting column combined with a 50 μl column. The final buffer was exchanged into 10 mM histidine, 40 mM NaCl, 6% trehalose, 0.05% TWEEN, pH 5.5 by LC system (Cytiva).The final drug substance (BDS) was filtered through a 0.22 μm sterile filtration unit (Millipore) and stored frozen (shock) in liquid nitrogen until further use.

[0483] 3.2 Quality attributes

[0484] ADC7-M

[0485] Concentration [mg / ml] 7.11 Drug-antibody ratio 3.87 PuritySEC[%] 99.9 Free drug content [%] 0.12 Endotoxin content [EU / mg] 0.34

[0486] ADC2-M

[0487] Concentration [mg / ml] 6.9 Drug-antibody ratio 3.86 PuritySEC[%] 99.7 Free drug content [%] 0.03 Endotoxin content [EU / mg] 0.19

[0488] ADC3-M

[0489] Concentration [mg / ml] 7.98 Drug-antibody ratio 4.2 PuritySEC[%] 100 Free drug content [%] 0.13 Endotoxin content [EU / mg] 0.14

[0490] ADC5-M

[0491]

[0492]

[0493] 3.3. Methods: Drug substance characterization

[0494] Size Exclusion Chromatography (SEC) Method

[0495] Monomer content and purity were assessed by size exclusion chromatography on a TOSOH TSKgel column. The injection volume range was 1-5 μl (up to 10 μg protein).

[0496]

[0497] Typical SEC chromatograms showing the purity of the input mAb and final BDS are shown in Figure 1 middle.

[0498] Reverse Phase HPLC (RP HPLC) Method

[0499] RP HPLC method parameters

[0500]

[0501] A typical RP-HPLC chromatogram illustrating the DAR determination of the final BDS is shown in Figure 2 middle.

[0502] Free Drug Method

[0503]

[0504]

[0505] gradient

[0506]

[0507] Injection volume 10.00 μL

[0508] Column temperature 50℃

[0509] Flow rate 0.75mL / min

[0510] Sample preparation Protein drop: 100 μL of API + 250 μL of cold MeOH + 50 μL of 3M MgCl 2. Spin at 20,000 rpm for 10 min.

[0511] Standard Preparation Mixture 20 μL of 20 mM DL (MS C2702209A in DMSO) + 20 μL DMSO + 40 μL MeOH + 20 μL of 200 mM NAC in diafiltration buffer. Incubate overnight to provide 4 mM DL-NAC. 4 mM DL-NAC was diluted in MeOH to provide 4 μM DL-NA C standard.

[0512] Endotoxin characterization

[0513] Endotoxin was determined by kinetic chromogenic LAL assay using the Endosafe PTS endotoxin system (Charles River). Buffer and antibody were diluted 10-fold in LAL reagent water. ADC was diluted 10-fold in LAL reagent water. All samples were analyzed on a 0.01-1 EU / mL kit. EU / mL values ​​were converted to EU / mg by dividing by ADC[P]mg / mL.

[0514] Example 4: ADC2-M specifically kills cancer cells in vitro with high potency

[0515] Human cancer cell lines were used to evaluate the potential of ADC2-M to kill cancer cells. ADC2-M showed subnanomolar and subnanomolar to single-digit nanomolar in vitro potency against different CEACAM5-positive cell lines, respectively (Table 3). In contrast, ADC2-M had a weak effect on the CEACAM5-negative cell line MDA-MB-231 (Table 3). As shown in the exemplary dose-response curves, ADC2-M was very potent against the CEACAM5-positive cell lines SK-CO-1, SNU-16, MKN-45, and LS174T (Table 3). Figure 3 ad). In contrast, AD C2-M had only a weak effect on the viability of antigen-negative MDA-MB-231 cells ( Figure 3 e).

[0516] An isotype control ADC utilizing the same linker payload as ADC2-M showed much lower effects on the CEACAM5-positive cell lines tested ( Figure 3 ).

[0517] In conclusion, ADC2-M specifically kills CEACAM5-expressing human cancer cell lines in vitro with high potency.

[0518]

[0519] Table 3. Efficacy of ADC2-M and free payload against various human cell lines. The maximum effect compared to untreated controls at the highest tested compound concentration is indicated in parentheses. The cutoff for efficacy reports -50% effect, otherwise stated as "n / a". For each cell line, CEACAM5 expression is indicated.

[0520] Methods – Viability assay:

[0521] The cytotoxic effect of ADC on cancer cell lines was measured by cell viability assay. One day before treatment, cells were seeded in a 96-well plate in a volume of 90 μL. Test compounds (ADC or free effective load) were prepared in cell culture medium at 10 times the starting concentration. The test compounds were serially diluted (1:4) and 10 μL of each dilution was added to the cells in triplicate. The plates were cultured at 37°C in a CO2 incubator for six days. For cell viability measurements, Cell Reagents (Promega TM Company, Madison, WI) was added to each well, and the plate was processed according to the manufacturer's instructions. The luminescent signal was measured using a Varioskan plate reader (Thermo Fisher). The luminescent readings were converted into % activity relative to untreated cells. Nonlinear regression analysis was performed using logarithm (inhibitor) compared to reaction, variable slope, 4-parameter fitting equation using Genedata Screener or GraphPad Prism to fit the data. Data are displayed as % relative cell viability compared to molar compound concentration, and error bars represent the standard deviation (SD) of duplicate or triplicate. Calculations derive from the arithmetic mean of the IC50 of multiple experiments.

[0522] ADC2-M was also compared with ADC SAR DM4 in terms of its cytotoxic effects on antigen-positive SK-CO-1 and antigen-negative MDA-MB-231 cell lines. Against SK-CO-1 cancer cells, ADC2-M showed similar potency to ADC SAR DM4 ( Figure 3 a compared with Figure 26a). Compared with ADC2-M, the non-specific effect of ADC SAR DM4 against antigen-negative MDA-MB-231 was higher ( Figure 3 e compared with Figure 26b).

[0523] ADC2-M was also generated using an antibody backbone lacking the YTE mutation. This ADC (ADC7-M) showed comparable results similar to the respective ADC with the YTE mutation (ADC2-M).

[0524] Example 5: Pharmacokinetic study of ADC

[0525] Pharmacokinetic studies were performed in human FcRn transgenic (276 hemizygous model) mice after a single intravenous administration of 3 mg / kg of ADC1, ADC1-M, ADC6-M, ADC7-M, ADC 2-M. Samples were collected from 6 animals / treatment group for ADC1-M, A DC6-M, ADC7-M and ADC2-M at 1, 24, 48, 72, 144, 168, 240, 336 and 504 hours after dosing, and samples were collected from 9 animals for ADC1 at 0.08 (about 5 minutes), 4, 24, 48, 72, 168, 240, 336 and 504 hours after dosing. Data were pooled for PK analysis, see for plasma concentration / treatment Figure 4 Back extrapolation of plasma concentrations at time 0 (C0) yielded concentrations slightly below the theoretical maximum for ADC1-M, ADC7-M, and ADC2-M and close to the theoretical maximum for ADC6-M and ADC1 (assuming dilution at 40 mL / kg plasma volume). Plasma concentrations decreased with a biphasic profile: a sharp decrease in plasma concentrations was observed up to 24 hours in all molecule groups, followed by a slower decline up to the last sampling time (504 hours), with the exception of animals treated with ADC6-M that were quantifiable up to 336 hours.

[0526] The longest terminal half-life (t1 / 2) values ​​were calculated for the Lala-YTE-mutant ADC2-M (190 h) with DAR=4, followed by the Lala-mutant ADC7-M (122 h) with DAR=4, the Lala-YTE-mutant and ADC1-M (64.5 h) with DAR=8, and the Lala-mutant ADC6-M (33.8 h) with DAR=8, and finally the IgG1.4 ADC1 (29.6 h) with DAR=8. The % of the extrapolated AUC infinity (inf) was less than 20%, allowing reliable calculation of AUC0-infinity and derived parameters (Cl, Vz and Vss). The highest AUC0-infinity and lowest Cl values ​​were calculated for ADC2-M (L ala-YTE-mutant, DAR=4) followed by ADC7-M, ADC6-M, ADC1-M and ADC1 in transgenic mice. AUC0-infinity and Cl values ​​ranged from 1360000 h*ng / mL to 10200000 h*ng / mL and 0.293 mL / h / kg to 1.16 mL / h / kg, respectively. No relevant differences in volume of distribution (Vss) were observed, with Vss ranging from 49.8 mL / kg to 113 mL / kg.

[0527] In summary, ADC2-M, which had the highest plasma exposure, lowest CI, and longest t1 / 2, showed the best PK profile after a single intravenous administration of 3 mg / kg in transgenic mice. Moving from IgG1.4 to Lala-mutants, the PK profile improved, further including the YTE-mutant, with the strongest effect observed by reducing the DAR from 8 to 4.

[0528] Table 5: PK parameters of exemplary ADCs after 3 mg / kg iv administration. Antibody-drug-conjugate (ADC) and drug-antibody ratio (DAR) are also indicated.

[0529]

[0530] Example 6: Efficacy of ADC1-M and ADC2-M in a pancreatic cell line-derived tumor model

[0531] The efficacy of ADC1-M and ADC2-M was evaluated in a human pancreatic cell line-derived xenograft model, BxPC3 (AT CC, CRL-1687). 6 BxPC3 cells were injected subcutaneously into the right flank of six to eight week old immunodeficient female mice (Hsd: Athymic Nude-Foxn1nu, Envigo). 3 When the mean volume of 10 mice / group was 2.5 μg / kg, 10 mice / group were treated intravenously once with vehicle (saline solution) or ADC1-M (5 mg / kg; day 0) or once with ADC2-M (5 mg / kg or 10 mg / kg; day 0). Tumor length (L) and width (W) were measured with calipers, and tumor volume was calculated using L×(W^2) / 2.

[0532] A single treatment with ADC1-M at a dose of 5 mg / kg and a single treatment with ADC2-M at a dose of 5 mg / kg or 10 mg / kg induced a significant anti-tumor effect. Both ADCs had comparable efficacy in this model ( Figure 5 There was no significant effect of treatment on body weight (data not shown).

[0533] Example 7: Preparation of ADC labetuzumab govitecan analogue AD C8

[0534] 7.1 Antibodies

[0535] For the purpose of additional comparative experiments, additional analogs of the ADC labetuzumab govenotecan were prepared based on a monoclonal antibody with the following sequence:

[0536] Heavy chain: DIQLTQSPSS LSASVGDRVT ITCKASQDVG TSVAWYQQKP GKA PKLLIYWTSTRHTGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCQQ YSLY RSFGQG TKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYP REAK VQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTLSKADYEKHKV YA CEVTHQGL SSPVTKSFNR GECEVQLVES GGGVVQPGRS LRLSCSASGF DFTTYWMSWV RQAPGKGLEW IGEIHPDSST INYAPSLKDR FTISRDNAKN TLFLQMDSLR PEDTGVYFCASLYFGFPWFA YWGQGTPVTV SSASTKGPS V FPLAPSSKST SGGTAALGCL VKDYFPEPVT VSWNSGALTSGVHTFPAV LQ SSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKRV EPKSCDKT HTCPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEV KF NWYVDGVEVH NAKTKPREEQYNSTYRVVSV LTVLHQDWLN GKEY KCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSLTCLV KGFYPSDIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQ QGNVFSC SVMHEALHNHYTQKSLSLSP GK(SEQ ID NO:62)

[0537] Light chain: DIQLTQSPSS LSASVGDRVT ITCKASQDVG TSVAWYQQKP GKA PKLLIYWTSTRHTGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCQQ YSLY RSFGQG TKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYP REAK VQWKVD NALQSGNSQE SVTEQDSKDS TYSLSTLTLSKADYEKHKV YA CEVTHQGL SSPVTKSFNR GEC(SEQ ID NO:63)

[0538] 7.2 Drug-Linker Compounds

[0539] As the drug-linker molecule to be conjugated to the above-mentioned antibody, a molecule having the following structure was used:

[0540]

[0541] This drug-linker molecule was purchased from SyntaBio LLC, 10239 Flanders Ct, San Diego, CA 92121. Lot No. S041070422.

[0542] 7.3 Conjugation

[0543] Monoclonal antibodies (mAb) were thawed at 2-8°C up to 3 days before conjugation, and stored in PBS at pH 6.8 at 2-8°C until further use. On the day of conjugation, the pH of the mAb solution was adjusted to a final concentration of 5% (v / v) by adding 0.5M Tris, 0.025M EDTA, pH 8.5. After pH adjustment, 10 molar equivalents of TCEP and an incubation of 120 min at 20°C were used to reduce mAb. Subsequently, mA b solution was diluted 1:1 with 20mM histidine, 80mM NaCl, pH 5.5, DMSO concentration was adjusted to 10% (v / v), and 16 molar equivalents of the above-mentioned drug-linker were added to start the reaction. The reaction was incubated at 20°C for 60min and finally quenched by adding 100mM NAC (n-acetyl-cysteine). The conjugated mAb (ie, ADC) was then processed via preparative size exclusion chromatography.

[0544] 7.4 Preparative Size Exclusion Chromatography, Desalting, and Filtration

[0545] The reaction mixture was purified using preparative size exclusion chromatography. A GE Hi Load 26 / 60 Superdex S200 column was connected to the Avant 25 system (GE Healthcare) and 20mM histidine, 80mM NaCl, pH 5.5 balance. Subsequently, the reaction mixture was injected and run through the column, wherein the flow rate was 5ml / min, using 20mM histidine, 80mM NaCl, pH 5.5 as running buffer. The fractions containing ADC were measured, combined and concentrated via UV light absorption at 280nm. The ADC material was concentrated using Vivaspin VS2022 equipment (Sartorius UK Co., Ltd.) according to the manufacturer's instructions. According to the manufacturer's instructions at The concentrated A DC material was transferred to a formulation buffer (10 mM histidine, 100 mM NaCl, 3% trehalose, 0.05% (w / v) PS20, pH 5.5) using a HiPrep 26 / 10 desalting column (GE Healthcare) at a flow rate of 10 ml / min on an Avant 25 system (GE Healthcare). The final ADC material was filtered using a 0.2 μm filter (0.2 μm PES filter, Merck Millipore), aliquoted, and then shock 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 resulting ADC from this work is referred to herein as "A DC8"; this ADC is an analog of labetuzumab govitecan.

[0546] 7.5 Further Characterization of ADC8 API

[0547] The ADC8 drug substance obtained above was further analyzed by: (a) size exclusion chromatography (SEC), which showed a monomer purity of 99.3%, (b) reverse phase HPLC (RP HPLC), which showed a DAR of 7.7, and (c) RP HPLC-based free drug method, which showed that the residual free drug level was less than 0.02% (on a molar basis).

[0548] Example 8: ADC1-M, ADC2-M, ADC6-M and ADC7-M kill cancer cells with higher specificity than ADC SAR D M4 (analog of tusamitamabravtansine) and ADC8 (analog of labetuzumab govenotecan)

[0549] Using the same method as 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 ADC SAR DM4 and ADC8, against cancer cells with CEACAM5 expression relative to the effects against cancer cells lacking CEACAM5 expression. The fold reduction in IC50, defined as the specificity factor, was calculated by dividing the IC50 against CEACAM5-negative MDA-MB-231 cells by the IC50 against each CEACAM5-positive cell line (see Table 6). The larger the value of the specificity factor, the more specific the ADC tested. ADC1-M, ADC2-M, ADC 6-M and ADC7-M showed much lower IC50s in CEACAM5-positive SK-CO-1, SNU-16 and MKN-45 cells than in CEACAM5-negative MDA-MB-231 cells, resulting in specificity factors ranging from 116 to 874. Due to the lack of effect on MDA-MB-231 cells in the concentration range tested (as shown in Tables 3 and Figure 3 e for ADC2-M), the specificity factors of ADC2-M and ADC7-M may be underestimated. For this reason, only for ADC2-M and ADC7-M, the IC50 was set to the highest tested concentration of 100 nM to calculate the specificity factor. In contrast, ADC SAR DM4 and ADC 8 showed lower specificity factors in the range of 26 to 73 and 1.8 to 4.8, respectively. In summary, more selective killing of CEACAM5-positive cancer cells in vitro by ADC1-M, ADC2-M, ADC6-M and ADC7-M was shown compared to ADC SAR DM4 and ADC8.

[0550]

[0551] Table 6. Relative potency increase (specificity factor) of ADCs in CEACAM5-positive cell lines compared to CEACAM5-negative MDA-MB-231 cell lines. The IC50 in MDA-MB-231 was divided by the IC50 in each of the three CEACAM5-positive cell lines to calculate the specificity factor for each ADC (the theoretical factor for MDA-MB-231 is reported as 1 for illustration only). The calculations were based on the geometric mean of IC50s from 2 to 4 individual experiments.

[0552] Example 9: ADC1-M, ADC2-M, ADC6-M, ADC7-M mediate a more potent bystander effect than ADC SAR DM4 for antigen-negative cells co-cultured with antigen-positive cells

[0553] The potential of ADC1-M, ADC2-M, ADC6-M, ADC7-M and ADC SAR DM4 to mediate bystander effects on antigen-negative cells of neighboring antigen-positive cells was evaluated in a bystander assay using the same method as in Example 10. In the presence of CEACAM5-positive SK-CO-1, ADC1-M, ADC2-M, ADC6-M and ADC7-M showed potent bystander effects against CEACAM5-negative MDA-MB-231 cells ( Figure 6 ). Co-culture experiments were performed at 1 nM ADC concentration, which caused maximal inhibition of CEACAM5-positive SK-CO-1 cell viability for ADC1 ( FIG. 26A ), but had no effect on CEACAM5-negative MDA-MB-231 cells alone ( FIG. 26B ). Consistent with these findings, no nonspecific effects were observed for ADC1-M, ADC2-M, ADC6-M, ADC7-M, and ADC SAR DM4 in the bystander assay setting for the MDA-MB-231 only control.

[0554] In summary, ADC1-M, ADC2-M, ADC6-M, ADC7-M mediate a potent bystander effect against antigen-negative cancer cells co-cultured with antigen-positive cells. These findings suggest the potential to effectively target tumors with heterogeneous target expression.

[0555] Compared with ADC SAR DM4, ADC1-M, ADC2-M, ADC6-M, and ADC7-M mediated a much more potent bystander effect against antigen-negative cells co-cultured with antigen-positive cells ( Figure 6 ).

[0556] For all ADCs tested, the extent of the bystander effect increased with the number of antigen-positive cells added to a constant number of antigen-negative cells. Without wishing to be bound by theory, this may be a result of more A DCs being processed by a higher number of antigen-positive cells to release more free payload, which is responsible for the bystander effect against antigen-negative cells.

[0557] In the bystander assay, no non-specific effects of the tested ADCs against MDA-MB-231 cells alone were observed for all tested ADCs at 1E-9 M tested concentration.

[0558] Example 10: Efficacy of ADC1-M and ADC3-M compared to ADC8

[0559] The efficacy of ADC1-M and ADC3-M compared to ADC8 was evaluated in a xenograft model derived from a human pancreatic adenocarcinoma cell line, HPAF-II (ATCC, CRL-1997). Six to eight-week-old immunodeficient female mice (Hsd: AthymicNude-Foxn1nu, Envigo) were inoculated with 5x10 6 HPAF-II cells were injected subcutaneously. When the tumor reached 150 mm 3 When the average volume of 10 mice / group was 100 μg / kg, 100 μg / kg of 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) was treated intravenously once. Tumor length (L) and width (W) were measured with calipers, and tumor volume was calculated using the formula L×W^2 / 2.

[0560] Single treatment with ADC1-M or ADC3-M at a dose of 1 mg / kg or 6 mg / kg induced significant anti-tumor effects. The effect was dose-dependent, as a single treatment with 1 mg / kg induced only a mild and transient anti-tumor effect, while higher doses showed much stronger anti-tumor effects. In contrast, single treatment with the same dose of ADC8 did not show significant anti-tumor effects at either dose ( Figure 7 None of the treatments had a significant effect on body weight (data not shown).

[0561] Example 11: Development of improved conjugation reaction conditions

[0562] 11.1mTG variants

[0563] Site-specific conjugation to position Q295 of the native mAb via commonly used mTG conjugation technology (see also: Fig. 9 ) is not very efficient and does not allow the generation of a homogeneous ADC with DAR = 2 (see Fig. 9 A).

[0564] Even when using alternative mTG variants such as from S. ladakhii, the results using prior art buffer systems were ineffective (see Fig. 9 B).

[0565] 11.2 Buffer composition, excipients and pH

[0566] As mentioned, although mTG from S. ladakhii showed better efficiency of conjugation to position Q295 compared to mTG from S. mobara, the conjugation efficiency using the published reaction conditions was still not optimal. Therefore, a high throughput (HT) assay was developed that enables efficient screening of improved reaction conditions with respect to, for example, buffer composition, excipients, pH, etc. This assay ( Fig.10 ) comprises a first conjugation step, in which an mTG-compatible substrate comprising biotin is conjugated using mTG under different reaction conditions. Afterwards, the efficacy of the conjugation is determined via a homogeneous time-resolved fluorescence (HTRF) assay that allows the assay to be performed at high throughput.

[0567] More than 10,000 different reaction conditions were screened and reaction parameters that increased conjugation efficiency were identified. The procedure for the conjugation reaction and the evaluation of conjugation efficiency are described in 11.3.1 to 11.3.3 Figure 11 (A), (B) and (C) show the effects of pH (A), salt (B) and buffer concentration (C) on mTG conjugation efficiency.

[0568] It was found that a pH of about 5.9-8.7 was beneficial in the conjugation reaction (FIG. 11(A)). In addition, it was found that reducing the sodium chloride concentration improved the conjugation efficiency (FIG. 11(B)). With regard to the concentration of the buffer substance, it is exemplarily shown that for experiments using HEPES, it was found that a lower concentration of the buffer was beneficial.

[0569] The finding that the most suitable reaction buffers are those that contain no sodium chloride and contain low concentrations of buffering agents is surprising since physiological concentrations of salts are typically used in such reactions.

[0570] In terms of normalized conjugation efficiency, the type and pH of buffer unexpectedly also appeared to play a role, as shown in the following table (extracted from Fig.11A ):

[0571]

[0572]

[0573] As shown above, too acidic pH or too much sodium chloride leads to poor conjugation efficiency. In addition, the following types of buffers with a pH of 7 to 8.5 give high conjugation efficiency: HEPES, Tris-HCl, MOPS / Na HEPES, BICINE, BICINE / Tris, Imidazole and Tricine.

[0574] The conjugation of drug-linker 4M was then evaluated using an appropriate buffer selected from the high throughput screening results. The procedure for the conjugation reaction and the evaluation of the conjugation efficiency are described in 11.3.4 and 11.3.5The results confirmed highly improved conjugation efficiencies for all selected buffers compared to the previously used buffers (see Fig. 12A and 12B ). Two buffers that performed very well included a pH 7.5 buffer containing 24 mM HEPES (see Fig. 12A ) and a buffer containing 24 mM Tris-HCl at pH 8. These buffers provided much higher DARs compared to reactions in prior art buffers (25 mM Tris-HCl, 150 mM NaCl, pH 8.0).

[0575] With the improved reaction conditions, a reduced amount of mTG enzyme is necessary for efficient conjugation of the linker to the antibody compared to when the reaction is carried out in a prior art buffer system.

[0576] 11.3 Method Summary

[0577] The methods used for expression and purification of transglutaminase are described above in Example 2.

[0578] 11.3.1 Conjugation Reaction Methods for High-Throughput Bioconjugation Screening

[0579] 2 μL of 5X concentrated buffer plate (shown in Table 7) (Molecular Dimensions) was mixed with 6 μL of pre-diluted additives (shown in Table 8) (Jena Bioscience). Herceptin (Trastuzumab, Gentech / Roche) and N-(biotinyl)cadaverine (B002, Zedira) were pre-mixed and 1 μL of the mixture was added to the buffer diluent to produce a final concentration of 5 mg / mL Herceptin and 687.6 μM N-(biotinyl)cadaverine in the reaction mixture. Enzyme "MTG_1" (mTG enzyme according to SEQ ID NO: 45) was added at 1 μL to produce a final concentration of 12.5 U / ml. After incubation at 37°C for 16 hours, transglutaminase inhibitor C102 (Z edira) was added to a final concentration of 0.1 mM to stop the reaction. The transglutaminase reaction was transferred to a high throughput screening system and evaluated with HTRF readings.

[0580] Controls for screening: Prior to the addition of MTG_1, add inhibitor to one well / plate as a negative control to measure background during the HTRF read.

[0581] Table 7: Custom buffers used for screening reaction conditions. All buffers are 5X concentrated:

[0582]

[0583]

[0584]

[0585]

[0586] Table 8: Concentrations of pre-diluted additives in the screening:

[0587]

[0588]

[0589]

[0590] 11.3.2 HTRF Procedure

[0591] Streptavidin-d2 (SA-d2, 610SADLA, Cisbio) and pAb anti-human IgG-Tb cryptate (IgG-Tb, 61HFCTAA, Cisbio) were diluted to CSA-d2 = 1500 ng / mL and CIgG-Tb = 150 ng / mL final concentrations in 9 μL of terbium buffer (61DB10RDF) in HTRF 96-well low volume white plates (66PL96100). The transglutaminase conjugation reaction was diluted in PBS pH 7.4 (Gibco) to produce a 10 nM antibody stock solution, and 1 μL of the dilution was added to the HTRF reagent mixture. It was carefully pipetted to avoid bubbles in the wells. The plate was sealed with adhesive tape to avoid evaporation and incubated for 4 h at room temperature. Background fluorescence was detected by two blank measurements with PBS as sample volume. TR-FRET signal detection was performed using EnVision 2104 multiplate reader, using excitation filter UV2 (320 nm) and emission filters APC (665 nm) and Europium (615 nm). Data processing was performed using EnVision workstation version 1.12.

[0592] 11.3.3 Data Reduction

[0593] Step 1: The ratio for each well was calculated as shown below in the equation Equation 1. This calculation minimizes well-to-well variation because the background noise of the donor is normalized for each well.

[0594]

[0595] Step 2: The calculation given in Equation 2 below normalizes the measurements for each well so that plate-to-plate variation is minimized.

[0596]

[0597] 11.3.4 mTG-mediated drug-linker conjugation and titration experiments

[0598] Typically, Herceptin / Trastuzumab was conjugated to the linker-payload Gly3-VC-MMAE1 (G3-MMAE, Moradec LLC) using increasing concentrations of MTG_1 in different buffer systems and otherwise constant reaction conditions. DAR was determined using reverse phase chromatography as described in 11.3.5.

[0599] 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 to produce a final concentration of 5 mg / mL, followed by the addition of 1.46 μL of 10 mM Gly3-VC-MMAE to produce 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 pre-prepared reagent mixture. The conjugation reaction was carried out at 37°C for 16 h while shaking at 600 rpm. The reaction was stopped by adding 2.5 μL of the transglutaminase inhibitor B102 (Zedira).

[0600] 11.3.5 Drug-Antibody Ratio (DAR) Analysis

[0601] A PLRP-S was run at 1.0 mL / min and 65 °C. Reverse phase HPLC was performed on a (50 x 2.1 mm, 8 μm) column (A gilent). 5-7.5 μg of antibody or ADC was injected and chromatographed, running a 7.5 minute linear gradient from 30% to 45% solvent B (solvent A: Lichrosolv H2O + 0.1% TFA; solvent B: 100% AcN + 0.1% TFA) relative to solvent A, while monitoring the absorbance at a wavelength of 214 nm.

[0602] MTG sequence

[0603] Herein, the mTG enzymes with the following names have their respective amino acid sequences shown below:

[0604] MTG_zymogen (= SEQ ID NO 44).

[0605] MTG_1 (activated) (=SEQ ID NO: 45)

[0606] MTG_2 (activated) (=SEQ ID NO: 73)

[0607] MTG_3 (activated) (=SEQ ID NO: 48)

[0608] MTG_4 (activated) (=SEQ ID NO: 49)

[0609] The following mTG enzymes from Streptoverticillium mobara can also be used in the methods of the present invention:

[0610] MTG_5 (from Streptomyces mohara) (=SEQ ID NO:81):

[0611] DSDDRV TPPAEPLDRM PDPYRPSYGR AETVVNNYIR KWQQVYSHRD GRKQQMTEEQREWLSYGCVG VTWVNSGQYP TNRLAFASFD EDRFKNELKNGRPRSGETRA EFEGRVAKES FDEEKGFQRAREVASVMNRA LENAHDESAY LDNLKKELAN GNDALRNEDA RSPFYSALRN TPSFKERNGG NHDPSRMKAVIYSKHFWSGQ DRSSSADKRK YGDPDAFRPA PGTGLVDMSR DRNIPRSPTS PGEGFVNFDY GWFGAQTEADADKTVWTHGNhYHAPNGSLG AMHVYESKFR NWSEGYSDFD RGAYVITFIP KSWNTAPDKV KQGWP

[0612] MTG_6 (from Streptomyces mohara) (=SEQ ID NO:82):

[0613] DSDDRV TPPAEPLDRM PDPYRPSYGR AETVVNNYIR KWQQVYSHRD GRKQQMTEEQREWLSYGCVG VTWVNSGQYP TNRLAFASFD EDRFKNELKNGRPRSGETRA EFEGRVAKES FDEEKGFQRAREVASVMNRA LENAHDESAY LDNLKKELAN GNDALRNEDA RSPFYSALRN TPSFKERNGG NHDPSRMKAVIYSKHFWSGQ DRSSSADKRK YGDPDAFRPA PGTGLVDMSR DRNIPRSPTS PGEGFVNFDY GWFGAQTEADADKTVWTHGNhYHAPNGSLG AMHVYESKFR NWSEGYSDFD RGAYVITFIP KSWNTAPDKV KQGWPLE

[0614] MTG_7 (from Streptomyces mobaraensis) (= SEQ ID NO:83):

[0615] FRAPDSDDRV TPPAEPLDRM PDPYRPSYGR AETVVNNYIR KWQQVYSHRD GRKQQMTEEQREWLSYGCVG VTWVNSGQYP TNRLAFASFD EDRFKNELKN GRPRSGETRA EFEGRVAKES FDEEKGFQRAREVASVMNRA LENAHDESAY LDNLKKELAN GNDALRNEDA RSPFYSALRN TPSFKERNGG NHDPSRMKAVIYSKHFWSGQ DRSSSADKRK YGDPDAFRPA PGTGLVDMSRDRNIPRSPTS PGEGFVNFDY GWFGAQTEADADKTVWTHGNhYHAPNGSLGAMHVYESKFR NWSEGYSDFD RGAYVITFIP KSWNTAPDKV KQGWP

[0616] MTG_8 (from Streptomyces mobaraensis) (= SEQ ID NO:84):

[0617] FRAPDSDDRV TPPAEPLDRM PDPYRPSYGR AETVVNNYIR KWQQVYSHRD GRKQQMTEEQREWLSYGCVG VTWVNSGQYP TNRLAFASFD EDRFKNELKN GRPRSGETRA EFEGRVAKES FDEEKGFQRAREVASVMNRA LENAHDESAY LDNLKKELAN GNDALRNEDA RSPFYSALRN TPSFKERNGG NHDPSRMKAVIYSKHFWSGQ DRSSSADKRK YGDPDAFRPA PGTGLVDMSRDRNIPRSPTS PGEGFVNFDY GWFGAQTEADADKTVWTHGNhYHAPNGSLGAMHVYESKFR NWSEGYSDFD RGAYVITFIP KSWNTAPDKV KQGWPLE

[0618] Example 12: mTG recognition tag

[0619] In order to achieve even higher DARs with the mTG conjugation method described above (i.e., two drugs were previously conjugated to the respective positions Q295 (EU numbering) of the two heavy chains of the mAb), certain peptide motifs were genetically fused to the mAb of interest to enable further conjugation at these motifs. Some examples of such peptide tags have been described in the prior art. However, unexpectedly, new and functional additional peptide sequences that can be used in a similar manner were identified (e.g., GGTLQSPP and TLQS). Conjugation of antibodies fused to such peptide motifs using mTG from ladaka num resulted in ADCs with a DAR of about 4 (see Figure 17).

[0620] 12.1 Identification and evaluation of MTG identification tags

[0621] Conjugation of mAb_Tag 1 antibody

[0622] The antibody containing the amino acid sequence "TLQS" within the light chain of the antibody was recombinantly expressed using a pTT5-based expression vector and HEK293 expression system (GibcoTM, Thermo Fisher Scientific Inc.), followed by purification using protein A chromatography. The antibody was conjugated to the drug-linker 4M using microbial transglutaminase (T001, Zedira Gmb H).

[0623] 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. ADCs were separated from G3-MMAE 4 M and possible high molecular weight species (HMWS) via size exclusion chromatography (SEC). SEC was performed using a Superdex 200increase 10 / 300 column with PBS, pH 7.0 as a running buffer. Fractionated samples containing ADC material were combined and concentrated to >1 mg / ml using an Amicon Ultra 15 50K centrifuge (Millipore).

[0624] Analysis of the ADC using hydrophobic interaction chromatography (HIC) and mass spectrometry (MS) revealed a drug-antibody ratio (DAR) of 1.7 (see Fig.13 ).

[0625] The conjugated and unconjugated antibodies were digested via chymotrypsin digestion using Smart Digest kit (Thermo Fisher Scientific), and subsequently analyzed by MALDI-MS using Bruker Ultraflex MALDI TOF / TOF mass spectrometer and α-cyano-4 hydroxyl-cinnamic acid (cinamic acid) matrix. The comparison of the detected mass reveals a peak (see Figure 14) that exists for mAb but does not exist for ADC at 877.368Da. The theoretical mass (876.968Da) of this mass-matching light chain peptide QSGVPSRF. The mass of the comparative detection reveals a peak (see Figure 14) at 2154.126Da that exists for ADC but does not exist for mAb. This mass closely matches the theoretical mass (2154.968Da) of the light chain peptide QSGVPSRF conjugated with a Gly3-VC-MMAE peptide. These results confirm that Gly3-VC-MMAE joint-payload can be successfully conjugated to the peptide QSGVPSRF of antibody light chain.

[0626] Conjugation of mAb Tag 2, mAb Tag 3 and mAb Tag 4 antibodies

[0627] The peptide identified above is located in the light chain of the antibody as a part of the sequence YTSTLQSGVPS. Its core sequence TLQSG is fused to the sequence of the HER2-binding antibody derived from trastuzumab, replacing the lysine 447 (mAb_tag 2) of the heavy chain. The antibody is recombinantly expressed using a pTT5-based plasmid using the Expi293 expression system (GibcoTM), and purified using protein A and size exclusion chromatography. Mass spectrometry reveals the partial truncation of the C-terminal QSG peptide. The same procedure is used to produce other antibodies with the sequence GGTLQSG (mAb_tag 3) and LC-GGTLQSPP (mAb_tag 4) fused to the C-terminal of the light chain. Mass spectrometry reveals the truncation of the peptide tag that is not present for mAb_tag 4.

[0628] The antibody was similarly expressed as described above using mTGase from Streptoverticillus mobara for antibodies containing the "TLQS" peptide. mAb_tag2, mAb_tag3, and mAb_tag4 Conjugation and purification. The drug-antibody ratio (DAR) was evaluated using HIC and MS and revealed that the DAR values ​​for mAb_tag2,_tag3 and_tag4 were 1.9, 1.7 and 1.9, respectively (see Figure 15).

[0629] 12.2 Conjugation of mAbs equipped with recognition tags (mAb_Tag 5 and mA b7-M) using native mAb conjugation technology

[0630] Antibodies containing a glutamine-containing recognition tag were conjugated using native mAb conjugation via mTG from Streptoverticillus ladakhii in combination with reaction conditions determined to be suitable for simultaneous conjugation of four drug-linkers / antibodies.

[0631] The tag sequence GGTLQSPP was fused to the C-terminal sequence of the light chain of CD20-specific mAbs derived from rituximab, mAb_tag 5 and ceacam-5 specific mAb 7-M. Antibodies were recombinantly expressed using the Expi293 expression system (Gibco™) using a pTT5-based plasmid and purified using protein A and size exclusion chromatography. Drug-linker 4M or 1M was conjugated to the antibody, followed by SEC purification as described above and DAR determination using RP-HPLC.

[0632] For this purpose, HiTrap desalting columns were used in combination with The buffer of mAb was exchanged to 24mM HEPES, pH 7.0 by a liquid chromatography (LC) system (Cytiva). The conjugation mixture was set as follows: 5mg / ml mAb, 5 molar equivalents of drug-linker 4M or 1M / conjugation site, 20U / ml MTG_1, 4% DMSO, 24mM HEPES, pH 7.0. The reaction was carried out at 37°C for 18h. ADCs were separated from drug-linker and mTG via size exclusion chromatography (SEC). Before SEC purification, the NaCl concentration of the sample was adjusted to 100mM using a 5M NaCl stock solution. SEC was implemented using a HiLoad Superdex 200 10 / 30increase column with 10mM histidine, 100mM NaCl, 3% trehalose, pH 7.0 as a running buffer. The fractionated samples containing ADC material were combined and concentrated to >1mg / ml using an Amicon Ultra 15 50K centrifuge (Millipore). Drug-to-antibody ratio and purity were assessed using reverse phase (RP) chromatography.

[0633] Efficient conjugation to both the light and heavy chains was observed in all cases studied, resulting in a drug-to-antibody ratio of 3.9 (see Figure 17), confirming that despite the naturally occurring glycan at N297, the drug-linker was successfully conjugated to Q295 in the heavy chain, and an additional drug-linker was attached to the LC-fused glutamine-tag GGTLQSPP.

[0634] Antibody sequences used in conjugation experiments:

[0635] >mAb_Tag2_LC (Trastuzumab)

[0636] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:66)

[0637] >mAb_Tag 2_HC(Trastuzumab - HC - Tag)

[0638] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEAYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG TLQSG( SEQ ID NO:67)

[0639] >mAb_Tag 3_LC(Trastuzumab - LC - Tag 1)

[0640] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC QTG ( SEQ ID NO:68)

[0641] >mAb_tag4_LC(Trastuzumab-LC-tag2)

[0642] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC QTQ PP (SEQ ID NO:69)

[0643] >mAb_tag3_and_4_HC(Trastuzumab HC)

[0644] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDRTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEAYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR E E M TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:70)

[0645] >mAb_tag5_HC(~Rituximab HC = SEQ ID NO:40)

[0646] QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:40)

[0647] >mAb_5_LC (Rituximab-LC-Tag)

[0648] QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC GGTLQSPP ( SEQ ID NO:71)

[0649] >mAb7-M_HC (ceacam-HC)

[0650] EVQLQESGPGLVKPSQTLSLTCTVSDGSVSRGGYYLTWIRQHPGKGLEWIGYIYYSGSTYFNPSLRSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARGIAVAPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:72)

[0651] >mAb7-M_LC(ceacam LC label = SEQ ID NO:36)

[0652] EIVLTQSPATLSVSPGERATLSCRTSQSVRSNLAWYQQKPGQAPRLLIYAASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYTNWPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC QTQ PP( SEQ ID NO:36)

Claims

1. A method for producing an antibody-linker-conjugate comprising the following steps: (1) providing an antibody, which optionally comprises an amino acid sequence selected from the group consisting of GGTLQSPP, TLQSG, TLQSPP, GGT LQSG and TLQSA and more preferably comprises the amino acid sequence TLQSPP or GGTLQS PP (most preferably comprises the amino acid sequence GGTLQSPP) in at least one of its light chain constant regions (CL) and preferably in both of its light chain constant regions (CL) and / or in at least one of its heavy chain constant regions (CH) and preferably in both of its heavy chain constant regions (CH); (2) Mix together 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 and 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, and wherein said linker is preferably a drug-linker, wherein said linker is covalently attached to a drug; (3) separating the antibody-linker-conjugate produced in step (2) from unreacted linker and from the transglutaminase, preferably by subjecting the mixture from step (2) to size exclusion chromatography.

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

5.

3. The method of claim 1 or 2, wherein the antibody comprises a glutamine at position 295 of the heavy chain of the antibody, whereby the antibody is a monoclonal antibody and wherein EU numbering is used to define the position of the glutamine at position 295, and / or wherein 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 wherein the reaction mixture comprises less than 10 mM NaCl and wherein the reaction buffer comprises one or more of the following buffers adjusted to a pH of 7 to pH 8.8: BICINE, BICINE / Tris, Tris-HCl, HEPES, and / or Tricine.

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

5. The method of claim 4, wherein in step (2) the mixture comprises the following drug-linker:

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

7. The method of any one of claims 1 to 5, wherein the antibody is a monoclonal antibody and preferably an anti-CEACAM5 antibody and / or wherein the drug is a growth inhibitor as defined in (a), (b) or (c), wherein (a), (b) or (c) is as specified below: (a) The growth inhibitor is a cytotoxic drug or a radioactive moiety. ) The growth inhibitor is selected from the group consisting of a chemotherapeutic agent, an enzyme, an antibiotic, a toxin such as a small molecule toxin or an enzyme-active toxin, a toxoid, a vinca alkaloid, a taxane, a maytansine or a maytansine analog, tomamycin or a pyrrolobenzodiazepine derivatives, Nostoc cyclic peptide derivatives, leptomycin derivatives, auristatin or dolastatin analogs, prodrugs, topoisomerase I inhibitors, topoisomerase II inhibitors, DNA alkylating agents, anti-tubulin agents, CC-1065 and CC-1065 analogs; (c) The growth inhibitor is exatecan.

8. An antibody-linker conjugate producible according to the method of any one of claims 1 to 7, wherein the linker is preferably a linker or a drug linker as defined below: wherein n is the number of [(linker)-(growth inhibitory agent)] moieties covalently attached to the antibody; or wherein n is the number of [(linker)—(exetatecan)] moieties covalently attached to the antibody; and wherein n is preferably 1 to 10 and more preferably 4.

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

10. The method of claim 4, wherein in step (2), the mixture comprises the following drug-linker:

11. The method of any of the preceding claims, wherein the glutamine of the amino acid sequence of the antibody selected from the group consisting of GGTLQSPP, TLQSG, TLQSPP, GGTLQSG and TLQSA reacts in step (2) of the method in the presence of the microbial transglutaminase to form a covalent bond with the H2N-part of the linker.

12. The method of any one of the preceding claims, wherein the antibody is a monoclonal antibody.

13. The method of any one of the preceding claims, wherein the microbial transglutaminase is a microbial transglutaminase from Streptoverticillium ladakhii or from Streptoverticillium mobara.

14. The method according to any of the preceding claims, wherein the reaction mixture obtained in step (2) is incubated at a temperature of 20°C to 38°C until the antibody-linker-conjugate is formed and can be isolated in method step (3).

15. The method of any one of the preceding claims, wherein the reaction buffer comprises one or more of the following buffers adjusted to a pH of 7 to pH 8.8: BICINE, BICINE / Tris, Tris-HCl, HEPES, and / or Tricine.

16. The method of any one of the preceding claims, wherein the reaction buffer comprises Tris-HCl adjusted to a pH of 7.7 to 8.5; or comprising BICINE and / or BICINE / Tris adjusted to a pH of 7.8 to 8.8; or Contains Tricine adjusted to a pH of 7.8 to 8.

0.

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

18. The method of any one of the preceding claims, wherein the reaction buffer is a reaction buffer comprising 0% to 10% DMSO, 5 mM to 60 mM HEPES and comprising less than 100 mM NaCl (preferably no NaCl) and having a pH of 6 to 7.5; The antibody further comprises GGTLQSPP, TLQSG, TLQSPP, GGTLQSG and TLQSA sequences in both light chain constant regions (CL) and / or in both heavy chain constant regions (CH); and The linker is a linker having the following formula: wherein R is the remainder of the linker and may optionally further comprise a drug.

19. The method of any one of the preceding claims, 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, most preferably wherein the reaction buffer does not comprise NaCl.

20. The method according to any one of the preceding claims, wherein the total amount of all buffering agents contained in the reaction buffer is below 160 mM and preferably below 155 mM.

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

22. The method of any one of the preceding claims, wherein the total amount of all buffers contained in the reaction buffer is less than 155 mM; and wherein the reaction buffer comprises less than 10 mM NaCl and wherein the total amount of the microbial transglutaminase is 2.5 U / ml to 12 U / ml.

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

1.

24. The method of any one of the preceding claims, wherein the antibody is trastuzumab, rituximab, or labetuzumab; or wherein the antibody comprises 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; or wherein the antibody comprises 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; and wherein the antibody is an IgG monoclonal antibody.

25. The method of any one of the preceding claims, wherein the reaction buffer comprises at least one buffer selected from imidazole, HEPES, BICINE, Tris-HCl, MOPS / Bis-Tris propane and MOPS / sodium HEPES, and wherein the buffer concentration is selected in the range of 10-160 mM, preferably wherein the buffer is HEPES at a concentration of 10 mM to 30 mM, more preferably at a concentration of 24 mM HEPES.

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

27. The method of any of the preceding claims, wherein the reaction buffer comprises about 0.5% to 8% DMSO and preferably about 4% to 7% DMSO and most preferably at most 5% DMSO.

28. The method of any one of the preceding claims, 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 contained in the mixture in step (2) of the method at a concentration of 5 U / mL to 25 U / mL and preferably at a concentration of 10 U / mL to 20 U / mL.

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

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

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

32. The method of any of the preceding claims, wherein the antibody comprises the amino acid sequence TLQSG, GGTLQSPP or GGTLQSG at the C-terminus of its light chain constant region (CL) and 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. The method of any one of the preceding claims, wherein the antibody-linker-conjugate produced in step (2) has a drug to antibody ratio (DAR) of 3 to 4.5, preferably 3.8 to 4.

1.

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

35. The method of claim 34, wherein the antibody comprises 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; and wherein the antibody is an IgG monoclonal antibody.

36. The method of claim 34, wherein the mTG enzyme is from Streptoverticillus ladakhii; wherein the antibody is a monoclonal antibody comprising glutamine at position 295 and wherein the reaction buffer comprises HEPES in an amount of about 20 mM to 30 mM; wherein the reaction buffer does not comprise NaCl and wherein the pH of the reaction buffer is about pH 7 to pH 7.5; and wherein EU numbering is used to define the position of the glutamine at position 295.

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

38. The method of any of the preceding claims, wherein the linker has structure (A) or (B) shown below, wherein R is a therapeutic agent or another payload:

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

40. The method of any one of the preceding claims, wherein a molar excess of drug-linker / conjugation site and preferably at least 4 molar equivalents of drug-linker / conjugation site are used in step (2).

41. An antibody-drug-conjugate or antibody-linker conjugate producible by the method of any preceding claim.

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

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 a microbial transglutaminase and wherein the linker is preferably further covalently attached to a payload.

44. The kit of parts according to claim 42 or 43, further comprising an instruction manual with instructions on 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, GGT LQSG and TLQSA at the C-terminus of the heavy chain of the antibody and / or an amino acid sequence selected from the group consisting of G GTLQSPP, GGTLQSG and TLQSA at the C-terminus of the light chain of the antibody.

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