Conjugates of modified antibody formats

Abstract

The present invention provides conjugates wherein a cargo is conjugated to an antibody or an antibody fragment via glycan structures at artificial N-glycosylation sites within peptide linkers. Furthermore, the present invention provides medical uses of said conjugates and methods for producing said conjugates.

Description

[0001] HOF FMAN N E ITLE

[0002] October 14, 2025 065966 K

[0003] Pentixapharm AG

[0004] BismarckstraBe 13, 97080 Wurzburg

[0005] “Conjugates of modified antibody formats”

[0006] FIELD OF THE INVENTION

[0007] The present invention provides conjugates wherein a cargo is conjugated to an antibody or antibody fragment via glycan structures at artificial N-glycosylation sites within peptide linkers. Furthermore, the present invention provides medical uses of said conjugates and methods for producing said conjugates.

[0008] BACKGROUND OF THE INVENTION

[0009] Antibody-conjugates (ACs) are a well-known and effective way to target cargo compounds to specific sites or cell types within the human body. Such antibody conjugates may be radioimmunoconjugates, antibody-drug conjugates as well as other antibody-conjugates which may be used for therapeutic or diagnostic purposes by specifically transporting an active agent or an imaging agent to cells associated with the disease. For example, for the treatment of cancer cytotoxic agents are coupled to antibodies directed against cancer-associated antigens.

[0010] To form the conjugate, the cargo compound may be coupled to the antibody molecule via a random process, using the available reactive groups of the side chains and the N and C termini of the antibody. For example, cargo compounds may be coupled to the amine group of the lysines of the antibody’s polypeptide chain. However, this process provides a mixture of different ACs having different ratios of cargo compounds to antibody molecules. Furthermore, coupled cargo compounds may interfere with the antibody:antigen interaction. This in turn leads to varying therapeutic efficacies of the ACs. In addition, this process yields rather large conjugates which may not have access to every target site or epitope on the antigens.

[0011] Therefore, there is a need in the art to provide smaller conjugates with an improved composition of the antibody-cargo mixture and efficient delivery of the cargo compound to the target sites, e.g. tumor cells. SUMMARY OF THE INVENTION

[0012] The present invention solves this problem by providing conjugates wherein a cargo compound is coupled at defined sites to antibodies or small antibody fragments with a more homogenous loading of cargo compounds to the antibody molecules. This is achieved by coupling the cargo compound to the antibody via glycan structures attached to artificial N-glycosylation sites within peptide linkers present in the antibody or antibody fragment.

[0013] According to a first aspect, the present invention provides a conjugate comprising

[0014] (i) an antibody module comprising

[0015] (a) at least one pair of heavy chain variable domain and light chain variable domain capable of specifically binding to a target antigen; and

[0016] (b) a peptide linker comprising an N-glycosylation site;

[0017] (ii) a glycan structure attached to the N-glycosylation site of the peptide linker; and

[0018] (iii) a cargo compound coupled to the glycan structure.

[0019] According to a second aspect, the present invention provides a pharmaceutical composition comprising the conjugate according to the first aspect.

[0020] According to a third aspect, the present invention provides the conjugate according to the first aspect for use in the diagnosis, treatment, prevention, prognosis and / or monitoring of a disease or condition.

[0021] According to a fourth aspect, the present invention provides a method for producing the conjugate according to the first aspect, comprising the steps of

[0022] (a) providing a composition of glycosylated antibody modules, wherein the glycosylated antibody module consists of an antibody module as defined in the first aspect, and glycan structures attached to the N-glycosylation sites of the peptide linkers of the antibody module;

[0023] (b) contacting the composition of glycosylated antibody modules with a glycoside hydrolase under conditions where the glycoside hydrolase cleaves the glycan structures so that unitary non-reducing ends are present at the glycan structures;

[0024] (c) contacting the composition of glycosylated antibody modules with a glycosyltransferase which is capable of transferring a modified monosaccharide to the unitary non-reducing ends of the glycan structures, and a corresponding modified nucleotide monosaccharide, under conditions where the modified monosaccharide is coupled to the unitary non-reducing ends of the glycan structures;

[0025] (d) reacting the composition of glycosylated antibody modules with a cargo compound under conditions where the cargo compound is coupled to the modified monosaccharide attached to the glycan structures, providing a conjugate according to the first aspect.

[0026] Other objects, features, advantages and aspects of the present invention will become apparent to those skilled in the art from the following description and appended claims. It should be understood, however, that the following description, appended claims, and specific examples, which indicate preferred embodiments of the application, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following.

[0027] DEFINITIONS

[0028] As used herein, the following expressions are generally intended to preferably have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise.

[0029] The expression "comprise", as used herein, besides its literal meaning also includes and specifically refers to the expressions "consist essentially of" and "consist of". Thus, the expression "comprise" refers to embodiments wherein the subject-matter which "comprises" specifically listed elements does not comprise further elements as well as embodiments wherein the subject-matter which "comprises" specifically listed elements may and / or indeed does encompass further elements. Likewise, the expression "have" is to be understood as the expression "comprise", also including and specifically referring to the expressions "consist essentially of' and "consist of". The term "consist essentially of", where possible, in particular refers to embodiments wherein the subject-matter comprises 20% or less, in particular 15% or less, 10% or less or especially 5% or less further elements in addition to the specifically listed elements of which the subject-matter consists essentially of. The indefinite article “a” or “an” as used herein is not limited to one instance of the item but also refers to multiple instances. For example, “an N-glycosylation site” refers to one or more N-glycosylation sites.

[0030] The term "antibody" in particular refers to a protein comprising at least two heavy chains and two light chains connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable domain (VH) and a heavy chain constant region (CH). Each light chain is comprised of a light chain variable domain (VL) and a light chain constant region (CL). The heavy chainconstant region comprises three or - in the case of antibodies of the IgM- or IgE-type - four heavy chain constant domains (CH1 , CH2, CH3 and CH4) wherein the first constant domain CH1 is adjacent to the variable region and may be connected to the second constant domain CH2 by a hinge region. The light chain constant region consists only of one light chain constant domain. The variable domains can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR), wherein each variable domain comprises three CDRs and four FRs. The amino acid residues of the CDRs are in particular determined based on the CDR localization according to the IMGT system.

[0031] The terms “light chain variable domain”, “light chain constant domain”, “heavy chain variable domain” and “heavy chain constant domain” and their abbreviations VL, CL, VH, CH, CH1 , CH2, CH3 and CH4, as used herein, refer to immunoglobin domains derived from antibodies, in particular derived from human IgG 1 antibodies. For example, “light chain variable domain” refers to an antibody light chain variable domain, and “heavy chain variable domain” refers to an antibody heavy chain variable domain. Light chain variable domains and heavy chain variable domains are collectively referred to herein as “antibody variable domains”. Light chain constant domains and heavy chain constant domains are collectively referred to herein as “antibody constant domains”.

[0032] The variable domains of the heavy and light chains contain a binding region that interacts with an antigen. The heavy chain constant region may be of any type such as y-, 5-, a , p- or e-type heavy chains. Preferably, the heavy chain of the antibody is a y-chain. Furthermore, the light chain constant region may also be of any type such as K- or A-type light chains. Preferably, the light chain of the antibody is a K-chain. The terms "y- (5-, a-, p- or £-) type heavy chain" and "K- (A-) type light chain" refer to antibody heavy chains or antibody light chains, respectively, which have constant region amino acid sequences derived from naturally occurring heavy or light chain constant region amino acid sequences, especially human heavy or light chain constant region amino acid sequences. In particular, the amino acid sequence of the constant domains of a y-type (especially y1-type) heavy chain is at least 95%, especially at least 98%, identical to the amino acid sequence of the constant domains of a human y (especially one of the allotypes of the human y1) antibody heavy chain. Furthermore, the amino acid sequence of the constant domain of a K-type light chain is in particular at least 95%, especially at least 98%, identical to the amino acid sequence of the constant domain of one of the allotypes of the human K antibody light chain. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The antibody can be, e.g., a humanized, human or chimeric antibody.

[0033] The antigen-binding portion of an antibody usually refers to fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments of an antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments, each of which binds to the same antigen, linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CH1 domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; and a dAb fragment, which consists of a VH domain.

[0034] The "Fab part" of an antibody in particular refers to a part of the antibody comprising the heavy and light chain variable domains (VH and VL) and the first domains of the heavy and light chain constant regions (CH1 and CL). In cases where the antibody does not comprise all of these regions, then the term "Fab part" only refers to those of the regions VH, VL, CH1 and CL which are present in the antibody. Preferably, "Fab part" refers to that part of an antibody corresponding to the fragment obtained by digesting a natural antibody with papain which contains the antigen binding activity of the antibody. In particular, the Fab part of an antibody encompasses the antigen binding site or antigen binding ability thereof. Preferably, the Fab part comprises at least the VH region of the antibody.

[0035] The "Fc part" of an antibody in particular refers to a part of the antibody comprising the heavy chain constant regions 2, 3 and - where applicable - 4 (CH2, CH3 and CH4). In particular, the Fc part comprises two of each of these regions. In cases where the antibody does not comprise all of these regions, then the term "Fc part" only refers to those of the regions CH2, CH3 and CH4 which are present in the antibody. Preferably, the Fc part comprises at least the CH2 region of the antibody. Preferably, "Fc part" refers to that part of an antibody corresponding to the fragment obtained by digesting a natural antibody with papain which does not contain the antigen binding activity of the antibody. In particular, the Fc part of an antibody is capable of binding to the Fc receptor and thus, e.g. comprises an Fc receptor binding site or an Fc receptor binding ability.

[0036] The term "antibody", as used herein, refers in certain embodiments to a population of antibodies of the same kind. In particular, all antibodies of the population of the antibody exhibit the features used for defining the antibody. In certain embodiments, all antibodies in the population of the antibody have the same amino acid sequence. Reference to a specific kind of antibody, such as a specific antibody fragment, in particular refers to a population of this kind of antibody.

[0037] The term "antibody" as used herein includes the full-length antibody as well as fragments and derivatives of said antibody. A "fragment or derivative" of an antibody in particular is a protein or glycoprotein which is derived from said antibody and is capable of binding to the same antigen, in particular to the same epitope as the antibody. Thus, a fragment or derivative of an antibody herein generally refers to a functional fragment or derivative. In particularly preferred embodiments, the fragment or derivative of an antibody comprises a heavy chain variable domain. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody or derivatives thereof. Examples of fragments of an antibody include (i) Fab fragments, monovalent fragments consisting of the variable region and the first constant domain of each the heavy and the light chain; (ii) F(ab)2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting of the variable region and the first constant domain CH1 of the heavy chain; (iv) Fv fragments consisting of the heavy chain and light chain variable region of a single arm of an antibody; (v) scFv fragments, Fv fragments consisting of a single polypeptide chain; (vi) (FV)2fragments consisting of two Fv fragments covalently linked together; (vii) VHH or nanobodies consisting of one heavy chain variable domain; and (viii) multibodies consisting of a heavy chain variable region and a light chain variable region covalently linked together in such a manner that association of the heavy chain and light chain variable regions can only occur intermolecular but not intramolecular. Derivatives of an antibody in particular include antibodies which bind to the same antigen as the parent antibody, but which have a different amino acid sequence than the parent antibody from which it is derived. These antibody fragments and derivatives are obtained using conventional techniques known to those with skill in the art.

[0038] A target amino acid sequence is "derived" from or "corresponds" to a reference amino acid sequence if the target amino acid sequence shares a homology or identity over its entire length with a corresponding part of the reference amino acid sequence of at least 75%, more preferably at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98% or at least 99%. The "corresponding part" means that, for example, framework region 1 of a heavy chain variable domain (FRH1) of a target antibody corresponds to framework region 1 of the heavy chain variable domain of the reference antibody. In particular embodiments, a target amino acid sequence which is "derived" from or "corresponds" to a reference amino acid sequence is 100% homologous, or in particular 100% identical, over its entire length with a corresponding part of the reference amino acid sequence. A "homology" or "identity" of an amino acid sequence or nucleotide sequence is preferably determined according to the invention over the entire length of the reference sequence or over the entire length of the corresponding part of the reference sequence which corresponds to the sequence which homology or identity is defined. An antibody derived from a parent antibody which is defined by one or more amino acid sequences, such as specific CDR sequences or specific variable region sequences, in particular is an antibody having amino acid sequences, such as CDR sequences or variable region sequences, which are at least 75%, preferably at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98% or at least 99% homologous or identical, especially identical, to the respective amino acid sequences of the parent antibody. In certain embodiments, the antibody derived from (i.e. derivative of) a parent antibody comprises the same CDR sequences as the parent antibody, but differs in the remaining sequences of the variable regions.

[0039] The term "antibody" as used herein also refers to multivalent and multispecific antibodies, i.e. antibody constructs which have more than two binding sites each binding to the same epitope and antibody constructs which have one or more binding sites binding to a first epitope and one or more binding sites binding to a second epitope, and optionally even further binding sites binding to further epitopes.

[0040] "Specific binding" preferably means that an agent such as an antibody binds stronger to a target such as an epitope for which it is specific compared to the binding to another target. An agent binds stronger to a first target compared to a second target if it binds to the first target with a dissociation constant (Kd) which is lower than the dissociation constant for the second target. Preferably the dissociation constant for the target to which the agent binds specifically is more than 10-fold, 30-fold, 100-fold or more than 500-fold lower than the dissociation constant for the target to which the agent does not bind specifically. Furthermore, the term "specific binding" in particular indicates a binding affinity between the binding partners with an affinity constant Kaof at least 105M’1, preferably at least 106M’1, more preferably at least 107M’1, for example at least 108M’1. An antibody specific for a certain antigen in particular refers to an antibody which is capable of binding to said antigen with an affinity having a Kaof at least 105M’1, preferably at least 106M’1, more preferably at least 107M’1.

[0041] The term "epitope" as used herein refers to the amino acid residues and glycan structures on the antigen of an antibody which are either directly contacted by the amino acids of the antibody, in particular the amino acids of the CDRs of the antibody, or which are in direct vicinity thereof and influence the binding of the antibody to its antigen.

[0042] The term “N-glycosylation site” as used herein refers to an amino acid sequence within a polypeptide chain of a peptide, polypeptide or protein which comprises an asparagine residue to which a glycan structure can be coupled enzymatically. An N-glycosylation site generally has the amino acid sequence of Asn-Xaa-Ser / Thr, wherein Asn is the amino acid asparagine, Xaa is any amino acid except for proline, and Ser / Thr is either serine or threonine.

[0043] The term "N-glycosylation" refers to all glycans attached to asparagine residues of the polypeptide chain of a peptide, polypeptide or protein. These asparagine residues are part of N-glycosylation sites. Likewise, "N-glycans" are glycans attached to asparagine residues of a polypeptide chain. The terms "glycan", "glycan structure", "carbohydrate", "carbohydrate chain" and "carbohydrate structure" are generally used synonymously herein. N-glycans generally have a common core structure consisting of two N-acetylglucosamine (GIcNAc) residues and three mannose residues, having the structure Mana1 ,6-(Mana1 ,3-)Manpi ,4- GlcNAcpi ,4-GlcNAcpi-Asn with Asn being the asparagine residue of the polypeptide chain.

[0044] In a "conjugate" two or more compounds are linked together. In certain embodiments, at least some of the properties from each compound are retained in the conjugate. Linking may be achieved by a covalent or non-covalent bond. Preferably, the compounds of the conjugate are linked via a covalent bond. The different compounds of a conjugate may be directly bound to each other via one or more covalent bonds between atoms of the compounds. Alternatively, the compounds may be bound to each other via a chemical moiety such as a linker molecule wherein the linker is covalently attached to atoms of the compounds. If the conjugate is composed of more than two compounds, then these compounds may, for example, be linked in a chain conformation, one compound attached to the next compound, or several compounds each may be attached to one central compound.

[0045] The term "patient" means according to the invention a human being, a nonhuman primate or another animal, in particular a mammal such as a cow, horse, pig, sheep, goat, dog, cat or a rodent such as a mouse and rat. In a particularly preferred embodiment, the patient is a human being.

[0046] The term "cancer" according to the invention in particular comprises leukemias, seminomas, melanomas, teratomas, lymphomas, neuroblastomas, gliomas, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, cancer of the brain, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, intestine cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophagus cancer, colorectal cancer, pancreas cancer, ear, nose and throat (ENT) cancer, bladder cancer, breast cancer, prostate cancer, cancer of the uterus, ovarian cancer and lung cancer and the metastases thereof. The term cancer according to the invention also comprises cancer metastases. The term cancer further also refers to and / or includes cancer stem cells, especially the cancer stem cells of the specific types of cancer listed above.

[0047] By "tumor" is meant a group of cells or tissue that is formed by misregulated cellular proliferation. Tumors may show partial or complete lack of structural organization and functional coordination with the normal tissue, and usually form a distinct mass of tissue, which may be either benign or malignant.

[0048] By "metastasis" is meant the spread of cancer cells from its original site to another part of the body. The formation of metastasis is a very complex process and normally involves detachment of cancer cells from a primary tumor, entering the body circulation and settling down to grow within normal tissues elsewhere in the body. When tumor cells metastasize, the new tumor is called a secondary or metastatic tumor, and its cells normally resemble those in the original tumor. This means, for example, that, if breast cancer metastasizes to the lungs, the secondary tumor is made up of abnormal breast cells, not of abnormal lung cells. The tumor in the lung is then called metastatic breast cancer, not lung cancer.

[0049] The term "pharmaceutical composition" particularly refers to a composition suitable for administering to a human or animal, i.e., a composition containing components which are pharmaceutically acceptable. Preferably, a pharmaceutical composition comprises an active compound or a salt or prodrug thereof together with a carrier, diluent or pharmaceutical excipient such as buffer, preservative and tonicity modifier. DETAILED DESCRIPTION OF THE INVENTION

[0050] The different aspects and embodiments of the invention disclosed herein make important contributions to the art as is also explained in the following.

[0051] The conjugate according to the first aspect

[0052] According to a first aspect, the present invention provides a conjugate comprising

[0053] (i) an antibody module comprising

[0054] (a) at least one pair of heavy chain variable domain and light chain variable domain capable of specifically binding to a target antigen; and

[0055] (b) a peptide linker comprising an N-glycosylation site;

[0056] (ii) a glycan structure attached to the N-glycosylation site of the peptide linker; and

[0057] (iii) a cargo compound coupled to the glycan structure.

[0058] In certain embodiments, the conjugate consists of the antibody module, one or more glycan structures, and one or more cargo compounds coupled to said glycan structures. At least one of the glycan structures is attached to the N-glycosylation site of a peptide linker of the antibody module. In certain embodiments, all of the glycan structures of the conjugate are attached to N-glycosylation sites of peptide linkers of the antibody module. In embodiments where the antibody module comprises one or more CH2 heavy chain constant domains, the conjugate may comprise further glycan structures attached to the N-glycosylation site present in said CH2 domains. In certain embodiments, the conjugate does not comprise a whole antibody.

[0059] The antibody domains of the antibody module

[0060] The antibody module is capable of specifically binding to a target antigen. Specific binding to the target antigen is mediated by the at least one pair of heavy chain variable domain and light chain variable domain.

[0061] In embodiments wherein the antibody module comprises more than one pair of heavy chain variable domain and light chain variable domain, the different pairs may be capable of specifically binding to the same target antigen or to different target antigens. In preferred embodiments, all of the pairs are capable of specifically binding to the same target antigen. In certain embodiments, each pair of heavy chain variable domain and light chain variable domain present in the antibody module has the same set of CDRs. In certain embodiments, the antibody module consists of one or more polypeptide chains. In particular, the antibody module consists of one to four polypeptide chains. In specific embodiments, the antibody module consists of one or two polypeptide chains.

[0062] In certain embodiments, the antibody module comprises between one and six pairs of heavy chain variable domain and light chain variable domain, in particular between one and four pairs. In preferred embodiments, the antibody module comprises one or two pairs of heavy chain variable domain and light chain variable domain. In certain embodiments, the antibody module comprises exactly one pair of heavy chain variable domain and light chain variable domain. In other embodiments, the antibody module comprises exactly two pairs of heavy chain variable domain and light chain variable domain.

[0063] In certain embodiments, the antibody module comprises no more than four antibody constant domains. In preferred embodiments, the antibody module comprises either no antibody constant domains, two antibody constant domains, or four antibody constant domains. In certain embodiments, the antibody module comprises no antibody constant domains. In other embodiments, the antibody module comprises two antibody constant domains. In even other embodiments, the antibody module comprises four antibody constant domains.

[0064] In certain embodiments, the antibody module is an antibody fragment. In particular, the antibody module does not comprise a whole antibody.

[0065] In certain embodiments, the antibody module does not comprise any N-glycosylation sites apart from the N-glycosylation sites in the peptide linkers. In certain embodiments, all N- glycosylation sites of the antibody module are artificial N-glycosylation sites.

[0066] The peptide linkers of the antibody module

[0067] In certain embodiments, the peptide linker is present on the same polypeptide chain as the heavy chain variable domain. In certain embodiments, the peptide linker is present on the same polypeptide chain as the light chain variable domain. In certain embodiments, the peptide linker is present on the same polypeptide chain as the heavy chain variable domain and the light chain variable domain.

[0068] In certain embodiments, the peptide linker is directly adjacent to the heavy chain variable domain on the polypeptide chain. In certain embodiments, the peptide linker is directly adjacent to the light chain variable domain on the polypeptide chain. In certain embodiments, the peptide linker is directly adjacent to the heavy chain variable domain and the light chain variable domain on the polypeptide chain. In certain embodiments, the peptide linker is located between the heavy chain variable domain and the light chain variable domain on the polypeptide chain. In certain embodiments, the peptide linker directly links the heavy chain variable domain and the light chain variable domain. In certain embodiments, the peptide linker is located at the N terminus or at the C terminus of the polypeptide chain.

[0069] In certain embodiments, the antibody module comprises more than one peptide linker each comprising an N-glycosylation site. Each of the peptide linker in particular is located within the antibody module as disclosed above. Two peptide linkers, however, are not located directly adjacent to each other on the same polypeptide chain. In certain embodiments, the antibody module comprises between one and eight peptide linkers each comprising an N-glycosylation site. In certain embodiments, the antibody module comprises between two and six peptide linkers each comprising an N-glycosylation site. In certain embodiments, the antibody module comprises one peptide linker comprising an N-glycosylation site. In certain embodiments, the antibody module comprises two peptide linkers each comprising an N-glycosylation site. In certain embodiments, the antibody module comprises three peptide linkers each comprising an N-glycosylation site. In certain embodiments, the antibody module comprises four peptide linkers each comprising an N-glycosylation site. In certain embodiments, the antibody module comprises six peptide linkers each comprising an N-glycosylation site.

[0070] In certain embodiments, the antibody module further comprises one or more peptide linkers without an N-glycosylation site.

[0071] In certain embodiments, one or more of the peptide linkers comprising an N-glycosylation site comprise two or more N-glycosylation sites. Especially, one or two of the peptide linkers comprise two or more N-glycosylation sites. In other embodiments, all of the peptide linkers comprising an N-glycosylation site comprise two or more N-glycosylation sites. In particular, peptide linkers comprising two or more N-glycosylation sites comprise exactly two N- glycosylation sites.

[0072] The N-glycosylation sites in the peptide linkers have the amino acid sequence of Asn-Xaa- Ser / Thr, wherein Asn is the asparagine, Xaa is any amino acid except for proline, and Ser / Thr is either serine or threonine. In certain embodiments, Xaa is selected from the group consisting of glycine, serine, valine and alanine. In particular, Xaa may be serine. In certain embodiments, Ser / Thr is threonine. In certain embodiments, the N-glycosylation site comprises an additional amino acid directly N-terminal of the Asn-Xaa-Ser / Thr motif, wherein said additional amino acid is glycine, serine or leucine, in particular glycine. The N-glycosylation site thus may have an amino acid sequence selected from the group consisting of the sequences of table 1 , wherein Xaa is any amino acid except for proline.

[0073] Table 1 - Amino acid sequences of the glycosylation site

[0074] Different N-glycosylation sites on one peptide linker as well as on different peptide linkers may have the same or different amino acid sequences. In certain embodiments, the N-glycosylation sites present on the peptide linkers of the antibody module all have the amino acid sequence of Asn-Ser-Thr.

[0075] In certain embodiments, the peptide linker has a length of between 5 and 50 amino acids. In certain embodiments, the peptide linker has a length of between 5 and 40 amino acids. In certain embodiments, the peptide linker is a short linker having between 5 and 15 amino acids. In certain embodiments, the peptide linker is a long linker having between 16 and 50 amino acids.

[0076] The peptide linker in particular is an artificial peptide linker. The artificial peptide linker is a peptide linker which is not naturally present in the context of the at least one pair of heavy chain variable domain and light chain variable domain of the antibody module. The artificial peptide linker especially has an amino acid sequence which is not present in and not derived from natural antibodies. The peptide linkers in the antibody module may be flexible or rigid linkers or a mixture thereof. Flexible linkers do not have a defined three-dimensional structure and adopt any structure fitting for the antibody module. Rigid linkers have a preferred three- dimensional structure, such as an elongated structure. The antibody module preferably comprises flexible linkers. A flexible linker may for example be of the GS type. GS type linkers comprise one or more, in particular 1 to 4, of the amino acid motif Gly-Gly-Gly-Ser (G3S, SEQ ID NO: 19) or Gly-Gly-Gly-Gly-Ser (G4S, SEQ ID NO: 20) with Gly being glycine and Ser being serine. Further exemplary amino acid sequences of flexible linker are SEQ ID NOs: 21 to 26. A rigid linker may for example be of the PAPAP type or EAK type. PAPAP type linkers comprise one or more, in particular 1 to 4, of the amino acid motif Pro-Ala-Pro-Ala-Pro (SEQ ID NO: 27) with Pro being proline and Ala being alanine. EAK type linkers comprise one or more, in particular 1 to 8, of the amino acid motif Glu-Ala-Ala-Ala-Lys (SEQ ID NO: 28) with Glu being glutamic acid, Lys being lysine and Ala being alanine. Further exemplary amino acid sequences of rigid linker are SEQ ID NOs: 29 to 31.

[0077] The one or more N-glycosylation sites are introduced into the peptide linkers by adding the respective amino acid sequence to the linker sequence and / or by substituting one or more amino acids of the linker sequence with those of the N-glycosylation site. In certain embodiments, an N-glycosylation site is not located directly at the N or C terminus of a peptide linker. In particular, the peptide linker comprises at least one amino acid, especially at least two amino acids, between its N terminus and the N-glycosylation site and between its C terminus and the N-glycosylation site.

[0078] In certain embodiments, the peptide linker comprises a peptide tag. Peptide tags are in particular peptides which can be specifically recognized and / or bound by a ligand. Exemplary peptide tags include a His tag having the amino acid sequence of SEQ ID NO: 32, a Strep tag having the amino acid sequence of SEQ ID NO: 33, a Flag tag having the amino acid sequence of SEQ ID NO: 34, a HA tag having the amino acid sequence of SEQ ID NO: 35, and a Myc tag having the amino acid sequence of SEQ ID NO: 36. In certain embodiments, the peptide tag is present at the N terminus or at the C terminus of the peptide linker. In certain embodiments, a peptide linker comprises a peptide tag is located at the N terminus or at the C terminus of the polypeptide chain. In certain embodiments, the peptide tag is located at the N terminus or at the C terminus of the polypeptide chain.

[0079] In certain embodiments, the antibody module comprises a peptide linker which comprises a His tag, wherein the His tag is located at the N terminus or the C terminus of the polypeptide chain. In certain embodiments, the antibody module comprises a peptide linker which comprises two Strep tags, wherein the peptide linker is located at the N terminus or the C terminus of the polypeptide chain. In these embodiments, one Strep tag may be at the N terminus of the peptide linker and the other Strep tag may be at the C terminus of the peptide linker.

[0080] In certain embodiments, a peptide linker which comprises a peptide tag and is located at the N terminus or at the C terminus of the polypeptide chain further comprises a protease cleavage site between the peptide tag and the attachment point to the remaining polypeptide chain of the antibody module. Exemplary protease cleavage sites include cleavage sites of thrombin, furin, cathepsin B, TEV (tobacco etch virus) protease, human rhinovirus protease 3C, factor Vila, factor Xa, factor Xia, matrix metalloprotease-1 , HIV-1 protease and HCV protease.

[0081] In certain embodiments, the peptide linker comprises an amino acid sequence according to any one of the sequences of table 2: Table 2 - Amino acid motifs of the peptide linkers

[0082] In certain embodiments, the peptide linker comprises exactly one amino acid sequence according to any one of the sequences of table 2. In other embodiments, the peptide linker comprises exactly two identical amino acid sequences according to any one of the sequences of table 2.

[0083] In certain embodiments, the peptide linker comprises, in particular consists of, an amino acid sequence according to any one of the sequences of table 3:

[0084] Table 3 - Amino acid sequences of peptide linkers

[0085] Different peptide linkers within the antibody module may have the same or different amino acid sequences. In certain embodiments, a peptide linker directly linking two antibody variable domains has a different amino acid sequence than a peptide linker at the N terminus or C terminus of the polypeptide chain.

[0086] The overall structure of the antibody module

[0087] In certain embodiments, the antibody module consists of (i) one or two pairs of heavy chain variable domain and light chain variable domain capable of specifically binding to a target antigen;

[0088] (ii) one to six peptide linkers, each comprising one or two N-glycosylation sites;

[0089] (iii) optionally up to six heavy chain constant domains;

[0090] (iv) optionally up to two hinge regions;

[0091] (v) optionally up to two light chain constant domains; and

[0092] (vi) optionally up to two peptide linkers without an N-glycosylation site

[0093] In certain embodiments, the antibody module is selected from the group consisting of scFv, diabody, single chain diabody, Fab, minibody, scFv-Fc, F(ab)2 and whole antibody.

[0094] In certain embodiments, the antibody module comprises only one pair of heavy chain variable domain and light chain variable domain capable of specifically binding to a target antigen.

[0095] In specific embodiments, the antibody module is a scFv fragment consisting of one polypeptide chain, wherein the heavy chain variable domain is connected by the peptide linker comprising an N-glycosylation site to the light chain variable domain. In these embodiments, the light chain variable domain is located N terminally of the heavy chain variable domain, or the heavy chain variable domain is located N terminally of the light chain variable domain. The antibody module may comprise an additional peptide linker attached to the N terminus or to the C terminus, or the antibody module may comprise an additional peptide linker attached to the N terminus and an additional peptide linker attached to the C terminus. In certain embodiments, said additional peptide linker(s) comprise one or two N-glycosylation sites. Optionally, said additional peptide linker(s) may comprise a peptide tag as described herein, for example a His tag or a Strep tag. In a specific embodiment, the polypeptide chain of the antibody module consists of, in the direction from the N terminus to the C terminus, the light chain variable domain, a first peptide linker comprising one or two N-glycosylation sites, the heavy chain variable domain, and a second peptide linker comprising one or two N-glycosylation sites and optionally one or two Strep tags or one His tag. In another specific embodiment, the polypeptide chain of the antibody module consists of, in the direction from the N terminus to the C terminus, a second peptide linker comprising one or two N-glycosylation sites and optionally one or two Strep tags or one His tag, the light chain variable domain, a first peptide linker comprising one or two N- glycosylation sites, and the heavy chain variable domain. In another specific embodiment, the polypeptide chain of the antibody module consists of, in the direction from the N terminus to the C terminus, a second peptide linker comprising one or two N-glycosylation sites, the light chain variable domain, a first peptide linker comprising one or two N-glycosylation sites, the heavy chain variable domain, and a third peptide linker comprising one or two N-glycosylation sites, wherein optionally the second or the third peptide linker comprises one or two Strep tags or one His tag. The first peptide linker in particular is a long flexible linker, for example a GS type linker having at least 20 amino acids, such as a linker comprising 4 G4S motifs, for example a linker having the amino acid sequence according to SEQ ID NO: 40 or 46.

[0096] In specific embodiments, the antibody module is a Fab fragment consisting of two polypeptide chains, wherein the first polypeptide chain comprises the heavy chain variable domain and a heavy chain constant domain, and the second polypeptide chain comprises the light chain variable domain and a light chain constant domain. Preferably, the variable domain is directly N terminal of the constant domain on both polypeptide chains. The peptide linker comprising an N-glycosylation site may be located at the C terminus or the N terminus of the first or the second polypeptide chain. In certain embodiments, a peptide linker comprising an N- glycosylation site is located at the C terminus of each of the first polypeptide chain and the second polypeptide chain. In further embodiments, a peptide linker comprising an N- glycosylation site is located at the N terminus of each of the first polypeptide chain and the second polypeptide chain. In even further embodiments, a peptide linker comprising an N- glycosylation site is located at each of the N terminus and the C terminus of each of the first polypeptide chain and the second polypeptide chain. The peptide linkers in particular are short linkers comprising one or two N-glycosylation sites. The peptide may be GS type linkers, for example peptide linkers having the amino acid sequence according to any one or SEQ ID NOs: 41 , 42 and 47. In a specific embodiment, the first polypeptide chain of the antibody module consists of, in the direction from the N terminus to the C terminus, optionally a second peptide linker comprising one or two N-glycosylation sites, the heavy chain variable domain, a heavy chain constant domain, and the first peptide linker comprising one or two N-glycosylation sites, and the second polypeptide chain of the antibody module consists of, in the direction from the N terminus to the C terminus, optionally a fourth peptide linker comprising one or two N- glycosylation sites, the light chain variable domain, a light chain constant domain and a third peptide linker comprising one or two N-glycosylation sites.

[0097] In certain embodiments, the antibody module comprises a first pair of first heavy chain variable domain and first light chain variable domain, and a second pair of second heavy chain variable domain and second light chain variable domain, each pair being capable of specifically binding to the same target antigen.

[0098] In specific embodiments, the antibody module is a diabody consisting of two polypeptide chains, wherein each polypeptide chain comprises a heavy chain variable domain and a light chain variable domain connected by the peptide linker comprising an N-glycosylation site. The heavy chain variable domain of one polypeptide chain binds to the light chain variable domain of the other polypeptide chain, and vice versa. Thereby, two pairs of heavy chain variable domain and light chain variable domain are formed. The light chain variable domain is located N terminally of the heavy chain variable domain on both polypeptide chains. Alternatively, the heavy chain variable domain is located N terminally of the light chain variable domain on both polypeptide chains. Preferably, both polypeptide chains have the same amino acid sequence. The peptide linker in particular is a short linker. The peptide linker may be of the GS type, in particular a linker comprising 2 G4S motifs, for example a linker having the amino acid sequence according to any one of SEQ ID NOs: 41 , 42 and 47. Each polypeptide chain may comprise an additional peptide linker attached to the N terminus or the C terminus, wherein said additional peptide linker comprises one or more N-glycosylation sites. Alternatively, each polypeptide chain may comprise two additional peptide linkers, one attached to the N terminus and one attached to the C terminus, wherein said additional peptide linkers comprises one or more N-glycosylation sites. In a specific embodiment, the polypeptide chains of the antibody module each consists of, in the direction from the N terminus to the C terminus, optionally a third peptide linker comprising one or two N-glycosylation sites, the light chain variable domain, a first peptide linker of 5 to 15 amino acids length comprising one or two N-glycosylation sites, the heavy chain variable domain, and a second peptide linker comprising zero, one or two N- glycosylation sites and optionally one or two Strep tags or one His tag. In another specific embodiment, the polypeptide chains of the antibody module each consists of, in the direction from the N terminus to the C terminus, optionally a third peptide linker comprising one or two N-glycosylation sites, the heavy chain variable domain, a first peptide linker of 5 to 15 amino acids length comprising one or two N-glycosylation sites, the light chain variable domain, and a second peptide linker comprising zero, one or two N-glycosylation sites and optionally one or two Strep tags or one His tag.

[0099] In specific embodiments, the antibody module is a single chain diabody consisting of one polypeptide chain, comprises from N terminus to C terminus

[0100] (i) a first heavy chain variable domain, a first peptide linker, a second light chain variable domain, a second peptide linker, a second heavy chain variable domain, a third peptide linker, a first light chain variable domain, or

[0101] (ii) a first light chain variable domain, a first peptide linker, a second heavy chain variable domain, a second peptide linker, a second light chain variable domain, a third peptide linker, a first heavy chain variable domain; wherein the first heavy chain variable domain and the first light chain variable domain form a first pair and the second heavy chain variable domain and the second light chain variable domain form a second pair, each pair being capable of binding to the target antigen. Both heavy chain variable domains may have the same amino acid sequence. Both light chain variable domains may have the same amino acid sequence. The first and the third peptide linker may have the same amino acid sequence. At least one of the peptide linkers comprises an N-glycosylation site. In certain embodiments, the first and the third peptide linker each comprise one or two N-glycosylation sites, especially one N-glycosylation site. In certain embodiments, the second peptide linker comprises one or two N-glycosylation sites. The first and third peptide linker in particular are short linkers. The first and third peptide linker may be of the GS type, in particular linkers comprising 2 G4S motifs, for example linkers having the amino acid sequence according to any one of SEQ ID NOs: 41 , 42 and 47. The second peptide linker in particular is a long linker. The second peptide linker may be of the GS type, in particular a linker comprising 4 G4S motifs, for example a linker having the amino acid sequence according to SEQ ID NO: 40 or 46. The polypeptide chain may comprise an additional peptide linker attached to the N terminus or the C terminus, wherein said additional peptide linker may or may not comprise one or more N-glycosylation sites.

[0102] In a specific embodiment, the polypeptide chain of the antibody module consists of, in the direction from the N terminus to the C terminus, optionally a fourth peptide linker comprising one or two N-glycosylation sites, a first light chain variable domain, a first peptide linker of 5 to 15 amino acids length comprising one N-glycosylation sites, a second heavy chain variable domain, a second peptide linker comprising zero, one or two N-glycosylation sites, a second light chain variable domain, a third peptide linker of 5 to 15 amino acids length comprising one N-glycosylation sites, a first heavy chain variable domain, and a fifth peptide linker comprising zero, one or two N-glycosylation sites and optionally one or two Strep tags or one His tag. In another specific embodiment, the polypeptide chain of the antibody module consists of, in the direction from the N terminus to the C terminus, optionally a fourth peptide linker comprising one or two N-glycosylation sites, a first heavy chain variable domain, a first peptide linker of 5 to 15 amino acids length comprising one N-glycosylation sites, a second light chain variable domain, a second peptide linker comprising zero, one or two N-glycosylation sites, a second heavy chain variable domain, a third peptide linker of 5 to 15 amino acids length comprising one N-glycosylation sites, a first light chain variable domain, and a fifth peptide linker comprising zero, one or two N-glycosylation sites and optionally one or two Strep tags or one His tag.

[0103] In specific embodiments, the antibody module is a minibody or a scFv-Fc fragment consisting of two polypeptide chains, wherein each polypeptide chain comprises, from N terminus to C terminus, the light chain variable domain, the peptide linker comprising an N-glycosylation site, the heavy chain variable domain, a hinge region, and a heavy chain constant domain. In certain embodiments, the peptide linker has one glycosylation site. The peptide linker in particular is a long flexible linker, for example a GS type linker having at least 20 amino acids, such as a linker comprising 4 G4S motifs, for example a linker having the amino acid sequence according to SEQ ID NO: 40 or 46. Minibodies only comprise one heavy chain constant domain per polypeptide chain, while scFv-Fc fragments additionally comprise a second heavy chain constant domain per polypeptide chain. In certain embodiments, the two polypeptide chains have the same amino acid sequence. In certain embodiments, the two polypeptide chains are connected to each other via one or more, especially two, disulfide bridges formed between the hinge regions. In certain embodiments of the minibody, the heavy chain constant domain is a CH2 domain. In alternative embodiments of the minibody, the heavy chain constant domain is a CH3 domain. In certain embodiments of the scFv-Fc fragment, the first heavy chain constant domain C terminal of the hinge region is a CH2 domain and the second heavy chain constant domain C terminal of the first heavy chain constant domain is a CH3 domain. The antibody module may comprise an additional peptide linker attached to the N terminus or to the C terminus, or the antibody module may comprise an additional peptide linker attached to the N terminus and an additional peptide linker attached to the C terminus. In certain embodiments, said additional peptide linker(s) comprise one or two N-glycosylation sites. Said additional peptide linker(s) may have the amino acid sequence according to any one of SEQ ID NOs: 41 , 42 and 47. In a specific embodiment, the polypeptide chain of the antibody module consists of, in the direction from the N terminus to the C terminus, optionally a second peptide linker optionally comprising one or two N-glycosylation sites, the light chain variable domain, a first peptide linker comprising one or two N-glycosylation sites, the heavy chain variable domain, a hinge region, a first heavy chain constant domain, optionally a second heavy chain constant domain, and optionally a third peptide linker optionally comprising one or two N-glycosylation sites. The first peptide linker in particular is a long flexible linker, for example a GS type linker having at least 20 amino acids, such as a linker comprising 4 G4S motifs, for example a linker having the amino acid sequence according to SEQ ID NO: 40 or 46.

[0104] In specific embodiments, the antibody module is a F(ab)2 fragment (also called F(ab’)2 fragment) consisting of four polypeptide chains, wherein the first and the second polypeptide chain each comprises, from N terminus to C terminus, the heavy chain variable domain, a heavy chain constant domain, a hinge region, and the peptide linker comprising an N- glycosylation site; and the third and the fourth polypeptide chain each comprises, from N terminus to C terminus, the light chain variable domain, and a light chain constant domain. The heavy chain constant domain in particular is a CH1 domain. In certain embodiments, the first and second polypeptide chains have the same amino acid sequence. In certain embodiments, the third and fourth polypeptide chains have the same amino acid sequence. In certain embodiments, first and second polypeptide chains are connected to each other via one or more, especially two, disulfide bridges formed between the hinge regions. In certain embodiments, the peptide linker has one or two glycosylation sites. In certain embodiments, the third and fourth polypeptide chains each comprise a peptide linker comprising an N- glycosylation site at the C terminus. The peptide linkers may have the amino acid sequence according to any one of SEQ ID NOs: 41 , 42 and 47.

[0105] In specific embodiments, the antibody module is whole antibody consisting of four polypeptide chains, wherein the first and the second polypeptide chain each comprises, from N terminus to C terminus, the heavy chain variable domain, a CH1 heavy chain constant domain, a hinge region, a CH2 heavy chain constant domain, and a CH3 heavy chain constant domain; and the third and the fourth polypeptide chain each comprises, from N terminus to C terminus, the light chain variable domain, and a light chain constant domain. The peptide linker comprising an N-glycosylation site may be located at the C terminus of the first and second polypeptide chains and / or at the C terminus of the third and fourth polypeptide chains. In certain embodiments, a peptide linker comprising an N-glycosylation site is located at the C terminus of each of the first polypeptide chain and the second polypeptide chain. In further embodiments, a peptide linker comprising an N-glycosylation site is located at the C terminus of each of the third polypeptide chain and the fourth polypeptide chain. In even further embodiments, a peptide linker comprising an N-glycosylation site is located at the C terminus of each of the first polypeptide chain, the second polypeptide chain, the third polypeptide chain and the fourth polypeptide chain. The peptide linkers in particular are short linkers comprising one or two N-glycosylation sites. The peptide may be GS type linkers, for example peptide linkers having the amino acid sequence according to any one or SEQ ID NOs: 41 , 42 and 47.

[0106] In certain embodiments, the first and second polypeptide chains have the same amino acid sequence. In certain embodiments, the third and fourth polypeptide chains have the same amino acid sequence. In certain embodiments, first and second polypeptide chains are connected to each other via one or more, especially two, disulfide bridges formed between the hinge regions. In a specific embodiment, the first and second polypeptide chain of the antibody module consists of, in the direction from the N terminus to the C terminus, the heavy chain variable domain, a CH1 heavy chain constant domain, a hinge region, a CH2 heavy chain constant domain, a CH3 heavy chain constant domain, and the first peptide linker comprising one or two N-glycosylation sites, and the third and fourth polypeptide chain of the antibody module consists of, in the direction from the N terminus to the C terminus, the light chain variable domain, a light chain constant domain and the second peptide linker comprising one or two N-glycosylation sites.

[0107] The glycan structure

[0108] The glycan structure is attached to the N-glycosylation site of the peptide linker. In embodiments where the antibody module comprises more than one N-glycosylation site, a glycan structure may be attached to essentially every N-glycosylation site of the antibody module.

[0109] In certain embodiments, the glycan structure comprises at least one N-acetylglucosamine residue (GIcNAc) which is directly attached to the asparagine residue of the N-glycosylation site.

[0110] In certain embodiments, the glycan structure comprises a modified monosaccharide unit at at least one non-reducing end. In specific embodiments, the glycan structure comprises only one non-reducing end. The modified monosaccharide unit in particular serves as coupling point for the cargo compound. The modified monosaccharide unit in particular is a monosaccharide unit which is not found in the naturally occurring glycosylation of antibodies. The modified monosaccharide unit in particular comprises a reactive chemical group which is capable of reacting with the cargo compound. The modified monosaccharide unit especially comprises a unique reactive chemical group which is capable of reacting with the cargo compound and is not present in naturally occurring glycosylation of antibodies. An exemplary reactive chemical group is an azido group. A suitable modified monosaccharide unit is for example N- azidoacetylgalactosamine (GalNAz). A further suitable modified monosaccharide unit is for example tetraacetylated N-azidoacetylgalactosamine (Ac4GalNAz).

[0111] In certain embodiments, the glycan structure consists of an N-acetylglucosamine residue (GIcNAc) which is directly attached to the asparagine residue of the N-glycosylation site, and a modified monosaccharide residue which is attached to the GIcNAc residue, and optionally a fucose residue (Fuc) which is attached to the GIcNAc residue.

[0112] In certain embodiments, the glycan structure comprises the structure of -GIcNAc-GIcNAc-Man- Man-GIcNAc-modMS-, wherein GIcNAc is an N-acetylglucosamine residue, Man is a mannose residue, and modMS is a modified monosaccharide unit. In particular, the first GIcNAc residue is attached to the asparagine residue of the N-glycosylation site, and the modified monosaccharide unit is attached to the cargo compound. In these embodiments, the glycan structure may further comprise one or more of a fucose residue attached to the first GIcNAc residue, a further -GIcNAc-modMS- structure attached to the second Man residue, a further - Man-GIcNAc-modMS- structure or -Man(-GlcNAc-modMS-)-GlcNAc-modMS- structure or - Man residue attached to the first Man residue, and a further GIcNAc residue attached to the first Man residue.

[0113] In certain embodiments, the modified monosaccharide unit is N-azidoacetylgalactoamine residue (GalNAz) or tetraacetylated N-azidoacetylgalactoamine residue (Ac4GalNAz). In these embodiments, the glycan structure may have the structure of [Asp]-GlcNAc-GalNAz-[cargo] or [Asp]-GlcNAc(-Fuc)-GalNAz-[cargo] or [Asp]-GlcNAc-Ac4GalNAz-[cargo] or [Asp]-GlcNAc(- Fuc)-Ac4GalNAz-[cargo], wherein [Asp]- and -[cargo] denote the attachment points to the N- glycosylation site of the antibody module and the cargo compound, respectively. In embodiments wherein the antibody module comprises more than one N-glycosylation sites, a mixture of these glycan structures may be attached thereto. Furthermore, in embodiments wherein the antibody module comprises more than one N-glycosylation sites, a mixture of glycan structures carrying a cargo compound and glycan structures not carrying a cargo compound may be attached thereto.

[0114] In preferred embodiments, the glycan structure is obtained by modifying the carbohydrate chains present at the N-glycosylation sites of the antibody module after production in host cells. Modification may be done by digesting the carbohydrate chains with a glycoside hydrolase which cleaves the carbohydrate chains between two specific monosaccharide units, and the optionally attaching a modified monosaccharide residue or oligosaccharide structure to the terminal monosaccharide unit of the carbohydrate chains. The glycoside hydrolase may in particular be able to cleave the glycan structure behind the GIcNAc residue at the reducing end or behind the GIcNAc residues in the antennae of complex-type or hybrid-type N-glycans. The cargo compound

[0115] The cargo compound may be any compound which can be coupled to the glycan structure attached to the N-glycosylation site of the antibody module. If more than one cargo compound is present in the conjugate, these cargo compounds may be identical or different, and in particular are all identical. In certain embodiments, a cargo compound is coupled to essentially every glycan structure attached to the N-glycosylation sites of the antibody module. Coupling of the cargo compound to the glycan structure of the conjugate can be achieved using any methods known in the art. The cargo compound is covalently attached to the glycan structure.

[0116] In certain embodiments, the cargo compound is useful in therapy, diagnosis, prognosis and / or monitoring of a disease, in particular cancer. For example, the cargo compound may be selected from the group consisting of radionuclides, chelators for radionuclides, chemotherapeutic agents, detectable labels, toxins, cytolytic components, immunomodulators, immunoeffectors, and liposomes. A particular preferred cargo compound is a chelator for a radionuclide or a chelator comprising a radionuclide or a cytotoxic agent capable of killing cancer cells, such as a chemotherapeutic agent.

[0117] The chelator may in particular be selected from the group consisting of bis(carboxymethyl)- 1 ,4, 8,11-tetraazabicyclo[6.6.2]hexadecane (CBTE2a), cyclohexyl- 1 ,2-diaminetetraacetic acid (CDTA), 4-(1 ,4,8,11-tetraazacyclotetradec-1-yl)-methylbenzoic acid (CPTA), N'-[5- [acetyl(hydroxy)amino]pentyl]-N-[5-[[4-[5-aminopentyl-(hydroxy)amino]-4-oxobutanoyl]amino] pentyl]-N-hydroxybutandiamide (DFO), 4,11-bis(carboxymethyl)-1 , 4, 8, 11 -tetraazabicycle [6.6.2]hexadecan (DO2A), 1 ,4,7,10-Tetraazacyclododecane-1 ,4,7-triacetic acid trisodium salt (DO3A), 2-(4,7,10-tris(2-amino-2-oxoethyl)-1 ,4,7,10-tetraazacyclododecan-1-yl)acetic acid (D03AM), 1 ,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA), 1 ,4,7,10- tetraazacyclododecane-1 ,4,7,10-tetraacetic amide (DOTAM), 1 ,4,7,10-tetraaza- cyclododecane-1 ,4,7,10-tetrakis[methylene(2-carboxyethyl) phosphinic acid] (DOTPI), 1 ,4,7, 10-tetraazacyclododecane-1 ,4,7, 10-tetrakis[methylene(2-carboxyethyl)phosphonic acid] (DOTP), 2-[1 ,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid]-pentanedioic acid (DOTAGA or DOTA-GA), N,N'-dipyridoxyfethylendiamine-N,N'-diacetate-5,5'-bis(phosphat) (DPDP), dipropylenetriamine (DPTA), diethylenetriaminepentaacetic acid (DTPA), ethylenediamine-N,N'-tetraacetic acid (EDTA), ethyleneglykol-O,O-bis(2-aminoethyl)- N,N,N',N'-tetraacetic acid (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), hydroxyethyldiaminetnacetic acid (HEDTA), 1-(p-nitrobenzyl)-1 ,4,7,10- tetraazacyclodecan-4,7,10-triacetate (HP-DOA3), 6-hydrazinyl-N-methylpyridine-3- carboxamide (HYNIC), mercaptoacetyltriglycine (MAG3), mercaptoacetyltriserine (MAS3), 4- Amino-6-((16-((6-carboxypyridin-2-yl)methyl)-1 ,4,10,13-tetraoxa-7, 16-diazacyclooctadecan- 7-yl)methyl)picolinic acid (MACROPA-NH2), 6-((16-((6-Carboxypyridin-2-yl)methyl)-1 ,4,10,13- tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)-4-isothiocyanatopicolinic acid (MACROPA- NCS), 3-(2-aminoethylamino)-2-[(2-aminoethylamino)methyl]propanoic acid (N4 chelator, 6- carboxy-1 ,4,8,11 -tetraazaundecane), 2,2'-(7-(4-isothiocyanatobenzyl)-1 ,4,7-triazonane-1 ,4- diyl) diacetic acid (NCS-MP-NODA), 2,2'-(7-(4-(2-((2-aminoethyl)amino)-2-oxoethyl)benzyl)-

[0118] 1.4.7-tri-azonane-1 ,4-diyl)diacetic acid (NH2-MPAA-NODA), 1 ,4,7-triazacyclononan-1- succinic acid-4, 7-diacetic acid (NODASA), 1-(1-carboxy-3-carboxypropyl)-4,7-(carboxy)-1 ,4,7- triazacyclononane (NODAGA), 1 ,4,7-triazacyclononanetriacetic acid (NOTA), 2, 2', 2"- [3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11 , 13-triene-3,6,9-triyl]triacetic acid (PCTA), S-2-(4-isothiocyanatobenzyl)-1 ,4,7,10-tetraazacyclododecane-tetraacetic acid (p-SCN-Bn- DOTA), 4,11-bis(carboxymethyl)-1 ,4,8,11-tetraaza-bicyclo[6.6.2]hexadecane (TE2A), 1 ,4,8,11-tetraazacyclododecane-1 ,4,8,11-tetraacetic acid (TETA), terpyridine-bis (methyleneamine) tetraacetic acid (TMT), 1 ,4,7,10-tetraazacyclotridecan-N,N',N",N"'- tetraacetic acid (TRITA), triethylenetetra-aminehexaacetie acid (TTHA), 4-amino-4-(2-[(3- hydroxy-1 ,6-dimethyl-4-oxo-1 ,4-dihydro-pyridin-2-ylmethyl)-carbamoyl]-ethyl)heptanedioic acid bis-[(3-hydroxy-1 ,6-dimethyl-4-oxo-1 ,4-dihydro-pyridin-2-ylmethyl)-amide] (THP), and

[0119] 1.4.7-triazacyclononane-1 ,4,7-tris [methylene(2-carboxyethyl)phosphinic acid (TRAP).

[0120] The radionuclide may be selected from the group consisting of11C,18F, AI18F2+,43Sc,44Sc,

[0121] Specific examples of chemotherapeutic agents include alkylating agents such as cisplatin, antimetabolites, plant alkaloids and terpenoids, vinca alkaloids, podophyllotoxin, taxanes such as taxol, topoisomerase inhibitors such as irinotecan and topotecan, antineoplastics such as doxorubicin or microtubule inhibitors such as auristatins and maytansin / maytansinoids.

[0122] The chemotherapeutic agent may in particular be selected from a group consisting of a V- ATPase inhibitor, a pro-apoptotic agent, a Bcl2 inhibitor, an MCL1 inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, an auristatin, a dolastatin, a maytansin, a maytansinoid, amatoxin, a methionine aminopeptidase, an inhibitor of nuclear export of proteins CRM1 , a DPPIV inhibitor, proteasome inhibitors, inhibitors of phosphoryl transfer reactions in mitochondria, protein phosphatase inhibitors, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a kinesin inhibitor, an HDAC inhibitor, a topoisomerase I inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder, a DHFR inhibitor, an inhibitor of microtubule formation, a stabilizer of microtubuli, a stabilizer of actin, a topoisomerase II inhibitor, a platinum compound, a ribosome inhibitor, an RNA polymerase II inhibitor and a bacterial toxin. The pharmaceutical composition according to the second aspect

[0123] In another aspect, the present invention provides a composition comprising the conjugate according to the invention. The composition may comprise one or more further components selected from the group consisting of solvents, diluents, and excipients. Preferably, the composition is a pharmaceutical composition. In this embodiment, the components of the composition preferably are all pharmaceutically acceptable. The composition may be a solid or fluid composition, in particular a - preferably aqueous - solution, emulsion or suspension or a lyophilized powder.

[0124] In certain embodiments, at least 90% of the N-glycosylation sites in the peptide linkers of the conjugates in the composition carry a glycan structure. In particular, at least 95% of the N- glycosylation sites in the peptide linkers of the conjugates in the composition carry a glycan structure. In certain embodiments, at least 90% of the N-glycosylation sites in the peptide linkers of the conjugates in the composition carry a glycan structure coupled to a cargo compound. In particular, at least 95% of the N-glycosylation sites in the peptide linkers of the conjugates in the composition carry a glycan structure coupled to a cargo compound.

[0125] The medical use according to the third aspect

[0126] The conjugate in particular is useful in medicine, in particular in therapy, prevention, diagnosis, prognosis and / or monitoring of a disease or condition, in particular a disease as described herein. Therefore, in a further aspect, the invention provides the conjugate or the composition for use in medicine. Preferably, the use in medicine is a use in the treatment, prevention, prognosis, diagnosis and / or monitoring of a disease or condition, especially a disease or condition associated with the antigen of the antibody module of the conjugate. Exemplary diseases include diseases associated with abnormal cell growth such as cancer, arthritis, bacterial and viral infections, inflammatory diseases, graft-versus-host disease, immunodeficiencies, interstitial lung disease, vascular disease such as hypertension, and scoliosis. Diagnosis, prognosis and / or monitoring of a disease or condition may include imaging and / or staging of the disease or condition, especially imaging and / or staging of cancer.

[0127] In a preferred embodiment, the disease is cancer. The cancer may be selected from the group consisting of lymphoma, myeloma and leukemia. The cancer may for example be non-Hodgkin lymphoma, central nervous system lymphoma, multiple myeloma, or histiocytic neoplasm. The cancer is in particular positive for the antigen of the antibody module and in particular comprises cancer cells which carry said antigen on their cell surface.

[0128] In specific embodiments, the conjugate is used in combination with another therapeutic or diagnostic agent. Said further agent may be any known agent useful in the therapy, prevention, diagnosis, prognosis and / or monitoring of the disease or condition. The method according to the fourth aspect

[0129] According to a further aspect, the present invention provides a method for producing the conjugate according to the first aspect, comprising the steps of

[0130] (a) providing a composition of glycosylated antibody modules, wherein the glycosylated antibody module consists of an antibody module as defined in the first aspect, and glycan structures attached to the N-glycosylation sites of the peptide linkers of the antibody module;

[0131] (b) contacting the composition of glycosylated antibody modules with a glycoside hydrolase under conditions where the glycoside hydrolase cleaves the glycan structures so that unitary non-reducing ends are present at the glycan structures;

[0132] (c) contacting the composition of glycosylated antibody modules with a glycosyltransferase which is capable of transferring a modified monosaccharide unit to the unitary non-reducing ends of the glycan structures, and a corresponding modified nucleotide monosaccharide, under conditions where the modified monosaccharide unit is coupled to the unitary non-reducing ends of the glycan structures;

[0133] (d) reacting the composition of glycosylated antibody modules with a cargo compound under conditions where the cargo compound is coupled to the modified monosaccharide attached to the glycan structures, providing a conjugate according to the first aspect.

[0134] The composition of glycosylated antibody modules provided in step (a) may be obtained from any suitable source. In certain embodiments, the glycosylated antibody modules were produced in host cells, especially in eukaryotic host cells such as mammalian host cells.

[0135] The glycosylated antibody modules are antibody modules as described herein comprising carbohydrate chains attached to their N-glycosylation sites. In certain embodiments, the glycosylated antibody modules in step (a) comprise complex-type glycan structures.

[0136] In step (b), the glycoside hydrolase may in particular be able to cleave the glycan structure behind the GIcNAc residue at the reducing end or behind the GIcNAc residues in the antennae of complex-type of hybrid-type N-glycans. For example, the glycoside hydrolase is selected from the group of p-1 ,4-galactosidase, endoS2, EndoS, Endo D, Endo F2, Endo F3, Endo H and Endo Hf. In certain embodiments, the unitary non-reducing ends present at the glycan structures are GIcNAc residues.

[0137] In step (c), the glycosyltransferase may in particular be able to transfer a modified monosaccharide unit to the unitary non-reducing ends present at the glycan structures. A suitable glycosyltransferase is for example p-1 ,4-galactosyltransferase (Y289L). The modified monosaccharide unit in particular comprises a reactive chemical group which is capable of reacting with the cargo compound in step (d). The modified monosaccharide unit especially comprises a unique reactive chemical group which is capable of reacting with the cargo compound in step (d) and is not present in naturally occurring glycosylation of antibodies. An exemplary reactive chemical group is an azido group. A suitable modified monosaccharide unit is for example N-azidoacetylgalactosamine (GalNAz). The corresponding modified nucleotide monosaccharide in this case is UDP-N-azidoacetylgalactosamine (UDP-GalNAz). A further suitable modified monosaccharide unit is for example tetraacetylated N- azidoacetylgalactosamine (Ac4GalNAz). The corresponding modified nucleotide monosaccharide in this case is II DP-tetraacetylated N-azidoacetylgalactosamine (UDP- Ac4GalNAz).

[0138] In step (d), the cargo compound which is to be reacted with the glycosylated antibody modules in the composition in particular comprises a reactive chemical group which is capable of reacting with the modified monosaccharide attached to the glycan structures. A suitable reactive chemical group is for example a dibenzocyclooctyne (DBCO) group in embodiments wherein the modified monosaccharide unit comprises an azido group.

[0139] In certain embodiments, the glycoside hydrolase and the residual glycan cleaved off from the glycan structures are removed from the composition between steps (b) and (c). In certain embodiments, the glycosyltransferase and residual modified nucleotide monosaccharides are removed from the composition between steps (c) and (d). In certain embodiments, uncoupled cargo compound is removed from the composition after step (d).

[0140] In certain embodiments, the composition of glycosylated antibody modules is contacted with a sialidase prior to step (b) under conditions where sialic acids are cleaved off of the glycan structures. A suitable sialidase is for example sialidase A.

[0141] BRIEF DESCRIPTION OF THE FIGURES

[0142] Figure 1 shows antibody module designs.

[0143] Figure 2 shows the structures of linker payloads A) DBCO-PEG(4)-vc-PAB-MMAE, B) DBCO- C6-PEG(8)-vc-PAB-MMAE, and C) representation for DBCO-C6-PEG(8)-vc-PAB linker variants comprising amatoxin or microcystins. Chemical structure of (A) derived from www.chemsrc.com, CAS Number 2129164-91-4. Structures of (B) and (C) provided by manufacturers TBD Biodiscovery (Tartuu, Estonia) / ! RIS Biotech (Marktredwitz, Germany).

[0144] Figure 3 shows a schematic representation of two complex type N-glycans with their respective denotation (based on the Oxford notation). Figure 4 shows a schematic representation of glycomodification and conjugation reactions of antibody modules using SiteClick™ and GlyCLICK® and of lgG1 antibody using GlyCLICK®. ScFvGlyc is shown exemplary for fragment conjugates.

[0145] Figure 5 shows the productivity of transfected cell pools. Amplification pools were seeded at 2x105cells / mL in fresh medium and after three-day-culture, cells were counted and antibody titers were determined by ELISA. Standard curves of Th69scFvWT, Th69diaGlyc and Th69scDbWT were used for the respective formats.

[0146] Figure 6 shows antibody titers of scDb, minibody (Mb) and F(ab’2) variants analyzed from CHO cell culture supernatant by LabChip microfluidic CE-SDS.

[0147] Figure 7 shows a qualitative analysis of antibody fragments by SDS-PAGE. Coomassie- stained SDS-Gel. Samples were prepared under non-reducing conditions. Glycosylated formats were additionally incubated with PNGase F prior to analysis.

[0148] Figure 8 shows qualitative analysis of antibody fragments by SDS-PAGE and CE-SDS. A) SDS-PAGE of scDb, minibody (Mb) and F(ab’2) variants under reducing conditions. B) SDS- PAGE of glycosylated formats with and without prior incubation with PNGase F, analyzed under reducing conditions. C) CE-SDS analysis of cell culture supernatants of PXP0071-Mb- NH1-glyc and PXP0069-Mb-NH1 under non-reducing (NR) and reducing (R) conditions.

[0149] Figure 9 shows N-glycoprofiling of antibody fragments. A) N-glycan profile of Th69scFvGlyc with annotation of the most abundant structures. N-glycans were released from intact protein by PNGase F, labelled with RapiFluor-MS™ reagent and separated by HILIC-UPLC; chromatogram with fluorescence signal 425nm emission wavelength is shown. B-D) Relative quantification of N-glycan parameters of released N-glycans of Th69scFvGlyc, Th69dia1Glyc, Th69diaGlyc, Th69scDbGlyc. (F - core fucose; oF - outer-arm fucose; B - bi-secting GIcNAc; LacNAc - N-acetyllactosamine; A - antennary GIcNAc; S - sialic acid(NeuAc)).

[0150] Figure 10 shows N-glycoprofiling of antibody formats. A) PXP0056-scDb-ABD-glyc; B) PXP0059-Mb-ABD-glyc; C) PXP0062-F(ab’)2-ABD-glyc. D) Relative quantification of N-glycan antennarity (A0-A4; A - antennary GIcNAc).

[0151] Figure 11 shows size exclusion chromatography of affinity purified antibodies on Superdex 200 10 / 300 GL. Chromatogram overlay of Th69scFvWT, Th69diaWT and Th69scDbWT in comparison to a standard protein mixture of Thyroglobulin (669 kDa), y-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), vitamin B12 (1.3 kDa) (A). Chromatogram overlays of glycosylated antibodies and their respective wildtype formats (B).

[0152] Figure 12 shows size exclusion chromatography of antibody formats PXP0056-scDb-ABD- glyc (A); PXP0059-Mb-ABD-glyc (B); and PXP0062-F(ab’)2-ABD-glyc (C). Figure 13 shows the binding of antibody fragments to target antigen on DERL-7 cell line and in ELISA. Binding of antibody fragments to DERL-7 cells was analyzed by flow cytometry. Detection via Streptavidin-PECy7. Positive cells (A) and median fluorescence intensity (B). Binding to target antigen in ELISA. Detection via Strep-Tactin®-HRP (C). Dashed lines in Figure 9 A and C indicate glycosylated antibody formats. For determination of median fluorescence intensity in flow cytometry and absorbance in ELISA, antibody concentrations were corrected for the number of Twin-Strep-tags® per molecule. Data show mean±SD of duplicates of one representative experiment.

[0153] Figure 14 shows the binding of antibody fragments to target antigens in ELISA. A) ScDb and minibody (Mb) formats were detected with StrepTactin®. B) F(ab’)2 and lgG1 were detected with anti-F(ab’)2 detection reagent.

[0154] Figure 15 shows analysis of cell binding of (A) scDb and minibody (Mb) formats detected via StrepMAB-lmmo, (B) F(ab’)2 formats and reference lgG1 detected via anti-F(ab’)2, (C) reference lgG1 detected via anti-hlgG+M, and (D) target control antibody and TF antibody detected via anti-hlgG+M. Shown are signal-to-noise ratios of stained cells: MFI (median fluorescence intensity) of stained cells divided by MFI of either secondary-reagent stained cells or isotype-stained cells, as indicated.

[0155] Figure 16 shows titration of antibody fragments on target-expressing MCF-7 tumor cells. Cells were stained with different concentrations of antibody fragments and detected with fluorophore-coupled StrepMAB-lmmo. Shown is the MFI of live cells.

[0156] Figure 17 shows the glycomodification of Th69scFvGlyc - Th69scFvGlyc after Sialidase A digestion. Analysis of glycan pattern for intermediate steps in azide activation of intact proteins by LC-MS. Bottom arrows indicate N-glycan variants of accordingly numbered stars with additional O-glycosylation (mass addition is 340Da). Calculated MW of Th69scFvGlyc after after Sialidase A digestion with diverse possible N-glycan combinations are shown in Table 9.

[0157] Figure 18 shows the glycomodification of Th69scFvGlyc - Th69scFvGlyc after SiaA and (3-1 ,4- galactosidase digestion. Bottom arrows indicate N-glycan variants of accordingly numbered stars with additional O-glycosylation (mass addition is 340Da). Calculated MW of Th69scFvGlyc after p-1 ,4-galactosidase digestion with diverse possible N-glycan combinations are shown in Table 10.

[0158] Figure 19 shows the glycomodification of Th69scFvGlyc - Th69scFvGlyc after SiteClick™ activation with GalT(Y289L) and dUTP-GalNAz. Bottom arrows indicate N-glycan variants of accordingly numbered stars with additional O-glycosylation (mass addition is 340Da). Calculated MW of Th69scFvGlyc after SiteClick™ activation with diverse possible N-glycan combinations are shown in Table 11. Figure 20 shows the establishment of conjugation reaction for Th69scFvGlyc with DBCO- PEG4-vc-PAB-MMAE. Th69scFvGlyc was incubated with 2eq. (light grey trace), 8eq. (dark grey trace) and 5eq. (grey trace) of DBCO-PEG4-vc-PAB-MMAE for 16 h. Reaction mixtures were analyzed. DAR-measurement by LC-MS analysis with BEH-C4-RPC, shown are UV (280nm)-chromatograms. LP: linker-payload.

[0159] Figure 21 shows the establishment of RPC analysis of Th69scFvGlyc-MMAE. DAR- measurement by LC-MS analysis with BEH-C4-RPC, shown are UV(280nm)-chromatograms. A) Comparison of RP-LC analysis methods. Improvement in LC gradient and column temperature for Th69scFvGlyc-MMAE, reaction mixture 2. Reactions in mixture 1 (upper) and mixture 2 (lower) were set up in separate experiments but with the same protocol, ratio LP:antibody 5:1. B) Chromatogram overlay of Th69scFvGlyc-MMAE in reaction mixture 2 (solid line) and after purification (dashed line).

[0160] Figure 22 shows ADC analysis of Th69scFvGlyc-MMAE by LC-MS. Intact protein was subjected to BEH-C4-RPC coupled to ESI-MS. Shown are UV(280nm)-chromatogram with peak annotation (upper left) and deconvoluted mass spectra of indicated peaks from chromatogram with schematic annotation of corresponding glycan residues. In mass spectra C and E, the different possible combinations of N-glycans for the same mass peak are annotated and indicated with grey lines. “Explosion” symbols indicate linker-payload residues. +O-glyc: additional O-linked glycosylation in Th69scFvGlyc.

[0161] Figure 23 shows RPC analysis for Th69scDb-format. RP chromatograms and different gradients tested for A) Th69scDbWT (20 pg), B) Th69scDbWT (24 pg), C) Th69scDbGlyc- MMAE SiteClick (13.5 pg). UV280nm chromatogram (dark grey lines); proportion of solvent A (acetonitrile +0.1 % formic acid, light grey lines).

[0162] Figure 24 shows the FDC development of antibody fragments with SiteClick™. Th69scFvGlyc (first row), Th69dia1Glyc (second row), Th69scDbGlyc (third row) generated by SiteClick™ were analyzed in LC-MS. Deconvoluted mass spectra of azide-acivated antibodies prior to conjugation (A), UV(280nm)-RPC-chromatograms of ADC preparations (B) and calculated drug load distribution (C) are shown.

[0163] Figure 25 shows the FDC development of antibody fragments with GlyCLICK®. Th69scFvGlyc (first row), Th69dia1Glyc (second row), Th69scDbGlyc (third row) generated by GlyCLICK® were analyzed in LC-MS. Deconvoluted mass spectra of azide-acivated antibodies prior to conjugation (A), UV(280nm)-RPC-chromatograms of ADC preparations (B) and calculated drug load distribution (C) are shown.

[0164] Figure 26 shows the ADC generation of cCD7Th69n(G1) using GlyCLICK® and DAR analysis. A) RPC chromatogram overlay of cCD7Th69n(G1), cCD7Th69n(G1)-azide (GlyCLICK) and cCD7Th69n(G1)-DBCO-MMAE. Reversed-phase chromatography after IdeS digest and reduction. B) Deconvoluted mass spectra of Fc / 2, LC and Fd fragments of cCD7Th69n(G1) (first row), cCD7Th69n(G1)-azide (GlyCLICK) (second row) and cCD7Th69n(G1)-DBCO- MMAE (third row); A762.1 Da: Fragmentation of DBCO-PEG4-vc-PAB-MMAE due to in-source CiD, 762Da=MMAE. C) RPC chromatograms of two separate conjugation reactions; respective solvent gradient depicted as solvent A [%]. D) Drug load distribution of cCD7Th69n(G1)-DBCO-MMAE from two separate conjugation reactions.

[0165] Figure 27 shows the characterization of DAR of MMAE-, amatoxin and microcystin-conjugated cCD7Th69n(G1)-ADCs by reversed-phase chromatography after IdeS digest and reduction of the samples. Conjugation with linker-payloads increased the hydrophobicity of the Fc / 2 fragment, according to the payload.

[0166] Figure 28 shows the target-binding of antibody fragment-drug conjugates in ELISA. Binding of A) scFv, B) diabody, C) scDb antibody fragment-drug conjugates in comparison to unconjugated formats. Data show mean±SD.

[0167] Figure 29 shows the target-binding of antibody-drug conjugates in ELISA. Binding of cCD7Th69n(G1)-DBCO-PEG8-vc-PAB-amatoxin (cCD7Th69n(G1)-vc-PAB-Amatoxin) and cCD7Th69n(G1)-DBCO-PEG8-vc-PAB-MMAE (cCD7Th69n(G1)- vc-PAB-MMAE) in comparison to isotype-ADCs and unconjugated cCD7Th69n(G1). Data show mean±SD of duplicates of one experiment.

[0168] Figure 30 shows the in vitro cytotoxicity comparison of FDCs, ADC and unmodified antibodies, free linker-payload and a CD20-targeted ADC in A) Jurkat cells, B) HSB-2 cells, C) ALL-SI L cells, D) HUVEC cells. Concentrations indicated are the relevant ADC concentrations (absolute concentration is increased by 9.3% to adjust the ratio of unconjugated:conjugated antibody molecules).

[0169] Figure 31 shows the relative quantification of N-glycan parameters of released N-glycans of Th69scFvGlyc, Th69dia1Glyc, Th69diaGlyc, Th69scDbGlyc. (A - antennary GIcNAc; B - bisecting GIcNAc; F - core fucose; oF - outer-arm fucose; G - Galactose; tG - terminal galactose; HB - hybrid type; LacNAc - N-acetyllactosamine; LeA - Lewis A; LeB - Lewis B; LeX - Lewis X; LeY - Lewis Y; M - high-mannose type; phos - phosphorylated; S - sialic acid(NeuAc); sulf - sulfation).

[0170] EXAMPLES

[0171] It should be understood that the following examples are for illustrative purpose only and are not to be construed as limiting this invention in any manner. The following examples include different antibody formats which target different antigens. Examplary antibody formats derived from the model antibody Th69 target CD7 and are described in Part 1 of each example. Further antibody formats derived from model antibodies PXP0056, PXP0059, PXP0062 and PXP0071 are described in Part 2 of each example.

[0172] Example 1 : Generation of antibodies and antibody fragments

[0173] Part 1

[0174] Single-chain variable fragment (scFv), diabody (dia) and single-chain diabody (scDb) were selected as alternative formats to IgG. In order to evaluate the use of antibody fragment-drug conjugates for therapeutic or diagnostic purposes, it was necessary to develop suitable antibodies and antibody fragments. In this respect, antibodies and antibody formats were engineered with additional glycosylation motifs in the linker sequences to introduce the cytotoxic handle. Formats are depicted in Figure 1.

[0175] Producibility was analyzed by Western Blot and ELISA (Figure 5). To verify the use of the antibody fragments for FDC generation, they needed to be characterized regarding their expression, biochemical integrity and antigen binding ability. Based on these results, the suitability for site-specific drug conjugation was evaluated.

[0176] All antibodies were produced in spinner cultures at 200 nM or 400 nM MTXand purified from supernatants by affinity chromatography with Strep-Tactin® Superflow® high-capacity cartridges on Akta FPLC (see below Table 4). Protein amounts determined after affinity chromatography via Strep-Tactin® Superflow® high-capacity cartridges from cell culture supernatant. ‘Pool’ indicates that supernatants of two cultivations were pooled for purification.

[0177] Table 4 - Purified amounts and titers of antibody fragment formats.

[0178] Protein amplification Cultivation Supernatant Protein Titer according

[0179] (MTX [days] [mL] amount to purified concentration) purified amount

[0180] Th69scFvWT 200 nM 8 860 mL 3.7 mg 4.3 pg / mL

[0181] Th69scFvGlyc 200 nM 10 1210 mL 3.2 mg 2.64 pg / mL

[0182] 10 910 L 2.37 mg 2.6 pg / mL

[0183] Th69diaWT Pool 200&400 nM 8 / 7 1130 mL 4.92 mg 4.35 pg / mL

[0184] Th69dialGlyc 200 nM 10 1125 mL 5.15 mg 4.58 pg / mL

[0185] Th69diaGlyc Pool 200&400 nM 8 / 7 1105 mL 5.26 mg 4.76 pg / mL

[0186] Th69scDbWT 200 nM 7 875 mL 3.0 mg 3.42 pg / mL

[0187] 10 860 mL 7.2 mg 8.37 pg / mL

[0188] Th69scDbGlyc 200 nM 10 1180 mL 4.67 mg 3.96 pg / mL

[0189] Whereas the increase of MTX concentration from 200 nM to 400 nM on average did not result in reduced cell growth, the cell number was lower in the 800 nM MTX pools than in the 400 nM and 200 nM MTX pools for six out of seven transfection pools. Overall, antibody titers increased with an increase in selection pressure. For Th69scFvWT and Th69diaWT, antibody titers were especially increased in 800 nM cell pools in comparison to the other formats. Put into correlation, antibody titers were either higher or at least comparable to the other pressure steps in all cell pools, despite the mostly lower cell numbers. Especially Th69scFvWT-, Th69dia1Glyc-, Th69diaGlyc- and Th69scDWT-transfectants showed similar or increased productivity at a clearly lower cell concentration.

[0190] Between 2.4 mg and 7.2 mg antibody were be purified from a culture volume of 860 mL - 1.2 L (see Table 4). Antibody titers of cell pools were assessed by back-calculation of purified protein amounts in supernatants. Antibody titers calculated from purified protein amounts ranged between 2.6 pg / mL (Th69scFvGlyc) and 8.37 pg / mL (Th69scDbWT). For Th69scDbWT, antibody titers varied by factor ~2 between two cultivations (3.42 pg / mL vs. 8.37 pg / mL), most probably due to different duration of cultivations (7 vs. 10 days). Thus, antibody titers between the distinct antibody formats and also between the respective glycosylated and wildtype formats can be faced as comparable.

[0191] Antibodies and antibody fragments were generated as follows. If not indicated otherwise, all molecular biology kits were used according to the manufacturer’s protocol. DNA cloning

[0192] For IgG cloning, the sequences of VH and VL flanked by Hindi 11 and BamHI restriction enzyme sites were purchased as plasmids from GeneArt (Thermo Fisher Scientific, Dreieich, Germany). Sequences of antibody fragments were flanked by Hindi I l / Xbal restriction sites. The encoding plasmids further contained an ampicillin resistance cassette. For enrichment of insert DNA, dam- / dcm-competent E. coli (New England Biolabs, Ipswich, USA) were transformed with plasmid DNA, plated onto LB agar plates containing 100 pg / mL ampicillin. Single colonies were picked to inoculate 5 mL LB medium supplemented with 100 pg / mL ampicillin (LB / amp). After shaking at 37 °C overnight, the plasmids were purified using the GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific, Dreieich, Germany). Insert DNA was prepared by restriction with respective Fast Digest restriction enzymes (Thermo Fisher Scientific, Dreieich, Germany) and isolation from agarose gels using the GeneJET Gel Extraction Kit (Thermo Fisher Scientific, Dreieich, Germany).

[0193] Plasmids (available at Glycotope GmbH) encoding the constant light and heavy chain regions of IgG were flanked by BamHI and Xbal restriction enzyme sites and included the dhfr gene (light chain) or the puromycin resistance gene pac (heavy chain).

[0194] Sequences for antibody fragments were inserted into vector backbones including the dhfr gene (but without light chain / heavy chain). Ligation was performed using the Rapid DNA Dephos & Ligation Kit (Roche, Mannheim, Germany) and T4 DNA Ligase (Invitrogen, Carlsbad, USA).

[0195] Transformation and isolation of plasmid DNA

[0196] Plasmids were transformed into dam+ NEB 5-alpha competent E. coli (New England Biolabs, Ipswich, USA) according to the manufacturer’s high efficiency protocol. Plasmid DNA was prepared as described above, using the GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific, Dreieich, Germany). For large scale preparation of plasmids, 100 mL LB / amp medium were inoculated with 100pL of 5 mL overnight cultures and left shaking at 37 °C overnight. Plasmids were then isolated using the NucleoBond Xtra Maxi Plus EF (Macherey- Nagel, Duren, Germany).

[0197] DNA ligation was verified by restriction control and the sequence of resulting plasmid DNA was verified by sequence analysis (Microsynth Seqlab GmbH, Gottingen, Germany).

[0198] Plasmid linearization for stable transfections

[0199] Expression vectors were linearized using their single Pvul restriction enzyme site prior to stable transfection into expression cells. 100 pg plasmid DNA were digested with 5 pL Pvul (Life Technologies, Carlsbad, USA) for 16 - 20 h at 37 °C in Digestion buffer R (Life Technologies, Carlsbad, USA) added to reach a final reaction volume of 200 pL. Nucleic acid extraction was performed by phenol / chloroform extraction (phenol / chloroform / isoamyl alcohol (Carl Roth, Karlsruhe, Germany), trichlormethane / chloroform (Carl Roth, Karlsruhe, Germany)) and subsequent ethanol precipitation of DNA (ethanol (Carl Roth, Karlsruhe, Germany), sodium acetate solution 3M (Sigma Aldrich, Steinheim, Germany)). DNA concentration was measured using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Dreieich, Germany) and the success of the linearization assessed by agarose gel electrophoresis.

[0200] Isolation of PBMCs from leukapheresis

[0201] Human primary peripheral blood mononuclear cells were derived from leukapheresis (Haema AG, Haema Blutspendezentrum, Rostock; or Institute for Transfusion medicine, Charite Berlin). PBMCs were isolated via ficoll-density centrifugation. 20 mL Biocoll Separating Solution (Biochrom, Berlin, Germany) were cautiously overlaid with 10 mL of leukopak and centrifuged at 1045 x g for 12 min (with low acceleration and brake). The overlying layer on top the Biocoll Separating Solution was aspirated, split to four 50 mL falcons, filled up with 25 mL sterile PBS (Biochrom, Berlin, Germany) and centrifuged at 700 x g for 10 min. After two additional washing steps with PBS, cells were counted.

[0202] Cell counting

[0203] Cells were counted using Guava ViaCount® reagent (Merck Millipore, Darmstadt, Germany) with the Guava® easyCyte (Merck Millipore, Darmstadt, Germany) or the Luna-FL Automated Cell Counter (Logos Biosystems, Villeneuve d’Ascq, France) using Trypanblau (Biochrom, Berlin, Germany) or AO / PI Cell Viability Kit (Logos Biosystems, Villeneuve d’Ascq, France).

[0204] Stable transfection

[0205] H9D8®-GEX were stably transfected by electroporation using Cell Line Nucleofector® Kit (Amaxa, Kbln). Prior to stable transfection with antibody-encoding plasmids, cells were seeded at a density of 2x105 cells / mL to keep them in a proliferating state. The following day, cells were counted to confirm viabilities over 85%. For each transfection, 9 pg of linearized DNA were mixed with 100 pL Nucleofector solution (Amaxa Cell Line Nucleofector Kit V, (Lonza, Basel, Switzerland)) and added to 2x106 previously pelleted cells (300 x g, 5 min). After careful resuspension, the cell mixture was transferred into a cuvette (Amaxa Cell Line Nucleofector Kit V, (Lonza, Basel, Switzerland) and subjected to electroporation.

[0206] For GEX® cells, program T-016 of the Amaxa Nucleofector (Lonza, Basel, Switzerland) was used. Electroporated cells were subsequently transferred into a 6-well plate containing 2.5 mL pre-warmed medium and cultivated under standard conditions. One day after transfection, the cells were counted again to assess the viability. Two days after transfection, cells were counted and seeded at a cell density of 2x105 cells / mL in culture medium containing the respective agents (puromycin (Takara Bio Eur., St. Germain-en-Laye, France) and / or methotrexate (Sigma Aldrich, Steinheim, Germany)) for selection pressure and gene amplification. Selection pressure was increased every 2 weeks in the according steps: 50 nM methotrexate / 0.4 pg / mL puromycin, 100 nM / 0.4 pg / mL, 200 nM / 0.8 pg / mL, 400 nM / 1.6 pg / mL to max. 800 nM / 3.2 pg / mL.

[0207] Production of cell culture supernatant in spinner flasks

[0208] Antibody-containing supernatants were produced in low-serum culture medium without selection pressure. Cells were grown in 0.5 - 1 L CELLSPIN glass spinner flasks (Pfeiffer GmbH, Lahnau) and stirred at 60 rpm under standard culture conditions. Cells were counted regularly to monitor viability and cell density. When cultures reached viabilities below 80%, supernatants were harvested by centrifugation of the cell suspension at 3,000 x g for 30 min. Residual cells and cell debris were separated by passing the supernatant through a 1.2 pm filter (Pall, Dreieich, Germany) before immediate purification of antibodies or storage of the supernatants at - 20 °C.

[0209] Titer measurement with the Octet® QKe system

[0210] The IgG-titers in cell culture supernatants were determined with Protein A sensors (ForteBio, Fremont, USA) using the Octet® QKe system. After centrifugation of the cell suspensions (2000 rpm, RT, 5 min), 200 pL of the supernatant (undiluted) and in multiple dilution steps was added to a 96-well plate for the determination of the titer. For quantification of antibody titers, a standard curve with Pankomab-GEX® in GTM 7.1 medium (comprising the same IgG-Fc portion) was employed.

[0211] Affinity-purification of IgG and antibody fragments from cell culture supernatant

[0212] IgG antibodies and antibody fragments were purified by affinity chromatography using the AKTA Prime plus or AKTA start system (GE Healthcare, Uppsala, Sweden). Supernatants stored at - 20 °C were thawed overnight at 4 °C or for 1-2 h at 37 °C. All buffers and supernatants were passed through a 0.22 pm filter before use.

[0213] Chimeric IgG was purified using HiTrap MabSelect SuRe protein A 1 mL or HiScreen MabSelect SuRe LX protein A 5 mL columns (GE Healthcare, Uppsala, Sweden). Supernatants were kept on ice while loading them onto the column with flow rates between 0.5 - 2 mL / min (depending on column volume). IgG was eluted with 0.1 M citrate, 0.15 M sodium chloride pH 3.6. Fractions were pooled and rebuffered in low-endotoxin D-PBS (Biochrom, Berlin, Germany) using Amicon Ultra 50 kDa filter units (Merck Millipore, Darmstadt, Germany). Before long-term storage at 4 °C, samples were passed through a 0.22 pm filter.

[0214] Purifications of antibody fragments were performed using Strep-Tactin® Superflow® high capacity cartridge columns with a column volume (CV) of 1 mL. After thawing, 200 pL BioLock Biotin blocking solution (IBA GmbH, Gottingen) was added to 1 L supernatant (in excess) and incubated under stirring conditions for 1 h at RT to prevent binding of possible biotin in the medium to the column. The pH of the supernatants was adjusted to pH 8.0 with 3 M NaOH prior to sterile filtration. Binding and elution was performed according to manufacturers protocols using binding, elution and column regeneration buffers (Buffer W, Buffer E, Buffer R; all IBA GmbH, Gottingen). Elution fractions were pooled and rebuffered in low-endotoxin D- PBS (Biochrom, Berlin, Germany) using Amicon Ultra 10 kDa filter units (Merck Millipore, Darmstadt, Germany). Before long-term storage at 4 °C, samples were passed through a 0.22 pm filter.

[0215] Part 2

[0216] In addition, different minibody design variants and F(ab’)2-fragments as well as singe-chain diabodies were designed (Figure 1) and expressed, namely PXP0056-scDb-ABD-glyc, PXP0059-Mb-ABD-glyc, PXP0071-Mb-NH1-glyc, PXP0062-F(ab’)2-ABD-glyc and the respective non-glycosylated formats.

[0217] Briefly, cDNA of antibody constructs was cloned into a mammalian expression vector, which was then transfected into CHO cells for transient production of the respective antibody formats. ScDb- and minibody constructs were recombinantly expressed with a tandem version of the Strep-tag® (Twin-Strep-tag®, IBA, Germany) to enable purification from cell supernatant by affinity chromatography and for detection. F(ab’)2-fragments were purified from cell culture supernatants via CaptureSelect CH1-XL (Thermo Scientific™, Germany) and preparative SEC.

[0218] Antibody titers were determined using LabChip microfluidic CE-SDS (LabChip GXII Touch HT Protein Characterization System (Perkin Elmer)).

[0219] For all formats, scDb, minibody and F(ab’)2, glycosylated antibody variants were successfully expressed in the CHO expression system (Figure 6). For scDb and F(ab’)2 the titers for glycosylated and wildtype formats were very similar, in the range of 300-400mg / L (Figure 6, Table 5). In the case of minibody variants, Mb-ABD-glyc and Mb-NH1-glyc even surpassed the expression levels of the non-glycosylated format with titers of ~360mg / L and ~275mg / L, respectively (Table 5). (Purified protein amounts of minibodies revealed that Labchip titer determination does possibly even underestimate the titer of minibody formats in supernatants.) Table 5 - Antibody titers ofscDb, minibody and F(ab’2) variants analyzed from CHO cell culture supernatant by LabChip microfluidic CE-SDS.

[0220] Sample Titer [mg / L]

[0221] PXP0055-scDb-ABD 330.0

[0222] PXP0056-scDb-ABD-glyc 391.0

[0223] PXP0058-Mb-ABD 11.0

[0224] PXP0059-Mb-ABD-glyc 148.0

[0225] PXP0069-Mb-NHl 82.4

[0226] PXP0071-Mb-NHl-glyc 275.1

[0227] PXP0061-F(ab')2-ABD 354.0

[0228] PXP0062-F(ab')2-ABD-glyc 362.0

[0229] Example 2: Qualitative analysis of antibody fragments by SDS-PAGE

[0230] Part 1

[0231] The purity of antibody preparations was analyzed by SDS-PAGE (Figure 7). Of major interest in the antibody fragment generation was also the occupancy of glyco-variants with N-glycans. Initially, the presence of N-glycans was investigated with PNGase F treatment and the thereby potentially emerging shift in the molecular weight. PNGase F cleaves N-linked oligosaccharides of high mannose, hybrid, and complex type between the innermost GIcNAc and asparagine residues.

[0232] Due to the denaturing character of SDS the non-covalent interactions in the diabody molecules are disturbed and the unlinked diabodies are separated into the monomer chains (Figure 7).

[0233] Th69diaWT and Th69scFvWT show a double band with a lower, more prominent band which matches the respective calculated molecular weight of ~28 kDa, and a less prominent band at ~30 kDa (data not shown). For Th69scDbWT this separation is not visible as good.

[0234] Analysis of the upper vs. lower bands of Th69scFvWT and Th69diaWT was performed from preparative SDS-PAGE, tryptic-in-gel-digestion / extraction and subsequent LC-MS as well as direct tryptic digest from samples and LC-MS. It revealed a lysine-hydroxylation in the Twin- Strep-tag® sequence adjacent to the G4S-sequence and subsequent O-glycosylation comprising 2 hexose residues for the proteins with a higher molecular weight (data not shown).

[0235] For Th69scFvWT the proportion of hydroxylated and glycosylated lysine amounted to 33%; this corresponds to the band intensities. That modification, resulting in a mass addition of +340 Da (hydroxylation +16 Da, 2x hexose +162Da), was also prominent by intact mass analysis (see Figure 17, Figure 25). A second, considerably less prominent post-translational modification is O-glycosylation in the C-terminal peptide with a di-sialylated HexHexNAc tetrasaccharide. These two post-translational modificaions (PTMs) are not expected to impact on functionality of the antibodies.

[0236] In comparison to the Th69diaWT, Th69dia1Glyc with one N-glycosylation site per monomer shows an increase in molecular weight of a few kilo-Daltons. Th69diaGlyc carrying two N- glycosylation sites per monomer shows an accordingly further increase of ~6-8 kDa in molecular weight, indicating a complete occupancy of both N-glycosylation motifs. This same increase of ~6-8 kDa was observed with Th69scFvGlyc and Th69scDbGlyc, also suggesting the occupancy of both N-glycosylation sites. The bands of the glycosylated molecules without PNGase F treatment appear more diffuse indicating very different N-glycan variants and respective heterogeneity in the molecules molecular weight.

[0237] After treatment with PNGase F the samples showed a decrease in the molecular weights to their respective wildtype formats, confirming the occupancy of all glycosites that were introduced. Intensities of the upper bands (with hydroxylation and O-glycosylation) in the double bands differ. The intensity of the upper bands decreases with the number of N- glycosylation sites introduced. The proteins are of very high purity, no aggregates or protein fragments are visible. The PNGase F treated Th69scDbGlyc sample shows a faint band ~30 kDa for the PNGase F added.

[0238] SDS-PAGE was performed using Mini-PROTEAN TGX™ precast gels with 10% for IgGs and 20% for diabodies and gels with a gradient from 4 - 20% (Bio-Rad, Feldkirchen, Germany). 1 pg of each sample was loaded per lane in non-reducing SDS sample buffer (non-reducing 4x sample buffer: 375 mM Tris-HCI, 10% SDS, 50% glycerol, 0.03% bromophenol blue). Proteins were incubated at RT for 10 min. Pre-stained ROTIOMark TRICOLOR protein marker (Carl Roth, Karlsruhe, Germany) was used as a protein standard. Gels were run in 1x Rotiphorese Tris / Glycin / SDS running buffer (Carl Roth, Karlsruhe, Germany) at 70 V for 15 min and then for 130 V for 55 - 70 min. Proteins were visualized using Coomassie staining solution (0.4 g / L Coomassie Brilliant Blue G-250 (Carl Roth, Karlsruhe, Germany), 5% (v / v) ethanol, 3.5% (v / v) perchloric acid).

[0239] Part 2

[0240] Wildtype and glycosylated formats were analyzed by SDS-PAGE to assess purity and if the formats with artificial N-glycosylation sites indeed carry N-glycans (Figure 8). For analysis, the samples were treated with reducing agent and separated on NuPAGE 4-12 % Bis-Tris Plus SDS-PAGE gels. llnder reducing conditions, PXP0058-Mb-ABD shows its main band corresponding to the reduced monomer chain at ~55kD, and PXP0061-F(ab’)2-ABD its corresponding light chain at ~30kD and heavy chain part at ~35kD. PXP0055-scDb-ABD stays intact under reducing conditions and displays its corresponding band at ~62kD. For PXP0056-scDb-ABD-glyc and PXP0059-Mb-ABD-glyc the main bands shift to 3-5kD higher molecular weights, and for PXP0062-F(ab’)2-ABD-glyc also the heavy chain carrying the introduced N-glycosylation site shifts up to 3-5kD higher molecular weight (Figure 8 A).

[0241] As for Th69-antibody formats, occupancy of glycosylation site was also qualitatively analyzed by SDS-PAGE after PNGase F treatment (Figure 8 B). After treatment with PNGase F PXP0056-scDb-ABD-glyc and PXP0059-Mb-ABD-glyc as well as the heavy chain of PXP0062- F(ab’)2-ABD-glyc showed a decrease in the molecular weights to the molecular weight of their respective wildtype formats, confirming the occupancy of the glycosites that were introduced. For PXP0062-F(ab‘)2-ABD-glyc a faint band detected at ~35kD (marked with *) indicates a proportion of -10-20% of non-glycosylated heavy chain part.

[0242] Analysis of PXP0071-Mb-NH1-glyc and its respective non-glycosylated format PXP0069-Mb- NH1 from the cell culture supernatants in Labchip CE-SDS also indicates prominent N- glycosylation and no residual band for incompletely occupied N-glycosylation sites (Figure 8 C).

[0243] In summary, these observations confirm that antibody formats with artificial N-glycosylation sites can be expressed in both human as well as CHO expression systems.

[0244] Example 3: Site-Occupancy determination of N-glycosylation sites by Peptide Mapping

[0245] The actual occupancy of the N-glycosylation site is of special interest since only molecules carrying N-glycans can be labeled with a cytotoxic payload and be therapeutically active. Siteoccupancy of the N-glycosylation sites was determined on the peptide level by differentiating and quantifying non-glycosylated versus glycosylated peptides. Release of N-glycans from a glycosylation site by PNGase F results in asparagine deamidation to aspartate whereas the non-glycosylated asparagine residue remains unaffected. This allows for separation of the peptide variants on high resolution RP-UPLC and detection of the resulting mass shift Am / z of 0.98 Da for singly charged ions. For quantification extracted ion chromatograms were generated to calculate peak areas of the deamidated and corresponding native peptides. The relative peak area of the deamidated peptide was calculated with respect to the overall peak area of the deamidated and corresponding native peptide to result in the site-specific relative molar amount of deamidation. Table 6 summarizes the amino acid sequences, masses of the multiple charged ions used for generation of extracted ion chromatograms and the relative abundance of deglycosylated peptides. All antibody fragments show a complete occupancy of the introduced N-glycosylation sites. Only in Th69diaGlyc, the second N-glycosylation position closer to the C-terminus is marginally less occupied with 98,5%. Table 6 - Site occupancy of N-glycosylation sites in antibody formats.

[0246] 50 g of dried sample were applied to sample preparation. The sample was re-dissolved in 100 pL of denaturation buffer (6 M guanidine hydrochloride in 1 mM EDTA and 0.25M Tris pH 7.5) followed by incubation for 2 min at room temperature. Subsequently, 10 mM DTT was added followed by incubation for 30 min at 37 °C and 700 rpm.

[0247] For alkylation, 2 pL of 1 M IAA was added (final cone. 20 mM), followed by incubation for 30min at room temperature in the dark.

[0248] After denaturation the samples were purified with gelfiltration mini columns Centripure MINI Z- 25 (emp BIOTECH GmbH, Berlin, Germany). To 100 pL of the sample trypsin (1 :25 w / w) were added with subsequent incubation for 3 h at 37 °C. Samples were deglycosylated by addition of 5m II of N-glycanase F (PNGaseF) and incubation for 1 h at 37 °C. Deglycosylation was terminated by addition of 6 pL of 10% formic acid (v / v 1 :20). The samples were dried in a Speedvac and stored at -20 °C until LC-MS / MS measurement. The resulting mixture of peptides was separated by an Acquity CSH C18 reversed-phase column (2.1 mm x 150 mm, 1.7 u; (Waters, Milford, USA)) and analyzed by high-resolution ESI-QTOF MS / MS. Data were evaluated employing the software ProteinScape (Bruker Daltonik, Hamburg, Germany) and Mascot (Matrix Science, Boston, USA). Relative quantification of peptides was calculated using Microsoft Excel. Example 4: N-glycan profiling

[0249] Part 1

[0250] Besides the occupancy of the glycosites which determines the proportion of molecules to be labeled with a payload, the N-glycan pattern is another important parameter. As illustrated in Figure 4, the SiteClick™ Azido Modification introduces azide residues to the antennary GIcNAc, so that the N-glycan structure defines the possible conjugation positions and therewith the possible drug-to-antibody ratio.

[0251] Relative quantification of N-glycan structures was based on peak areas of fluorescence signals, which reflect relative molar amounts of N-glycans. In case of co-eluting structures additionally mass spectrometric quantification was used. Relative peak areas of peaks that could not be annotated with certainty ranged from 0.9% for Th69diaGlyc to maximum 3.7% for Th69scDbGlyc. The N-glycan profile of Th69scFvGlyc was annotated with significant and high abundant structures (Figure 9). N-glycan profiles for Th69dia1Glyc, Th69diaGlyc and Th69scDbGlyc were also analyzed and quantifications are summarized below. For all glycosylated antibody fragments, the N-glycoprofiles revealed a very comparable glycosylation with respect to the N-glycan structures attached. Abundance of distinct N-glycans differs between the samples. The predominant N-glycan in all preparations is the core-fucosylated biantennary, di-sialylated glycan (FA2G2S2), followed by core-fucosylated mono-sialylated glycans with and without bisecting GIcNAc or outer-arm fucose (FA2G2S1 , FA2BG2S1 , FA2BFG2S1 , FA2FG2S1). Only for the Th69scDGIyc a triantennary glycan (FA3G3S1) is of second highest abundance.

[0252] Relative molar amounts of individual N-glycan structures were summarized to distinct N-glycan parameters (Figure 9 B-D; Figure 31). Regarding the antennarity, about half of the N-glycans are bi-antennary, on average 30% triantennary and 15% tetraantennary (Figure 9 C). Th69dia1Glyc and Th69scDbGlyc show a slightly higher degree of biantennary glycans in comparison to the other two formats. In correlation, Th69scFvGlyc reaches the highest amount of four antennary GIcNAc residues with -20% of N-glycans, followed by Th69diaGlyc of -16%. Overall, all four glycosylated antibody constructs bear N-glycans with on average 2.5 GIcNAc residues that can be installed with linker payloads. Assuming complete modification and conjugation, this would generate a drug load of -5 for Th69scFvGlyc, Th69dia1Glyc and Th69scDbGlyc, and -10 for Th69diaGlyc.

[0253] Analysis of sialylation showed that N-glycans are predominantly mono- and di-sialylated, to a lower extent tri-sialylated and rarely tetra-sialylated (Figure 9 D). In practice, this means that antibody fragments require a desialylation step prior to glycomodification.

[0254] The degree of core-fucosylation varies from 90% to 97%, outer-arm fucosylation (oF; also termed as Lewisa / Lewisx epitope) from 11% to 18%. Presence of core-fucose (F; Figure 9 B) is not of special relevance, detection of outer-arm fucose however was relevant for interpreting the glycomodification with SiteClick™ . Branches with outer-arm fucose cannot be cleaved by P-1 ,4-galactosidase (see Figure 9B) and therefore cannot be activated with GalNAz, which may be beneficial in terms of the drug load. Among the four antibody fragments, the proportion of N-glycan structures with bisecting GIcNAc is similar with 27-31%. N-acetyllactosamine is only prominent in Th69scFvGlyc (Figure 9 B). Identification of all glycan structures and their quantification were essential to interpret mass spectra of glycoproteins in the analysis of the glycomodification and the drug conjugation.

[0255] Taken together, SDS-PAGE and peptide mapping showed a complete occupancy of the introduced N-glycosylation sites for all four glycosylated antibody fragments. This is the best possible starting point for generating fragment-drug conjugates. Glycan profiling revealed a relatively wide spectrum of N-glycans, the presence of sialylation, and a relative quantification of antennary GIcNAc residues that are indicative for the possible drug load of the antibody fragments.

[0256] N-glycan structures of IgG and antibody fragments were determined and quantified by HILIC- UPLC-FLD-ESI-QTOF-MS / MS (ultra- performance hydrophilic interaction chromatography with fluorescence detection coupled to electrospray injection quadrupole time-of-flight tandem mass spectrometry). The samples were denatured and reduced in the presence of TCEP. The N-glycans were released by action of PNGase F and subsequently fluorescence tagged with RapiFluor-MS™ (Waters, Milford, USA) (using a protocol proprietary to Glycotope GmbH). The mixture of fluorescence labeled N-glycans was separated by HILIC UPLC employing an Acquity l-Class UPLC instrument with fluorescence detection (Waters, Milford, USA) and the ACQUITY UPLC Glycan BEH Amide Column (130A, 1.7 pm, 2.1 mm X 150 mm) (Waters, Milford, USA). A gradient from 22-44 %B within 82 min (A: acetonitrile, B: 100 mM ammonium formate, pH 4.5) was applied.

[0257] Quantification of N-glycans was based on fluorescence signals. For structure identification online coupled high resolution ESI-QTOF MS / MS (Bruker Daltonik, Hamburg, Germany) was used in the positive ion mode. Relative quantification of N-glycan structures was based on peak areas of fluorescence signals, which reflect relative molar amounts of N-glycans. In case of co-eluting structures additionally mass spectrometric quantification was used.

[0258] Molar amounts of individual N-glycan structures were summarized to the following N-glycan parameters (Figure 9, Figure 31):

[0259] A1-A4: complex type N-glycans with mono- to tetraantennary N-acetylglucosamin

[0260] GO: non galactosylated complex type N-glycans

[0261] G1-4: mono- to tetragalactosylated complex type N-glycans SO: non sialylated complex type N-glycans

[0262] S1-S4: mono- to tetrasialylated complex type N-glycans

[0263] F: core-fucosylated N-glycans oF: outer-arm fucosylated N-glycans

[0264] B: complex N-glycans with bisecting GIcNAc tG: complex N-glycans with terminal galactose

[0265] HB: hybrid type N-glycans

[0266] LacNAc: complex N-glycans with N-acetyllactosamine

[0267] M: high mannose type N-glycans

[0268] NeuGc: complex N-glycans with N-glycolylneuraminic acid phos: phosphorylated N-glycans sulf: sulfated N-glycans

[0269] LeA / LeX: complex N-glycans with LewisA or LewisX

[0270] LeB / LeY: complex N-glycans with LewisB or LewisY

[0271] Part 2

[0272] As for Th69-antibody formats, also N-glycan profiles were determined for PXP0056-scDb- ABD-glyc, PXP0059-Mb-ABD-glyc and PXP0062-F(ab’)2-ABD-glyc (Figure 10) with the same method (see above).

[0273] For PXP0056-scDb-ABD-glyc and PXP0059-Mb-ABD-glyc the N-glycan profiles were very similar, carrying about 50-60% bi-antennary and -25% tri-antennary N-glycans (Figure 10 D). PXP0056-scDb-ABD-glyc shows a slightly higher degree of -12% tetra-antennary N-glycans. Highest complexity of N-glycosylation was found on PXP0062-F(ab’)2-ABD-glyc with more than 70% of tri- and tetraantennary N-glycans including also a higher degree of LacNAc structures (Figure 10 C).

[0274] Given the high occupancy of N-glycosylation sites and the observed N-glycan pattern, also minibody and F(ab’)2 formats - besides scFv and diabody and scDb - can be used to conjugate different compounds via either their core- or antennary N-glycans. Example 5: Evaluation of antigen binding

[0275] Part 1

[0276] Correct assembly of the VH and VL is essential for antigen binding. Therefore, the antibodies were analyzed by size exclusion chromatography under physiological conditions to assess the assembly of Fv-fragment of the scFv preparations and the association of complementary VH / VL pairs to form the bivalent dimers in diabody preparations.

[0277] Characterization by SEC

[0278] Chromatogram overlays of the wildtype formats show major peaks with the same retention for Th69diaWT and Th69scDbWT and a stronger retention for the Th69scFvWT format (Figure 11 A). This reveals that the Th69diaWT actually dimerizes to form intact diabody molecules and that the main proportion of Th69scFvWT forms a monovalent Fv fragment.

[0279] The overlays of the glycosylated and non-glycosylated formats show less retention of the glycosylated constructs in comparison to the wildtype variants (Figure 11 B). SDS-PAGE and Western Blot analysis rule out the possibility of immoderate dimer formation for all glycosylated formats. The actual degree of retention is higher than the 1-2 kDa per N-glycan should affect the size of the molecules. For instance, Th69scFvGlyc has about the same retention time as the diabody Th69diaWT which corresponds to a mass difference to Th69scFvWT of 28 kDa. So, the introduced N-glycans greatly influence the hydrodynamic radius of the antibody fragments. This may be relevant for in vivo plasma clearance and biodistribution.

[0280] Diabody assembly was very efficient, Th69diaWT preparations contained 99% of assembled chains (defined as monomers; Table 7, below). Glycosylated diabody molecules showed a decrease in this assembly. Th69scFvWT preparation contained 17.5% dimers and Th69scDbWT preparation contained 10.2% dimers / multimers. In comparison, Th69scFvGlyc sample comprised only 7.9% dimers and Th69scDbGlyc 5.1%. So, the glycosylation seemed to have a positive effect on monomer content for these two formats.

[0281] To be able to compare the antibody formats in functional assays, monomers were prepared by size exclusion chromatography to reach >95% monomers in preparations. Thereby, an influence of aggregates should be excluded. Table 7 - Size exclusion chromatography analysis of antibody fragments.

[0282] , Fragments /

[0283] SampleeSr„.. Dimers [%] Monomer [%] unpaired chains

[0284] Multimers [%] . ,

[0285] Th69scFvWT - 17.52 82.48

[0286] Th69scFvGlyc - 7.91 92.04

[0287] Th69diaWT - - 98.98 1.02

[0288] Th69dialGlyc - - 92.12 7.88

[0289] Th69diaGlyc - - 91.34 8.66

[0290] Th69scDbWT 3.36 6.87 89.77

[0291] Th69scDbGlyc - 5.12 94.88

[0292] Characterization of antigen binding

[0293] To examine possible differences in avidity of the antibody formats and how the introduction of the N-glycosylation may influence the affinity and avidity to the antigen, binding assays with DERL-7 cells using flow cytometry and with target antigen using ELISA were performed. In both assays, antibodies were detected by anti-StrepTag reagents. Since all diabody (dia) molecules contain two tags per molecule and thereby double the fluorescence or colorimetric signals in comparison to scFv and scDb molecules at the same number of bound molecules, antibody concentrations were adjusted to the respective molar concentrations of the Twin- Strep-tag® to be able to compare the values of the MFI (Figure 13 B) and absorbance in ELISA (Figure 13 C) for the different antibody formats.

[0294] Regarding the proportion of positive cells (Figure 13 A), wildtype formats showed antigen binding in the following order (as determined by non-linear regression analysis): diaWT (EC50=505 pM) > scDbWT (EC50=778 pM) > scFvWT (EC50=1104 pM). The respective glycosylated formats Th69dia1 Glyc and Th69scDbGlyc showed 1.3-1.45-fold decreased EC50 values, Th69scFvGlyc and Th69diaGlyc 1.8-fold. For Th69dia1Glyc and Th69scDbGlyc this implies a 1 .3-1.45-fold weaker antigen binding. For Th69scFvGlyc and Th69diaGlyc however, the increased differences may arise either from lower avidity or from hindered detection by the secondary reagent due to N-glycosylation in the Twin-Strep-tag® linker sequence. This constraint also applies to interpretation of the two next measurements.

[0295] Regarding the median fluorescence intensity (Figure 13 B), which reflects the absolute number of bound molecules, different maximal intensities were observed. Th69scFvWT reached the highest intensity, followed by diaWT and scDbWT. This effect was even more prominent for Th69scFvWT and Th69scFvGlyc on HSB-2 cells with higher target expression (data not shown). At saturing antibody concentrations, more monovalent scFv formats than bivalent diabody formats can occupy the receptors on the cell surface. By evaluating the EC50 values, wildtype formats showed antigen binding in the following order: scDbWT (EC50=2796 pM) > diaWT (EC50=4675 pM) > scFvWT (EC50=5273 pM). Here, respective glycosylated formats showed 1.2-1.4-fold decreased binding.

[0296] Analysis of binding to immobilized target antigen in ELISA (Figure 13 C), scDbWT showed the best antigen binding (EC50=322 pM), followed by scFvWT (EC50=364 pM) and diaWT (EC50=385 pM) with respect to the concentration dependent binding curve. In a total of three ELISA assays, scFvWT and diaWT actually reveal an almost equal average EC50, so both have comparable antigen binding in ELISA. The glycosylated formats showed again 1.2-1.4- fold decreased binding compared to their wildtype formats.

[0297] In summary, the affinity of antigen binding is only minimally affected by the introduction of N- glycans in the linker sequences. Considering the three interpretations above, both diabody formats (dia, scDb) showed a slightly better binding than the scFv format, and the binding to DERL-7 cells indicated that bivalency of these antibodies may also result in doubled avidity to the target antigen. Nevertheless, a definite, quantitative assessment of avidity is not possible with this assay setup.

[0298] Assays were performed as follows:

[0299] Size-exclusion chromatography

[0300] IgG antibodies, antibody-drug conjugate (ADC) and antibody fragment-drug conjugate (FDC) constructs were analyzed sterile filtered (0.22 pm) in D-PBS (Biochrom, Berlin, Germany) using the HPLC 1200 / 1260 (Agilent Technologies, Waldbronn, Germany). Protein amounts of 10 - 30 pg in volumes of 8 - 30 pL were injected onto a Superdex 200 Increase 10 / 300 GL column (10x30 mm, (GE Healthcare, Uppsala, Sweden)). The proteins were eluted with an isocratic flow rate of 0.75 mL / min and D-PBS as the running buffer. For analytical SEC of antibody fragments, 100 - 150 pg antibody in 200 pL D-PBS (sterile) were injected onto a Superdex 200 10 / 300 GL column (GE Healthcare, Uppsala, Sweden), using AKTAprime plus chromatography system. The proteins were eluted with an isocratic flow rate of 0.5 mL / min and D-PBS as the running buffer. Monomer preparations of chimeric IgG or antibody fragments were produced by size exclusion chromatography (SEC) using the AKTAprime plus chromatography system with low-endotoxin D-PBS (Biochrom, Berlin, Germany) as running buffer. Up to 2 mg antibody in 500 pL was injected onto a Superdex 200 10 / 300 GL column (GE Healthcare, Uppsala, Sweden). Monomer fractions were pooled, concentrated in Amicon Ultra 10 kDa or 50 kDa filter units (Merck Millipore, Darmstadt, Germany) and passed through a 0.22 pm sterile filter before storage at 4 °C. Antigen ELISA

[0301] Nunc MaxiSorpTM F96 ELISA plates (Thermo Fisher Scientific, Dreieich, Germany) were coated with 1 pg / mL target antigen in 0.1 M carbonate buffer pH 9.6 and incubated overnight at 4 °C. After each incubation, plates were washed three times with a buffer containing PBS and 0.05% (v / v) Tween 20 (Carl Roth, Karlsruhe, Germany). Unspecific binding was blocked with 2% (v / v) bovine serum albumin (BSA) in PBS for 2 h at RT. Serially decreasing dilutions (usually 1 :2) of either antibodies, antibody fragments, ADCs or FDCs in 1% (v / v) BSA / PBS were incubated for 1 h at RT.

[0302] For IgG detection, horse-radish peroxidase (HRP)-conjugated goat anti-human (IgG-Fcy) antibody (Jackson ImmunoResearch, Ely, UK) was used diluted 1 :20 000 - 1 :40 000 in 1% (v / v) BSA / PBS. Antibody fragments were detected with Strep-Tactin-HRP (IBA GmbH, Gottingen), diluted 1 :2000 in 1% (v / v) BSA / PBS. For peroxidase activity detection, TMB One Component HRP Microwell Substrate (Tebu-bio laboratories, Offenbach, Germany) diluted 1 :1 with ultra-pure water (Biochrom, Berlin, Germany) was added and the reaction was stopped by addition of 1.25 M sulfuric acid. Absorbance was measured at 450 nm and 620 nm using the EnSpire 2300 Multilabel reader (Perkin Elmer, Waltham, USA) or Spark® Multimode Microplate Reader (Tecan, Crailsheim, Germany).

[0303] Part 2

[0304] Characterization by SEC

[0305] Correct assembly and integrity of the molecules was further assessed by analytical size exclusion chromatography using Waters SEC Column ACQUITY UPLC Protein BEH SEC (200A, 1 ,7 pm, 4,6 mm x 300 mm) on an Agilent 1290 device. Overall, all molecules show a proper assembly and good purity (Figure 12). Both scDb-formats show a low percentage of dimers / multimers (Table 8). Both F(ab’)2 formats show a certain degree of fragments, namely unpaired heavy chain and light chain (as determined by SDS-PAGE under non-reducing conditions, data not shown). Overall, introduced N-glycosylation does not show negative influence on conformation of antibody formats. Table 8 - Size exclusion chromatography analysis ofscDb, minibody and F(ab’2) variants.

[0306] Sample Multimers / Dimers [%] Monomers [%] Fragments [%]

[0307] PXP0055-scDb-ABD 3.4 96.6

[0308] PXP0056-scDb-ABD-glyc 6.9 93.10

[0309] PXP0058-Mb-ABD 2.95 97.05

[0310] PXP0059-Mb-ABD-glyc 0.81 99.19

[0311] PXP0061-F(ab')2-ABD 0.17 95.32 4.51

[0312] PXP0062-F(ab')2-ABD-glyc - 92.91 7.09

[0313] Characterization of antigen binding

[0314] Since the position of glycosylation sites is different between the formats, analysis of antigen binding is of particular interest to not disrupt antigen binding. In scDb and minibody formats, N-glycosylation site was introduced close to the binding regions in the connecting linkers between the VH-VL binding domains whereas in the F(ab’)2-fragment the linker comprising the glycosylation site is introduced between the hinge-region and the C-terminal albuminbinding domain.

[0315] Antibodies were first analyzed in antigen ELISA assays for specific binding to its target-proteins A and B at two concentrations (35nM and 1.75nM). To control off-target binding, exemplary irrelevant proteins C and D were used (Figure 14). Briefly, selected protein antigens were coated to 96-well plates overnight, unspecific binding was blocked and test antibody samples were added. ScDbs and minibodies were detected using StrepTactin reagent, whereas F(ab’)2 and reference lgG1 were detected using anti-F(ab’)2 detection reagent for comparison. Reference IgG is the natural IgG from which scDb, Minibody and F(ab’)2 fragments were derived.

[0316] All constructs showed the expected binding pattern on on / off-target proteins - with stronger binding to protein A than to protein B and no significant binding to off-target proteins C and D (Figure 14). In particular, binding of PXP0056-scDb-ABD-glyc was very similar to its nonglycosylated counterpart, as well as PXP0059-Mb-ABD-glyc binding very similar to PXP0058- Mb-ABD. Moreover, both PXP0061-F(ab’)2-ABD and PXP0062-F(ab’)2-ABD-glyc bound very comparable to the reference IgG 1 antibody.

[0317] Binding of all formats to on- and off-target cell lines was next also tested (Figure 15). Respective cell lines were stained with the antibody formats and subsequently scDbs and minibodies were detected with StrepMAB-lmmo reagent, and F(ab’)2 fragments and reference lgG1 were detected with anti-F(ab’)2 detection reagent. Further controls included a control antibody for target antigen expression and for the TF glycan, both detected with an anti- hlgG+IgM detection reagent. As read-out signal-to-noise ratios of antibody staining vs. secondary reagent or isotype controls were calculated. In addition, titration of scDb and minibody formats was performed on MCF-7 cells with detection via StrepMAB-lmmo reagent.

[0318] As determined for the reference antibody (lgG1), the constructs are expected to show binding to the target-expressing F9 cells (carrying the TF O-glycan), and no or low binding to 1) F9cosmcKO cell line carrying Tn glycans but lacking target expression, 2) to the targetexpressing HEK cells but lacking O-glycan expression and 3) HL-60 cells carrying TF glycans but lacking target expression (Figure 15C).

[0319] All targeting constructs showed desirable binding to on-target- F9 cells (TF+), but not to HL- 60 and target-expressing HEK but non-O-glycosylated cells (Figure 15 A and B). Low binding to F9cosmcKO cells was observed with PXP0056-scDb-ABD-glyc and PXP0059-Mb-ABD-glyc constructs. However, no signs of higher background binding were observed on other off-target cell lines.

[0320] S / N ratios of the scDbs were reduced by half to two third compared to Mbs, however it is not clear if this is due to lower binding, or reduced detectability within the TwinStrep-Tag of scDb vs minibodies as it was not observed in ELISA binding. F(ab’)2 constructs again bound comparable to the reference lgG1.

[0321] Within each group of glycosylated vs. non-glycosylated compounds, the fragments bound very comparable to each other, resulting also in similar EC50 values for binding on MCF7 tumor cells as shown for PXP0056-scDb-ABD-glyc vs. PXP0055-scDb-ABD and PXP0059-Mb-ABD- glyc vs. PXP0058-Mb-ABD (Figure 16).

[0322] Example 6: Development of antibody- and fragment-drug conjugates

[0323] Two different site-specific labelling techniques were examined for the antibody fragments, as depicted in Figure 4. The SiteClick™ glycan modification (Figure 4) employs (3-1 ,4- galactosidase and trims the N-glycans to the antennary GIcNAc. GlyCLICK® modification by contrast employs the endoglycosidase EndoS2 (Figure 4). EndoS and EndoS2 both catalyze the hydrolysis of the p-1 ,4 linkage between the two GIcNAcs in the core of the N-linked glycan of human IgG.

[0324] The specificity of EndoS and EndoS2 has so far been described to be limited to IgG molecules, and biantennary N-glycans in the acute phase protein a1-acid glycoprotein in the case of EndoS2. Both glycan-modifications have only been evaluated for IgGs so far. The IgG was only modified via GlyCLICK®. Glycomodification using GlyCLICK® and SiteClick™ Glycomodification of antibody fragments

[0325] Efficient incorporation of N-acetylazidogalactosamine by enzymatic modification is necessary to enable drug conjugation to the antibody fragments. The efficiency of these reactions should in particular be evaluated because the amount of antibody was increased 3-fold (with respect to glycosylation sites) in contrast to manufacturers specification in order to limit costs. In detail, the reactions were scaled up from designated 1.75 nmol IgG (corresponding to 250 pg) to 26 nmol scFv, and 14 nmol scDb or diabody (corresponding to -800 pg each). Each step in the glycomodification process was monitored for all formats, but exemplified for Th69scFvGlyc. As the antibody fragments were highly sialylated and p-1 ,4-galactosidase is only active on terminal galactose residues, N-acetylneuraminic acid was first removed by Sialidase A, a Sialidase that removes 2,3- and 2,6-linked N-acetylneuraminic acid. Unmodified Th69scFvGlyc was not subjected to LC-MS analysis due to its very complex glycosylation pattern and 2 glycosylation sites in one molecule. Th69scFvGlyc was monitored for completeness of the reactions after Sialidase A treatment (Figure 17), p-1 ,4- galactosidase treatment (Figure 18) and subsequent activation reaction with p-1 ,4- galactosyltransfrase (Y289L) (Figure 17). For Figure 17 and Figure 18 the 16 and 18 most abundant peaks were annotated with the respective possible glycan combinations - in accordance with the abundance of N-glycans as determined by glycan profiling.

[0326] The mass spectra show very efficient modification in trimming of the N-glycans. No abundant peaks were detected for residual sialylated or terminal galactosyl N-glycan structures (Figure 17 and Figure 18). In the subsequent activation reaction, GalT(Y289L) introduced the N- azidoacetylgalactosamine to all available N-acetylglucosamine residues (Figure 19). Again, no abundant peaks were detected for incompletely activated N-glycans. The trimming and GalNAz attachment simplify the glycan pattern and generate a more homogenous glycoprotein mixture. Overall, the enzymatic modifications proceed very completely, also with increased amounts of starting antibody material.

[0327] The calculated molecular weights of Th69scFvGlyc with diverse possible N-glycan combinations after Sialidase A treatment is shown in Table 9 (MWof Th69scFvGlyc = 29465.5 Da). Table 9 - Calculated molecular weights of Th69scFvGlyc after Sialidase A treatment.

[0328] The calculated MW of Th69scFvGlyc with diverse possible N-glycan combinations after p-1 , 4- galactosidase treatment is shown in Table 10 (MW of Th69scFvGlyc = 29465.5 Da). Table 10 - Calculated molecular weights of Th69scFvGlyc after / 3-1 ,4-galactosidase treatment.

[0329] The calculated MW of Th69scFvGlyc with diverse possible N-glycan combinations after p-1 , 4- galactosidase treatment after SiteClick™ azide activation (with GalNaz) is shown in Table 11 .

[0330] Masses marked indicate most abundant glycan combinations (MW of Th69scFvGlyc = 29465.5 Da).

[0331] Table 11 - Calculated molecular weights of Th69scFvGlyc after SiteClick™ azide activation. Establishment of reaction conditions for conjugation of Th69scFvGlyc-MMAE

[0332] After successful azide-activation, conjugation reaction of antibody fragments with linkerpayload was established. The proof-of-concept evaluation was performed with a commercially available linker-payload DBCO-PEG4-vc-PABC-MMAE (Figure 2 A). Setting up conjugation reactions of proteins, which require mild physiological conditions, with a hydrophobic and bulky DBCO-linking-group and additionally hydrophobic toxin MMAE is a challenging task. First, the solubility of the linker-payload and the protein was tested in a mixture of buffer and organic solvent, more precisely PBS and a varying proportion of DMSO. Next, the reaction was optimized with respect to conjugation efficiency and material input of costly linker-payload. Reaction mixtures of Th69scFvGlyc with 2 eq., 5 eq. and 8 eq. of linker-payload (which correspond to LP:azide ratios of 0.38:1 , 0.96:1 and 1.5:1) were analyzed by reversed phase chromatography (Figure 20). The differentially conjugated molecules can be separated due to differences in the degree of hydrophobicity.

[0333] Th69scFvGlyc is conjugated with up to 7 linker-payload molecules. The identification of the drug loaded species is depicted in Figure 22. The reaction ratio of 2:1 led to a high proportion of 17% unconjugated scFv-molecules (Table 12), whereas with a ratio of 8:1 the proportion was below 1 .5%. Unfavorable for this setup is the amount of residual linker-payload. The ratio of 5: 1 , led to the most suitable ratio between the input of linker-payload and generating a highly conjugated ADC with only 2.5% DARO molecules. The overall DAR of unpurified Th69scFvGlyc-MMAE with this conjugation reaction is 3.8 (Table 12). With regard to reaction efficiency, the copper-free click reaction with DBCO proves to be a very efficient reaction since with a ratio of ~1 :1 all of the linker payloads molecules are consumed in the reaction and no further increase in conjugation was observed with an excess of linker-payload.

[0334] Table 12 - Quantification of drug load distribution of Th69scFvGlyc conjugation reactions. LP: linker-payload (DBC0-PEG4-vc-PAB-MMAE). ratio ratio , , average distribution of drug load [%] LP:mAb LP:azide DAR

[0335] 0 1 2 3 4 5 6 7

[0336] 2:1 0.38:1 17 20 23 13 21 5 0 0 2.17

[0337] 5:1 0.96:1 2.5 7 14 13 29 20 11 5 3.83

[0338] 8:1 1.5:1 1.4 5 12 16 33 23 10 0 3.80

[0339] Establishment of analytical methods for FDC characterization

[0340] As can be seen in chromatograms in Figure 20, the separation of conjugates antibody fragments is not sufficient to allow for accurate quantification. To solve this issue, chromatography parameters were adjusted to increase the resolution on reversed phase. Besides an increase in column temperature from 50 °C to 65 °C, also the LC-gradient was adapted (see method section below for specification). Previous analysis was performed with a steeper gradient (Figure 20) which resulted in a mountain-like peak resolution without a proper baseline separation.

[0341] When analyzing the same Th69scFvGlyc-MMAE conjugation reaction with the newly established gradient and temperature, the different drug load variants could not be absolutely baseline-separated with this column resin and eluent system but a considerably improved peak resolution was achieved (Figure 21A). The established method allows a proper quantification for evaluating the ADC conjugation reactions.

[0342] During conjugation reaction, a certain turbity could be observed in the mixture. After purification by ultrafiltration and sterile filtration, the final ADC preparations were clear solutions. Comparison of purified Th69scFvGlyc-MMAE sample with the reaction mixture in RPC shows an elimination of the peaks with higher drug load (Figure 21 B). This reveals that highly conjugated molecules with 5, 6 and 7 payloads per Th69scFvGlyc are prone to form irreversible aggregates. These aggregates could successfully be removed by purification.

[0343] DAR determination

[0344] Determination of the drug load distribution was performed with RP-LC-MS as depicted in Figure 22. Differentially conjugated molecules are separated based on hydrophobicity of the introduced payload. Respective drug-loaded variants were identified by mass spectra. N- glycan combinations were assigned in accordance with the abundance of N-glycans as determined by glycan profiling.

[0345] In purified ADC preparation, Th69scFvGlyc is conjugated with up to 4 payloads. In the unconjugated fraction (peak #0), Th69scFvGlyc-MMAE molecules with rather rare core N- glycans are included; the mass peak with the highest intensity could not be identified as by now (mass spectrum Figure 22 B). All identified glycosylation variants in Th69scFv-Glyc- MMAE comprised only biantennary structures. Molecules with tri- and tetraantennary N- glycans were not identified. Due to higher drug load, they obviously tend to stronger aggregation and are eliminated from ADC mixture by purification.

[0346] Initially, RPC analysis method of Th69scDb-formats was inapplicable for DAR determination. In order to assign and quantify differentially conjugated molecules via LC-MS, reversed-phase chromatography needed to be established.

[0347] Using a method previously applied for analysis of IgG light chain and heavy chain fragments, the Th69scDbWT could not be resolved as a distinct peak (gradient 20%-50% in 10 min; Figure 23 A). A first attempt to more efficiently elute the 58 kDa large Th69scDbWT was to increase the steepness of the gradient (gradient 25%-35% in 5.5 min, followed by 35%-80% in 3 min; Figure 23 B). However, resolution was still insufficient to be able to resolve differentially loaded drug conjugates. Indeed, elution of the scDb required a shallower gradient (28%-60% in 14.5 min), as shown for the drug conjugate Th69scDbGlyc-MMAE (Figure 23 C). Based on the satisfying peak shape, the drug to antibody ratio could be determined properly for the Th69scDbGlyc-MMAE SiteClick with the established elution profile. Site-specifc antibody fragment-drug conjugation using SiteClick™

[0348] The outcome of SiteClick™ conjugation was evaluated for Th69scFvGlyc, Th69dia1Glyc and Th69scDbGlyc. Th69dia1Glyc was chosen as the appropriate equivalent for scFv and scDb with respect to glycosylation sites. Figure 24 shows the results of antibody fragment-drug conjugate preparation with DBC0-PEG4-vc-PABC-MMAE by SiteClick™.

[0349] Mass spectra of the azide-activated antibody molecules depict the glycan pattern before conjugation (Figure 24A). All paramount detected masses could be assigned to glycosylated antibodies with introduced azide functionality. Th69dia1Glyc-MMAE elutes as monomer in RPC. So, for this diabody-ADC the drug load distribution and DAR are determined by combination of occupancy on the two monomer chains. Generated ADCs of all three formats showed a very comparable distribution of drug loading. Th69scFvGlyc and Th69scDbGlyc reached a very similar DAR of 2.27 and 2.23, respectively (Table 13). In comparison, the drug- to-antibody ratio for Th69dia1Glyc-MMAE was slightly higher with 2.47. The proportion of unconjugated molecules in the ADC preparations accounts for 5-10% (Figure 24 C).

[0350] Site-specifc antibody fragment-drug conjugation using GlyCLICK®

[0351] As introduced above, GlyCLICK® modification of antibody fragments was an exploratory approach since enzymatic activity of EndoS2 towards N-glycoylated antibody fragments was not expected. Figure 25 shows the results of antibody fragment-drug conjugate preparation with DBCO-PEG4-VC-PABC-MMAE by GlyCLICK®.

[0352] Analysis of EndoS2 digested antibody fragments revealed a strikingly less complex N-glycan pattern (Figure 25 A). The most abundant peak in the respective mass spectrum each corresponds to the antibody fragment with only fucosylated core-GIcNAc residue attached and activated with GalNAz, as depicted (Figure 4). The second most abundant peak corresponds to the same species with the additional lysine hydroxylation and O-glycosylation. The cluster of low-abundant peaks and of higher masses were assigned to antibody from which one N- glycan was hydrolyzed and the second one was not, in the case of Th69scFvGlyc and Th69scDbGlyc. For Th69dia1Glyc, this peak cluster comprises the monomer with uncleaved N-glycans. Assignment of the residual N-glycans in combination with the achieved drug load revealed, that tri- and tetraantennary N-glycans were not cleaved. Besides EndoS2, also EndoS was tested for trimming of N-glycans and showed a similar activity but slightly higher signal intensities for residual glycans (data not shown). These data show that EndoS2 can efficiently hydrolyze bi-antennary N-glycans in the antibody fragments.

[0353] Subsequent conjugation to the core-glycan resulted in drug conjugates with a reduced drug load distribution of 0-2. In comparison to the SiteClick™ activation and conjugation, the proportion of unconjugated molecules is increased and accounts for 30-36% (Figure 25 C). Respective DARs reached are 0.95 (Th69scFvGlyc), 0.9 (Th69dia1Glyc) and 0.83 (Th69scDbGlyc) and are very similar for the three antibody fragments (Table 13).

[0354] Notably, this is the first report, that EndoS2 can actually hydrolyze N-glycans in glycoproteins other than IgG and a1-acid glycoprotein.

[0355] Table 13 - Overview of drug-to-antibody ratios for fragment-drug conjugates. sample DAR

[0356] Th69scFvGlyc-MMAE SiteClick 2.27

[0357] Th69dialGlyc-MMAE SiteClick 2.47

[0358] Th69scDbGlyc-MMAE SiteClick 2.23

[0359] Th69scFvGlyc-MMAE GlyClick 0.95

[0360] Th69dialGlyc-MMAE GlyClick 0.90

[0361] Th69scDbGlyc-MMAE GlyClick 0.83

[0362] Site-specific conjugation of cCD7Th69n(G1) using GlyCLICK®

[0363] To achieve a site-specific labelling for cCD7Th69n(G1), the N-glycosylation position at N297 was selected as conjugation site and the GlyCLICK® technique to achieve a DAR of ~2.0. The modification steps with EndoS2 and GalT (GlyCLICK®, see above) and conjugation reaction were established and upscaled. Figure 26 A shows the chromatogram overlay of unmodified cCD7Th69n(G1), azide-activated IgG and the purified ADC after conjugation with DBCO- PEG4-VC-PAB-MMAE.

[0364] The glycomodification of the Fc / 2 has no influence on the retention of the Fc-part. Introduction of the cytotoxic handle changed the retention to a higher retention time of the Fc / 2 due to hydrophobicity of the DBCO-PEG4-vc-PAB-MMAE. Mass spectra of Fc / 2 (Figure 26 B) identify the N-glycan pattern of Fc glycosylation (glycan specification) before cleavage, the GalNAz introduction by two-step enzymatic modulation of the Fc-glycans and the addition of the linkerpayload (+1684 Da). For the Fc / 2-GalNAz the actual mass is 24333Da but the azide-residue can be reduced in LC-MS sample preparation, resulting in the mass of 24308Da. Subsequent reaction with DBCO-PEG4-vc-PAB-MMAE resulted in highly efficient conjugation (mass 25992Da). The respective mass spectrum shows two major peaks of 25992Da and 25992Da- 762Da. Due to collision induced dissociation (CID) in the MS, fragmentation of the linkerpayload occurs, which has been previously reported for MMAE-LP (Bruins et al., 2018).

[0365] The drug load distribution and DAR were determined by peak area quantification of unconjugated and conjugated Fc / 2 in the UV280nm-chromatograms. To achieve a better peak separation and baseline for quantification of peak areas, the solvent gradient was adjusted (Figure 26 C, bottom UV chromatogram). Figure 26 D shows the drug load distribution of cCD7Th69n(G1)-DBCOMMAE from two separate conjugation reactions that were quantified according to depiction in Figure 26 C. Conjugation of cCD7Th69n(G1) using GlyCLICK® and the DBCO-PEG4-vc-PAB-MMAE linker payload resulted in homogenous conjugates with -14% DAR1 and 85% DAR2 species. The overall DAR of the conjugate preparation was 1.84 (Table 15).

[0366] GlyCLICK® conjugation of IgG with different linker-payloads

[0367] Two novel payload classes with different modes of action than MMAE were evaluated for their activity against leukemic and T-cell lymphoma cells. For good comparability of payload effects, GlyCLICK® conjugation with cCD7Th69n(G1) was chosen to generate ADCs with a homogenous DAR distribution. With the established reaction conditions, cCD7Th69n(G1) was conjugated to two microcystin variants, an amatoxin derivative and again MMAE, and analyzed for the drug load. The linker-payload compounds comprised the same SPAAC- reactive group, the amino acid valine-citrulline protease cleavage motif, but a PEG8-spacer instead of a PEG4- spacer as in the previous conjugations. Due to decreased hydrophobicity of the two new payload classes, a reduction in organic solvent content from 20% to 10% in the conjugation reaction was tested and proved as beneficial. For the sample preparation, soluble IdeS enzyme was used instead of immobilized IdeS which - judging from increased peak areas for Fc / 2 - seemed to increase the recovery of Fc / 2 fragments (compare Figure 26 C and Figure 27).

[0368] Due to the rather hydrophobic linker-payload conjugated to the Fc-part in the ADCs, the retention shifts towards higher retention times. Judging from retention times on the RP phase, amatoxin and MC-1 are less hydrophobic than MMAE, whereas MC-2 occurred as hydrophobic as MMAE (Figure 27).

[0369] Table 14 - Overview of generated antibody-drug conjugates based on cCD7Th69n(G1) or isotype control antibody and the resulting DAR Successful conjugation with almost equal drug-to-antibody ratios of 1.97-1 .99 for all conjugates was demonstrated by RPC-HPLC (Table 14).

[0370] Assays were performed as follows:

[0371] Glycomodification using GlyCLICK® and SiteClick™

[0372] Removal of sialic acid in antibody fragments was performed using Sialidase A (Prozyme, San Leandro, USA). 800 pg of antibody fragment were digested with 20 pL of Sialidase A in 1x Reaction Buffer (supplied with SiaA, (Prozyme, San Leandro, USA)). Digest was performed at 37 °C, 500 rpm, overnight (20h).

[0373] For glycomodification SiteClick™ Antibody Azido Modification Kit (Thermo Fisher Scientific, Dreieich, Germany) and GlyCLICK® Azide Activation kit (Genovis, Lund, Sweden) were used. The respective manufacturer’s protocols were adjusted, as described: Azide-activation of antibody fragments using SiteClick were scaled up from designated 1.75 nmol IgG (corresponding to 250 pg) to 26 nmol scFv, and 14 nmol scDb or diabody (corresponding to -800 pg each). Therefore, respective buffer volumes were increased. Moreover, supplied 2 mL 50 kDa centrifugal filters were not used, but instead Amicon Ultra 10 kDa (Merck Millipore, Darmstadt, Germany).

[0374] After azide-activation, samples were buffer exchanged in Amicon Ultra 10 kDa (for antibody fragments) or 50 kDa filter (for IgG) units (Merck Millipore, Darmstadt, Germany) and passed through a 0.22 pm sterile filter before storage at 4 °C until use for analysis or conjugation.

[0375] Click conjugation reaction and purification

[0376] All linker-payload (LP) variants (Figure 2) were dissolved in 100% DMSO (Sigma Aldrich, Steinheim, Germany) to a final concentration of 10 mM and stored in aliquots at -80 °C. For preparation of conjugation reactions, LP were diluted to 1 mM - 2 mM in DMSO. To avoid precipitation of LP due to poor solubility in aqueous solution, and of antibodies in organic solvent, predilutions of LP and antibody were established. LPs were prediluted with PBS to reach in 60%DMSO in PBS. Antibodies were diluted with DMSO to reach 5-10%DMSO in PBS. Both predilutions were incubated for 10-15 min at RT, before mixing and incubation of SPAAC click reactions for 16-24 h at RT, without shaking.

[0377] For conjugation with DBCO-C6-PEG8-vc-PAB-microcystin 1 , DBCO-C6-PEG8-vc-PAB- microcystin 2, DBCO-C6-PEG8-vc-PAB-amatoxin final DMSO concentration was 10%, for DBOO-O6-PEG8-VC-PAB-MMAE 20%. For the establishment of SPAAC click reactions, LP to azide molar ratios were tested as indicated in the Results section. For preparative conjugation of IgG (200 pg - 600 pg), reactions were set up with LP to azide molar ratios of 2.5-5 to 1 (5-10:1 molar ratio of linker-payload: IgG). For preparative conjugation of antibody fragments (200 pg - 600 pg), reactions were set up with LP to azide molar ratios of 2-3.2 to 1 (10:1 molar ratio of Hnker-payload:Th69scFvGlyc SiteClick, 4:1 molar ratio of Hnker-payload:Th69scFvGlyc GlyClick, etc.).

[0378] After incubation, samples were diluted in PBS and gently buffer exchanged in multiple centrifugation steps via Amicon Ultra 10 kDa (antibody fragments) or 50 kDa (IgG) filter units (Merck Millipore, Darmstadt, Germany) and passed through a 0.22 pm sterile filter before storage at 4 °C.

[0379] Sample preparation

[0380] Before LC-MS analysis of the IgG / ADC samples, the samples were digested with FragITTM spin columns (immobilized FabRICATOR® (IdeS) enzyme)(Genovis, Lund, Sweden) to F(ab’)2 and Fc / 2 fragments. The manufacturer’s protocol was modified. Sample amounts of 10-50 pg were loaded in either recommended buffer (10 mM sodium phosphate and 0.15 M NaCI (pH 6.9)) or respective sample buffer (PBS) or sample diluted in digestion buffer (approximately 1 :2). The column was incubated at 37 °C and 700 rpm for 15 - 60 min. As described in the results section, sample preparation of ADCs was improved by utilizing soluble FabRICATOR® enzyme (IdeS) (Genovis, Lund, Sweden) for Fab / Fc separation. 25 pg of ADC were digested with 30U FabRICATOR®, for 30-40 min at 37 °C, shaking at 450 rpm.

[0381] For the reduction of the disulfide bonds, the eluted samples were subsequently incubated with 50 mM DTT at 50 °C for 5 - 10 min. The following reversed-phase chromatography method was established for the analysis of the drug load distribution of the generated ADCs and FDCs.

[0382] DAR determination

[0383] The following reversed-phase chromatography method was established for the analysis of the drug load distribution of the generated ADCs and FDCs.

[0384] Analyses of all MMAE-conjugated ADCs and FDCs were performed by RP-UPLC-MS on an AQUITY UPLC (Waters, Milford, USA) coupled to an ESI-QTOF Impact HD (Bruker Daltonik, Hamburg, Germany). cCD7Th69n(G1)-DBCO-PEG8-vc-PAB-amatoxin, - MMAE, -MC1 , -MC2 and lso(hlgG1)-DBCO-PEG8-vc-PAB-amatoxin, -MMAE, -MC1 , -MC2 were analyzed on an Agilent 1290 Infinity UHPLC (Agilent Technologies, Waldbronn, Germany) without mass spectrometry, otherwise using the same parameters as defined in the following. 6 pg - 30 pg of antibody fragment-drug conjugates in respective sample buffer (conjugation mixture or purified sample in PBS pH7.4) were loaded onto a ACQUITY LIPLC Protein BEH C4 column (300 A, 1.7 pm, 2.1 mm x 150 mm, (Waters, Milford, USA)) and eluted at a flow rate of 0.3 mL / min using solvent A: ULC / MS acetonitrile containing 0.1% formic acid; and solvent B: ULC / MS water containing 0.1 % formic acid (ULC / MS water, ULC / MS AcN and formic acid (99% ULC / MS) (BioSolve, Dieuze, France)).

[0385] Column temperature was 60 °C for all IgG-drug conjugates; for Th69scFv-MMAE (SiteClick) analysis establishment: 50 °C; for establishment of RPC analysis for Th69scDbWT 60 °C; for final drug load analysis of Th69scFv-MMAE (SiteClick and GlyClick), Th69dia1Glyc-MMAE (SiteClick and GlyClick), Th69scDbGlyc-MMAE (SiteClick and GlyClick): 65 °C.

[0386] Tested gradients for Th69scDbWT / Th69scDbGlyc-MMAE were: Gradient 1 : 20-50% A over 10 min; gradient 2: 25%-35% A over 5.5 min, followed by 35%-80% A over 3 min; gradient 3: 28%-60% A over 14.5 min.

[0387] Gradient 3 was selected for drug load analysis. Gradient for Th69scFv-MMAE (SiteClick) analysis establishment was: 20%-90% solvent A over 10 min (compare Figure 21).

[0388] Gradient for final drug load analysis of Th69scFv-MMAE (SiteClick and GlyClick) and Th69dia1Glyc-MMAE (SiteClick and GlyClick) was: 25-75% solvent A over 14.5 min (compare Figure 21).

[0389] Gradients for ADC drug load analysis: Gradient 1 : 20-50% A over 10 min; gradient 2: 25%- 35% A over 5.5min, followed by 35%-80% A over 3 min; gradient 3: 25-40% A over 15 min, followed by 45-80% A over 4 min. Gradient 3 was selected for drug load analysis of cCD7Th69n(G1)-DBCO-PEG8-vc-PAB-amatoxin, -MMAE, -MC1 , -MC2 and Iso(hlgGI)- DBCO-PEG8-vc-PAB-amatoxin, -MMAE, -MC1 , -MC2.

[0390] The HyStar Software (version 3.2, Bruker Daltonik, Hamburg, Germany) was used for chromatography and data acquisition. Evaluation of the data was conducted manually using DataAnalysis 4.4 (Bruker Daltonik, Hamburg, Germany).

[0391] Example 7: Evaluation of ADC and FDC target binding

[0392] The conjugation of a hydrophobic drug to the antibody may increase aggregation of the antibody conjugate. Additionally, multiple rounds of buffer exchange and incubations during modification steps may induce aggregation or fragmentation of the antibody. To allow a comparative functional examination of the ADCs, the ADC preparations were analyzed for their monomer content by size exclusion chromatography after the conjugation reaction and purification. In all ADC preparations the proportion of dimers and multimers in sum amounted to less than 1.6% (Table 15). In particular, cCD7Th69n(G1)-MMAE GlyClick did essentially not contain multimer aggregates. Th69scFvGlyc-MMAE generated by both SiteClick™ and GlyCLICK® showed the highest monomer content, which was also improved compared to the Th69scFvGlyc antibody prior to monomer preparation (Table 7, Example 5). Fragments were 5 only apparent in the diabody and IgG drug conjugate preparations to different extent. The determined fragment proportions in Th69dia1Glyc-MMAE correspond to unpaired monomer chains, according to the retention time. These proportions were strikingly different between the Th69dia1Glyc-MMAE SiteClick (37%) and the Th69dia1Glyc-MMAE GlyClick (5%).

[0393] The results reveal that Th69scFvGlyc, Th69dia1Glyc, Th69scDbGlyc and the cCD7Th69n(G1) 10 are not prone to aggregation or fragmentation throughout the modification, conjugation and purification process. However, for Th69dia1Glyc-MMAE generated by SiteClick™ conjugation the dimer association seems to be diminished. Therefore, Th69dia1Glyc-MMAE SiteClick was precluded from the in vitro cytotoxicity determination.

[0394] Table 15 - Size exclusion chromatography results of the ADC and FDCs.

[0395] Sample DAR Multimer Dimer Monomer Fragment

[0396] % % % %

[0397] Th69scFvGlyc-MMAE SiteClick 2.27 0.96 0.6 98.4

[0398] Th69dialGlyc-MMAE SiteClick 2.47 0.5 0.4 62.0 37.1

[0399] Th69scDbGlyc-MMAE SiteClick 2.23 0.5 0.8 94.3 4.3

[0400] Th69scFvGlyc-MMAE GlyClick 0.95 0.4 0.5 99.1

[0401] Th69dialGlyc-MMAE GlyClick 0.90 0.4 - 94.8 4.8

[0402] Th69scDbGlyc-MMAE GlyClick 0.83 n.d. n.d. n.d n.d.qcCD7Th69n(Gl)-DBCO-MMAE GlyClick 1.84 0.06 0.3 96.3 3.4

[0403] Table 16 - Overview of size exclusion chromatography analysis of antibody-drug conjugates and reference cCD7Th69n(G1).

[0404] Antibody Linker-Payload DAR Multimer Dimer Monomer Fragment

[0405] [%] [%] [%] [%] cCD7Th69n(Gl) - _ Q 2 94.7 5.1 cCD7Th69n(Gl) DBCO-PEG8-vc-PAB-amatoxin 1.98 - 0.4 96.6 3.0 cCD7Th69n(Gl) DBCO-PEG8-VC-PAB-MMAE 1.97 0.1 0.2 96.9 2.8 cCD7Th69n(Gl) DBCO-PEG8-VC-PAB-MC1 1.99 - 0.5 97.1 2.0 cCD7Th69n(Gl) DBCO-PEG8-vc-PAB-MC2 1.99 - 0.6 96.8 2.2

[0406] Iso(hlgGl) DBCO-PEG8-vc-PAB-amatoxin 1.97 - 1.9 97.0 1.0

[0407] Iso(hlgGl) DBCO-PEG8-VC-PAB-MMAE 1.97 1.2 1.8 95.6 1.0

[0408] Iso(hlgGl) DBCO-PEG8-vc-PAB-MCl 1.98 - 1.9 97.0 0.92

[0409] Iso(hlgGl) DBCO-PEG8-vc-PAB-MC2 1.98 - 1.8 97.1 0.95 In analytical SEC-HPLC all cCD7Th69n(G1)-conjugates showed a high level of purity with about 97% monomers (Table 16). No precipitations were observed during conjugation and concentration of the samples or isotype controls and no significant increase of dimers and multimers was determined for the cCD7Th69n(G1) antibody after conjugation. So, introduction of neither payload affected the stability of the antibody.

[0410] To characterize the impact of drug conjugation on the binding of the antibody fragments to their target, the different fragment-drug conjugates generated via GlyCLICK® and SiteClick™ technique were compared for their antigen-binding ability by ELISA.

[0411] Table 17- Antigen binding analysis by ELISA.

[0412] EC50 values as determined by non-linear regression analysis.

[0413] ECso antibody [nM] ECso ADC [nM]

[0414] Format

[0415] GlyClick conjugate SiteClick conjugate

[0416] Th69scFvGlyc 1.46 1.46 2.29

[0417] Th69dialGlyc 0.94 1.28 2.22

[0418] Th69scDbGlyc 0.83 1.15

[0419] The binding activities of the Th69scFvGlyc-conjugates, ranked by EC50 values, were as follows: Th69scFvGlyc (1.46 nM), Th69scFvGlyc-MMAE GlyClick (1.46 nM), Th69scFvGlyc- MMAE SiteClick (2.29 nM) (Table 17). For all fragment conjugates generated by GlyCLICK® (EndoS2), the maximum target binding is slightly increased compared to the unmodified antibodies (Figure 28). The strongest effect on target binding was observed for the SiteClick- conjugated Th69dia1 Glyc with a 2.4-fold decreased EC50 value but this sample also contained 37% of unpaired diabody monomer chains (see Table 15). The Th69scDbGlyc-MMAE SiteClick sample was not available for this comparative assay.

[0420] Overall, the glycan modification and drug conjugation hardly affect the antigen binding of the tested fragment-drug conjugates.

[0421] Subsequently, also the newly generated amatoxin- and MMAE-ADCs were evaluated for their target-binding ability (Figure 29).

[0422] As anticipated, the isotype controls showed no binding to the target antigen aside from slight background signals starting at 7 nM. The cCD7Th69n(G1)-DBCO-PEG8-vc-PAB-MMAE (EC50=0.076 nM) and cCD7Th69n(G1)-DBCO-PEG8-vc-PAB-amatoxin (EC50=0.059 nM) ADCs showed similar affinities towards their target antigen as the unconjugated antibody (EC50=0.041 nM). So, no effect of the Fc-modification and introduction of the payload-moieties possessing more or less hydrophobic features, was observed.

[0423] Assays were performed as described in Example 5. Example 8: In vitro activity

[0424] The cytotoxicity of Th69scFvGlyc-MMAE SiteClick, Th69scDbGlycMMAE SiteClick and cCD7Th69n(G1)- DBCO-MMAE GlyClick was measured in vitro on Jurkat, HSB-2 and ALLSIL cell lines expressing low, medium and high amounts of target antigen, respectively (Figure 30 A-C). In addition, off-target effects of the FDCs and the ADC were evaluated on targetnegative HUVEC cells, primary normal endothelial cells (Figure 30 D). Since FDC and ADC samples varied in the proportion of unconjugated molecules, all FDC and ADC samples were adjusted to the same proportion of unconjugated molecules (9.3%) to allow a comparison of cytotoxic effects. Due to this adjustment, the resulting DAR of Th69scFvGlyc-MMAE SiteClick in the assay is 2.19 and for cCD7Th69n(G1)-DBCO-MMAE GlyClick the DAR is 1.68 instead of 1.84 in the starting material. Cytotoxic effects were confirmed in repetitive assays for Jurkat and HSB-2 cells.

[0425] In vitro cell viability values of ADCs and FDCs are provided in Table 18.

[0426] Table 18 - In vitro cell viability values of ADCs and FDCs.

[0427] IC values [nM] Fynrpccinn50

[0428] Cell line [CD7-ABC per Th69scFvGlyc- ™^DbGlvC’cCD7Th69n(Gl)- DBCO-PEG4- aCD20-mc- cell] MMAE SiteClick 1" , DBCO-MMAE vc-PAB-MMAE vc-MMAE

[0429] SiteClick

[0430] ALL-SIL 201979 0.43 0.22 0.29 55.64 112.60

[0431] HSB-2 96085 0.60 0.21 0.23 27.35 74.54

[0432] Jurkat 25895 0.87 4.59 0.39 48.45 n.a.

[0433] HUVEC no expression >200.00 >200.00 >400.00 89.09 221.00

[0434] The calculation of the therapeutic range of the ADC and FDCs is provided in Table 19. For the calculation of the therapeutic range, the lower limit of IC50 values observed on HUVEC cells (namely 200 nM and 400 nM) were used.

[0435] Table 19 - Calculation of the therapeutic range of the ADC and FDCs.

[0436] For the calculation of the therapeutic range, the lower limit of IC50 values observed on HUVEC cells (namely 200 nM and 400 nM) were used. therapeutic range (IC50[HUVEC] / IC50[T-ALL])

[0437] Expression

[0438] Cell line [CD7-ABC / cell] Th69scFvGlyc- Th69scDbGlyc- cCD7Th69n(Gl)-

[0439] MMAE SiteClick MMAE SiteClick DBCO-MMAE

[0440] ALL-SIL 201979 >465 >908 >1388

[0441] HSB-2 96085 >332 >944 >1713

[0442] Jurkat 25895 >231 >44 >1026 no

[0443] HUVEC - expression

[0444] All three generated drug conjugate formats were highly potent in killing leukemic cells with similar IC50 values across the three cell lines (Figure 30) with very different target antigen expression levels, as shown in Table 18. Looking at Th69scFvGlyc-MMAE, IC50 values of 0.43 nM with ALL-SI L cells, 0.60 nM with HSB-2 cells and 0.87 nM with Jurkat cells were determined. This reveals an only 2.0-fold decrease in potency with an ~8-fold decreased ABC when comparing potency in ALL-SI L and Jurkat cells. Th69scDbGlyc-MMAE SiteClick and cCD7Th69n(G1)-DBCO-MMAE GlyClick exhibited very similar potencies of 0.21- 0.29 nM in both cell lines with medium and high CD7 expression. Only in the low CD7-expressing Jurkat cells Th69scDbGlyc-MMAE showed a distinctly lower potency, which was ten times lower than that of cCD7Th69n(G1)-DBCO-MMAE.

[0445] A substantial difference between the fragment conjugates and the IgG-ADC is the absolute cytotoxicity. Whereas scFv and scDb conjugates decrease the viability in HSB-2 cells to 0.2% and 0.5%, respectively, the IgG-ADC only reaches 20.6% viability. This behaviour is observed in all three target cell lines tested and also repeatedly in additional assays (not shown). The respective unmodified antibodies did not show antiproliferative effects on the target cells or HUVEC cells.

[0446] A CD20-targeting lgG1-ADC comprising also of the cleavable MMAE-linker-payload but conjugated via hinge cysteines with a DAR of 4.1 was included for comparison. It showed off- target cytotoxic effects in the T-ALL cell lines and also in the HUVEC cells but to a lesser extent. The non-targeted DBCO-PEG4- vc-PAB-MMAE linker-payload showed IC50 values between 27 nM and 56 nM in the T-ALL cell lines and 89 nM in HUVEC cells. The primary endothelial cells are slightly less sensitive towards the aCD20-ADC and the linker-payload than the leukemic cells.

[0447] Among the CD7-targeted ADC and FDCs, Th69scFvGlyc-MMAE SiteClick and Th69scDbGlyc- MMAE SiteClick showed a slight cytotoxic effect on HUVEC cells in the highest concentration tested whereas the cCD7Th69n(G1)-DBCO-MMAE did not (Figure 30). These off-target effects of the fragment-drug conjugates were lower than the ones observed with CD20(lgG)-mc-vc- MMAE.

[0448] Even though size-related in vivo kinetics of the formats will differentially influence the actual therapeutic window of the ADC and FDCs, an estimation of a potential therapeutic window of the generated drug conjugate formats was calculated based solely on the performed cellular cytotoxicity assays towards on-target cells vs. off-target cells. For a quantitative comparison, the ratio of IC50[HUVEC] (off-target) to IC50 [T-ALL] (on-target) was calculated and termed ‘therapeutic range’ (Table 19). With a factor greater than 1000, the cCD7Th69n(G1)-DBCO- MMAE shows the widest therapeutic range. Th69scDbGlyc-MMAE SiteClick and Th69scFvGlyc-MMAE SiteClick, with median ratios of >632 and >340, still show a sufficient window, suggesting great therapeutic potential for the IgG-based ADC, as well as the scFv- and scDb-FDCs.

[0449] In summary, the three drug conjugate formats showed a similar potency across the T-ALL cell lines, showing that the generated ADC and FDCs are effectively targeting and killing T-ALL cells of a wide range of CD7 expression levels. However, the absolute cytotoxicity was strongly increased for scFv-and scDb-conjugates versus the IgG-ADC.

[0450] In vitro cytotoxicity assay with ADCs and FDCs was performed as follows:

[0451] Cytotoxicity of ADCs and FDCs was assessed according to the receptor mediated internalization assay as proliferation assay over 96 h and subsequent CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Madison, USA). Here, 75 pL of each target cells and ADC / FDC dilution (or respective control antibodies, linker-payloads or medium control) was mixed. ADC and FDC dilutions (as well as controls) were accordingly prepared as 2x concentrated dilutions in culture medium. For comparative cytotoxicity analysis of ADCs and FDCs, the respective antibody and drug conjugate concentrations were adjusted as described in the results section (to adjust the ratio of unconjugated:conjugated antibody molecules), Figure 30.

[0452] SEQUENCE LISTING

[0453]

Claims

- 67 -CLAIMS1 . A conjugate comprising:(i) an antibody module comprising(a) at least one pair of heavy chain variable domain and light chain variable domain capable of specifically binding to a target antigen; and(b) an artificial peptide linker comprising an N-glycosylation site;(ii) a glycan structure attached to the N-glycosylation site of the peptide linker; and(iii) a cargo compound coupled to the glycan structure.

2. The conjugate according to claim 1 , wherein the peptide linker(i) is present on the same polypeptide chain of the antibody module as the heavy chain variable domain and / or the light chain variable domain;(ii) is directly adjacent to the heavy chain variable domain and / or the light chain variable domain on a polypeptide chain of the antibody module;(iii) is located between the heavy chain variable domain and the light chain variable domain on a polypeptide chain of the antibody module;(iv) directly links the heavy chain variable domain and the light chain variable domain; and / or(v) is located at the N terminus or at the C terminus of a polypeptide chain of the antibody module.

3. The conjugate according to claim 1 or 2, wherein the antibody module(i) comprises one or more additional peptide linkers each comprising one or more N- glycosylation sites; and / or(ii) further comprises one or more peptide linkers without an N-glycosylation site.

4. The conjugate according to any one of claims 1 to 3, consisting of the antibody module, one or more of the glycan structures, and one or more of the cargo compounds.

5. The conjugate according to any one of claims 1 to 4, wherein the conjugate does not comprise a whole antibody.- 68 -6. The conjugate according to any one of claims 1 to 5, wherein the antibody module consists of(i) one pair of heavy chain variable domain and a light chain variable domain capable of specifically binding to a target antigen, or two pairs of heavy chain variable domain and light chain variable domain capable of specifically binding to a target antigen;(ii) one to six peptide linkers, each comprising one or two N-glycosylation sites;(iii) optionally up to four heavy chain constant domains;(iv) optionally up to two hinge regions;(v) optionally up to two light chain constant domains; and(vi) optionally up to two peptide linkers without an N-glycosylation site.

7. The conjugate according to any one of claims 1 to 6, wherein the peptide linker(i) consists of 40 or less amino acids;(ii) comprises the amino acid sequence of any one of SEQ ID NOs: 37 to 39;(iii) comprises the amino acid sequence of any one of SEQ ID NOs: 19, 20, 27 and 28; and / or(iv) comprises the amino acid sequence of any one of SEQ ID NOs: 40 to 50.

8. The conjugate according to any one of claims 1 to 7, wherein the glycan structure(i) comprises an N-acetylglucosamine residue (GIcNAc) which is directly attached to the asparagine residue of the N-glycosylation site;(ii) comprises a modified monosaccharide unit at at least one non-reducing end, wherein the modified monosaccharide unit is optionally an N-azidoacetylgalactosamine residue (GalNAz) or a tetraacetylated N-azidoacetylgalactosamine residue (Ac4GalNAz);(iii) comprises the structure of -GIcNAc-modMS-, wherein GIcNAc is an N- acetylglucosamine residue which is attached to the asparagine residue of the N- glycosylation site, and modMS is a modified monosaccharide unit which is attached to the cargo compound; and / or(iv) comprises the structure of -GIcNAc-GIcNAc-Man-Man-GIcNAc-modMS-, wherein GIcNAc is an N-acetylglucosamine residue, Man is a mannose residue, and modMS- 69 - is a modified monosaccharide unit, and wherein the first GIcNAc residue is attached to the asparagine residue of the N-glycosylation site, and the modified monosaccharide unit is attached to the cargo compound.

9. The conjugate according to any one of claims 1 to 8, wherein the cargo compound is selected from the group consisting of radionuclides, chelators for radionuclides, chemotherapeutic agents, detectable labels, toxins, cytolytic components, immunomodulators, immunoeffectors, and liposomes.

10. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 9 and optionally one or more solvents, diluents and / or excipients.11 . The conjugate according to any one of claims 1 to 9 for use in medicine.

12. The conjugate according to any one of claims 1 to 9 for use in the treatment, prevention, prognosis, diagnosis and / or monitoring of a disease or condition.

13. The conjugate according to any one of claims 1 to 9 for use in the treatment, prevention, prognosis, diagnosis and / or monitoring of a disease or condition, wherein the disease or condition is selected from the group consisting of diseases associated with abnormal cell growth such as cancer, arthritis, bacterial and viral infections, inflammatory diseases, graft-versus-host disease, immunodeficiencies, interstitial lung disease, vascular disease such as hypertension, and scoliosis.

14. A method for producing the conjugate according to any one of claims 1 to 9, comprising the steps of(a) providing a composition of glycosylated antibody modules, wherein the glycosylated antibody module consists of an antibody module as defined in any one of claims 1 to 9, and glycan structures attached to the N-glycosylation sites of the peptide linkers of the antibody module;(b) contacting the composition of glycosylated antibody modules with a glycoside hydrolase under conditions where the glycoside hydrolase cleaves the glycan structures so that unitary non-reducing ends are present at the glycan structures;(c) contacting the composition of glycosylated antibody modules with a glycosyltransferase which is capable of transferring a modified monosaccharide to the unitary non-reducing ends of the glycan structures, and a corresponding modified nucleotide monosaccharide, under conditions where the modified monosaccharide is coupled to the unitary non-reducing ends of the glycan structures; and- 70 -(d) reacting the composition of glycosylated antibody modules with a cargo compound under conditions where the cargo compound is coupled to the modified monosaccharide attached to the glycan structures, providing a conjugate according to any one of claims 1 to 9.

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