Novel TCO-functionalized glycan engineered immunoglobulin molecules, novel TCO-functionalized saccharide compounds for preparing the same, and novel antibody payload conjugates

Abstract

The invention relates to the field of bioconjugation of functional entities, such as payloads and targeting agents, mediated by trans-cyclooctene (TCO) compounds. More particularly the present invention relates to novel TCO functionalized saccharide compounds, applicable in the glycan engineering of immunoglobulin molecules. The invention also relates to targeting agents, in particular immunoglobulin molecules, TCO-functionalized by glycan engineering of their natural glycan residues. The invention further relates to bioconjugates, like antibody payload conjugates (APCs) obtainable by covalently linking said TCO-functionalized targeting agent with a second functionalized molecule comprising a docking group (DG), in particular a tri- or tetrazine-type group, capable of reacting with the TCO-type functional group of the first functionalized molecule. The present invention further relates to preparation methods of TCO functionalized, glycan-engineered immunoglobulins and bioconjugates thereof. The invention also relates to the use of such conjugates of the present invention for use in medicine, to corresponding pharmaceutical compositions as well as to corresponding diagnostic and analytical kits.

Description

[0001] Novel TCO-functionalized glycan engineered immunoglobulin molecules, novel TCO-functionalized saccharide compounds for preparing the same, and novel antibody payload conjugates

[0002] FIELD OF THE INVENTION

[0003] The invention relates to the field of bioconjugation of functional entities (such as payloads and targeting agents) mediated by trans-cyclooctene (TCO) compounds. More particularly the present invention relates to novel TCO functionalized saccharide compounds, applicable in the glycan engineering of immunoglobulin molecules. The invention also relates to targeting agents, in particular immunoglobulin molecules, TCO-functionalized by glycan engineering of their natural glycan residues.

[0004] The invention further relates to bioconjugates, like antibody payload conjugates (APCs) obtainable by covalently linking, in particular, via biorthogonal bioconjugation via a Diels-Alder-type cycloaddition, said TCO-functionalized targeting agent, particularly selected from said trans-cyclooctene-functionalized immunoglobulins with a second functionalized molecule comprising a docking group (DG), in particular a tri- or tetrazine-type group, capable of reacting with the trans-cyclooctene-type functional group of the first functionalized molecule.

[0005] The present invention further relates to preparation methods of TCO functionalized, glycan-engineered immunoglobulins and bioconjugates thereof.

[0006] The invention also relates to the use of such conjugates of the present invention for use in medicine, to corresponding pharmaceutical compositions as well as to corresponding diagnostic and analytical kits.

[0007] BACKGROUND OF THE INVENTION

[0008] The strain promoted inverse electron demand Diels-Alder reaction (SPIEDAC) is among the fastest bioorthogonal reactions - several orders of magnitude faster than azide based click reactions - and broadly applied for various applications in chemical biology, life science, and nuclear medicine. It enables quantitative bioconjugations without much influence of the utilized buffer, pH or added solvents. The fast reaction kinetics of this bioorthogonal click reaction enable the use at extremly low concentrations, e.g. in living systems like cells or even in vivo click applications, like pretargeting in nuclear medicine.

[0009] Antibody-drug conjugates (ADCs) are a class of targeted chemotherapy which has seen incredible growth recently, which is reflected in the growing number of approved ADC drugs and increasing numbers of clinical trials, and which are therefore well known in the art.

[0010] M / 61204VERAXA 2

[0011] Through increasing innovation in the field ADCs have become a highly optimized class of anti-tumor therapeutics regarding their targeting moieties, bioconjugation method, linkers, and payloads. The former stochastic lysine or cysteine conjugation is shifting more towards site-specific methods, which circumvent issues caused by heterogeneity, like uneven drug-to-antibody ratios (DARs) causing possible aggregation and concomitant side effects like fast clearance, immunogenicity or hepatoxicity.

[0012] Site-specific bioconjugation methods include incorporation of non-canonical amino acids, introduction of peptide tags for enzymatic ligations, or glycan engineering introducing biorthogonal click-chemistry. While the first two methods require adjustment to each individual antibody via engineering of its sequence, glycan engineering offers a universally applicable bioconjugation method without any modification of the antibody or expression system. This is enabled by remodelling of the N-glycans at the conserved N-glycosylation site, present on all IgGs at position N297. (S. J. Walsh, et al. Chem. Soc. Rev., 2021,50, 1305)

[0013] The Fc glycan can be modified either chemically or enzymatically. Chemical methods are limited to oxidation of native or introduced fucose (Zuberbuhler K, Casi G, Bernardes GJ, et al. Fucose-specific conjugation of hydrazide derivatives to a vascular-targeting monoclonal antibody in IgG format. Chem Common 2012, 48, 7100) or sialic acid residues (Zhou Q, Stefano JE, Manning C, et al. Site-specific antibody-drug conjugation through glycoengineering. Bioconjugate Chem 2014, 25, 510-520) forming aldehydes, which are subsequently conjugated to hydroxyl-amine or hydrazine payloads forming oximes or hydrazones, respectively. The high amount of oxidant can lead to unwanted side reactions and the applicability is limited by the heterogeneity of antibody glycans.

[0014] Enzymatical glycan engineering can be achieved via deglycosylation with an endoglycosidase and subsequent transglycosidation with glycans bearing biorthogonal reactive groups, like azides, alkynes or ketones. In 2012, Wang and co-workers introduced an endoglycosidase based bioconjugation method utilizing mutants of Endo S (Huang W, Giddens J, Fan SQ, et al. Chemoenzymatic glycoengineering of intact IgG antibodies for gain of functions. J. Am. Chem. Soc. 2012, 134, 12308). This was later expanded by Huang and co-workers, which described the use of two enzymes (Endo-M and Endo-S-D233Q) to enable efficient incorporation of azide-modified sialic acid moieties (F. Tang, Y. Yang, Y. Tang, S. Tang, L. Yang, B. Sun, B. Jiang, J. Dong, H. Liu, M. Huang, M. Y. Geng and W. Huang, Org. Biomol. Chem., 2016, 14, 9501). Davis and co-workers incorporated biantennary sugar oxazolines with alkyne, azide, disulfide and phenyliodide moieties onto trastuzumab (T. B. Parsons, W. B. Struwe, J. Gault, K. Yamamoto, T. A. Taylor, R. Raj, K. Wais, S. Mohammed, C. V. Robinson, J. L. P. Benesch and B. G. Davis

[0015] M / 65047-PCTVERAXA 3

[0016] , Angew. Chem., Int. Ed., 2016, 55, 2361). The main disadvantage of such transglycosidation methods is the very complex and expensive synthesis of the bioorthogonal reactive groups containing oligosaccharide oxazolines.

[0017] Another widely used glycan engineering method utilizes evolved (3-1,4-galactosyltransferase mutants to transfer chemically modified N-acetylgalactosamine (GalNAc) moieties onto terminal N-acetylglucosamine (GlcNAc) residues. This can either be done on GOF glycoforms to include 4 reactive GalNAc moieties (Z. Zhu, B. Ramakrishnan, J. Li, Y. Wang, Y. Feng, P. Prabakaran, S. Colantonio, M. A. Dyba, P. K. Qasba and D. S. Dimitrov, mAbs, 2014, 6, 1190; P. Thompson, E. Ezeadi, I. Hutchinson, R. Fleming, B. Bezabeh, J. Lin, S. Mao, C. Chen, L. Masterson, H. Zhong, D. Toader, P. Howard, H. Wu, C. Gao and N. Dimasi, ACS Med. Chem. Lett., 2016, 7, 1005), or after deglycosylation to the innermost GlcNAc by endoglycosidases and introduction of only one N-azidoacetylgalactosamine (GalNAz) to each antibody glycan (R. van Geel, M. A. Wijdeven, R. Heesbeen, J. M. M. Verkade, A. A. Wasiel, S. S. van Berkel and F. L. van Delft, Bioconjugate Chem., 2015, 26, 2233; W02016170186 A1). This has the advantage of removing glycan heterogeneity and producing a homogeneous ADC. Despite their great utility, the above-mentioned methods are utilizing either slow oxime-ligation reactions or azide-alkyne click chemistry for subsequent bioconjugation to payloads, which require an excess of payload reagent for efficient conjugation and produce suboptimal conjugates in terms of e.g. efficacy and pharmacokinetics.

[0018] Amongst the various candidates to be possibly selected as bioconjugation mediating moieties, trans-cyclooctene (TCO) derivatives, in particular TCO based ncAA (TCO-ncAA), were shown to have a much faster reaction kinetics as compared to other candidate molecules (N. K. Devaraj, et. al 2009).

[0019] The applicability of TCO-ncAAs was further improved by the present applicant who successfully developed so-called hydrophilic TCOs (hyTCOs) as described in PCT / EP2024 / 071144.

[0020] By introducing hydrophilic substituents into the TCO moiety its lipophilic character, which causes unspecific binding and long washout times in fluorescent labeling applications and causes certain negative impact on the hydrophilicity of bioconjugates in targeted therapy, could be significantly reduced.

[0021] Moreover, the intrinsic instability of conventional TCOs towards isomerization from the click-reactive trans- to the unreactive c / s-isomers also could concomitantly be addressed by introducing such hydrophilic substituents in particular ring positions relative to the carbon-

[0022] M / 65047-PCTVERAXA 4

[0023] carbon double bond of the TCO moiety. Additionally, previously introduced ncAA, bearing the amino acid handle at the 2-position are prone to eliminating the amino acid moiety after click reaction and are therefore not suited for modification of biomolecules [(Fan et al., 2016)].

[0024] For applications in in vivo labeling applications very fast reaction kinetics are of utmost importance, though.

[0025] The use of TCO moieties, and more particularly of hydrophilic TCO moieties, linked to glycan residues of an antibody molecues for the preparation of corresponding antibody payload conjugates, so far has not been suggested.

[0026] There is therefore a significant need for the provision of improved glycan engineered immunoglobulin molecules or corresponding glycan engineered fragments thereof which allow a more efficient coupling of payload molecules.

[0027] SUMMARY OF THE INVENTION

[0028] The above-mentioned problem was surprisingly solved by the provision of novel glycan engineered immunoglobulin molecules, in particular IgGs, and corresponding glycan engineered fragments thereof carrying a glycan engineered group in an amino acid sequence position corresponding to position N297 (according to Eu numbering) present on all IgGs, terminally functionalised at its glycan side chain by a terminal TCO moiety, more preferably a hydrophilic TCO (hyTCO) that does not undergo trans-lo-cis isomerization and allows for near quantitative conjugation with diene-bearing derivatives or conjugates (such as 1,2,4,5-tetrazine derivatives) and, due to their increased hydrophilicity, the conjugation of more hydrophobic payloads, and, in the case of pharmacologically active conjugates provide for a better functional profile of such conjugate in vivo.

[0029] More specifically the above mentioned problem was solved by the provision of glycan engineered IgG molecules carrying hyTCO derivatives, bearing a geminal polar, particularly hydrophilic, substituent at the 3-position (also designated homoallyl position) in combination with a polar, in particular hydrophilic moiety at the 2-position (also designated allyl-position) of the TCO moiety attached via the homoallyl position to the terminal monosaccharide moiety of the glycan side chain.

[0030] The invention enables the use of such hyTCOs functionalized immunoglobulins for prolonged application in biological relevant conditions, specifically during the employment for bioconjugation in a highly efficient manner.

[0031] The invention also favours the use of hyTCO-modified glycan engineered immunoglobulin moieties for in vivo click applications.

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[0033] Additionally, it aids in optimizing the bioconjugation handle for ADCs towards hydrophilic properties, influencing pharmacokinetics, solubility and aggregation of such ADCs. The increased hydrophilicity should also aid in reduction of unspecific binding.

[0034] Surprisingly, although the hyTCO derivatives possess increased steric hindrance, they display very fast reaction kinetics, even outmatching that of previously known TCO-based compounds.

[0035] More particularly, the above-mentioned problem is solved by the provision of:

[0036] a) novel hyTCO-modified glycan derivatives (represented by general formula I (as referred to herein below);

[0037] b) novel hyTCO-functionalized homogeneous glycan engineered immunoglobulin molecules obtained by binding said hyTCO compounds to a shortened, engineered glycan side chain of said immunoglobulin molecule; and

[0038] c) conjugates obtainable by covalently linking said novel glycan engineered and hyTCO- functionalized immunoglobulin with a second functionalized molecule comprising a docking group (DG), in particular triazin or tetrazin, and more particular tetrazin moiety, capable of reacting with the hyTCO-type functional group of the immunoglobulin.

[0039] BRIEF DESCRIPTION OF THE FIGURES

[0040] Figure 1 A schematically illustrates the conversion of Neu5Ac to a TCO functionalized CMP derivative of formula I of the invention as well as the glycan engineering concept of the present invention starting out from a glycosylated immunoglobulin molecule (showing, in the same molecule two different exemplary glycan structures, namely optionally fucosylated G2A1 and Man9N2), the partial degradation of the glycan side chains and the enzyme catalyzed introduction of a TCO-functionalised / V-acetylneuraminic acid moiety from a TCO functionalized CMP derivative of formula I as activated enzyme substrate, which allows via click reaction of a tetrazine functionalized payload molecule with said TCO moiety the generation of antibody payload conjugates (APCs); Figure 1B shows some nonlimiting examples of isoforms of the largest N-linked oligosaccharide structure (G2A2) found in human IgG, which also may be remodelled according to the invention; Figure 1C shows some examples for isoforms of a high mannose glycan of the type Man9N2, which also may be remodelled according to the invention

[0041] Figure 2 illustrates the advantages of an antibody drug conjugate generated from a glycan engineered TRASTUZUMAB antibody and a tetrazine functionalized prodrug (ADC 26), relative to a conventional ADC (Enhertu ®), wherein a very similar prodrug was attached to the antibody moiety via amino acid residues.

[0042] M / 65047-PCTVERAXA 6

[0043] Figure 3 illustrates the experimental results of subunit analysis of the heavy chain via LC-MS for intermediates 17 - 18 and final ADC 21, i.e. for the consecutive steps after deglycosylation

[0044] Figure 4 illustrates the results of hydrophobic interaction chromatography of TCO-modified Trastuzumab overlayed with final ADC after conjugation with compound 12.

[0045] Figure 5 illustrates the amino acid sequences of TRASTUZUMAB heavy chain and light chain.

[0046] Figure 6 illustrates the labeling and subsequent click reaction with a fluorescent dye (Cy5) of TROP2 hybridoma cells in a one pot protocol, employing neuramidase in parallel to the labeling mixture containing a CMP-sialic acid-9-TCO derivative of Example 9.

[0047] DETAILED DESCRIPTION OF THE INVENTION

[0048] A. ABBREVIATIONS

[0049] AcOH = acetic acid

[0050] ADC = antibody drug conjugates

[0051] APC = antibody payload conjugates

[0052] aq.= aqueous

[0053] conc.= concentrated

[0054] CMP = cytidine monophosphate

[0055] CTP = cytidine triphosphate

[0056] Cy5-HTet = a fluorophore derivative with a terminal tetrazine group which allows TCO-linking suitable of labeling; CAS-Nr.: 1801695-57-7, Click Chemistry Tools) DAR = Drug-to-antibody ratio

[0057] DOL = Degree of labelling

[0058] DCM = dichloromethane

[0059] DIPEA= A / , / V-diisopropylethylamine

[0060] DMF = dimethylformamide

[0061] DMSO = dimethylsulfoxide

[0062] eq.= equivalent(s)

[0063] EtOAc = ethyl acetate

[0064] EtOH = ethanol

[0065] Fuc = fucose, fucosyl

[0066] Gal = galactose, galactosyl

[0067] M / 65047-PCTVERAXA

[0068] GlcNAc = N-acetyl glucosamine (2-Acetamido-2-desoxy-D-glucopyranose (IUPAC)) GCE = genetic code expansion

[0069] h = hour(s)

[0070] HATU = 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate, a coupling agent

[0071] IEDDA = Inverse Electron-Demand Diels-Alder Cycloaddition

[0072] kDa = kilo Dalton

[0073] IMGT = the international ImMunoGenetics information system

[0074] Man =mannose, mannosyl

[0075] MeCN = acetonitrile

[0076] min = minutes

[0077] MMAE = Monomethyl auristatin E ((S)- / V-((3 / :?,4S,5S)-1-((S)-2-((1 f?,2 / :?)-3-(((1 S,2R)-1-hydroxy-1 -phenylpropan-2-yl)amino)-1 -methoxy-2-methyl-3-oxopropyl)pyrrolidin-1 -yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)- / V,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamide), a anti-neoplastic agent

[0078] MeOH = methanol

[0079] ncAA = non-canonical amino acid

[0080] Neu5Ac = N-acetyl neuraminic acid; CAS-Nummer: 21646-00-4

[0081] PBS = phosphate buffered saline

[0082] POI = polypeptide of interest,

[0083] RP-HPLC = reversed phase high-performance liquid chromatography

[0084] rpm = rounds per minute

[0085] rt = room / ambient temperature (20-25°C)

[0086] SDS-PAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis

[0087] Sia = sialic acid, sialyl

[0088] SPIEDAC = Strain-promoted Inverse Electron-Demand Diels-Alder cycloaddition TCO = a core structure (as encompassed by “TCO- type” moieties of the invention) comprising a mo no unsaturated cyclic structure of the following formula; the numbering given therein identifies the respective carbon atoms designated herein as 1-position, 2-position (i.e. allyl position) or 3-position (i.e. homoallyl position)

[0089]

[0090] M / 65047-PCTVERAXA 8

[0091] hyTCO = hydrophilic TCO as used preferably for the present invention, it contains at least one hydrophilic ring substituent in the TCO ring structure, preferably in 2- position and / or 3-position

[0092] TFA = trifluoroacetic acid

[0093] THF = tetrahydrofurane

[0094] TLC = thin layer chromatography

[0095] TsCI = tosyl chloride

[0096] UDP = uridine diphosphate

[0097] B. DEFINITIONS

[0098] B.1 General Definitions

[0099] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0100] The terms "purified", "substantially purified," and "isolated" as used herein refer to the state of being free of other, dissimilar compounds with which a compound of the invention is normally associated in its natural state, so that the "purified", "substantially purified," and "isolated" subject comprises at least 0.5%, 1%, 5%, 10%, or 20%, or at least 50% or 75% of the mass, by weight, of a given sample. In one embodiment, these terms refer to the compound of the invention comprising at least 95, 96, 97, 98, 99 or 100%, of the mass, by weight, of a given sample. As used herein, the terms "purified", "substantially purified," and "isolated" when referring to a nucleic acid or protein, also refers to a state of purification or concentration different than that which occurs naturally, for example in a prokaryotic or eukaryotic environment, like, for example in a bacterial or fungal cell, or in the mammalian organism, especially human body. Any degree of purification or concentration greater than that which occurs naturally, including (1) the purification from other associated structures or compounds or (2) the association with structures or compounds to which it is not normally associated in said prokaryotic or eukaryotic environment, are within the meaning of "isolated”. The nucleic acid or protein or classes of nucleic acids or proteins, described herein, may be isolated, or otherwise associated with structures or compounds to which they

[0101] M / 65047-PCTVERAXA 9

[0102] are not normally associated in nature, according to a variety of methods and processes known to those of skill in the art.

[0103] In the context of the descriptions provided herein and of the appended claims, the use of “or” means “and / or” unless stated otherwise.

[0104] Similarly, “comprise,” “comprises,” “comprising”, “include,” “includes,” and “including” are interchangeable and not intended to be limiting.

[0105] It is to be further understood that where descriptions of various embodiments use the term "comprising," those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language "consisting essentially of" or "consisting of.”

[0106] The term “one or more” or the similar term “at least one” refers to e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0107] When the lower and upper limits of a numerical range are disclosed, any numerical value and any inclusive range falling within that range is specifically disclosed, including its upper and lower end value. In particular, every range of values disclosed herein should be understood to mean every value and narrower range that falls within the broader range The term “about” indicates a potential variation of ± 25% of the stated value, in particular ± 15%, ±10 %, more particularly ± 5%, ± 2% or ± 1 %.

[0108] The term "substantially" describes a range of values of from about 80 to 100%, such as, for example, 85-99.9%, in particular 90 to 99.9%, more particularly 95 to 99.9%, or 98 to 99.9% and especially 99 to 99.9%.

[0109] “Predominantly” refers to a proportion in the range of above 50%, as for example in the range of 51 to 100%, particularly in the range of 75 to 99,9%; more particularly 85 to 98,5%, like 95 to 99%.

[0110] If the present disclosure refers to features, parameters and ranges thereof of different degree of preference (including general, not explicitly preferred features, parameters and ranges thereof) then, unless otherwise stated, any combination of two or more of such features, parameters and ranges thereof, irrespective of their respective degree of preference, is encompassed by the disclosure of the present description.

[0111] Compounds as herein described may contain one or more asymmetric elements such as stereogenic centers, stereogenic axes and the like, e.g. asymmetric carbon atoms, so that the compounds can exist in different stereoisomeric forms. These compounds can be, for example, racemates or optically active forms. All stereoisomers, diastereomers, Z- and E-forms, in purified and mixture forms are included. Accordingly, when a compound is recited

[0112] M / 65047-PCTVERAXA 10

[0113] by specific name or a class of compounds is recited, all these forms are intended to be included.

[0114] Compounds as herein described may also exist in more than one form of structural isomers also designated as constitutional isomers or regioisomers. These are molecules that differ only in the different sequence of their atoms or atomic groups while having the same gross formula.

[0115] Therefore, unless otherwise stated, for each of the compounds, biomolecules and conjugates as described herein, any such potential stereo- or regiosomeric form or mixture of more than one stereo- and / or regioisomeric form is within the scope of the present invention.

[0116] A “pharmaceutical composition" comprises in addition to an active ingredient, in particular an ADC of the invention, one or more substances such as selected from the group consisting of pharmaceutically acceptable preservatives, pharmaceutically acceptable colorants, pharmaceutically acceptable protective colloids, pharmaceutically acceptable pH regulators and pharmaceutically acceptable osmotic pressure regulators. Such substances are described in the art. A more detailed description of pharmaceutical compositions of the invention is provided below.

[0117] As used herein, the term "effective amount" refers to the amount of a therapy which is sufficient to reduce or ameliorate the severity and / or duration of a disorder or one or more symptoms thereof, prevent the advancement of a disorder, cause regression of a disorder, prevent the recurrence, development, onset or progression of one or more symptoms associated with a disorder, detect a disorder, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy (e.g., prophylactic or therapeutic agent).

[0118] B.2 Immunology

[0119] The term "antibody”, as used herein, broadly refers to any immunoglobulin (Ig) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such functional fragment, mutant, variant, or derivative antibody formats are known in the art. Nonlimiting embodiments of which are discussed below. A “full-length antibody”, as used herein, refers to an Ig molecule comprising four polypeptide chains, two heavy chains and two light chains. The chains are usually linked to one another via disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (also referred to herein as “variable heavy chain”, or abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light

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[0121] chain variable region (also referred to herein as “variable light chain”, or abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class e.g., IgG 1, lgG2, lgG3, lgG4, Ig A1 and lgA2) or subclass.

[0122] The term “IgG” identifies is the most abundant antibody in normal human serum, accounting for 70-85% of the total immunoglobulin pool. It is monomeric with a molecular weight of approximately 150 kDa, is the major antibody of the secondary immune response and has the longest half-life (20-24 days) of the five immunoglobulin classes. IgG consists of four human subclasses (lgG1, lgG2, lgG3 and lgG4) each containing a different heavy chain. They are highly homologous and differ mainly in the hinge region and the extent to which they activate the host immune system. lgG1 and lgG4 contain two inter-chain disulphide bonds in the hinge region, lgG2 has 4 and lgG3 has 11. In mice the IgG class is divided into five sub-classes (lgG1, lgG2A, lgG2B, lgG2C and lgG3) and in rat there are four (lgG1, lgG2A, lgG2B, lgG2C). Sub-class nomenclature has arisen independently for each species and so there is no general relationship between the sub-classes from each species.

[0123] The terms "antigen-binding portion" of an antibody (or simply "antibody portion"), “antigen-binding moiety” of an antibody (or simply “antibody moiety”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen ( / .e. the immunogenic product of the invention), i.e. are functional fragments of an antibody. It has been shown that the antigen-binding function of an antibody can be performed by one or more fragments of a full-length antibody. Such antibody embodiments may also be bispecific, dual specific, or multi-specific, specifically binding to two or more different antigens. Examples of binding fragments encompassed within the term "antigenbinding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341: 544-546, 1989; Winter et al., WO 90 / 05144 A1, herein incorporated by reference), which comprises a single variable domain; and (vi) an isolated complementarity determining region

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[0125] (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., Science 242: 423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883, 1988). Such single chain antibodies are also encompassed within the term "antigen-binding portion" of an antibody. Other forms of single chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448, 1993; Poljak et al., Structure 2: 1121-1123, 1994). Such antibody binding portions are known in the art (Kontermann and Dubel eds., Antibody Engineering, Springer-Verlag. New York. 790 pp., 2001, ISBN 3-540-41354-5).

[0126] The term "antibody”, as used herein, also comprises antibody constructs. The term “antibody construct” as used herein refers to a polypeptide comprising one or more of the antigen-binding portions of the invention linked to a linker polypeptide or an immunoglobulin constant domain. Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions. Such linker polypeptides are well known in the art (see e.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448, 1993; Poljak et al., Structure 2: 1121 -1123, 1994).

[0127] An immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences are known in the art.

[0128] Still further, a binding protein of the present invention e.g. an antibody) may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the binding protein of the invention with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov et a!., Human Antibodies and Hybridomas 6: 93-101, 1995) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov et a!., Mol. Immunol. 31: 1047-1058, 1994). Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and

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[0130] immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.

[0131] An "isolated antibody", as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities. An isolated antibody that specifically binds the immunogenic product of the invention may, however, have cross-reactivity to other antigens, such as Ap globulomers, e.g. A (20-42) globulomer or other Ap forms. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals and / or any other targeted AB form.

[0132] The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences {e.g. mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular in CDR3. However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0133] The term "recombinant human antibody", as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further in Section B, below), antibodies isolated from a recombinant, combinatorial human antibody library (Hoogenboom, TIB Tech. 15: 62-70, 1997; Azzazy and Highsmith, Clin. Biochem. 35: 425-445, 2002; Gavilondo J. V., and Larrick J. W. (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), antibodies isolated from an animal e.g. a mouse) that is transgenic for human immunoglobulin genes (see e.g. Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A., and Green L. L. (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al (2000) Immunology Today 21:364-370) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human

[0134] M / 65047-PCTVERAXA 14

[0135] germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0136] The term “chimeric antibody” refers to antibodies which comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies having murine heavy and light chain variable regions linked to human constant regions.

[0137] The term “CDR-grafted antibody” refers to antibodies which comprise heavy and light chain variable region sequences from one species but in which the sequences of one or more of the CDR regions of VH and / or VL are replaced with CDR sequences of another species, such as antibodies having murine CDRs (e.g., CDR3) in which one or more of the murine variable heavy and light chain regions has been replaced with human variable heavy and light chain sequences.

[0138] The terms " Kabat numbering", " Kabat definitions and " Kabat labeling" are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable ( / .e. hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U. S. Department of Health and Human Services, NIH Publication No. 91-3242). For the heavy chain variable region, the hypervariable region ranges from amino acid positions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, and amino acid positions 95 to 102 for CDR3. For the light chain variable region, the hypervariable region ranges from amino acid positions 24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3.

[0139] As used herein, the terms "acceptor" and "acceptor antibody" refer to the antibody or nucleic acid sequence providing or encoding at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% of the amino acid sequences of one or more of the framework regions. In some embodiments, the term "acceptor" refers to the antibody amino acid or nucleic acid sequence providing or encoding the constant region(s). In yet another embodiment, the term "acceptor" refers to the antibody amino acid or nucleic acid sequence providing or encoding one or more of the framework regions and the constant region(s). In a specific embodiment, the term "acceptor" refers to a human antibody amino acid or nucleic acid sequence that provides or encodes at least 80%, for example at least 85%, at least 90%, at least 95%, at least 98%, or 100% of the amino acid sequences of one or more of the framework regions. In accordance with this embodiment, an acceptor may contain at least 1, at least 2, at least 3, least 4, at least 5, or at least 10 amino acid residues that does (do) not

[0140] M / 65047-PCTVERAXA 15

[0141] occur at one or more specific positions of a human antibody. An acceptor framework region and / or acceptor constant region(s) may be, e.g., derived or obtained from a germline antibody gene, a mature antibody gene, a functional antibody e.g., antibodies well-known in the art, antibodies in development, or antibodies commercially available).

[0142] As used herein, the term " CDR" refers to the complementarity determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable regions. The term “CDR set” as used herein refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) found that certain sub- portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as L1, L2 and L3 or H1, H2 and H3 where the " L" and the " H" designates the light chain and the heavy chains regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)). Still other CDR boundary definitions may not strictly follow one of the above systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, particular embodiments use Kabat or Chothia or by EU numbering defined CDRs.

[0143] As used herein, the term "canonical" residue refers to a residue in a CDR or framework that defines a particular canonical CDR structure as defined by Chothia et al. (J. Mol. Biol. 196:901-907 (1987); Chothia et al., J. Mol. Biol. 227:799 (1992), both are incorporated herein by reference). According to Chothia etal., critical portions of the CDRs of many antibodies have nearly identical peptide backbone confirmations despite great diversity at the level of amino acid sequence. Each canonical structure specifies primarily a set of

[0144] M / 65047-PCTVERAXA 16

[0145] peptide backbone torsion angles for a contiguous segment of amino acid residues forming a loop.

[0146] As used herein, the terms "donor" and "donor antibody" refer to an antibody providing one or more CDRs. In one embodiment, the donor antibody is an antibody from a species different from the antibody from which the framework regions are obtained or derived. In the context of a humanized antibody, the term "donor antibody" refers to a non-human antibody providing one or more CDRs.

[0147] As used herein, the term "framework" or "framework sequence" refers to the remaining sequences of a variable region minus the CDRs. Because the exact definition of a CDR sequence can be determined by different systems, the meaning of a framework sequence is subject to correspondingly different interpretations. The six CDRs (CDR-L1, -L2, and -L3 of light chain and CDR-H1, -H2, and -H3 of heavy chain) also divide the framework regions on the light chain and the heavy chain into four sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying the particular subregions as FR1, FR2, FR3 or FR4, a framework region, as referred by others, represents the combined FR's within the variable region of a single, naturally occurring immunoglobulin chain. As used herein, a FR represents one of the four sub-regions, and FRs represents two or more of the four sub- regions constituting a framework region.

[0148] Human heavy chain and light chain acceptor sequences are known in the art.

[0149] As used herein, the term "germline antibody gene" or "gene fragment" refers to an immunoglobulin sequence encoded by non-lymphoid cells that have not undergone the maturation process that leads to genetic rearrangement and mutation for expression of a particular immunoglobulin. (See, e.g., Shapiro et al., Crit. Rev. Immunol. 22(3): 183-200 (2002); Marchalonis et al., Adv Exp Med Biol. 484:13-30 (2001)). One of the advantages provided by various embodiments of the present invention stems from the recognition that germline antibody genes are more likely than mature antibody genes to conserve essential amino acid sequence structures characteristic of individuals in the species, hence less likely to be recognized as from a foreign source when used therapeutically in that species.

[0150] As used herein, the term "key residues” refer to certain residues within the variable region that have more impact on the binding specificity and / or affinity of an antibody, in particular a humanized antibody. A key residue includes, but is not limited to, one or more of the following: a residue that is adjacent to a CDR, a potential glycosylation site (can be either N- or O-glycosylation site), a rare residue, a residue capable of interacting with the antigen, a residue capable of interacting with a CDR, a canonical residue, a contact residue between heavy chain variable region and light chain variable region, a residue within the Vernier zone,

[0151] M / 65047-PCTVERAXA 17

[0152] and a residue in the region that overlaps between the Chothia definition of a variable heavy chain CDR1 and the Kabat definition of the first heavy chain framework.

[0153] As used herein, the term "humanized antibody" is an antibody or a variant, derivative, analog or portion thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in the context of a CDR refers to a CDR having an amino acid sequence at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a non-human antibody CDR. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin ( / .e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. According to one aspect, a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In some embodiments, a humanized antibody contains both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, a humanized antibody only contains a humanized light chain. In some embodiments, a humanized antibody only contains a humanized heavy chain. In specific embodiments, a humanized antibody only contains a humanized variable domain of a light chain and / or of a heavy chain.

[0154] The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including without limitation IgG 1, lgG2, lgG3 and lgG4. The humanized antibody may comprise sequences from more than one class or isotype, and particular constant domains may be selected to optimize desired effector functions using techniques well-known in the art.

[0155] The framework and CDR regions of a humanized antibody need not correspond precisely to the parental sequences, e.g., the donor antibody CDR or the consensus framework may be mutagenized by substitution, insertion and / or deletion of at least one amino acid residue so that the CDR or framework residue at that site does not correspond to either the donor antibody or the consensus framework. In one embodiment, such mutations, however, will not be extensive. Usually, at least 90%, at least 95%, at least 98%, or at least 99% of the humanized antibody residues will correspond to those of the parental FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework region in the consensus immunoglobulin sequence. As used herein, the term "consensus

[0156] M / 65047-PCTVERAXA 18

[0157] immunoglobulin sequence" refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In a family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence.

[0158] As used herein, " Vernier" zone refers to a subset of framework residues that may adjust CDR structure and fine-tune the fit to antigen as described by Foote and Winter (1992, J. Mol. Biol. 224:487-499, which is incorporated herein by reference). Vernier zone residues form a layer underlying the CDRs and may impact on the structure of CDRs and the affinity of the antibody.

[0159] The term “antibody”, as used herein, also comprises multivalent binding proteins. The term "multivalent binding protein" is used in this specification to denote a binding protein comprising two or more antigen binding sites. The multivalent binding protein is engineered to have the three or more antigen binding sites, and is generally not a naturally occurring antibody. The term “multispecific binding protein” refers to a binding protein capable of binding two or more related or unrelated targets. Dual variable domain (DVD) binding proteins as used herein, are binding proteins that comprise two or more antigen binding sites and are tetravalent or multivalent binding proteins. Such DVDs may be monospecific, i.e. capable of binding one antigen or multispecific, i.e. capable of binding two or more antigens. DVD binding proteins comprising two heavy chain DVD polypeptides and two light chain DVD polypeptides are refered to a DVD Ig. Each half of a DVD Ig comprises a heavy chain DVD polypeptide, and a light chain DVD polypeptide, and two antigen binding sites. Each binding site comprises a heavy chain variable domain and a light chain variable domain with a total of 6 CDRs involved in antigen binding per antigen binding site. DVD binding proteins and methods of making DVD binding proteins are disclosed in US. Patent Application No.

[0160] 11 / 507,050 and incorporated herein by reference.

[0161] The term “labeled binding protein”, as used herein, refers to a binding protein with a label incorporated that provides for the identification of the binding protein. Likewise, the term “labeled antibody” as used herein, refers to an antibody with a label incorporated that provides for the identification of the antibody. In one aspect, the label is a detectable marker, e.g., incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin {e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides e.g.,3H,14C,35S,90Y, " Tc,111In,125l,1311,177Lu,166Ho, or

[0162] M / 65047-PCTVERAXA 19

[0163] 153Sm); fluorescent labels {e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels {e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by a secondary reporter {e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags); and magnetic agents, such as gadolinium chelates.

[0164] The term "antibody”, as used herein, also comprises antibody conjugates. The term “antibody conjugate” refers to a binding protein, such as an antibody, chemically linked to a second chemical moiety, such as a therapeutic agent.

[0165] The term " KD" (also " Kd" or “KD”), as used herein, is intended to refer to the "equilibrium dissociation constant", and refers to the value obtained in a titration measurement at equilibrium, or by dividing the dissociation rate constant (koff) by the association rate constant (kon). The association rate constant (kon), the dissociation rate constant (koff), and the equilibrium dissociation constant (KD) are used to represent the binding affinity of a binding protein e.g., an antibody) to an antigen. Methods for determining association and dissociation rate constants are well known in the art. Using fluorescencebased techniques offers high sensitivity and the ability to examine samples in physiological buffers at equilibrium. Other experimental approaches and instruments such as a BIAcore® (biomolecular interaction analysis) assay can be used {e.g., instrument available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden). Additionally, a KinExA® (Kinetic Exclusion Assay) assay, available from Sapidyne Instruments (Boise, Idaho) can also be used.

[0166] “Internalize” or “internalization” of an immunoglobulin molecule relates to the ability of an immunoglobulin or ADC or APC as described herein binding to a cell surface receptor to induce a receptor-mediated endocytosis upon binding.

[0167] “De-glycosylated” or “de-glycosylation” relates to the, partial and in particular complete, removal of one or more glycosyl-residues from a glycosylated species of a biomolecule, as for example a glycosylated immunoglobulin molecule.

[0168] B.3 Chemical definitions

[0169] The term “halogen” denotes a fluorine, bromine, chlorine or iodine radical, in particular a fluorine radical.

[0170] A “hydrocarbyl group” relates to an optionally substituted, straight-chain or branched, non-cyclic or cyclic, homo- or hetero- hydrocarbyl groups having a hydrocarbon chain comprising 1 to 50, 1 to 25, 1 to 12 or in particular 1 to 6 carbon atoms. A “hetero- hydrocarbyl” group may carry within its hydrocarbon chain one or more, as for example 1, 2,

[0171] M / 65047-PCTVERAXA 20

[0172] 3, 4 or 5, more particular 1 or 2 identical or different heteroatoms, in particular selected from O, S and NH. Particular examples of such hydrocarbyl groups are optionally substituted, linear or branched (hetero)alkyl-, (hetero)alkenyl-, (hetero)alkinyl, (hetero)cycloalkyl- and (hetero)cycloalkenyl-groups.

[0173] A “hydrocarbylene group” relates to an optionally substituted, straight-chain or branched, homo- or hetero-hydrocarbylene bridging group having a hydrocarbon chain comprising 1 to 50, 1 to 25, 1 to 12 or in particular 1 to 6 carbon atoms. A “hetero- hydrocarbylene” group may carry within its hydrocarbon chain one or more, as for example 1, 2, 3, 4 or 5, more particular 1 or 2 identical or different heteroatoms, in particular selected from O, S and NH. Particular examples of such hydrocarbylene groups are optionally substituted, linear or branched (hetero)alkylene-, (hetero)alkenylene-, (hetero)alkinylene, (hetero)cycloalkylene- and (hetero)cycloalkenylene-groups.

[0174] A “spacer group” in the context of the invention is to be interpreted di- or polyvalent, in particular divalent group. A particular example of a “spacer group” are hydrocarbylene groups, in particular alkyene groups. Particularly encompassed are linear or branched C1-C4-alkylene residues selected from are -CH2-, -(CH2)2-, -(CH2)3-,-(CH2)4-, -(CH2)2-CH(CH3)-, -CH2-CH(CH3)-CH2- -CH2-CH(CH3)-, -CH(CH3)-CH2-, -CH(CH3)-CH(CH3)-, -C(CH3)2-CH2-, -CH2-C(CH3)2-, -CH2-CH(CH2CH3)- and -CH(CH2CH3)-CH2-.

[0175] “Alkyl" or “alkanyl” refers to straight-chain or branched homo-hydrocarbyl residues, selected from methyl, ethyl, n-propyl, 1 -methylethyl, n-butyl, 1 -methylpropyl, 2-methylpropyl, 1,1 -dimethylethyl, n-pentyl, 1 -methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1 -ethylpropyl, 1,1 -dimethylpropyl, 1,2-dimethylpropyl, n-hexyl, 1 -methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1 -dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1 -ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1 -methylpropyl und 1-ethyl-2-methylpropyl; n-neptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecyl, n-eicosyl, n-hencosyl, n-docosyl, n-tricosyl, n-tetracosyl, n-pentacosyl, n-hexacosyl, n-heptacosyl, n-octacosyl, n-nonacosyl, squalyl, as well as the branched isomers thereof. Particularly, encompassed are linear or branched Ci-C4-alkyl residues selected from methyl, ethyl, n-propyl, 1 -methylethyl, n-butyl, 1 -methylpropyl, 2-methylpropyl and 1,1 -dimethylethyl.

[0176] “Alkenyl” relates to the mono-unsaturated analogs of the abovementioned alkyl groups comprising a single carbon-carbon double bond. As non-limiting examples there may be mentioned C2-C6-alkenyls, like Ethenyl, 1 -Propenyl, 2-Propenyl, 1 -Methylethenyl, 1-

[0177] M / 65047-PCTVERAXA 21

[0178] Butenyl, 2-Butenyl, 3-Butenyl, 1-Methyl-1 -propenyl, 2-Methyl-1 -propenyl, 1-Methyl-2-propenyl, 2-Methyl-2-propenyl, 1 -Pentenyl, 2-Pentenyl, 3-Pentenyl, 4-Pentenyl, 1-Methyl-1-butenyl, 2-Methyl-1 -butenyl, 3-Methyl-1 -butenyl, 1-Methyl-2-butenyl, 2-Methyl-2-butenyl, 3-Methyl-2-butenyl, 1-Methyl-3-butenyl, 2-Methyl-3-butenyl, 3-Methyl-3-butenyl, 1,1-Dimethyl-2-propenyl, 1,2-Dimethyl-1 -propenyl, 1,2-Dimethyl-2-propenyl, 1-Ethyl-1 -propenyl, 1 -Ethyl-2-propenyl, 1 -Hexenyl, 2-Hexenyl, 3-Hexenyl, 4-Hexenyl, 5-Hexenyl, 1-Methyl-1 -pentenyl, 2-Methyl-1 -pentenyl, 3-Methyl-1 -pentenyl, 4-Methyl-1 -pentenyl, 1-Methyl-2-pentenyl, 2-Methyl-2-pentenyl, 3-Methyl-2-pentenyl, 4-Methyl-2-pentenyl, 1-Methyl-3-pentenyl, 2-Methyl-3pentenyl, 3-Methyl-3-pentenyl, 4-Methyl-3-pentenyl, 1-Methyl-4-pentenyl, 2-Methyl-4-pentenyl, 3-Methyl-4-pentenyl, 4-Methyl-4-pentenyl, 1,1-Dimethyl-2-butenyl, 1,1-Dimethyl-3-butenyl, 1,2-Dimethyl-1 -butenyl, 1,2-Dimethyl-2-butenyl, 1,2-Dimethyl-3-butenyl, 1,3-Dimethyl-1 -butenyl, 1,3-Dimethyl-2-butenyl, 1,3-Dimethyl-3-butenyl, 2,2-Dimethyl-3-butenyl, 2, 3-Dimethyl-1 -butenyl, 2,3-Dimethyl-2-butenyl, 2,3-Dimethyl-3-butenyl, 3,3-Dimethyl-1-butenyl, 3,3-Dimethyl-2-butenyl, 1-Ethyl-1 -butenyl, 1 -Ethyl-2-butenyl, 1 -Ethyl-3-butenyl, 2-Ethyl-1 -butenyl, 2-Ethyl-2-butenyl, 2-Ethyl-3-butenyl, 1,1,2-Trimethyl-2-propenyl, 1-Ethyl-1-methyl-2-propenyl, 1 -Ethyl-2-methyl-1 -propenyl and 1-Ethyl-2-methyl-2-propenyl. Particularly there may be mentioned C2-C4-alkenyls, like ethenyl, 1 -propenyl, 2-propenyl, 1-methylethenyl, 1 -butenyl, 2-butenyl, 3-butenyl, 1-methyl-1 -propenyl, 2-methyl-1 -propenyl, 1-methyl-2-propenyl and 2-methyl-2-propenyl.

[0179] The term “cycloalkyl” relates to carbocyclic residues having 3 to 20 carbon atoms, and more particularly to C3-C12-cycloalkyl, residues, in particular cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl und cyclododecyl, cyclopropyl-methyl, cyclopropyl-ethyl, cyclobutyl-methyl, cyclobutyl-ethyl, cyclopentyl-methyl, cyclopentyl-ethyl, and cyclohexyl-methyl. Particularly there may be mentioned to C3-C6-cycloalkyl residues selected from cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

[0180] The term “Cycloalkane” relates to saturated carbocyclic ring having 3 to 6 carbon atoms, and more particularly to C3-C6-cycloalkane rings, selected from cyclopropane, cyclobutane, cyclopentane and cyclohexane rings.

[0181] The term “Cycloalkene” relates to mono- or polyunsaturated carbocyclic ring having 3 to 6 carbon atoms, and more particularly to monounsaturated carbocyclic to C3-C6-cycloalkene rings, selected from cyclopropene, cyclobutene, cyclopentene and cyclohexene rings.

[0182] M / 65047-PCTVERAXA 22

[0183] The term “cycloalkenyl” relates to mono- or polyunsaturated analogs of the above-memntioned cycloakyl residues; as non-limiting examples there may be mentioned „mono-unsaturated residues having at least 5 carbon atoms such as cyclopenten-1-yl, cyclopenten-3-yl, cyclohexen-1-yl, cyclohexen-3-yl and cyclohexen-4-yl-.

[0184] The term “alkanoyl” refers to acyl analogues of the type R(C=O)-, wherein R represents an alkyl or cycloalkyl group as defined above.

[0185] The term “alkanoyloxy” refers to residus of the type R(C=O)-O-, wherein R represents an alkyl or cycloalkyl group as defined above.

[0186] The term “alkylene" refers to straight chain or branched hydrocarbon bridges having 1 to 10 particularly 1 to 7 carbon atoms and more particularly 1 to 4 carbon atoms; non-limiting examples thereof are -CH2-, -(CH2)2-, -(CH2)3-,-(CH2)4-, -(CH2)2-CH(CH3)-, -CH2-CH(CH3)-CH2- -CH2-CH(CH3)-, -CH(CH3)-CH2-, -CH(CH3)-CH(CH3)-, -C(CH3)2-CH2-, -CH2-C(CH3)2-, -CH2-CH(CH2CH3)-, -CH(CH2CH3)-CH2-, -(CH2)5-,-C(CH3)2-CH(CH3)-, -CH(CH3)-C(CH3)2-, -(CH2)6, -(CH2)7-, -CH(CH3)-CH2-CH2-CH(CH3)- or -CH(CH3)-CH2-CH2-CH2-CH(CH3)-; -CH(CH2CH3)-CH(CH2CH3)-, -C(CH2CH3)2-CH2-, -CH2-C(CH2CH3)2-, -CH2-CH(n-Propyl)-, -CH(n-Propyl)-CH2-, -CH(n-Propyl)-CH(CH3)-, -CH2-CH(n-Butyl)-, -CH(n-Butyl)-CH2-, -CH(CH3)-CH(CH2CH3)-, -CH(CH3)-CH(n-Propyl)-, -CH(CH2CH3)-CH(CH3)-, -CH(CH3)-CH(CH2CH3)-. Particularly encompassed are linear or branched C1-C4-alkylene residues selected from -CH2-, -(CH2)2-, -(CH2)3-,-(CH2)4-, -(CH2)2-CH(CH3)-, -CH2-CH(CH3)-CH2- -CH2-CH(CH3)-, -CH(CH3)-CH2-, -CH(CH3)-CH(CH3)-, -C(CH3)2-CH2-, -CH2-C(CH3)2-, -CH2-CH(CH2CH3)- and -CH(CH2CH3)-CH2-.

[0187] “Oxyalkylene” or “alkyleneoxy” or refers to straight chain or branched hydrocarbon bridges having 1 to 10 particularly 1 to 7 carbon atoms and more particularly 1 to 4 carbon atoms as defined above, but terminated by -O- at on of the two ends of the bridge.. Particularly encompassed are linear or branched oxy-C1-C4-alkylene residues selected from -O-CH2-, -O-(CH2)2-, -O-(CH2)3-, -O-(CH2)4-, -O-(CH2)2-CH(CH3)-, -O-CH2-CH(CH3)-CH2-, -O-CH2-CH(CH3)-, -O-CH(CH3)-CH2-, -O-CH(CH3)-CH(CH3)-, -O-C(CH3)2-CH2-, -O-CH2-C(CH3)2-, -O-CH2-CH(CH2CH3)- and -OCH(CH2CH3)-CH2-; and the corresponding -C1-C4-alkyleneoxy residues selected from -CH2-O-, -(CH2)2-O-, -(CH2)3-,-(CH2)4-O-, -(CH2)2-CH(CH3)-O-, -CH2-CH(CH3)-CH2-O-, -CH2-CH(CH3)-O-, -CH(CH3)-CH2-O-, -CH(CH3)-CH(CH3)-O-, -C(CH3)2-CH2-O-, -CH2-C(CH3)2-O-, -CH2-CH(CH2CH3)-O- and -CH(CH2CH3)-CH2-O-.

[0188] “Alkenylene” represent the monounsaturated analogs of the above alkylene groups having 2 to 10, particularly 2 to 7 carbon atoms and more particularly 2 to 4 carbon atoms.

[0189] M / 65047-PCTVERAXA 23

[0190] “Alkoxy” relates to a radical of the formula R-O-, wherein R is a straight-chain or branched alkyl group as defined above. Particular alkoxy groups have from 1 to 6, in particular 1 to 4, like 1, 2 or 3 carbon atoms. Non-limiting examples are methoxy, ethoxy, n-propoxy, 1 -methylethoxy, n-butoxy, 1 -methylpropoxy, 2-methylpropoxy, 1,1 -dimethylethoxy, n-pentyloxy, 1 -methylbutyoxy, 2-methylbutoxy, 3-methylbutoxy, 2,2-dimethylpropoxy, 1-ethylpropoxy, 1,1 -dimethylpropoxy, 1,2-dimethylpropoxy, n-hexyloxy, 1 -methylpentyloxy, 2-methylpentyloxy, 3-methylpentyloxy, 4-methylpentyloxy, 1,1 -dimethylbutoxy, 1,2-dimethylbutoxy, 1,3-dimethylbutoxy, 2,2-dimethylbutoxy, 2,3-dimethylbutoxy, 3,3-dimethylbutoxy, 1 -ethylbutoxy, 2-ethylbutoxy, 1,1,2-trimethylpropoxy, 1,2,2-trimethylpropoxy, 1 -ethyl- 1 -methylpropoxy und 1 -ethyl-2-methylpropoxy. “Alkenoxy” relates to a radical of the formula R-O-, wherein R is a straight-chain or branched alkenyl group a defined above. Particular alkenoxy groups have from 2 to 6, in particular 2 to 4, more particularly 2 or 3 carbon atoms as defined herein.

[0191] “Aryl” relates to mono- or polycyclic aromatic, particularly monocyclic moieties, having 6 to 26, in particular 6 to 14, more particularly 6 ring carbon atoms, in particular, phenyl, fluorenyl, naphthenyl and phenantrenyl moieties.

[0192] “Heteroaryl” relates to analogues of the above-mentioned aryl groups additionally comprising at least one, like 1 to 10, particularly 1 to 5, more particularly 1, 2 or 3 identical or different ring-heteroatoms, selected from O, S and N.

[0193] “Aralkyl” relates to an aryl residue linked to an alkyl group, each as defined above. In particular, such aralkyl groups comprise 7 to 24 carbon atoms. As non-limiting examples there may be mentioned phenylmethyl or phenylethyl.

[0194] Unless indicated otherwise, the term “substituents” is selected from halogen, linear or branched C1-C4-alkyl, linear or branched C2-C4-alkenyl, CN, CF3, hydroxyl, -O-CF3, linear or branched C1-C4-alkoxy, linear or branched C2-C4-alkeneoxy, C2-C4-alkanoyloxy, -amino, or -N(C1-C4-alkyl)2, -O-CF3, linear or branched C2-C5-alkanoyloxy, linear or branched C1-C4-alkylaminocarbonyloxy, or linear or branched C1-C4-alkylthio, -NR4R5, carboxy, or linear or branched C1-C4-alkyl carboxylate groups,

[0195] Unless indicated otherwise, the term "substituted" means that a radical is substituted with 1, 2 or 3, especially 1 or 2, substituent(s).

[0196] A “linkage” is formed between two neighbored structural motifs of a compound of the invention and is, unless otherwise indicated, either a chemical bond, or is a “linking group” selected from a ether, thioether, ester, amide, carbamate, dicarbamate, carbonate,

[0197] M / 65047-PCTVERAXA 24

[0198] hydrazine, urea, alkylene oxide linkage in any possible orientation. Nonlimiting examples are the following linking groups:

[0199] An “ether” linking group contains at least one group of the type: (-O-).

[0200] A “thioether” linking group contains at least one group of the type: (-S-).

[0201] An “amide” linking group contains at least one group of the type: -C(=O)N(R)- or - (R)N-C(=O)-.

[0202] A “carbamate” linking group contains at least one group of the type: -O-C(=O)-N(R)- or -N(R)- C(=O)-O-.

[0203] A “hydrazine” linking group contains at least one group of the type: -NH-NH-.

[0204] An “imine” linking group contains at least one group of the type: =N-, or =N-NR-, each in any orientation.

[0205] An ’’oxime” linking group contains at least one group of the type =N-0 in any orientation.

[0206] An “amine” linkage contains at least one group of the type -NR-; it also encompasses the corresponding “ammonium” linkage of the type_-N+RR-.

[0207] Further examples of linking groups are asymmetric linking groups selected from -NR- CH(NR2)-, -NR-C(NR)-NR-, -C(O)-NR-CR(NR2)-, -C(O)-NR-C(NR)-NR-, -NR-CH(NR2)-C(O)-, -NR-C(NR)-NR-C(O)-, each in any orientation.

[0208] In the above-mentioned chemical formulae of particular linkers residues R independently of each other may represent H or linear or branched Ci-C4-alkyl.

[0209] A “cleavable group” encompasses any group, which may be cleaved enzymatically or chemically, in particular under in vivo or ex vivo conditions; an enzymatic cleavage may be effected, for example, through the action of a protease; a chemical cleavage, may be effected for example through hydrolytic cleavage or reductive cleavage of S-S bonds.

[0210] Drug-to-antibody ratio (DAR) relates to the distribution of different payload, such as drugs or the cytotoxic group to an antibody-drug conjugate (ADC), or the average of different drug to antibody loads for an ADC.

[0211] A “tetrazine” or “tetrazinyl” group according to the present invention represents, unless otherwise defined, a residue that consists of a six-membered aromatic ring containing four nitrogen atoms with the molecular formula -C2N4-, in particular derived from the 1,2,4,5- tetrazine or s-tetrazine isomer, and linked to neighboring groups via ring carbon positions 3 and 6.

[0212] A “TCO-type” molecule or moiety as herein described encompasses a chemical structure of the formula:

[0213] M / 65047-PCTVERAXA 25

[0214]

[0215] which may be further mono- or polysubstituted, and / or may be incorporated in an optionally mono- or polysubstituted polycyclic, in particular bicyclic, ring structure. Such “TCO-type” molecule or moiety is preferably (directly or indirectly via a linking / spacer moiety as defined below) bound to the carbon atom at position 9 of the carbon structure of Neu5Ac via a chemical bond formed at anyone of the carbon atoms at anyone of the TCO ring positions 2, 3, 4, 5, 6 or 7, in particular ring positions 2, 3 or 4.

[0216] A “bioorthogonal” reaction or “click reaction” refers to any chemical reaction that can occur inside of living systems without reacting with native components / substrates or interfering with native biochemical processes.

[0217] A “docking group” (DG) is a chemical moiety that has the ability to chemically react, in particular, via a so-called “bioorthogonal” or “click reaction”, with a trans-cyclooctene group as defined herein. In particular, such docking groups are preferably trans-cyclooctene reactive groups selected from dienes. More particularly, it is selected from dienes having the ability to react in a biological environment with the trans-cyclooctene group. As non-limiting examples there may be mentioned, groups comprising optionally substituted triazinyl or tetrazinyl residues and 1,2-quinones. Such groups are well known in the prior art [Yang et al., Angew. Chem. Int. Ed. 2012, 51, 5222 -5225; Fan et al., Angew. Chem. Int. Ed. 2016, 55, 14046 -14050; Mao et al., Angew. Chem. Int. Ed. 2019, 58, 1106 -1109; Qu et al., Angew. Chem. Int. Ed. 2018, 57, 12057 -12061; Eising et al., Bioconjugate Chem. 2018, 29, 3054-3059; Meng et al., J. Org. Chem. 2017, 82, 1676-1687; Xie et al., Angew. Chem. Int. Ed. 2020, 59, 16967-16973; Lambert et al., J. Am. Chem. Soc. 2019, 141, 17068-17074; Jemas et al., J. Am. Chem. Soc. 2022, 144, 1647-1662; Selvaraj et al., Tetrahedron Letters 2014, 55, 4795-4797; Dowling et al., J. Org. Chem. 2018, 83, 4229-4238; Battisti et al., Bioconjugate Chem. 2022, 33, 4, 608-624; Karver et al., Bioconjugate Chem. 2011, 22, 2263-2270; Bender et al., Org. Lett. 2017, 19, 5693-5696; Kamber et al., J. Am. Chem. Soc.

[0218] 2015, 137, 8388-8391; Ros et al., Bioconjugate Chem. 2020, 31, 933-938; Ros et al., Chem. Commun., 2020, 56, 11086; van Onzen et al., J. Am. Chem. Soc. 2020, 142, 10955-10963; Carlson et al., J. Am. Chem. Soc. 2018, 140, 3603-3612.]

[0219] “Tetrazine ligation” refers to the reaction of a trans-cyclooctene and an s-tetrazine in an inverse-demand Diels Alder reaction followed by retro Diels Alder reaction to eliminate

[0220] M / 65047-PCTVERAXA 26

[0221] nitrogen (N2). A reaction of this type proceeds with high velocity, allowing bio molecule modification at extremely low concentrations.

[0222] An “inverse electron-demand Diels-Alder (I EDDA) cycloaddition” is a reaction between an electron-poor diene and an electron-rich dienophile and represents only one example of different types of “bioorthogonal reactions”. The diene used may be, for example, a 1,2,4,5-tetrazine or a 1,2,4-triazine. The dienophiles encompass a variety of molecules including strained cyclic alkenes, such as trans-cyclooctenes (TCO, norbornenes, cyclopropenes or azetines). Of these, the reaction between a tetrazine and TCO is the fastest reported to date and suitable for in vivo applications (Smeek et al, Current Opinion in Chemical Biology Volume 60, February 2021, Pages 79-88).

[0223] “Acid or base addition salts” of compounds of the invention are especially addition salts with physiologically tolerated acids or bases. Physiologically tolerated acid addition salts can be formed by treatment of the base form of a compound of the invention with appropriate organic or inorganic acids. Compounds of the invention containing an acidic proton may be converted into their non-toxic metal or amine addition salt forms by treatment with appropriate organic and inorganic bases. The compounds and salts of the invention also comprise the hydrates and solvent addition forms thereof, e.g. hydrates, alcoholates and the like.

[0224] “Physiologically tolerated” acids or bases are in particular those which are tolerated by the system used for the incorporation of the first and second dienophiles (e.g. a biological system such as a translation system used for preparation of polypeptides with trans-cyclooctenyl or cyclooctynyl groups), e.g. which are substantially non-toxic to living cells.

[0225] Compounds as herein described may contain one or more asymmetric elements such as stereogenic centers, stereogenic axes and the like, e.g. asymmetric carbon atoms, so that the compounds can exist in different stereoisomeric forms. These compounds can be, for example, racemates or optically active forms. All stereoisomers, diastereomers, in purified and mixture forms are included. Accordingly, when a compound is recited by specific name or a class of compounds is recited, all these forms are intended to be included. In the case of hyTCO residues of the present invention Z- and E-forms thereof are excluded.

[0226] Compounds as herein described may also exist in more than one form of structural isomers also designated as constitutional isomers or regioisomers. These are molecules that differ only in the different sequence of their atoms or atomic groups while having the same gross formula.

[0227] M / 65047-PCTVERAXA 27

[0228] Therefore, unless otherwise stated, for each of the compounds, biomolecules and conjugates as described herein, any such potential stereo- or regiosomeric form or mixture of more than one stereo- and / or regiosomeric form is within the scope of the present invention.

[0229] B.4 Biochemical definitions

[0230] A “polypeptide” is any oligomer of amino acid residues (natural or unnatural, or a combination thereof), of any length, typically but not exclusively joined by covalent peptide bonds. A polypeptide can be from any source, e.g., a naturally occurring polypeptide, a polypeptide produced by recombinant molecular genetic techniques, a polypeptide from a cell or translation system, or a polypeptide produced by cell-free synthetic means. A polypeptide is characterized by its amino acid sequence, e.g., the primary structure of its component amino acid residues. As used herein, the amino acid sequence of a polypeptide is not limited to full-length sequences, but can be partial or complete sequences. Furthermore, it is not intended that a polypeptide be limited by possessing or not possessing any particular biological activity.

[0231] As used herein, the term "protein" is synonymous with polypeptide. The term "peptide" refers to a small polypeptide, for example but not limited to, from 2-25 amino acids in length.

[0232] The term “bioorthogonal” reaction or “click reaction” refers to any chemical reaction that can occur inside of living systems, i.e. in aqueous environment, without reacting with native compounds / substrates or interfering biochemical processes. “Tetrazine ligation” may for example be mentioned as one type of bioorthogonal reaction. Bioorthogonal chemistry typically proceeds in two steps. First, a cellular substrate is modified with a bioorthogonal functional group (also designated chemical reporter) as for example a trans-cyclooctene compound described herein. Cellular substrates include for example immunoglobulins, like natural or recombinant antibodies, etc. The chemical reporter must not alter the structure of the substrate dramatically to avoid affecting its bioactivity. In a second step, a probe, such as a functionalized molecule containing a docking group, such as a complementary functional group, as for example a tetrazine group, is introduced to react and label the substrate.

[0233] As used herein, the term “host cell” or “transformed cell” refers to a cell (or organism) altered to harbor at least one nucleic acid molecule, for instance, a recombinant gene encoding a desired protein or nucleic acid sequence which upon transcription yields a polypeptide for use as described herein. The host cell is a prokaryotic or eukaryotic cell, like a bacterial cell, a fungal cell, a plant cell, an insect cell or mammalian cell. The host cell may contain a recombinant gene which has been integrated into the nuclear or organelle

[0234] M / 65047-PCTVERAXA 28

[0235] genomes of the host cell. Alternatively, the host may contain the recombinant gene extra-chromosomally.

[0236] A particular organism or cell is meant to be “capable of producing a protein of interest (POI)” when it produces a POI naturally or when it does not produce said POI naturally but is transformed to produce said POI.

[0237] The terms “glycan” and “polysaccharides” are defined by IUPAC as synonyms meaning "compounds consisting of a large number of monosaccharides linked glycosidically". Glycans can be homo- or heteropolymers of monosaccharide residues, and can be linear or branched. In practice the term “glycan” may also be used to refer to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, or a proteoglycan, even if the carbohydrate is only an oligosaccharide. Glycans usually consist solely of O-glycosidic linkages of monosaccharides. For example, cellulose is a glycan (or, to be more specific, a glucan) composed of (3-1,4-linked D-glucose, and chitin is a glycan composed of p-1,4-linked N-acetyl-D-glucosamine. Glycans can be homo- or heteropolymers of monosaccharide residues, and can be linear or branched.

[0238] An endoglycosidase is an enzyme that releases oligosaccharides from glycoproteins or glycolipids. It may also cleave polysaccharide chains between residues that are not the terminal residue, although releasing oligosaccharides from conjugated protein and lipid molecules is more common. Different endoglycosidase types exist. Two endoglycosidases from the human pathogen Streptococcus pyogenes, EndoS and EndoS2, have been shown to hydrolyze N-linked glycans of human immunoglobulin G (Sjögren, J., et al Glycobiology, 2015, 25(10):1053-1063).

[0239] The enzyme galactosyltransferase (EC 2.4.1.38) catalyzes the reaction involving UDP-galactose and N-acetylglucosamine for the production of galactose beta-1, 4-N-acetylglucosamine. B4GalT1 (Gene ID: 2683; of homo sapiens) defines a gene encoding one of seven beta-1,4-galactosyltransferases (beta4GalT). All genes encode type II membranebound glycoproteins that appear to have exclusive specificity for the donor substrate UDP-galactose; all transfer galactose in a beta1,4 linkage to similar acceptor sugars: GlcNAc, Glc, and Xyl;

[0240] (see also https: / / www.ncbi. nlm.nih.gov / gene? Db=gene& Cmd=DetailsSearch& Term=2683) The enzyme beta-galactoside alpha-2, 6-sialyltransferase (EC 2.4.3.1) (ST6Gal1) (UniProt P15907) is an enzyme that in humans is encoded by the ST6GAL1 gene. The protein encoded by this gene is a type II membrane protein that catalyzes the transfer of sialic acid from CMP-sialic acid to galactose-containing substrates. The encoded protein, which is normally found in the Golgi but which can be proteolytically processed to a soluble

[0241] M / 65047-PCTVERAXA 29

[0242] form, is involved in the generation of the cell-surface carbohydrate determinants and differentiation antigens HB-6, CDw75, and CD76. This protein is a member of glycosyltransferase family 29. Three transcript variants encoding two different isoforms have been found for this gene.

[0243] The enzyme alkaline phosphatase (ALP, alkaline phenyl phosphatase) (E. C.3.1.3.1.) is a phosphatase with the physiological role of dephosphorylating compounds. The enzyme is found across a multitude of organisms, prokaryotes and eukaryotes alike, with the same general function, but in different structural forms suitable to the environment they function in. Alkaline phosphatase is found in the periplasmic space of E. coli bacteria. This enzyme is heat stable and has its maximum activity at high pH. Alkaline phosphatase is a zinc-containing dimeric enzyme with the MW: 86,000 Da, each subunit containing 429 amino acids with four cysteine residues linking the two subunits. Alkaline phosphatase contains four Zn ions and two Mg ions, with Zn occupying active sites A and B, and Mg occupying site C, so the fully active native alkaline phosphatase is referred to as (ZnAZnBMgC)2 enzyme. The mechanism of action of alkaline phosphatase involves the geometric coordination of the substrate between the Zn ions in the active sites.

[0244] B.5 Oligosaccharide structures found in human IgG

[0245] The human glycans are mainly classified as 'biantennary complex' structure with a core fucose (Fuc) and are often terminated with N-acetylneuraminic acid (Neu5Ac), a sialic acid. Figure 1 B, left structure) shows the largest N-linked oligosaccharide structure found in human IgG. The third N-acetylglucosamine (GlcNAc,) bisecting arm represents around 10% of human IgGs glycoforms (see also IMGT Information system; IMGT Lexique; N-glycosylation of immunoglobulins (IG) or antibodies.

[0246] (https: / / www.imgt. Org / IMGTeducation / IMGTIexique / G / Glycosylation.html)

[0247] Fc glycosylation of monoclonal antibodies expressed in human cells are glycosylated on the IGHG CH2 N297 (Ell numbering). Said N-glycans are of the complex biantennary form. There is a total of 36 possible isoforms, divided up in 3 major classes GO, G1, G2, according to the number of terminal galactose.

[0248] Classes Glycan name Fucosylated Afucosylated

[0249] GO agalactosylated G0A0 +B+F -B+F +B-F -B-F asialylated G1 monogalactosylated G1A0 +B+F -B+F +B-F -B-F asialylated G1A1 +B+F -B+F +B-F -B-F monosialylated

[0250]

[0251] M / 65047-PCTVERAXA 30

[0252] G2 digalactosylated G2A0 +B+F -B+F +B-F -B-F asialylated G2A1 +B+F -B+F +B-F -B-F monosialylated G2A2 +B+F -B+F +B-F -B-F disialylated

[0253]

[0254] GO = agalactosylated, G1 = monogalactosylated, G2 = digalactosylated

[0255] AO = asialylated, A1 = monosialylated, A2 = disialylated.

[0256] +B, -B: with or without a 'bisecting' N-acetyl glucosamine, GlcNAc (between the two antennae of the glycan and attached to the position 4 of the branching mannose); +B = bisected; -B = not bisected

[0257] +F = fucosylated, -F = afucosylated.

[0258] Monosialylation and monogalactosylation may occur on either the α1-3 or α1-6 arm of the biantennary structures.

[0259] Non-limiting examples of another type of glycan structure are shown in Fig.lC which are so-called high mannose glycan structures comprising 6 to 9 mannose moieties (also designated Man6N2 to Man9N2), which may also be glycan engineered according to the method of the present invention.

[0260] C. PARTICULAR ASPECTS AND EMBODIMENTS

[0261] 1. The first aspect of the present invention

[0262] The first aspect of the present invention relates to particular / V-acetylneuraminic acid (Neu5Ac) derivatives, which are applicable in the biocatalytic preparation of glycan structures carrying a terminal clickable functional moiety.

[0263] A first embodiment of the invention relates to / V-acetylneuraminic acid (Neu5Ac) derivatives of the general formula I:

[0264] TCO-Neu5Ac-CMP

[0265] (I)

[0266] wherein

[0267] CMP represents a residue of the formula II

[0268] M / 65047-PCTVERAXA 31

[0269]

[0270] (II)

[0271] linked via its phosphoryl group to the hydroxyl group in position 2 of the of Neu5Ac ring structure;

[0272] and

[0273] TCO represents a trans-cyclooctenyl-type residue linked to the terminal carbon atom in position 9 of the Neu5Ac moiety.

[0274] According to a second, more particular embodiment the Neu5Ac derivative of the first embodiment is a CMP-p-D- / V-acetylneuraminic acid (CMP-Neu5Ac) derivative of the general formula III

[0275]

[0276] wherein

[0277] TCO represents a moiety of the general formula (V)

[0278] X1-X2a- X3- (V)

[0279] wherein

[0280] X1is a TCO-type moiety of the general formula (VI)

[0281] M / 65047-PCTVERAXA 32

[0282] Y6

[0283] 1Z

[0284] R1

[0285]

[0286] (VI)

[0287] wherein

[0288] Y1, Y2, Y3, Y4, Y5, Y6independently are substituted or non-substituted methylene (-CH2-) groups, keto groups, -NH-, -S- or -O-, provided that at least 4 of Y1, Y2, Y3, Y4, Y5, Y6are substituted or non-substituted methylene (-CH2-) groups, and wherein two neighbouring residues of Y1, Y2, Y3, Y4, Y5and Y6do not simultaneously represent a hetero group selected from -NH-, -S- or -O-; and wherein two neighbouring residues of Y1, Y2, Y3, Y4, Y5and Y6may also form a three- to sixmembered carbocyclic or heterocyclic, nonaromatic or aromatic, saturated or unsaturated ring, provided that two neighbouring residues of any of Y1, Y2, Y3, Y4, Y5and Y6which do not form said ring, do not simultaneously represent a hetero group selected from - NH-, -S- or -O-; and

[0289] wherein said ring is optionally carrying at least one ring substituents, selected from carboxylic acid groups, C1-C4 alkyl carboxylate groups and keto groups; wherein said ring substituents are either directly attached or attached via a C1-C4 alkylene group or a C2-C4alkyenylene group or an oxy-C1-C4-alkylene group or a C1-C4- alkyleneoxy group to said ring group;

[0290] substituents R1, R2and R3independently of each other are hydrogen, or substituents selected from halogen, linear or branched Ci-C4-alkyl, linear or branched C2-C4-alkenyl, CF3, CN, OH, linear or branched C1- C4-alkoxy, linear or branched C2-C4-alkeneoxy, -O-CF3, linear or branched C2-C5-alkanoyloxy, linear or branched C1-C4- alkylaminocarbonyloxy, or linear or branched Ci-C4-alkylthio, - NR4R4a, carboxy, or linear or branched C1-C4-alkyl carboxylate

[0291] M / 65047-PCTVERAXA

[0292] groups, particularly R1, R2and R3independently of each other are hydrogen, or substituents selected from a hydrophilic substituent, and more particularly R1, R2, R3are OH or linear or branched C1-C4- alkoxy, and even more particularly R1, R2are OH or linear or branched C1-C4-alkoxy ands R3is H;

[0293] wherein

[0294] R4and R4aindependently of each other represent H or linear or branched Ci-C4-alkyl;

[0295] X2is a spacer group selected from lower alkylene, preferably methylene;

[0296] a is 0 or 1;

[0297] X3is a linking group selected from -O-, -S-, -NR8-, -N+R8R8a-, -C(O)- NR9-, -NR9-C(O)-, -NH-CH(NH2)-, -CH(NH2)-NH-, -NH-C(NH)-NH-, - NR9-C(O)-O-, -O-C(O)-NR9-, -C(O)-NH-CH(NH2)-, -C(O)-NH-C(NH)- NH-, NH-CH(NH2)-C(O)-, -NH-C(NH)-NH-C(O)-, =N-, =N-O-, =N-NR8-;

[0298] wherein

[0299] R8, R8aund R9independently of each other represent H or linear or branched Ci-C4-alkyl, or linear or branched C2-C4-alkenyl; and

[0300]

[0301] represents the chemical bond linking the terminal group X1to spacer group X2or to the linking group X3.

[0302] According to non-limiting particular embodiments of the above Y1, Y2, Y3, Y4, Y5and Y6independently of each other have the following meanings:

[0303] Y1represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group, -NH-, -S- or -O-; and / or

[0304] Y2represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group, keto group, -NH-, -S- or -O-; and / or

[0305] Y3represents a substituted, in particular mono-substituted, or non-substituted methylene (- CH2-) group, keto group, -NH-, -S- or -O-; and / or

[0306] Y4represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group, keto group, -NH-, -S- or -O-; and / or

[0307] M / 65047-PCTVERAXA

[0308] Y5represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group, keto group, -NH-, -S- or -O-; and / or

[0309] Y6represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group, keto group, -NH-, -S- or -O-.

[0310] According to non-limiting even moreparticular embodiments of the above Y1, Y2, Y3, Y4, Y5and Y6independently of each other have the following meanings:

[0311] Y1represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group; and / or

[0312] Y2represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group, and / or

[0313] Y3represents a substituted, in particular mono-substituted, or non-substituted methylene (- CH2-) group, keto group, -NH-, -S- or -O-; and / or

[0314] Y4represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group; and / or

[0315] Y5represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group, keto group, -NH-, -S- or -O-; and / or

[0316] Y6represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group.

[0317] According to non-limiting even more particular embodiments of the above Y1, Y2, Y3, Y4, Y5and Y6independently of each other have the following meanings:

[0318] Y1represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group;

[0319] Y2represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group;

[0320] Y3represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group;

[0321] Y4represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group;

[0322] Y5represents a substituted, in particular mono-substituted, or non-substituted methylene (- CH2-) group, and

[0323] Y6represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group.

[0324] M / 65047-PCTVERAXA

[0325] According to other non-limiting more particular embodiments of the above Y1, Y2, Y3, Y4, Y5and Y6independently of each other have the following meanings:

[0326] Y1represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group, and / or

[0327] Y2represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group, and / or

[0328] Y3represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group, and / or

[0329] Y4represents a substituted, in particular mono-substituted, or non-substituted methylene (- CH2-) group, keto group, -NH-, -S- or -O-; and / or

[0330] Y5represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group, and / or

[0331] Y6represents a substituted, in particular mono-substituted, or non-substituted methylene (- CH2-) group, keto group, -NH-, -S- or -O-;

[0332] According to other non-limiting more particular embodiments of the above Y1, Y2, Y3, Y4, Y5and Y6independently of each other have the following meanings:

[0333] Y1represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group; and / or

[0334] Y2represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group; and / or

[0335] Y3and Y4together form a three- to six-membered carbocyclic or heterocyclic, nonaromatic or aromatic, saturated or unsaturated ring, optionally carrying at least one ring substituent; and / or

[0336] Y5represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group; and / or

[0337] Y6represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group.

[0338] According to other non-limiting more particular embodiments of the above Y1, Y2, Y3, Y4, Y5and Y6independently of each other have the following meanings:

[0339] Y1represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group, and / or

[0340] M / 65047-PCTVERAXA 36

[0341] Y2represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group, and / or

[0342] Y3represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group, and / or

[0343] Y4and Y5together form a three- to six-membered carbocyclic or heterocyclic, nonaromatic or aromatic, saturated or unsaturated ring, optionally carrying at least one ring substituent; and / or

[0344] Y6represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group.

[0345] According to other non-limiting more particular embodiments of the above Y1, Y2, Y3, Y4, Y5and Y6independently of each other have the following meanings:

[0346] Y1represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group, and / or

[0347] Y2represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group, and / or

[0348] Y3represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group, and / or

[0349] Y4represents a substituted, in particular mono-substituted, or non-substituted methylene (-CH2-) group, and / or

[0350] Y5ans Y6together form a three- to six-membered carbocyclic or heterocyclic, nonaromatic or aromatic, saturated or unsaturated ring, optionally carrying at least one ring substituent.

[0351] According to other non-limiting even more particular embodiments of the above Y1represents a mono-substituted methylene (-CH2-) group, and / or

[0352] Y2represents a mono-substituted methylene (-CH2-) group;

[0353] wherein the substituents independently are selected from OH, linear or branched C1-C4-alkoxy, linear or branched C2-C4-alkeneoxy, -O-CF3, carboxy or linear or branched C1-C4-alkyl carboxylate groups.

[0354] According to other non-limiting even more particular embodiments of the above Y1represents a mono-substituted methylene (-CH2-) group, and / or

[0355] Y2represents a mono-substituted methylene (-CH2-) group;

[0356] M / 65047-PCTVERAXA 37

[0357] wherein the substituents independently are selected from OH, linear or branched C1-C4-alkoxy, or linear or branched C1-C4-alkyl carboxylate groups.

[0358] According to other non-limiting even more particular embodiments of the above Y1represents a mono-substituted methylene (-CH2-) group, and

[0359] Y2represents a mono-substituted methylene (-CH2-) group;

[0360] wherein the substituents independently are selected from OH or linear or branched C1-C4-alkoxy groups.

[0361] According to other non-limiting even more particular embodiments of the above Y1represents a mono-substituted methylene (-CH2-) group, and / or

[0362] Y2represents a mono-substituted methylene (-CH2-) group;

[0363] wherein the substituents independently are selected from OH, linear or branched C1-C4-alkoxy, linear or branched C2-C4-alkeneoxy, -O-CF3, carboxy or linear or branched C1-C4-alkyl carboxylate groups; and

[0364] Y3, Y4, Y5and Y6independently of each other represent a substituted or non-substituted methylene (-CH2-) group, wherein the substituents independetly of each other are selected from halogen, linear or branched Ci-C4-alkyl, linear or branched C2-C4-alkenyl, CF3, CN, OH, linear or branched C1-C4-alkoxy, linear or branched C2-C4-alkeneoxy, -O-CF3, linear or branched C2-C5-alkanoyloxy, linear or branched Ci-C4-alkylaminocarbonyloxy, or linear or branched Ci-C4-alkylthio, -NR4R4a, carboxy, or linear or branched C1-C4-alkyl carboxylate groups.

[0365] According to other non-limiting even more particular embodiments of the above Y1represents a mono-substituted methylene (-CH2-) group, and / or

[0366] Y2represents a mono-substituted methylene (-CH2-) group;

[0367] wherein the substituents independently are selected from OH, linear or branched C1-C4-alkoxy, or linear or branched C1-C4-alkyl carboxylate groups; and

[0368] Y3, Y4, Y5and Y6independently of each other represent a substituted or non-substituted methylene (-CH2-) group, wherein the substituent sindependetly of each other are selected from halogen, linear or branched Ci-C4-alkyl, linear or branched C2-C4-alkenyl, CF3, CN, OH, linear or branched C1-C4-alkoxy, linear or branched C2-C4-alkeneoxy, -O-CF3, linear or branched C2-C5-alkanoyloxy, linear or branched Ci-C4-alkylaminocarbonyloxy, or linear or

[0369] M / 65047-PCTVERAXA 38

[0370] branched Ci-C4-alkylthio, -NR4R4a, carboxy, or linear or branched C1-C4-alkyl carboxylate groups.

[0371] According to other non-limiting even more particular embodiments of the above Y1represents a mono-substituted methylene (-CH2-) group, and

[0372] Y2represents a mono-substituted methylene (-CH2-) group;

[0373] wherein the substituents independently are selected from OH or linear or branched C1-C4-alkoxy groups; and

[0374] Y3, Y4, Y5and Y6independently of each other represent a substituted, in particular monosubstituted, or non-substituted methylene (-CH2-) group, wherein the substituents independetly of each other are selected from halogen, linear or branched Ci-C4-alkyl, linear or branched C2-C4-alkenyl, CF3, CN, OH, linear or branched C1-C4-alkoxy, linear or branched C2-C4-alkeneoxy, -O-CF3, linear or branched C2-C5-alkanoyloxy, linear or branched C1-C4-alkylaminocarbonyloxy, or linear or branched C1-C4-alkylthio, -NR4R4a, carboxy, or linear or branched C1-C4-alkyl carboxylate groups.

[0375] According to non-limiting even more particular embodiments of the above particular embodiments, anyone of Y1, Y2, Y3, Y4, Y5and Y6represents a mono-substituted or nonsubstituted methylene group, wherein in one of said groups Y1, Y2, Y3, Y4, Y5and Y6a hydrogen atom bond to its ring carbon atom is replaced by a chemical bond

[0376]

[0377] linking the terminal group X1to spacer group X2or to the linking group X3.

[0378] Accoding to another more particular embodiment Y3is -CH2-.

[0379] Accoding to another more particular embodiment Y3is -O-.

[0380] Accoding to still another more particular embodiment Y3is -S-.

[0381] Accoding to still another more particular embodiment Y3is -NH-.

[0382] Accoding to still another more particular embodiment Y3is > CH-NR4R4a.

[0383] Accoding to still another more particular embodiment Y3is > CH-OH.

[0384] Accoding to still another more particular embodiment Y3is > CH-CN.

[0385] Accoding to still another more particular embodiment Y3is > CH-OCI-C4 aklyl.

[0386] Accoding to still another more particular embodiment Y3is > CH-CF3.

[0387] Accoding to still another more particular embodiment Y3is > CH-OCF3.

[0388] Accoding to another more particular embodiment Y4is -CH2-.

[0389] M / 65047-PCTVERAXA 39

[0390] Accoding to another more particular embodiment Y4is -O-.

[0391] Accoding to still another more particular embodiment Y4is -S-.

[0392] Accoding to still another more particular embodiment Y4is -NH-.

[0393] Accoding to still another more particular embodiment Y4is > CH-NR4R4a.

[0394] Accoding to still another more particular embodiment Y4is > CH-OH

[0395] Accoding to still another more particular embodiment Y4is > CH-OCI-C4 aklyl.

[0396] Accoding to still another more particular embodiment Y4is > CH-CN.

[0397] Accoding to still another more particular embodiment Y4is > CH-CF3.

[0398] Accoding to still another more particular embodiment Y4is > CH-OCF3.

[0399] Accoding to another more particular embodiment Y5is -CH2-.

[0400] Accoding to another more particular embodiment Y5is -O-.

[0401] Accoding to still another more particular embodiment Y5is -S-.

[0402] Accoding to still another more particular embodiment Y5is -NH-.

[0403] Accoding to still another more particular embodiment Y5is > CH-NR4R4a.

[0404] Accoding to still another more particular embodiment Y5is > CH-OH.

[0405] Accoding to still another more particular embodiment Y5is > CH-CN.

[0406] Accoding to still another more particular embodiment Y5is > CH-OCI-C4 aklyl.

[0407] Accoding to still another more particular embodiment Y5is > CH-CF3.

[0408] Accoding to still another more particular embodiment Y5is > CH-OCF3.

[0409] Accoding to another more particular embodiment Y6is -CH2-.

[0410] Accoding to another more particular embodiment Y6is -O-.

[0411] Accoding to still another more particular embodiment Y6is -S-.

[0412] Accoding to still another more particular embodiment Y6is -NH-.

[0413] Accoding to another more particular embodiment Y6is > CH- NR4R4a.

[0414] Accoding to still another more particular embodiment Y6is > CH-OH

[0415] Accoding to still another more particular embodiment Y6is > CH-CN.

[0416] Accoding to still another more particular embodiment Y6is > CH-OCI-C4 aklyl.

[0417] Accoding to still another more particular embodiment Y6is > CH-CF3.

[0418] Accoding to still another more particular embodiment Y6is > CH-OCF3.

[0419] According to another particular embodiment compounds of general formula V are provided, wherein residues Y3to Y6are identical and each represent methylene (-CH2-).

[0420] M / 65047-PCTVERAXA

[0421] According to still another particular embodiment compounds of the general formula V are provided, wherein residues Y1and Y2each independetly represent > CH-OH, or > CH-OC1-C4 aklyl, wherein the hydrogen atom at the ring carbon of Y1or Y2is replaced by a

[0422] chemical bond

[0423]

[0424] According to other non-limiting particular embodiments of the above embodiments Y2represents mono-substituted or non-substituted methylene group, wherein a hydrogen atom bond to its ring carbon atom is replaced by a chemical bond

[0425]

[0426] linking the terminal group X1to spacer group X2or to the linking group X3.

[0427] According to a third embodiment, the Neu5Ac derivatives of embodiment two are selected from compounds, wherein X1is selected from a TCO-type residue of the general formula VII to X:

[0428] I— / R1JI O—- R3

[0429] (VII)

[0430]

[0431] wherein

[0432] R1, R2and R3are as defined above and may be bound to any ring position except for the C=C double bond of the cyclooctene ring,

[0433] M / 65047-PCTVERAXA

[0434] Cyc represents the three- to six-membered carbocyclic or heterocyclic, nonaromatic or aromatic, saturated or unsaturated ring, optionally carrying at least one ring substituents, in particular selected from OH, or linear or branched C1-C4 alkyl carboxylate groups, linear or branched C1-C4-alkoxy groups and keto groups;

[0435]

[0436] represents the chemical bond linking the terminal group X1to spacer group X2as defined in claim 2, and may be linked to any ring position within a X1moiety of any one of the formulae VII to XII except for the C=C double bond of the trans-cyclooctene ring.

[0437] More particulary, in anyone of the above formulae VII, VII, IX and X, the residues R1and R2are as defined above and may be bound to any ring position except for the C=C double bond of the cyclooctene ring, and R3is H.

[0438] According to non-limiting particular embodiments of the above

[0439]

[0440] represents a chemical bond at the homoallyl carbon atom linking the terminal group X1to spacer group X2or to the linking group X3.

[0441] In particular, / V-Acetyl neuraminic acid (Neu5Ac) derivatives of the general formula I:

[0442] TCO-Neu5Ac-CMP

[0443]

[0444] are provided,

[0445] wherein

[0446] Neu5Ac represents a moiety of the formula la

[0447] LOH

[0448] ,\OH

[0449] H

[0450] N

[0451]

[0452] M / 65047-PCTVERAXA 42

[0453] (la)

[0454] CMP represents a residue of the formula lb

[0455]

[0456] linked via its phosphoryl group to the oxyl group in position 2 of the of Neu5Ac moiety;

[0457] and

[0458] TCO represents a trans-cyclooctenyl-type residue linked to the terminal carbon atom in position 9 of the Neu5Ac moiety and represents a moiety of the general formula lc (corresponding to general formula V, above)

[0459] X1-X2a- X3- (lc)

[0460] wherein

[0461] X1is selected from a residue of the general formula VII to XII:

[0462]

[0463] M / 65047-PCTVERAXA 43

[0464]

[0465] wherein

[0466] R1, R2and R3may be bound to any ring position except for the carbon atoms of the C=C double bond of the cyclooctene ring, and are independently of each other selected from hydrogen, or substituents selected from halogen, linear or branched C1-C4- alkyl, linear or branched C2-C4-alkenyl, CF3, CN, OH, linear or branched C1-C4-alkoxy, linear or branched C2-C4-alkeneoxy, -O- CF3, linear or branched C2-C5-alkanoyloxy, linear or branched Ci-C4-alkylaminocarbonyloxy, or linear or branched C1-C4- alkylthio, -NR4R4a, carboxy, or linear or branched C1-C4-alkyl carboxylate groups, and particularly independently of each other selected from hydrogen, OH or linear or branched C1-C4- alkoxy, particularly R1, R2and R3independently of each other are hydrogen, or substituents selected from a hydrophilic substituent, and more particularly R1, R2, R3are OH or linear or branched C1-C4-alkoxy, and even more particularly R1, R2are OH or linear or branched C1-C4-alkoxy ands R3is H; wherein

[0467] R4and R4aindependently of each other represent H or linear or branched Ci-C4-alkyl;

[0468] Cyc represents together with the ring atoms to which it is attached form a three- to six-membered carbocyclic or heterocyclic, particularly carbocyclic, nonaromatic or aromatic, saturated or unsaturated ring, optionally carrying at least one ring substituent, selected from OH, carboxylic acid groups, or

[0469] M / 65047-PCTVERAXA 44

[0470] linear or branched C1-C4 alkyl carboxylate groups, linear or branched C1-C4-alkoxy groups and keto groups;

[0471]

[0472] represents the chemical bond linking the terminal group X1to spacer group X2or to the linking group X3as defined below, and may be linked to any ring position within a X1moiety of any one of the formulae VII to XII except for the carbon atoms of the C=C double bond of the trans-cyclooctene ring; X2is a spacer group selected from lower alkylene, preferably methylene; a is 0 or 1;

[0473] X3is a linking group selected from -O-, -S-,-NR8-, -N+R8R8a-, -C(O)-NR9-, - NR9-C(O)-, -NH-CH(NH2)-, -CH(NH2)-NH-, -NH-C(NH)-NH-, -NR9-C(O)-O-, -O-C(O)-NR9-, -C(O)-NH-CH(NH2)-, -C(O)-NH-C(NH)-NH-, NH-CH(NH2)- C(O)-, -NH-C(NH)-NH-C(O)-, =N-, =N-O-, =N-NR9;

[0474] wherein

[0475] R8, R8aand R9independently of each other represent H or linear or branched Ci-C4-alkyl or linear or branched C2-C4-alkenyl.

[0476] More particulary, in anyone of the above formulae VII, VII, IX and X, the residues R1and R2are as defined above and may be bound to any ring position except for the C=C double bond of the cyclooctene ring, and R3is H.

[0477] As particular examples of bicyclic moieties of the above formula VIII to X. the following nonlimiting structures are listed below:

[0478] Bicyclic structures comprising a three-ring:

[0479]

[0480] M / 65047-PCTVERAXA 45

[0481] (Villa) (IXa) (Xa)

[0482] wherein

[0483] R1and R2and are independely selected from OH or linear or brached C1-C4- alkoxy,

[0484] R3is hydrogen, or substituents selected from halogen, linear or branched C1-C4- alkyl, linear or branched C2-C4-alkenyl, CF3, CN, OH, linear or branched C1-C4-alkoxy, linear or branched C2-C4-alkeneoxy, -O-CF3, linear or branched C2-C5-alkanoyloxy, linear or branched Ci-C4-alkylaminocarbonyloxy, or linear or branched Ci-C4-alkylthio, -NR5aR5, carboxy, or linear or branched C1-C4-alkyl carboxylate groups, and more particularly R3is H;

[0485] wherein

[0486] R5and R5aindependently of each other represent H or linear or branched C1-C4-alkoxy

[0487] R4is absent or is selected from H, OH, keto or linear or branched C1-C4-alkoxy; > Z1is -O- if R4is absent; and

[0488] > Z1-R4is selected from > C=O, > CH-R4or > N-R4.

[0489] Bicyclic structures comprising a four-ring:

[0490] Carbocyclic:

[0491]

[0492] R1, R2and R3are as defined above for the bicyclic structures comprising a three-ring and more particularly R3is H; and

[0493] R5is selected from H, OH, keto or linear or branched C1-C4-alkoxy

[0494] Heterocyclic:

[0495] M / 65047-PCTVERAXA

[0496]

[0497] R1, R2and R3are as defined above for the bicyclic structures comprising a three-ring and more particularly R3is H;

[0498] R6is selected from H, OH or linear or branched C1-C4-alkoxy;

[0499] R7is absent or is selected from H, OH, keto or linear or branched C1-C4-alkoxy;

[0500] > Z1is -O- if R7is absent; and

[0501] > Z1-R7is selected from > C=O, > CH-R7or > N-R7;

[0502] Bicyclic structures comprising a five-ring:

[0503] M / 65047-PCTVERAXA

[0504]

[0505] (Ville) (IXe) (Xe)

[0506] wherein

[0507] R1, R2and R3are as defined above for the bicyclic structures comprising a three-ring and more particularly R3is H;

[0508] R6is absent or is selected from H, OH or linear or branched C1-C4-alkoxy;

[0509] R7is absent or is selected from H, OH, keto or linear or branched C1-C4-alkoxy; > Z1is -O- if R7is absent and if > Z2and > Z3are each > CH-R6;

[0510] > Z1-R7is selected from > C=O, > CH-R7or > N-R7;

[0511] > Z2-R6and > Z3-R7are independently selected from > C=O, > CH-OH, > CH-CI-C4- alkoxy or > N-H if > Z1-R7is > CH2, or

[0512] > Z2and > Z3are -O- if R6and R7are absent;

[0513] Bicyclic structures comprising a six-ring:

[0514] Aromatic:

[0515]

[0516] R1, R2and R3are as defined above for the bicyclic structures comprising a three-ring and more particularly R3is H; and

[0517] R6is selected from H, OH or linear or branched C1-C4-alkoxy

[0518] M / 65047-PCTVERAXA

[0519] Heteroaromatic:

[0520]

[0521] R1, R2and R3are as defined above for the bicyclic structures comprising a three-ring and more particularly R3is H;

[0522] R6is selected from H, OH or linear or branched C1-C4-alkoxy; and

[0523] Y7, Y8, Y9and Y10are independently selected from =CH-, =C-R6- or =N-; provided that one or two of Y7, Y8, Y9and Y10are =N-.

[0524] According to a fourth embodiment, the Neu5Ac derivatives of embodiment three are selected from compounds, wherein X1is selected from residues of the general formula XVI

[0525]

[0526] (XVI)

[0527] wherein

[0528] R1, R2and R3are as defined above for formula VI and more particularly R3is H.

[0529] According to a fifth embodiment, the Neu5Ac derivatives of embodiment four are selected from compounds, wherein X1is selected from a residue of the general formula XVII

[0530] M / 65047-PCTVERAXA

[0531] 49

[0532]

[0533] (XVII)

[0534] wherein

[0535] R1and R2independently of each other are OH or C1-C4-alkoxy.

[0536] 2. The second aspect of the present invention

[0537] According to the second aspect of of the present invention glycan-engineered immunoglobulin molecules are provided, comprising at least one glycan residue engineered by introducing a terminal clickable TCO moiety as herein defined above.

[0538] A sixth embodiment of the present invention provides a glycan-engineered immunoglobulin of the general formula XX

[0539] lg-[G cNAc(B1 -4)-Gal(a2-6)-Neu5Ac-TCO]n

[0540]

[0541] [(alpha 1-6)Fuc]m

[0542] (XX)

[0543] wherein

[0544] n represents an integer of at least 1, preferably 2;

[0545] m is 0 or 1

[0546] Ig represents an immunoglobulin; more particularly a monoclonal antibody

[0547] M / 65047-PCTVERAXA 50

[0548] TCO is as defined above according to the first aspect of the present invention;

[0549] GlcNAc represents an N-acetyl-glucosamin moiety, in particular N- acetyl-B-D-glucosamin;

[0550] Gal represents a galactosyl moiety, in particular p-D-galactosyl moiety;

[0551] Neu5Ac represents an / V-acetylneuraminic acid moiety, in particular a-D- acetyl neuraminic acid moiety; and

[0552] Fuc represents a fucosyl moiety, in particular 6-desoxy-L-galactosyl moiety.

[0553] According to a seventh embodiment of the invention embodiment, the glycan-engineered immunoglobulin of embodiment six is provided, which is of the general formula (XXI)

[0554] lg-[GlcNAc(B1-4)-Gal(a2-6)-Neu5Ac-TCO]n

[0555] (XXI)

[0556] wherein

[0557] n, Ig, TCO, GlcNAc, Gal and Neu5Ac are as defined herein above,

[0558] and wherein TCO is covalently attached to carbon atom C-9 of Neu5Ac, and whereinGlcNAc in said formula XXI is optionally linked via an (a1-6)-glycosidic linkage to a Fuc moiety as defined above.

[0559] According to an eighth embodiment of the invention, the glycan-engineered immunoglobulin of anyone of the embodiments six and seven is an antibody molecule or an Fc fragment thereof.

[0560] According to a nineth embodiment of the invention, the glycan-engineered immunoglobulin of embodiment eight comprises at least one glycan moiety, which is N-linked to the side chain of amino acid Asn in position 297 (according to Ell numbering of the C-domain of immunoglobulins) of the CH2 domain of the antibody heavy chain (HC) or the corresponding Fc fragment thereof.

[0561] M / 65047-PCTVERAXA 51

[0562] According to a tenth embodiment of the invention, the glycan-engineered immunoglobulin of anyone of the embodiments six to nine is selected from a polyclonal or monoclonal antibody, carrying at least one engineered glycan side chain.

[0563] According to an eleventh embodiment, the glycan-engineered immunoglobulin of anyone of the embodiments six to ten is an IgG antibody.

[0564] 3. The third aspect of the present invention

[0565] The third aspect of the present invention relates to particular methods for preparing glycan engineered immunoglobulin molecules as defined herein above.

[0566] According to a twelfth embodiment of the invention, a method of preparing a glycan-engineered immunoglobulin of anyone of the embodiments six 6 to eleven is provided, which method comprises

[0567] a) the enzyme-catalyzed conversion of a natural glycan side chain of the immunoglobulin molecule carrying at least one glycan side chain, which is attached to the Ig-heavy chain of the molecule via an optionally fucosylated GlucNAc moiety into an optionally fucosylated -GalNAc-Gal side chain,, thereby obtaining a first glycan-modified immunoglobulin; and

[0568] b) enzymatically attaching an / V-acetylneuraminic acid (Neu5Ac) derivative as defined in anyone of the above embodiments of the above first aspect of the invention, under conditions allowing the covalent binding of the TCO-Neu5Ac- moiety of said Neu5Ac-derivative to the terminal Gal moiety of said first glycan- modified immunoglobulin, thereby obtaining a second glycan-engineered immunoglobulin which is as defined in anyone of the embodiments of the second aspect of the invention,

[0569] According to a thirteenth embodiment of the invention, in the method of embodiment twelve the individual steps are performed as follows:

[0570] step a) is performed in the simultaneous presence of an enzyme having endoglycosidase activity, an enzyme having galactosyl transferase activity, and an enzyme having alkaline phosphatase activity, as well as in the presence of UDP Gal and MnCl2in a liquid buffered medium; and

[0571] M / 65047-PCTVERAXA 52

[0572] step b) is performed in the simultaneous presence of an enzyme having sialyl transferase activity and an Neu5Ac derivative as defined in anyone of the embodiments of the first aspect of the invention.

[0573] According to fourteenth embodiment of the invention, in the method of embodiment thirteen said enzyme having endoglycosidase activity is selected from EndoS or Endo S2, in particular EndoS2, and said enzyme having galactosyl transferase is B4GalT1; and said enzyme having sialyltransferase activity is a beta-galactoside alpha-2, 6-sialyltransferase (ST6Gal1).

[0574] The enzymatic 2-step synthesis of TCO-functionalized antibodies is explained in more detail in the following section:

[0575] Antibodies having the same protein sequence can be differentially glycosylated depending on many factors such as their environment, source, purification and storage conditions, etc. Even a single antibody preparation may comprise a multitude of differentially glycosylated antibody molecules. As shown in Fig. 1B, a typical glycan at position Asn297 (according to Eu numbering; cf. Edelman, G. M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969). PMID: 5257969; IMGT® (https: / / www.imgt.orq)) or https: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html) of human IgG contains a biantennary heptasaccharide core with extensions that are variable. Each arm of the biantennary heptasaccharide core terminates with an N-acetylglucosamine (GlcNAc) residue; if there is no extension, that glycoform is commonly referred to as a " GO" glycoform. If one of the arms is extended by a terminal galactose (Gal) residue, the glycoform is referred to as " G1". If both arms are extended by terminal galactose residues, the glycoform is referred to as " G2". For example, naturally occurring human IgG may contain a mixture of glycoforms at Asn297. Non-limiting examples of another type of glycan structure are shown in Fig.lC which are so-called high mannose glycan structures comprising 6 to 9 mannose moieties (also designated Man6N2 to Man9N2), which may also be glycan engineered according to the method of the present invention.

[0576] The present invention provides a method of making a functionalized antibody by enzymatically remodeling its glycans. The goal of glycan remodeling of the invention is to replace in a first step the biantennary heterogenous glycanstructure by a structurally homogenous, more simple glycan structure, and in a second step to attach a functionalized sialic acid residue, in particular Neu5Ac residue, to one single glycan terminus per Fc chain of the antibody molecule. A preferred enzyme for sialylation (i.e. a sialyltransferase) is one that utilizes galactosly residue as a preferred substrate.

[0577] M / 65047-PCTVERAXA 53

[0578] Step 1: Restructuring of heterogenous biantennary glycan side chains

[0579] Prior to sialylating the antibody or fragment thereof, the method for making the functionalized antibody or fragment includes the enzymatic remodeling of the heterogenous biantennary glycan structure of a particular antibody or fragment so that the resulting glycan(s) have a homogenous monoantennary structure containing a terminal galactose that thereby can serve as a better substrate for sialyltransferases such as a p-galactoside a-(2,6)-sialyltransferase 1 (ST6Gal 1). For example, the naturally occurring glycan may terminate with GlcNAc, which is not a preferred substrate for ST6Gal 1.

[0580] The starting antibody or fragment is thus, stepwise or preferably simultaneously, contacted (1) with an endoglucanase having the ability to degade the entire heterogenous native glycan structure down to the core glycan GlcNAc linked to the Asn297 amino acid residue of the Fc chain, (2) an galactosyltransferase (GalT), (3) UDP-galactose (UDP-Gal) under conditions and for a time sufficient to achieve galactosylation of core GlcNAc residue of Asn297 of the antibody's heavy chain to bind a galactosyl moiety to GlcNAc, and optionally (4) an alkaline phosphatase. In a particular embodiment endoglycosidases from Streptococcus pyogenes, EndoS and EndoS2, are preferably applied which have been shown to hydrolyze N-linked glycans of human immunoglobulin G. In another particular embodiment a galactosyl transferase is applied which has specificity for the donor substrate UDP-galactose and transfers galactose in a beta1,4 linkage to the acceptor sugars GlcNAc. B4GalT1 is particularly preferred. Alkaline phosphatase is provided in order to dephosphorylate formed UDP.

[0581] Step 2: Sialylation

[0582] The antibody or fragment thereof as obtained in step 1 is contacted by at least one sialyltransferase, under conditions and for a time sufficient to incorporate at least one functionalized sialic acid onto the terminus of at least one re-structured glycan of the antibody. More particularly, the re-structured antibody of step 1 and a TCO-functionalized CMP-sialic acid substrate of the invention, in particular a compound of the above Formula I, can be contacted by at least one sialyltransferase under conditions and for a time sufficient to incorporate said functionalized sialic acids onto the restructured glycan moiety of the antibody as obtauined in step 1. The sialyltransferase may be derived from mammals, fishes,

[0583] M / 65047-PCTVERAXA 54

[0584] amphibians, birds, invertebrates, or bacteria. In one embodiment, the sialyltransferase is an a-(2,3)-sialyltransferase. In another embodiment, the sialyltransferase is an a-(2,6)-sialyltransferase. In yet another embodiment, the sialyltransferase is an a-(2,8)-sialyltransferase. In a preferred embodiment, the sialyltransferase is an a-(2,6)-sialyltransferase, more preferably a p-galactoside a-(2,6)-sialyltransferase 1 (ST6Gal1). In a preferred embodiment, the sialyltransferase is a mammalian, like a rat p-galactoside a-2,6-sialyltransferase 1 (ST6Gal1).

[0585] The functionalized sialic acid substrate is typically a nucleotide associated sialic acid, preferably a CMP-sialic acid, which is also known as CMP-Sia. In a preferred embodiment, the functionalized CMP-sialic acid is a CMP-TCO-modified sialic acid, more preferably CMP-Neu5Ac-9-TCO. In a particularly preferred embodiment, the antibody is contacted with said CMP-sialic acid derivative in the presence of ST6Gal 1.

[0586] The thus obtained TCO-functionalized antibody may, or may not, contain a fucose as part of the core glycan structure at residue 297 (see, e.g., Fig. 1 A and B showing a fucose as part of the glycan). Optionally, the antibody can be treated with a fucosidase or a fucosyl transferase to remove or add a fucose residue, as desired.

[0587] 4. The fouth aspect of the present invention

[0588] The fourth aspect of the present invention provides conjugates of a glycan engineered immunoglobulin molecules of the third aspect of the invention combined via click reaction with a payload molecule functionalized with a docking group having the ability to react with the TCO-group of the glycan engineered immunoglobulin molecule of the invention According to a fifteenth embodiment of the invention, a conjugate of a glycan-engineered immunoglobulin of anyone of the embodiments of the second aspect of the invention and a payload molecule (P) functionalized with a terminal docking group selected from tetrazines or triazines is provided, wherein said terminal docking group is capable of covalently binding (i.e. being conjugated) in a copper-free strain promoted inverse-electrondemand Diels-Alder cycloaddition (SPIEDAC) to said TCO group of said glycan-engineered immunoglobulin anyone of the embodiments of the second aspect of the invention.

[0589] According to a sixteenth embodiment of the invention, said conjugate of embodiment fifteen is an antibody payload conjugate (APC), comprising an antibody molecule or Fc-fragment thereof as defined in anyone of the embodiments of the second aspect of the invention, covalently bound via its TCO group in a copper-free strain promoted inverseelectron-demand Diels-Alder cycloaddition (SPIEDAC) to said payload molecule (P) which is

[0590] M / 65047-PCTVERAXA 55

[0591] functionalized with a terminal docking group selected from triazines and tetrazines capable of covalently binding to said TCO group of the glycan engineered immunoglobulin moiety.

[0592] According to a seventeenth embodiment of the invention, the conjugate of anyone of the embodiments fifteen and sixteen comprises a payload molecule, which is selected from bio-active compounds, labeling agents, protein degraders, in particular payloads applicable in proteolysis targeting chimeras (PROTACs), photosensitizers, and chelators.

[0593] 5. The fifth aspect of the present invention

[0594] The fifth aspect of the present invention relates to aspects of the medical use, either diagnostic a linear or therapeutically of conjugates of the fouth aspect of the invention According to an eighteenth embodiment of the invention, a pharmaceutical composition is provided, comprising in a pharmaceutically acceptable carrier at least one conjugate as defined in anyone of the embodiments of the 4thaspect of the invention, or a diagnostic composition comprising in a diagnostically applicable carrier at least one conjugate as defined in anyone of the embodiments of the fourth aspect of the invention.

[0595] According to a ninteenth embodiment of the invention the present invention also relates to a conjugate as defined in anyone of the embodiments of the fourth aspect of the invention for use in medicine, as in particular in diagnosis and / or therapy.

[0596] According to a twentieth embodiment of the invention, the conjugate as defined in anyone of the embodiments of the fourth aspect of the invention, is an ADC for use in the diagnosis and / or treatment of cancers, in particular breast cancer, gastric cancer or other Her2 overexpressing tumors, as for example tumors of ovary, endometrium, bladder, lung, colon, and head and neck.

[0597] A final embodiment of the present invention relates to a diagnostic or analytical kit comprising at least one conjugate as defined in anyone of the embodiments of the fourth aspect of the invention.

[0598] 6. The sixth aspect of the present invention

[0599] According to another aspect of the invention novel bicyclic TCO compounds are provided, which are of the general formula (Va)

[0600] X1-X2a- X3-W

[0601] M / 65047-PCTVERAXA 56

[0602] (Va)

[0603] wherein

[0604] X1represents a bicyclic moiety of anyone of the above-identified Formulae VIII, IX, X, VI I la-VI llg, IXa - IXg, or Xa - Xg;

[0605]

[0606] , X2and a are as defined above for Formula V,

[0607] X3is missing or is defined above for Formula V, and

[0608] W represents H, linear or branched C1-C4 alkyl, or linear or branched Ci- 04 alkoxy; or, when X3is missing W may be selected from -OR10, -C(O)OR10, - NR102 or -C(O)NR102, wherein R10represents H, or linear or branched C1-C4 alkyl.

[0609] D. FURTHER EMBODIMENTS

[0610] 1. Payload molecules (PM)

[0611] 1.1 Bioactive compounds

[0612] Bioactive compounds include, but are not limited to, the following:

[0613] Bioactive compounds applicable according to the present invention include but are not limited to: small organic molecule drugs, steroids, lipids, proteins, aptamers, oligopeptides, oligonucleotides, oligosaccharides, as well as peptides, peptoids, amino acids, nucleotides, oligo- or polynucleotides, nucleosides, DNA, RNA, toxins, glycans and immunoglobulins.

[0614] Exemplary classes of bioactive compounds that can be used in the practice of the present invention include but are not limited to hormones, cytotoxins, antiproliferative / antitumor agents, antiviral agents, antibiotics, cytokines, anti-inflammatory agents, antihypertensive agents, chemosensitizing, photosensitizing and radiosensitizing agents, anti-AIDS substances, anti-viral agents, immunosuppressants, immunostimulants, enzyme inhibitors, anti-Parkinson agents, neurotoxins, channel blockers, modulators of cell-extracellular matrix interactions including cell growth inhibitors and anti-adhesion molecules, inhibitors of DNA, RNA or protein synthesis, steroidal and non-steriodal anti-inflammatory agents, anti- angiogenic factors, anti-Alzheimer agents.

[0615] In some embodiments, the bioactive compound is a low to medium molecular weight compound (e.g. about 200 to 5000 Da, about 200 to about 1500 Da, preferably about 300 to about 1000 Da).

[0616] M / 65047-PCTVERAXA 57

[0617] Exemplary cytotoxic drugs are particularly those which are used for cancer therapy. Such drugs include, in general, DNA damaging agents, anti-metabolites, natural products and their analogs, enzyme inhibitors such as dihydro folate reductase inhibitors and thymidylate synthase inhibitors, DNA binders, DNA alkylators, radiation sensitizers, DNA intercalators, DNA cleavers, microtubule stabilizing and destabilizing agents, topoisomerases inhibitors. Examples include but are not limited to platinum-based drugs, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, taxanes, lexitropsins, the pteridine family of drugs, diynenes, the podophyllotoxins, dolastatins, maytansinoids, differentiation inducers, and taxols. Particularly useful members of those classes include, for example, auristatins, maytansines, maytansinoids, calicheamicins, dactinomycines, duocarmycins, CC1065 and its analogs, camptothecin and its analogs, SN-38 and its analogs; DXd, tubulysin M, cryptophycins, pyrrolobenzodiazepines and pyrrolobenzodiazepine dimers (PBDs), pyridinobenzodiazepines (PDDs) and indolinobenzodiazepines (IBDs) (cf. US20210206763A1), methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, DNA minor groove binders, 6- mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin, anthracyclines (doxorubicin, epirubicin, idarubicin, daunorubicin, PNU-159682 (cf. US 10,288,745 B2.) and its analogs, mitomycin C, mitomycin A, caminomycin, aminopterin, tallysomycin, podophyllotoxin and;podophyllotoxin derivatives such as etoposide or etoposide phosphate, vinblastine, vincristine, vindesine, taxol, taxotere retinoic acid, butyric acid, N8-acetyl spermidine, staurosporin, colchicine, camptothecin, esperamicin, ene-diynes, and their analogues, hemiasterlin and its analogues.

[0618] Other exemplary drug classes are angiogenesis inhibitors, cell cycle progression inhibitors, P13K / m-TOR / AKT pathway inhibitors, MAPK signaling pathway inhibitors, kinase inhibitors, protein chaperones inhibitors, HDAC inhibitors, PARP inhibitors, Wnt / Hedgehog signaling pathway inhibitors, RNA polymerase inhibitors, and protein degraders (cf. https: / / pubs.acs.org / doi / 10.1021 / acschembio.0c00285).

[0619] Examples of auristatins include dolastatin 10, monomethyl auristatin E (MMAE), auristatin F, monomethyl auristatin F (MMAF), auristatin F hydroxypropylamide (AF HPA), auristatin F phenylene diamine (AFP), monomethyl auristatin D (MMAD), auristatin PE, auristatin EB, auristatin EFP, auristatin TP and auristatin AQ. Suitable auristatins are also described in U. S.; Publication Nos. 2003 / 0083263, 2011 / 0020343, and 2011 / 0070248; PCT Application publication Nos. WO09 / 117531, W02005 / 081711, W004 / 010957; W002 / 088172 and WO01 / 24763, and U. S. Patent Nos. 7,498,298; 6,884,869; 6,323,315;

[0620] M / 65047-PCTVERAXA 58

[0621] 6,239,104; 6,124,431;;6, 034, 065; 5,780,588; 5,767,237; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725;;5, 530, 097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744;;4, 879, 278; 4,879,278; 4,816,444; and 4,486,414, the disclosures of which are incorporated herein by reference in their entirety.

[0622] Exemplary drugs include the dolastatins and analogues thereof including: dolastatin A ( U. S. Pat No. 4,486,414), dolastatin B (U. S. Pat No. 4,486,414), dolastatin 10 (U. S. Pat No.

[0623] 4,486,444, 5,410,024, 5,504,191, 5,521,284, 5,530,097, 5,599,902, 5,635,483, 5,663,149, 5,665,860, 5,780,588, 6,034,065, 6,323,315), dolastatin 13 (U. S. Pat No. 4,986,988), dolastatin 14 (U. S. Pat No. 5,138,036), dolastatin 15 (U. S. Pat No. 4,879,278), dolastatin 16 (U. S. Pat No. 6,239,104), dolastatin 17 (U. S. Pat No.. 6,239,104), and dolastatin 18 (U. S. Pat No.. 6,239,104), each patent incorporated herein by reference in their entirety.

[0624] Exemplary maytansines, maytansinoids, such as DM-1 and DM-4, or maytansinoid analogs, including maytansinol and maytansinol analogs, are described in U. S. Patent Nos.

[0625] 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533; 5,208,020; 5,416,064; 5,475,092; 5,585,499; 5,846,545; 6,333,410; 6,441,163; 6,716,821 and 7,276,497.

[0626] Other examples include mertansine and ansamitocin.; Pyrrolobenzodiazepines (PBDs), which expressly include dimers and analogs, include but are not limited to those described in [Denny, Exp. Opin. Ther. Patents, 10(4):459-474 (2000)], [Hartley et al., Expert Opin Investig Drugs. 2011, 20(6):733-44], Antonow et al., Chem Rev. 2011, 111(4), 2815-64],

[0627] Calicheamicins include, e.g. enediynes, esperamicin, and those described in U. S. Patent Nos. 5,714,586 and 5,739,116.

[0628] Examples of duocarmycins and analogs include CC1065, duocarmycin SA, duocarmycin A, duocarmycin B I, duocarmycin B2, duocarmycin Cl, duocarmycin C2, duocarmycin D, DU- 86, KW-2189, adozelesin, bizelesin, carzelesin, seco- adozelesin. Other examples include those described in, for example, US Patent No. 5,070,092; 5,101,092; 5,187,186; 5,475,092; 5,595,499; 5,846,545; 6,534,660; 6,548,530; 6,586,618; 6,660,742; 6,756,397; 7,049,316; 7,553,816; 8,815,226; US20150104407; 61 / 988,011 filed may 2, 2014 and 62 / 010,972 filed June 11, 2014; the disclosure of each of which is incorporated herein in its entirety.

[0629] Exemplary vinca alkaloids include vincristine, vinblastine, vindesine, and navelbine, and those disclosed in U. S. Publication Nos. 2002 / 0103136 and 2010 / 0305149, and in U. S.

[0630] M / 65047-PCTVERAXA 59

[0631] Patent No. 7,303,749, the disclosures of which are incorporated herein by reference in their entirety.

[0632] Exemplary epothilone compounds include epothilone A, B, C, D, E, and F, and derivatives thereof. Suitable epothilone compounds and derivatives thereof are described, for example, in U. S. Patent Nos. 6,956,036; 6,989,450; 6,121,029; 6,117,659; 6,096,757; 6,043,372;; 5,969,145; and 5,886,026; and WO97 / 19086; WO98 / 08849; W098 / 22461; W098 / 25929; W098 / 38192; WO99 / 01124; WO99 / 02514; WO99 / 03848; WO99 / 07692; WO99 / 27890; and W099 / 28324; the disclosures of which are incorporated herein by reference in their entirety.

[0633] Exemplary cryptophycin compounds are described in U. S. Patent Nos. 6,680,311 and 6,747,021; the disclosures of which are incorporated herein by reference in their entirety.

[0634] Exemplary platinum compounds include cisplatin, carboplatin, oxaliplatin, iproplatin, ormaplatin, tetraplatin.

[0635] Exemplary DNA binding or alkylating drugs include CC-1065 and its analogs, anthracyclines, calicheamicins, dactinomycines, mitromycines, duocarmycins, seco-duocarmycins, CBIs, seco-CBIs, pyrrolobenzodiazepines, and the like.

[0636] Exemplary microtubule stabilizing and destabilizing agents include taxane compounds, such as paclitaxel, docetaxel, tesetaxel, and carbazitaxel; maytansinoids, auristatins and analogs thereof, vinca alkaloid derivatives, epothilones and cryptophycins.

[0637] Exemplary topoisomerase inhibitors include camptothecin and camptothecin derivatives, camptothecin analogs and non-natural camptothecins, such as, for example, CPT-11, SN-38, topotecan, 9-aminocamptothecin, rubitecan, gimatecan, karenitecin, silatecan, lurtotecan, exatecan, DXd, diflometotecan, belotecan, lurtotecan and S39625. Other camptothecin compounds that can be used in the present invention include those described in, for example, J. Med. Chem., 29:2358-2363 (1986); J. Med. Chem., 23:554 (1980); J. Med Chem., 30: 1774 (1987).

[0638] Angiogenesis inhibitors include, but are not limited to, MetAP2 inhibitors, VEGF inhibitors, PIGF inhibitors, VGFR inhibitors, PDGFR inhibitors, MetAP2 inhibitors. Exemplary VGFR and PDGFR inhibitors include sorafenib, sunitinib and vatalanib. Exemplary MetAP2 inhibitors include fumagillol analogs, meaning compounds that include the fumagillin core structure.

[0639] Exemplary cell cycle progression inhibitors include CDK inhibitors such as, for example, BMS-387032 and PD0332991; Rho-kinase inhibitors such as, for example, AZD7762; aurora kinase inhibitors such as, for example, AZD1152, MLN8054 and MLN8237;

[0640] M / 65047-PCTVERAXA 60

[0641] PLK inhibitors such as, for example, Bl 2536, BI6727, GSK461364, ON-01910; and KSP inhibitors such as, for example, SB 743921, SB 715992, MK-0731, AZD8477, AZ3146 and ARRY-520.

[0642] Exemplary P13K / m-TOR / AKT signalling pathway inhibitors include phosphoinositide 3- kinase (P13K) inhibitors, GSK-3 inhibitors, ATM inhibitors, DNA-PK inhibitors and PDK-1 inhibitors.

[0643] Exemplary P13 kinases are disclosed in U. S. Patent No. 6,608,053, and include BEZ235, BGT226, BKM120, CAL263, demethoxyviridin, GDC-0941, GSK615, IC87114, LY294002, Palomid 529, perifosine, PF-04691502, PX-866, SAR245408, SAR245409, SF1126, Wortmannin, XL147 and XL765.

[0644] Exemplary AKT inhibitors include, but are not limited to AT7867.

[0645] Exemplary MAPK signaling pathway inhibitors include MEK, Ras, JNK, B-Raf and p38 MAPK inhibitors.

[0646] Exemplary MEK inhibitors are disclosed in U. S. Patent No. 7,517,944 and include GDC-;0973, GSKI 120212, MSC1936369B, AS703026, R05126766 and R04987655, PD0325901, AZD6244, AZD8330 and GDC-0973.

[0647] Exemplary B-raf inhibitors include CDC-0879, PLX-4032, and SB590885.

[0648] Exemplary B p38 MAPK inhibitors include BIRB 796, LY2228820 and SB 202190. Exemplary receptor tyrosine kinases inhibitors include but are not limited to AEE788 (NVP-AEE 788), BIBW2992 (Afatinib), Lapatinib, Erlotinib (Tarceva), Gefitinib (Iressa), AP24534 (Ponatinib), ABT-869 (linifanib), AZD2171, CHR-258 (Dovitinib), Sunitinib (Sutent), Sorafenib (Nexavar), and Vatalinib.

[0649] Exemplary protein chaperon inhibitors include HSP90 inhibitors. Exemplary inhibitors include 17AAG derivatives, BIIB021, BIIB028, SNX-5422, NVP-AUY-922 and KW-2478.

[0650] Exemplary HDAC inhibitors include Belinostat (PXD101), CUDC-101, Droxinostat, ITF2357 (Givinostat, Gavinostat), JNJ-26481585, LAQ824 (NVP-LAQ824, Dacinostat), LBH-589 (Panobinostat), MC1568, MGCD0103 (Mocetinostat), MS-275 (Entinostat), PCI- 24781, Pyroxamide (NSC 696085), SB939, Trichostatin A and Vorinostat (SAHA). Exemplary PARP inhibitors include iniparib (BSI 201), olaparib (AZD-2281), ABT-888 (Veliparib), AG014699, CEP9722, MK 4827, KU-0059436 (AZD2281), LT-673, 3- aminobenzamide, A-966492, and AZD2461.

[0651] Exemplary Wnt / Hedgehog signalling pathway inhibitors include vismodegib, cyclopamine and XAV-939.

[0652] M / 65047-PCTVERAXA 61

[0653] Exemplary RNA polymerase inhibitors include amatoxins. Exemplary amatoxins include alpha-amanitins, beta amanitins, gamma amanitins, eta amanitins, amanullin, amanullic acid, amanisamide, amanon, and proamanullin.

[0654] Exemplary cytokines include IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, TNF.

[0655] As non-limiting examples of particular drugs there may be mentioned Auristatins, Maytansinoids, PBDs, topoisomerase inhibitors, anthracyclines

[0656] In another embodiment, a combination of two or more different drugs as described above are used.

[0657] According to another embodiment, the bioactive compound may be selected from any synthetic or naturally occurring compounds comprising one or more natural and / or nonnatural, proteinogenic and / or non-proteinogenic amino acid residues, such as in particular oligo- or polypeptides or proteins.

[0658] A particular group of such compounds comprises immunoglobulin molecules as for example antibodies, antibody derivatives, antibody fragments, antibody (fragment) fusions (e.g. bi-specific and tri-specific mAb fragments or derivatives), polyclonal or monoclonal antibodies, such as human, humanized, mouse or chimeric antibodies.

[0659] Typical non-limiting examples of antibodies for use in the present invention are selected form biologically, in particular pharmacologically active antibody molecules. Nonlimiting examples are selected form the following group: trastuzumab, bevacizumab, cetuximab, panitumumab, ipilimumab, rituximab, alemtuzumab, ofatumumab, gemtuzumab, brentuximab, ibritumomab, tositumomab, pertuzumab, adecatumumab, IGN101, INA01 labetuzumab, hua33, pemtumomab, oregovomab, minretumomab (CC49), cG250, J591, MOv-18, farletuzumab (MGRAb-003), 3F8, ch14,18, KW-2871, hu3S193, lgN31 1, IM- 2C6, CDP-791, etaracizumab, volociximab, nimotuzumab, MM-121, AMG 102, METMAB, SCH 900105, AVE1642, IMC-A12, MK-0646, R1507, CP 751871, KB004, III A4, mapatumumab, HGS-ETR2, CS-1008, denosumab, sibrotuzumab, F19, 81 C6, pinatuzumab, lifastuzumab, glembatumumab, coltuximab, lorvotuzumab, indatuximab, anti-PSMA, MLN-0264, ABT-414, milatuzumab, ramucirumab, abagovomab, abituzumab, adecatumumab, afutuzumab, altumomab pentetate, amatuximab, anatumomab, anetumab, apolizumab, arcitumomab, ascrinvacumab, atezolizumab, bavituximab, bectumomab, belimumab, bivatuzumab, brontictuzumab, cantuzumab, capromab, catumaxomab, citatuzumab, cixutumumab, clivatuzumab, codrituzumab, conatumumab, dacetuzumab, dallotuzumab, daratumumab, demcizumab, denintuzumab, depatuxizumab, derlotuximab, detumomab, dinutuximab, drozitumab, duligotumab, durvalumab, dusigitumab, ecromeximab, edrecolomab,

[0660] M / 65047-PCTVERAXA 62

[0661] elgemtumab, emactuzumab, enavatuzumab emibetuzumab, enfortumab, enoblituzumab, ensituximab, epratuzumab, ertumaxomab, etaracizumab, farletuzumab, ficlatuzumab, figitumumab, flanvotumab, futuximab, galiximab, ganitumab, icrucumab, igovomab, imalumab, imgatuzumab, indusatumab, inebilizumab, intetumumab, iratumumab, isatuximab, lexatuzumab, lilotomab, lintuzumab, lirilumab, lucatumumab, lumretuzumab, margetuximab, matuzumab, mirvetuximab, mitumomab, mogamulizumab, moxetumomab, nacolomab, naptumomab, narnatumab, necitumumab, nesvacumab, nimotuzumab, nivolumab, nofetumomab, obinutuzumab, ocaratuzumab, ofatumumab, olaratumab, onartuzumab, ontuxizumab, oportuzumab, oregovomab, otlertuzumab, pankomab, parsatuzumab, pasotuxizumab, patritumab, pembrolizumab, pemtumomab, pidilizumab, pintumomab, polatuzumab, pritumumab, quilizumab, racotumomab, ramucirumab, rilotumumab, robatumumab, sacituzumab, samalizumab, satumomab, seribantumab, siltuximab, sofituzumab, tacatuzumab, taplitumomab, tarextumab, tenatumomab, teprotumumab, tetulomab, ticilimumab, tigatuzumab, tositumomab, tovetumab, tremelimumab, tucotuzumab, ublituximab, ulocuplumab, urelumab, utomilumab, vadastuximab, vandortuzumab, vantictumab, vanucizumab, varlilumab, veltuzumab, vesencumab, volociximab, vorsetuzumab votumumab, zalutumumab, zatuxima, combination and derivatives thereof, as well as other monoclonal antibodies targeting CAI 25, CAI 5-3, CAI 9-9, L6, Lewis Y, Lewis X, alpha fetoprotein, CA 242, placental alkaline phosphatase, prostate specific antigen, prostate specific membrane antigen, prostatic acid phosphatase, epidermal growth factor, MAGE- 1, MAGE-2, MAGE-3, MAGE-4, transferrin receptor, p97, MUCI, CEA, gplOO, MARTI, IL-2 receptor, CD20, CD52, CD33, CD22, human chorionic gonadotropin, CD38, CD40, mucin, P21, MPG, and Neu oncogene product.

[0662] 1.2 Labeling Agents

[0663] Labeling agents which may be used according to the invention can comprise any type of label known in the art which does not inhibitor negatively affect reactivity of the tetrazine moiety.

[0664] Labels of the invention include, but are not limited to, dyes (e.g. fluorescent, luminescent, or phosphorescent dyes, such as dansyl, coumarin, fluorescein, acridine, rhodamine, silicon-rhodamine, BODIPY, or cyanine dyes), chromophores (e.g., phytochrome, phycobilin, bilirubin, etc.), radiolabels (e.g. radioactive forms of hydrogen, fluorine, carbon, phosphorous, sulphur, or iodine, such as tritium, fluorine-18, carbon-11, carbon-14, phosphorous-32, phosphorous-33, sulphur-33, sulphur-35, iodine-123, or iodine-125), MRI-sensitive spin labels, affinity tags (e.g. biotin, His-tag, Flag-tag, strep-tag, sugars, lipids,

[0665] M / 65047-PCTVERAXA 63

[0666] sterols, PEG-linkers, benzylguanines, benzylcytosines, or co-factors), polyethylene glycol groups (e.g., a branched PEG, a linear PEG, PEGs of different molecular weights, etc.), photocrosslinkers (such as p-azidoiodoacetanilide), NMR probes, X-ray probes, pH probes, IR probes, resins, solid supports.

[0667] In some embodiments, exemplary dyes can include an NIR contrast agent that fluoresces in the near infrared region of the spectrum. Exemplary near-infrared fluorophores can include dyes and other fluorophores with emission wavelengths (e.g., peak emission wavelengths) between about 630 and 1000 nm, e.g., between about 630 and 800 nm, between about 800 and 900 nm, between about 900 and 1000 nm, between about 680 and 750 nm, between about 750 and 800 nm, between about 800 and 850 nm, between about 850 and 900 nm, between about 900 and 950 nm, or between about 950 and 1000 nm. Fluorophores with emission wavelengths (e.g., peak emission wavelengths) greater than 1000 nm can also be used in the methods described herein.

[0668] In some embodiments, exemplary fluorophores include 7-amino-4-methylcoumarin-3 -acetic acid (AMCA), TEXAS RED™ (Molecular Probes, Inc., Eugene, Oreg.), 5-(and -6)-carboxy-X-rhodamine, lissamine rhodamine B, 5-(and -6)-carboxyfluorescein, fluorescein-5-isothiocyanate (FITC), 7-diethylaminocoumarin-3-carboxylic acid, tetramethylrhodamine-5-(and -6)-isothiocyanate, 5 -(and -6)-carboxytetramethylrhodamine, 7-hydroxycoumarin-3-carboxylic acid, 6-[fluorescein 5-(and -6)-carboxamido]hexanoic acid, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a diaza-3-indacenepropionic acid, eosin-5-isothiocyanate, erythrosin-5-isothiocyanate, and CASCADE™ blue acetylazide (Molecular Probes, Inc., Eugene, Oreg.) and ATTO dyes.

[0669] Other suitable fluorophores are for example described in EP3572468A1. Further labelling agents are 177-Lutetium, 89-Zirkonium, 131-lod, 68-Gallium, 99m-Technecium, 225-Actinium, 213-Bismut, 90-Ytrium, 212-Plumbum, 111-lndium, 64-Copper, 67-Copper, 124-lodine, 227-Thorium and 188-Rhenium.

[0670] 1.3 Chelators

[0671] Lists of typically applicable chelators and their short names are given below; Corresponding salts thereof are also applicable.:

[0672] Acetyl acetone (ACAC), ethylene diamine (EN), 2-(2-aminoethylamino)ethanol (AEEA), diethylene triamine (DIEN), iminodiacetate (IDA), triethylene tetramine (TRIEN), triaminotriethylamine, nitrilotriacetate (NTA) and its saltslike Na3NTA or FeNTA, ethylenediaminotriacetate (TED), ethylenediamine tetraacetate (EDTA) and its salts like Na2EDTA and CaNa2EDTA, diethylene triaminpentaacetate (DTPA), 1,4,7,10-

[0673] M / 65047-PCTVERAXA 64

[0674] ztetraazacyclododecane-1,4,7,10-tetraacetate (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), Oxalate (OX), tartrate (TART), citrate (CIT), dimethylglyoxime (DMG), 8-hydroxyquinoline, 2,2'-bipyridine (BPY), 1,10-phenanthroline (PHEN), dimercapto succinic acid (DMSA), 1,2-bis(diphenylphosphino)ethane (DPPE), sodium salicylate, methoxy salicylates, British anti-Lewisite or 2,3-dimercaprol (BAL), meso-2,3-dimercaptosuccinic acid (DMSA); Siderophores secreted by microorganisms, as for example desferrioxamine or deferoxamine B, also known as Deferral (Novartis), produced by Streptomyces spp. deferoxamine (DFO), a trihydroxamic acid secreted by Streptomyces pilosus; phytochemicals like curcuminoids and derivatives of mugineic acid, like 3-hydroxy-mugineic acid and 2'-deoxy-mugineic acid; synthetically produced chelators, like Ibuprofen; derivatives of catechol, hydroxamate and hydroxypyridinone, like hydroxamate desferal and hydroxypyridinone deferiprone; deferiprone (L1 or 1,2-dimethyl-3-hydroxypyrid-4-one); D-penicillamine (DPA or D-PEN) whoich is p-p-dimethylcysteine or 3-mercapto-D-valine; tetraethylenetetraamine (TETA) or trientine and its two major metabolites Ni -acetyltriethylenetetramine (MAT) and Ni, N -diacetyltriethylenetetramine (DAT); hydroxyquinolines; clioquinol, which is a halogenated derivative of 8-hydroxyquinoline; and 5,7-dichloro-2-[(dimethylamino)methyl]quinolin-8-ol (PBT2).

[0675] 1.4 Protein degraders

[0676] As non-limiting examples there may be mentioned PROTACs in general, but there is a plethora of different E3 ligase binding molecules in combination with specific targeted proteins to degrade (vgl WO2017201449A1). (Maneiro, M. et al ACS Chem. Biol. 2020, 15, 6, 1306-1312)

[0677] As protein degrades there should be mentioned compounds that are suitable in targeted protein degradation (TPD). A major class of molecules is known as proteolysistargeting chimeras (PROTACs). Reference can be made to Bekes, M., Langley, D. R. & Crews, C. M. PROTAC targeted protein degraders: the past is prologue. Nat Rev Drug Discov21, 181-200 (2022). These chimeras are heterobifunctional small molecules consisting of two ligands joined by a linker. One ligand recruits and binds a protein of interest (POI) while the other recruits and binds an E3 ubiquitin ligase. Simultaneous binding of the POI and ligase by the PROTAC induces ubiquitylation of the POI and its subsequent degradation by the ubiquitin-porteasome system, after which the PROTAC is recycled to target another copy of the POI.

[0678] 2. Targeting agents and their Targets

[0679] M / 65047-PCTVERAXA 65

[0680] The primary object of such targeting agent is the formation of a covalent or noncovalent linkage with a particular “target”. A secondary object of the targeting agent is the targeted transport of a “payload molecule” (PM) to said target. In order to achieve said second object the targeting agent (herein the glycan engineered immunoglobulin) has to be combined (reversibly or irreversibly) with at least one payload molecule. For this purpose said targeting agent has to be functionalised by introducing said at least one TCO The functionalized targeting agent carrying said at least one TCO may then be linked to said at least one payload molecule through bioconjugation via said TCO residue. Said TCO is reactive with a payload molecule which in turn carries a corresponding moiety reactive with said at least one TCO residue of the targeting agent. The thus obtained bioconjugate, in particuar the targeting agent portion thereof, allows the transfer of the payload molecule to the intended target.

[0681] For example, a “target” can be any molecule, which is present in and / or on an organism, tissue or cell. Such targets may be nonspecific or specific for a particular organism, tissue or cell. Targets include cell surface targets, e.g. receptors, glycoproteins, glycans, carbohydrates; structural proteins, e.g. amyloid plaques; abundant extracellular targets such as in stroma, extracellular matrix targets such as growth factors, and proteases; intracellular targets, e.g. surfaces of Golgi bodies, surfaces of mitochondria, RNA, DNA, enzymes, components of cell signaling pathways; and / or foreign bodies, e.g. pathogens such as viruses, bacteria, fungi, yeast or parts thereof.

[0682] Examples of targets include compounds such as proteins of which the presence or expression level is correlated with a certain tissue or cell type or of which the expression level is up- regulated ordown-regulated in a certain disorder.

[0683] In particular, such target is a protein such as a (internalizing or non- internalizing) receptor.

[0684] Targets can be selected from any suitable targets within the human or animal body or on a pathogen or parasite.

[0685] Non-limiting examples of suitable targets include but are not limited to a group comprising cellular components such as cell membranes and cell walls, receptors such as cell membrane receptors, intracellular structures such as Golgi bodies or mitochondria, enzymes, receptors, DNA, RNA, viruses or viral particles, macrophages, tumor-associated macrophages, antibodies, proteins, carbohydrates, monosaccharides, polysaccharides, cytokines, hormones, steroids, somatostatin receptor, monoamine oxidase, muscarinic receptors, myocardial sympatic nerve system, leukotriene receptors, e.g. on leukocytes, urokinase plasminogen activator receptor (uPAR), folate receptor, apoptosis marker, (anti-)

[0686] M / 65047-PCTVERAXA 66

[0687] angiogenesis marker, gastrin receptor, dopaminergic system, serotonergic system, GABAergic system, adrenergic system, cholinergic system, opioid receptors, GPIIb / llla receptor and other thrombus related receptors, fibrin, calcitonin receptor, tuftsin receptor, P-glycoprotein, neurotensin receptors, neuropeptide receptors, substance P receptors, NK receptor, CCK receptors, sigma receptors, interleukin receptors, herpes simplex virus tyrosine kinase, human tyrosine kinase, integrin receptor, fibronectin targets, AOC3, ALK, AXL, C242, CA-125, CCL11, CCR5, CD2, CD3, CD4, CD5, CD15, CA15-3, CD18, CD19, CA19-9, CD20, CD21, CD22, CD23, CD25, CD28, CD30, CD31, CD33, CD37, CD38, CD40, CD41, CD44v6, CD45, CD51, CD52, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD72, CD74, CD79-B, CD80, CD105, CD125, CD138, CD141, CD147, CD152, CD154, CD174, CD227, CD326, CD340, VEGF / EGF and VEGF / EGF receptors, VEGF-A, VEGFR2, VEGFR1, TAG72, CEA, CEACAM5, MUC1, MUC16, GPNMB, PSMA, Cripto, Tenascin C, Melanocortin-1 receptor, G250, HLA DR, ED-B, TMEFF2, EphB2, EphB4, EphA2, FAP, Mesothelin, GD2, GD3, CAIX, 5T4, clumping factor, CTLA-4, CXCR2, FGFRI, FGFR2, FGFR3, FGFR4, NaPi2b, NOTCHI, NOTCH2, NOTCH3, NOTCH4, NECTIN4, ErbB2, ErbB3, EpCAM, FLT3, HGF, HER2, HER3, TROP2, FOLR1, TAG-72, HMI24, ICAM, ICOS-L, IGF-1 receptor, TRPV1, CFTR, gdNMB, CA9, c-KIT, c-MET, ACE, APP, adrenergic receptor beta2, Claudin 3, Claudin 6, Claudin 18.2, RON, RORI, ROR1, PD-LI, PD-L2, B7-H3, B7-H4, IL-2 receptor, IL-4 receptor, IL-13 receptor, integrins, IFN-alpha, IFN-gamma, IgE, IGF-1 receptor, IL-1, IL-4, IL-5, IL-6, IL-12, IL-13, IL-22, IL-23, interferon receptor, ITGB2 (CD18), LFA-1 (CDI la), L-selectin, P-selectin, E-selectin, mucin, myostatin, NCA-90, NGF, PDGFR alpha, prostatic carcinoma cells, Pseudomonas aeruginosa, rabies, RANKL, respiratory syncytial virus, Rhesus factor, SLAMF7, sphingosine-1 -phosphate, TGF-1, TGFbeta2, TGFbeta, TNFalpha, TRAIL-R1, TRAIL-R2, CTAA 16.88, vimentin, matrix metalloproteinases (MMP) such as MMP2, MMP9, MMP14, LDL receptor, endoglins, polysialic acids and their corresponding lectins. An example of fibronectin targets are the alternatively spliced extra-domain-A (ED-A) and extra-domain-B (ED-B) of fibronectin. Non-limiting examples of targets in stroma can be found in V. Hofmeister, D. Schrama, J. C. Becker, Cancer Immun.;lmmunother. 2008, 57, 1, the contents of which are hereby incorporated by reference.

[0688] More particularly, in order to allow a (specific) targeting of the above-listed targets, the targeting agent can comprise compounds comprising an ncAA-functionalized peptide sequence. Such compounds include but are not limited to antibodies, antibody derivatives, antibody fusions (e.g. bi-specific and tri-specific mAbs.

[0689] In one particular embodiment, antibodies are used to form a targeting agent. While antibodies or immunoglobulins derived from IgG antibodies are particularly well-suited for use in this invention, immunoglobulins from any of the classes or subclasses may be

[0690] M / 65047-PCTVERAXA 67

[0691] selected, e.g. IgG, IgA, IgM, IgD and IgE. Suitably, the immunoglobulin is of the class IgG including but not limited to IgG subclasses (lgG1, 2, 3 and 4) or the class IgM which is able to specifically bind to a specific epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, camelized single domain antibodies, recombinant antibodies, anti-idiotype antibodies, multispecific antibodies, antibody fragments, such as, Fv, VHH, Fab, F(ab)2, Fab', Fab'-SH, F(ab')2, single chain variable fragment antibodies (scFv), tandem / bis-scFv, Fc, pFc', scFv-Fc, disulfide Fv (dsFv), bispecific antibodies (bc-scFv) such as BiTE antibodies, trispecific antibody derivatives such as tribodies, camelid antibodies, minibodies, nanobodies, resurfaced antibodies, humanized antibodies, fully human antibodies, single domain antibodies (sdAb, also known as Nanobody™), chimeric antibodies, chimeric antibodies comprising at least one human constant region, dual-affinity antibodies such as dual-affinity retargeting proteins (DART™), and multimers and derivatives thereof, such as divalent or multivalent single-chain variable fragments (e.g. di-scFvs, tri-scFvs) including but not limited to minibodies, diabodies, triabodies, tribodies, tetrabodies, and the like, and multivalent antibodies. Reference is made to [Trends in Biotechnology 2015, 33, 2, 65], [Trends Biotechnol. 2012, 30, 575-582], and [Cane. Gen. Prot. 2013 10, 1-18], and [BioDrugs 2014, 28, 331-343], the contents of which are hereby incorporated by reference.

[0692] " Antibody fragment" refers to at least a portion of the variable region of the immunoglobulin that binds to its target, i.e. the antigen-binding region.

[0693] Other embodiments use antibody mimetics as targeting agents, such as but not limited to Affimers, Anticalins, Avimers, Alphabodies, Affibodies, DARPins, and multimers and derivatives thereof; reference is made to [Trends in Biotechnology 2015, 33, 2, 65], the contents of which is hereby incorporated by reference.

[0694] For the avoidance of doubt, in the context of this invention the term "antibody" is meant to encompass all of the antibody variations, fragments, derivatives, fusions, analogs and mimetics outlined in this paragraph, unless specified otherwise.

[0695] Any antibody or fragment thereof as described hereinabove may be used in the context of the invention as long as it contains a glycan moiety, which can be subjected to a glycan engineering process of the invention.

[0696] Typical non-limiting examples of antibody molecules to be further modified to form glycan engineered, TCO modified targeting agents of the present invention are selected form biologically, in particular pharmacologically active antibody molecules. Nonlimiting examples are selected form the following group: trastuzumab, bevacizumab,

[0697] M / 65047-PCTVERAXA 68

[0698] cetuximab, panitumumab, ipilimumab, rituximab, alemtuzumab, ofatumumab, gemtuzumab, brentuximab, ibritumomab, tositumomab, pertuzumab, adecatumumab, IGN101, INA01 labetuzumab, hua33, pemtumomab, oregovomab, minretumomab (CC49), cG250, J591, MOv-18, farletuzumab (MGRAb-003), 3F8, ch14,18, KW-2871, hu3S193, lgN31 1, IM- 2C6, CDP-791, etaracizumab, volociximab, nimotuzumab, MM-121, AMG 102, METMAB, SCH 900105, AVE1642, IMC-A12, MK-0646, R1507, CP 751871, KB004, III A4, mapatumumab, HGS-ETR2, CS-1008, denosumab, sibrotuzumab, F19, 81 C6, pinatuzumab, lifastuzumab, glembatumumab, coltuximab, lorvotuzumab, indatuximab, anti-PSMA, MLN-0264, ABT-414, milatuzumab, ramucirumab, abagovomab, abituzumab, adecatumumab, afutuzumab, altumomab pentetate, amatuximab, anatumomab, anetumab, apolizumab, arcitumomab, ascrinvacumab, atezolizumab, bavituximab, bectumomab, belimumab, bivatuzumab, brontictuzumab, cantuzumab, capromab, catumaxomab, citatuzumab, cixutumumab, clivatuzumab, codrituzumab, conatumumab, dacetuzumab, dallotuzumab, daratumumab, demcizumab, denintuzumab, depatuxizumab, derlotuximab, detumomab, dinutuximab, drozitumab, duligotumab, durvalumab, dusigitumab, ecromeximab, edrecolomab, elgemtumab, emactuzumab, enavatuzumab emibetuzumab, enfortumab, enoblituzumab, ensituximab, epratuzumab, ertumaxomab, etaracizumab, farletuzumab, ficlatuzumab, figitumumab, flanvotumab, futuximab, galiximab, ganitumab, icrucumab, igovomab, imalumab, imgatuzumab, indusatumab, inebilizumab, intetumumab, iratumumab, isatuximab, lexatuzumab, lilotomab, lintuzumab, lirilumab, lucatumumab, lumretuzumab, margetuximab, matuzumab, mirvetuximab, mitumomab, mogamulizumab, moxetumomab, nacolomab, naptumomab, narnatumab, necitumumab, nesvacumab, nimotuzumab, nivolumab, nofetumomab, obinutuzumab, ocaratuzumab, ofatumumab, olaratumab, onartuzumab, ontuxizumab, oportuzumab, oregovomab, otlertuzumab, pankomab, parsatuzumab, pasotuxizumab, patritumab, pembrolizumab, pemtumomab, pidilizumab, pintumomab, polatuzumab, pritumumab, quilizumab, racotumomab, ramucirumab, rilotumumab, robatumumab, sacituzumab, samalizumab, satumomab, seribantumab, siltuximab, sofituzumab, tacatuzumab, taplitumomab, tarextumab, tenatumomab, teprotumumab, tetulomab, ticilimumab, tigatuzumab, tositumomab, tovetumab, tremelimumab, tucotuzumab, ublituximab, ulocuplumab, urelumab, utomilumab, vadastuximab, vandortuzumab, vantictumab, vanucizumab, varlilumab, veltuzumab, vesencumab, volociximab, vorsetuzumab votumumab, zalutumumab, zatuxima, combination and derivatives thereof, as well as other monoclonal antibodies targeting CAI 25, CAI 5-3, CAI 9-9, L6, Lewis Y, Lewis X, alpha fetoprotein, CA 242, placental alkaline phosphatase, prostate specific antigen,

[0699] M / 65047-PCTVERAXA 69

[0700] prostate specific membrane antigen, prostatic acid phosphatase, epidermal growth factor, MAGE- 1, MAGE-2, MAGE-3, MAGE-4, transferrin receptor, p97, MllCI, CEA, gplOO, MARTI, IL-2 receptor, CD20, CD52, CD33, CD22, human chorionic gonadotropin, CD38, CD40, mucin, P21, MPG, and Neu oncogene product.

[0701] According to a further particular embodiment of the invention, the target and targeting agent are selected so as to result in the specific or increased targeting of a tissue or disease, such as cancer, an inflammation, an infection, a cardiovascular disease, e.g. thrombus, atherosclerotic lesion, hypoxic site, e.g. stroke, tumor, cardiovascular disorder, brain disorder, apoptosis, angiogenesis, an organ, and reporter gene / enzyme. This can be achieved by selecting targets with tissue-, cell- or disease- specific expression.

[0702] By way of example, the targeting agent specifically binds or complexes with a cell surface molecule, such as a cell surface receptor or antigen, for a given cell population. Following specific binding or complexing of the targeting agent with the receptor, the drug will enter the cell.

[0703] As used herein, a targeting agent that "specifically binds or complexes with" or "targets" a cell surface molecule, an extracellular matrix target, or another target, preferentially associates with the target via intermolecular forces. For example, the ligand can preferentially associate with the target with a dissociation constant (Kd or KD) of less than about 50 nM, less than about 5 nM, or less than about 500 pM.

[0704] 3. Functionalized payload molecules (PM)

[0705] Functionalised payload molecules are provided in order to attach the payload, as for example a pharmaceutical drug or marker or chelator to the glycan engineered immunoglobulin molecule of the present invention. Such a functionalized PM may be further illustrated by the following general formula (XXX)

[0706] (DG)-[(Y1)a-(Y2)b-PM]c

[0707] (XXX)

[0708] wherein

[0709] a is 0 or represents an integer selected from 1 and 2,

[0710] b is 0 or represents an integer selected from 1 and 2,

[0711] c represents an integer selected of at least 1, particularly from 1 and 2,

[0712] Y1represents a cleavable moiety,

[0713] M / 65047-PCTVERAXA 70

[0714] Y2represents a self-immolative moiety,

[0715] PM is a payload molecule as defined herein above and

[0716] DG is a docking group, capable of reacting with a TCO moiety of a glycan engineered immunoglobulin molecule via click reaction as herein defined, wherein, when c represents an integer of more than 1, then the DG also comprises a branching moiety (BM).

[0717] Said BM covalently inserted at one end of the DG between DG and the PM portion which may be further illustrated by the following formula XXXa

[0718] (DG)-(BM)-[(Y1)a-(Y2)b-PM]c

[0719] (XXXa)

[0720] Particular examples of docking groups of the triazine or tetrazine -type are further described below.

[0721] A “branching moiety” represents an at least trivalent group, which allows the functionalization with more than one, as for example 1 or 2 payload molecules in a single step. Non-limiting examples of such branching groups are trivalent cycloaliphatic or cycloaromatic residues of the general formula

[0722]

[0723] or trivalent, non-cyclic, aliphatic groups or tertiary amino groups of the following formulae

[0724]

[0725] wherein one of the three valencies is attached to the DG.

[0726] 3.1 Enzymatically or chemically cleavable moiety (Y1)

[0727] M / 65047-PCTVERAXA 71

[0728] A “cleavable moiety” encompasses any group, which may be cleaved enzymatically or chemically, in particular under in vivo or ex vivo conditions. An enzymatic cleavage may be effected, for example, through the action of a protease. A chemical cleavage, may be effected for example through hydrolytic cleavage or reductive cleavage of S-S bonds.

[0729] Suitable cleavable moieties are well known from the prior art.

[0730] Reference can be made to: Bargh et al., Chem Soc Rev 2019, 48(16), 4361-4374; Poreba, FEBS J 2020, 287(10), 1936-1969 and Salomon et al., Mol Pharmaceutics 2019 16,(12), 4817-4825.

[0731] According to a particular embodiment of the present invention said moiety Y1is an enzymatically or chemically cleavable linker group, selected from

[0732] a) a peptidyl group, in particular di-, tri- or tetra-peptidyl group;

[0733] b) a disulfide group of the formula -(CR7R8)n2-S-S-(C R7R8)n2-X5- or X5-(CR7R8)n2-S-S-(C R7R8)n2-X5- wherein

[0734] n2 represents an integer from 1 to 4

[0735] residues R7and R8independently of each other are selected from H or lower alkyl, in particular methyl; or two residues R7and R8together with the carbon atom which they are attached to form a cyclic C4 -to C8-alkyl group; and

[0736] moiety X5is selected from -C(O)- and -O-;

[0737] moiety X5is selected from -C(O)- and -(O)C-(CH2)-NH-;

[0738] c) hydrazone groups selected from > C=N-N(R9)- and -N(R9)-N=C<

[0739] wherein

[0740] R9is H or lower alkyl; and

[0741] d) beta-glucuronidase-sensitive cleavable linker groups (glucuronide-linker groups), in particular carrying a beta-glucuronic acid derived trigger residue;

[0742] According to a particular embodiment thereof, the cleavable linker is a peptidyl group according to feature a).

[0743] According to another particular embodiment thereof, the cleavable linker is a glucuronide-linker group according to feature d).

[0744] As further examples there may be mentioned p-glucuronide linkers, carrying a betaglucuronic acid derived trigger residue. Non-limiting examples thereof are:

[0745] M / 65047-PCTVERAXA

[0746]

[0747] 3.2 Self-immolative moiety( Y2)

[0748] Suitable self-immolative moieties Y2are well known from the prior art.

[0749] M / 65047-PCTVERAXA 73

[0750] Reference can be made to: Santi et al., J Med Chem 2014, 57(6), 2303-2314; Alouane et al., Angew Chem Int Ed 2015, 54(26), 7492-7509; and Kolakowski et al., Angew Chem 2016, 128(28), 8080-8083.

[0751] 3.3 Docking groups (DG)

[0752] Suitable DG are in particular selected from tri- or tetrazine docking groups also known from the prior art. Particular examples are for example described in WO2023 / 10494 or different scientific articles [Yang et al., Angew. Chem. Int. Ed. 2012, 51, 5222 -5225; Fan et al., Angew. Chem. Int. Ed. 2016, 55, 14046 -14050; Mao et al., Angew. Chem. Int. Ed. 2019, 58, 1106 -1109; Qu et al., Angew. Chem. Int. Ed. 2018, 57, 12057 -12061; Eising et al., Bioconjugate Chem. 2018, 29, 3054-3059; Meng et al., J. Org. Chem. 2017, 82, 1676-1687; Xie et al., Angew. Chem. Int. Ed. 2020, 59, 16967-16973; Lambert et al., J. Am. Chem. Soc.

[0753] 2019, 141, 17068-17074; Jemas et al., J. Am. Chem. Soc. 2022, 144, 1647-1662; Selvaraj et al., Tetrahedron Letters 2014, 55, 4795-4797; Dowling et al., J. Org. Chem. 2018, 83, 4229-4238; Battisti et al., Bioconjugate Chem. 2022, 33, 4, 608-624; Karver et al., Bioconjugate Chem. 2011, 22, 2263-2270; Bender et al., Org. Lett. 2017, 19, 5693-5696; Kamber et al., J. Am. Chem. Soc. 2015, 137, 8388-8391; Ros et al., Bioconjugate Chem.

[0754] 2020, 31, 933-938; Ros et al., Chem. Commun., 2020, 56, 11086; van Onzen et al., J. Am. Chem. Soc. 2020, 142, 10955-10963; Carlson et al., J. Am. Chem. Soc. 2018, 140, 3603-3612.].

[0755] WO2023 / 10494 discloses phosphonate-terminated DGs of the formula XXXI N=N

[0756] R

[0757] -I ns

[0758]

[0759] (XXXI)

[0760] wherein

[0761] n3 represent an integer selected from 1 or 2

[0762] Sp1and Sp2are same or different spacer groups

[0763] linkages a, p, and y are independently from each other selected from a chemical bond, or an ether, thioether, ester, amide, carbonyl (in particular keto), carbamate, dicarbamate, carbonate, hydrazine, urea, alkylene oxide or linear or branched polyalkylene oxide linkage;

[0764] Z represents a phosphor containing hydrophilic group, in particular (R1O)2P(O)-, (R1aO)2P(O)-O-, and (R2O)3P-O-;

[0765] M / 65047-PCTVERAXA 74

[0766] wherein

[0767] R1, R1aand R2are same or different and independently of each other represent H or lower alkyl, in particular methyl or ethyl; and even more particularly H;

[0768] and

[0769] R represents H or a chemical group capable of forming a chemical bond, or capable of forming an ether, thioether, ester, such as active esters like succinimidyl- or pentafluorophenyl- ester, amide, carbamate, dicarbamate, carbonate, hydrazine, urea, alkylene oxide or linear or branched polyalkylene oxide linkage.

[0770] In said formula XXXI Sp2may be replaced by a branching moiety (BM) as defined above.

[0771] As examples of suitable spacer groups there may be mentioned

[0772] a) mono- or polycyclic optionally mono-or poly-substituted aromatic moieties having 6 to 14 ring carbon atoms, in particular 1,2-phenylene 1,3-phenylene or 1,4-phenylene; wherein said one or more optional substituents are independently of each other selected from -Hal, -CHal3, -OH, -SH,, -NR’2, NO2, -CN, -C(=O)R”, -C(=O)OR”’, alkyl, alkenyl, alkynyl, and alkoxy;

[0773] wherein

[0774] R’, R” and R’” independently of each other are selected from H and Ci -to C4-alkyl; (Moiety M1);

[0775] b) heterocyclic residues of the general formula XXXII

[0776]

[0777] (XXXII)

[0778] wherein

[0779] one, two or three of the ring moieties Xi to X4 represents N and the other represent > CH; (Moiety M2);

[0780] c) linear or branched lower-alkylene, in particular -(CH2)ni-,

[0781] M / 65047-PCTVERAXA 75

[0782] wherein n1 is an integer from 1 to 4; more particularly methylene; (Moiety M3);

[0783] d) linear or branched polyalkylene oxide moieties, in particular selected from linear the moieties -((CH2)xi-O)yi- or -(O-(CH2)xi)yi- and the branched analogues thereof;

[0784] wherein

[0785] x1 independently of each other represent an integer selected from 1, 2, 3 or 4; in particular 1 or 2; and

[0786] y1 independently of each other represent an integer from 1 to 20, in particular 1 to 4; (Moiety M4);

[0787] wherein Sp1is selected from M1, M2, M3 or combinations thereof; and Sp2is selected from M1 to M4 or combinations thereof.

[0788] In the following, particular preferred structures of DGs of general formula XXXI are displayed. In said formulae residues R1independently of each other are H or lower alkyl, in particular methyl or ethyl; and even more particularly H

[0789] 17a 18a 20a

[0790] RiO-PsO

[0791] OR-,

[0792]

[0793] 21a 24a

[0794] M / 65047-PCTVERAXA 76

[0795] 25a 26a 27a 28a

[0796] O H

[0797]

[0798] 29a 30a 31a

[0799] M / 65047-PCTVERAXA 77

[0800] ZZ- / =\

[0801] / \\ H / \ / _ _, _4

[0802] / / / \ \ / N CXk

[0803] J* Z Z=Z Tk -■ —

[0804] 11O> N

[0805] m X

[0806] O CM

[0807] / . CK X

[0808] ^ CM Z Z-Z 0- \ \ _ / _ / _ / / Vc O

[0809] zz\= / _ \ / V / / 1- - - 33a 36a

[0810]

[0811] 38a 39a 40a Further particular examples of DGs general formula XXXI are:

[0812] Formula Formula

[0813]

[0814] M / 65047-PCTVERAXA

[0815] 78

[0816]

[0817] M / 65047-PCTVERAXA

[0818] 79

[0819]

[0820] M / 65047-PCTVERAXA

[0821] 80

[0822]

[0823] M / 65047-PCTVERAXA

[0824] 81

[0825] Formula Formula X* JI: I D N N Z, J O 04 — - 5 _\ _ / \ _ 4 J / s J / '.. / J* \ _-' ' V} \Q £L — ' - —“" ^g $gi~ “ _ 3; 0 i

[0826] HO -P'-J

[0827] 0*1

[0828] 32 OH

[0829] r°

[0830] Hcr^

[0831] 33

[0832]

[0833] M / 65047-PCTVERAXA

[0834]

[0835] M / 65047-PCTVERAXA

[0836]

[0837] M / 65047-PCTVERAXA

[0838]

[0839] M / 65047-PCTVERAXA

[0840]

[0841] M / 65047-PCTVERAXA 86

[0842] Formula Formula

[0843] .0 0. 0 0

[0844] o o y-A

[0845] VAQA,--?H2A AA^NH2

[0846] N II I II^N

[0847] N^N i

[0848] N^N

[0849] N^N

[0850] ^p° o' °

[0851] HO'oh

[0852] 72 )

[0853] 75

[0854] 0 0

[0855] ^ N^ A^2

[0856] N^N

[0857] u i

[0858] N^N

[0859] HO'oh

[0860] 76

[0861]

[0862] As further tetrazines suitable as DGs there may be mentioned analogues of the above specific compounds wherein the phosphonate group is missing and replaced by H, or where the tetrazine moiety is replaced by an 1, 2, 4-triazine moiety.

[0863] 4. Conjugates of the present invention

[0864] The present invention also relates to conjugates, which are synthesized by forming a covalent linkage between a first functionalized molecule, which is a TCO-functionalized in engineered, immunoglobulin molecule, as described above with a second functionalized payload molecule comprising a docking group (DG) capable of reacting with said TCO functional group of the first molecule.

[0865] Methods of preparing such conjugates are in principle well known in the art. Biorthogonal bioconjugation via a Diels-Alder-type cycloaddition reaction of said two molecules may be performed in line with prior art teaching (see for example Oliveira et al,

[0866] M / 65047-PCTVERAXA 87

[0867] Chem Soc Rev, 2017, 46,4895-4950). More particularly, said docking group (DG) is selected from an optionally substituted triazinyl or optionally substituted tetrazinyl groups, capable of covalently reacting in a copper-free strain promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) with said TCO group of the invention.

[0868] 5. Pharmaceutical compositions

[0869] The conjugates or bioconjugates of the present invention, as for example APCs, in particular ADCs (i.e. the active agents or ingredients) of this invention are generally given as “pharmaceutical compositions” comprised of a therapeutically and / or prophylactically effective amount or a diagnostically effective amount of at least one such active ingredient or its pharmaceutically acceptable salt and optionally at least one pharmaceutically acceptable excipient.

[0870] Said pharmaceutical compositions may be delivered via suitable routes of administration such as via oral, rectal, transmucosal, topical, ophthalmic, otologic, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, as the case may be.

[0871] Depending on the nature or the mode of administration and dosage form said composition said at least one additional pharmaceutical excipient may be different.

[0872] An “excipient” is a substance formulated alongside the active ingredient and is included for different purpose, as for example for long-term stabilization, bulking up solid formulations that contain potent active ingredients in small amounts (thus often referred to as "bulking agents", "fillers", or "diluents"), or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as for example facilitating drug absorption, reducing viscosity, or enhancing solubility. Excipients can also be useful in the manufacturing process of the pharmaceutical composition, to aid in the handling of the active substance concerns such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation or aggregation over the expected shelf life. The selection of appropriate excipients not only depends upon the route of administration and the dosage form, but also on the particular active ingredient and other factors.

[0873] Excipients may be selected from the following classes: immunological adjuvants, antiadherents, binders, coatings, colours, disintegrant, flavours, glidants, lubricants, preservatives, sorbents, sweeteners, and vehicles.

[0874] Non limiting examples of excipients comprise diluents, preserving agents, stabilizers, emulsifying agents, like emulsifying polymers, such as polysorbates or poloxamers,

[0875] M / 65047-PCTVERAXA 88

[0876] antioxidants; anti-irritants, chelating agents and stabilizing salts, such as chlorides, sulfates, phosphates, diphosphates, hydrobromides and nitrates, suspending agents, antibacterial agents or antifungal agents. Further, buffering agents such as buffering systems of low molecular weight organic acids together with the respective salts, or inorganic buffering substances, such as phosphate buffers, can be used. Further suitable ingredients are also known from relevant pharmacological standard literature. Also the proportion of the various components will vary depending on the nature of the specific component used and is generally known to the person skilled in the art (Remington's Pharmaceutical science (" Handbook of Pharmaceutical Excipients", 2nd Edition, (1994), Edited by A Wade and PJ Weller or in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit.

[0877] 1985).

[0878] A pharmaceutical composition as used herein may be presented in the form of a “dosage form” or “unit dose” and may comprise one or more APC, in particular ADCs as described herein. Thus, a pharmaceutical composition as used herein could, for example, provide two active agents admixed together in a unit dose or provide two active agents combined in a dosage form wherein the active agents are physically separated.

[0879] Furthermore, one may administer the pharmaceutical composition in a targeted drug delivery system, for example, in a liposome coated with endothelial cell-specific antibody.

[0880] The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, emulsifying, encapsulating, entrapping or combinations thereof. Proper formulation is dependent upon the route of administration chosen.

[0881] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable risk / benefit ratio.

[0882] The invention includes all “pharmaceutically acceptable salt forms” of the active ingredient. Pharmaceutically acceptable salts are those in which the counter ions do not contribute significantly to the physiological activity or toxicity of the compounds and as such function as pharmacological equivalents. These salts can be made according to common organic techniques employing commercially available reagents. Some anionic salt forms include acetate, acistrate, besylate, bromide, chloride, citrate, fumarate, glucouronate, hydrobromide, hydrochloride, hydroiodide, iodide, lactate, maleate, mesylate, nitrate,

[0883] M / 65047-PCTVERAXA 89

[0884] pamoate, phosphate, succinate, sulfate, tartrate, tosylate, and xinofoate. Some cationic salt forms include ammonium, aluminum, benzathine, bismuth, calcium, choline, diethylamine, diethanolamine, lithium, magnesium, meglumine, 4-phenylcyclohexylamine, piperazine, potassium, sodium, tromethamine, and zinc.

[0885] A "therapeutically effective amount" and / or "prophylactically effective amount" means an amount effective, when administered to a human or non-human patient, to provide any therapeutic and / or prophylactic benefit. More particularly, a “therapeutically effective amount” is an amount of an active ingredient disclosed herein or a combination of two or more such active ingredients, which inhibits, totally or partially, the progression of the condition or alleviates, at least partially, one or more symptoms of the condition.

[0886] A "diagnostically effective amount" means an amount effective to allow obtaining from the patient a diagnostically valuable information on status or progression of a disease state.

[0887] A therapeutic benefit may be an amelioration of symptoms of a diseased patient, e.g., an amount effective to decrease the symptoms of a diseased patient. In certain circumstances a patient may not present symptoms of a condition for which the patient is being treated. Thus, a prophylactically effective amount of a compound is also an amount sufficient to provide a significant positive effect on any indicia of a disease, disorder or condition e.g. an amount sufficient to significantly reduce the frequency and severity of disease symptoms to occur.

[0888] A therapeutically effective amount can also be an amount, which is prophylactically effective.

[0889] A “patient” as used herein means human or non-human, in particular human, animals. A "dosage form" is any unit of administration (“unit dose”) of one or more active agents as described herein.

[0890] The term "treating" or “treatment” refers to: (i) preventing a disease, disorder or condition from occurring in a patient which may be predisposed to the disease, disorder and / or condition but has not yet been diagnosed as having it; (ii) inhibiting the disease, disorder or condition, i.e., arresting its development; and (iii) relieving the disease, disorder or condition, i.e., causing regression of the disease, disorder and / or condition. In particular it encompasses a prophylactic or therapeutic treatment or combinations thereof.

[0891] “Frequency” of dosage may vary depending on the compound used and the particular type of infection treated. A dosage regimen of once per day is possible. Dosage regimens in which the active agent is administered for several times daily, as for example 2 to 10 times, like 2, 3, 4, 5, 6, 7, 8, 9 or 10 times may occasionally be more helpful.

[0892] M / 65047-PCTVERAXA 90

[0893] It will be understood, however, that the specific dose level and frequency for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease in the patient undergoing therapy. Patients may generally be monitored for therapeutic or prophylactic effectiveness using assays suitable for the condition being treated or prevented, which will be familiar to those of ordinary skill in the art.

[0894] Particular examples of pharmaceutical compositions according to the present invention are liquid form preparations such as solutions, suspensions, and emulsions and comprise, a therapeutically effective amount of at least one APC, in particular ADC component as defined above, optionally together with at least one further pharmaceutically acceptable excipient as defined above and may be administered through any suitable route.

[0895] Further examples of pharmaceutical compositions according to the present invention are solid form preparations such as powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.

[0896] The numerous possible variations that will become immediately evident to a person skilled in the art after heaving considered the disclosure provided herein also fall within the scope of the invention.

[0897] The following examples are illustrative only and are not intended to limit the scope of the embodiments described herein.

[0898] EXPERIMENTAL PART

[0899] A) Materials and Methods

[0900] Reagents were purchased from commercial suppliers and used without further purification. All solvents, including anhydrous solvents, were used as obtained from the commercial sources. Air and water-sensitive reagents and reactions were generally handled under inert conditions, like argon or nitrogen atmosphere.

[0901] Cells:

[0902] MAXHER BT-474 cells were obtained from contract research organization Charles River Laboratories,

[0903] M / 65047-PCTVERAXA 91

[0904] Antibodies:

[0905] mAb Trastuzumab: a) expressed by Veraxa; b) Herceptin, Roche / EurimPharm mAb FLYSYN is commercially available and was obtained from Synlmmune mAb 20D9 is commercially available and was obtained from Proteogenix Enzymes:

[0906] The enzymes CSS (CMP-sialic acid synthetase from Neisseria meningitidis group B) (C1999), pyrophosphatase (inorganic from baker's yeast, S. cerevisiae) (11643), and alkaline phosphatase (from calf intestinal mucosa) (79390) were purchased from Sigma-Aldrich. The transferases B4GalT1 (11220-h07h) and ST6Gal1 (50740-m07h) were purchased from Sino Biological.

[0907] The endoglycosidase EndoS2 was either employed as soluble enzyme (GlycINATOR Lyophilized, A0-GL1-020) or immobilized enzyme (GlycINATOR immobilized) (microspin column, A0-GL6-010), which were purchased from Genovis.

[0908] Flash chromatographic purification was performed on a Biotage Isolera One purification system using silica gel (0.060-0.200 mm), KP-Sil cartridges.

[0909] Size exclusion chromatography of small molecules was performed employing Bio-Gel P-2, fine polyacrylamide beads (Bio-Rad) that were packed into Econo-Pac or Econo glass columns (Bio-Rad). The packed columns were operated with H2O as eluent and a flow rate either solely driven by gravity or supported by an LC pump (Agilent 1260 Infinity II Preparative binary pump) at 1.5 mL / min.

[0910] Preparative HPLC purification of sugar building blocks was performed on Agilent Infinity 1260 series equipment consisting of Agilent 1260 preparative pumps, a 1260 preparative autosampler, and a 1260 multiple wavelength detector VL. The preparative columns used were Waters XBridge Prep C18 OBD (5pm, 19x250 mm) and Waters XBridge BEH Amide OBD Prep Column (130 A, 5 pm, 10 x 100 mm) operated with a linear gradient of acetonitrile in H2O, and both solvents containing either 0.1% TFA for the C18 column or 0.1% NH3 for the amide column as modifier.

[0911] Affinity chromatography of antibodies was performed on an Äkta Pure system (Cytiva) employing either a HiTrap MabSelect SuRe (Cytiva, 11003494, 5 mL) or a HiTrap Fibro PrismA protein A column (Cytiva, 17549855, 0.4 mL), eluting with a linear gradient (0–100 %) of sodium citrate (0.1 M; pH 3.4) in PBS (pH 7.4). Upon elution, the antibodies were directly neutralized with Tris (1 M, pH 10).

[0912] Size exclusion chromatography of antibodies was performed on an Äkta Pure system (Cytiva) employing Superdex 200 columns (Cytiva) and eluting with PBS pH 7.4.

[0913] M / 65047-PCTVERAXA

[0914] B) Overview of synthetic procedures of the invention

[0915] Figure 1A schematically illustrates the conversion of Neu5Ac to a TCO functionalized CMP derivative of above formula I. It further illustrates the enzymatic remodeling of a biantennary, optionally fucosylated glycan moiety of the type G2A1 of a glycosylated antibody molecule, which method comprises a one-pot de-glycosylation / galactosylation of the glycan residue followed in a next step by sialylation with said TCO functionalized CMP derivative of formula I, and the subsequent coupling with a tetrazine-functionalised payload (as for example drug or dye) molecule. For illustrative purpose only, an analogous remodeling reaction of the invention is shown for a high mannose glycan residue of the type Man9N2. Figure 1 B show some examples of the largest N-linked oligosaccharide structure (G2A2) found in human IgG which also may be remodelled according to the invention. The third N-acetylglucosamine (GlcNAc, NAG) bisecting arm represents around 10% of human IgGs glycoforms. Figure 1C shows some examples for isoforms of the high mannose glycan of the type Man9N2, which also may be remodelled according to the invention, as all further common glycans, like for example GO, G0F (fucosylated), G1, G1F (fucosylated), G2, G2F (fucosylated) and others.

[0916] C) Synthesis of Compounds

[0917] EXAMPLE 1 - Synthesis of methyl (2S,4S,5R,6S)-5-acetamido-2,4-dihydroxy-6-((1 R,2R)-1,2,3-trihydroxypropyl)tetrahydro-2H-pyran-2-carboxylate (1 )

[0918] . OH

[0919] H N CO2Me

[0920]

[0921] N-Acetylneuraminic acid (Neu5Ac) (5.00 g, 16.2 mmol, 1.00 eq) was suspended in MeOH (300 mL). Amberlyst-15 (H+) (15 g) was added and the suspension was heated at 80 °C for 20 min. The solution was cooled to room temperature while continuing stirring, then filtered, and the solvent was removed under reduced pressure.

[0922] Compound 1 was obtained as beige solid (5.4 g, quant, yield).

[0923] M / 65047-PCTVERAXA 93

[0924] EXAMPLE 2 - Synthesis of methyl (2S,4S,5 / ?,6S)-5-acetamido-6-((1 / ?,2R)-1,2-dihydroxy-3-(tosyloxy)propyl)-2,4-dihydroxytetrahydro-2H-pyran-2-carboxylate (2)

[0925] TsO

[0926]

[0927] Compound 1 (1.50 g, 4.64 mmol, 1.00 eq) was dissolved in anhydrous pyridine (20 mL), and the solution was cooled to 0 °C. A solution of TsCI (1.30 g, 6.82 mmol, 1.47 eq) in anhydrous pyridine (5 mL) was added slowly, and the reaction mixture was stirred at room temperature for 14 h. After quenching with MeOH, the solvents were removed under reduced pressure. Purification via flash chromatography (DCM / MeOH 20:1— >3:1) yielded 2 as colorless foam (0.91 g, 41%).

[0928] EXAMPLE 3 - Synthesis of (2S,4S,5R,6S)-5-acetamido-6-((1 R,2R)-3-azido-1,2-dihydroxypropyl)-2,4-dihydroxytetrahydro-2H-pyran-2-carboxylic acid (3)

[0929]

[0930] Compound 2 (0.91 g, 1.90 mmol, 1.00 eq) and NaN3(0.50 g, 7.60 mmol, 4.00 eq) were dissolved in acetone (20 mL) and H2O (5 mL), and the solution refluxed for 14 h. The solvents were removed under reduced pressure.

[0931] M / 65047-PCTVERAXA

[0932] Purification via flash chromatography (DCM / MeOH 10:1— >1:1) yielded 3 as pale brown foam (0.27 g, 42%).

[0933] EXAMPLE 4 - Synthesis of (2S,4S,5 / ?,6S)-5-acetamido-6-((1 f?,2 / ?)-3-amino-1,2-dihydroxypropyl)-2,4-dihydroxytetrahydro-2H-pyran-2-carboxylic acid (4)

[0934] I2I

[0935] PH

[0936] . OH

[0937] H

[0938] N

[0939]

[0940] Compound 3 (0.90 g, 2.70 mmol, 1.00 eq) was dissolved in H2O (15 mL). A solution of PPh3(2.10 g, 8.10 mmol, 3.00 eq) in acetone (65 mL) was added, and the reaction mixture was refluxed for 18 h.

[0941] After removal of all solvents under reduced pressure, the crude was re-dissolved in H2O and filtered through Celite. The aqueous solution was washed with DCM. After reduction of the volume, the solution was filtered through an 0.45 μm membrane. Removal of all remaining solvent under reduced pressure yielded 4 as orange foam (0.81 g, 98%).

[0942] EXAMPLE 5 - Synthesis of (2R,4S,5R,6S)-5-acetamido-6-((1R,2R)-3-amino-1,2-dihydroxypropyl)-2-(((((2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-hydroxytetrahydro-2H-pyran-2-carboxylic acid (5)

[0943] M / 65047-PCTVERAXA

[0944] 95

[0945]

[0946] Compound 4 (11.0 mg, 35.7 μmol, 1.00 eq) was incubated with CTP (20.0 mg, 38.0 μmol, 1.06 eq), CSS (CMP-sialic acid synthetase from Neisseria meningitidis) (1 U) and pyrophosphatase (1 U) in Tris-HCI, pH 9.0 (100 mM) containing MgCl2 (20 mM) (total volume: 2 mL) at 37 °C shaking at 300 rpm for 2 h.

[0947] Purification via size exclusion chromatography (Bio-Gel P-2 resin; H2O) yielded 5 as colorless solid (16 mg, 73%).

[0948] EXAMPLE 6 - Synthesis of 2,5-dioxopyrrolidin-1-yl (E)-2-(1,2-dihydroxycyclooct-3-en-1-yl)acetate (6)

[0949] The compound was produced as previously described in PCT / EP2024 / 07114.

[0950] a) Synthesis of (Z)-9-Oxabicyclo[6.1.0]non-2-ene (V1)

[0951] ^:o

[0952]

[0953] V1

[0954] A solution of 3-chloroperbenzoic acid (12.7 g, 73.4 mmol, 1.00 eq) in CH3CI (220 mL) was added dropwise to 1,3-cyclooctadiene (11.4 mL, 9.93 g, 91.8 mmol, 1.25 eq) over 3 h with stirring. After complete addition the mixture was stirred at room temperature (rt) overnight. The formed precipitate was filtered off and the filter cake washed with CH2CI2 several times. The filtrate was washed with an aqueous solution of Na2SO3followed by a saturated aqueous

[0955] M / 65047-PCTVERAXA

[0956] solution of NaHCO3. The organic phase was dried over Na2SO4and the solvent removed under reduced pressure.

[0957] Purification via flash chromatography (cyclohexane / EtOAc 20:1— >10:1) yielded V1 as colorless oil (8.93 g, 98%).

[0958] b) Synthesis of (Z)-8-hydroxycyclooct-2-en-1-yl acetate (V2)

[0959] 0

[0960]

[0961] To a solution of acetic acid (5.80 mL, 6.09 g, 101 mmol, 1.50 eq) and tetrakis(triphenylphosphine)palladium(0) (7.82 g, 6.76 mmol, 0.100 eq) in THF (190 mL) was added V1 (8.40 g, 67.6 mmol, 1.00 eq) dropwise at 0 °C. After complete addition the mixture was stirred at this temperature for 2 h. The reaction was quenched by addition of 5 mL H2O2(30% in water). A saturated aqueous solution of NaHCO3was added and the mixture extracted with CH2Cl2. The organic phase was dried over Na2SO4and the solvent removed under reduced pressure.

[0962] Purification via flash chromatography (cyclohexane / EtOAc 10:1— >1:1) yielded V2 as colorless oil (10.9 g, 87%).

[0963] c) Synthesis of - (Z)-8-oxocyclooct-2-en-1-yl acetate (V3)

[0964]

[0965] M / 65047-PCTVERAXA 97

[0966] To a solution of V2 (9.96 g, 54.1 mmol, 1.00 eq) in CH2CI2 (540 mL) was added pyridinium chlorochromate (17.5 g, 81.1 mmol, 1.50 eq) and the mixture was stirred at rt for 8 h. The reaction mixture was filtered through a plug of silica and the solvent was removed under reduced pressure.

[0967] Purification via flash chromatography (cyclohexane / EtOAc 20:1— >5:1) yielded V3 as colorless oil (7.11 g, 72%).

[0968] d) Synthesis of methyl (Z)-2-(2-acetoxy-1-hydroxycyclooct-3-en-1-yl)acetate (V4)

[0969] O OMe

[0970]

[0971] V4

[0972] To a solution of V3 (7.11 g, 39.0 mmol, 1.00 eq) in CH2Cl2(390 mL) was added BF3·OEt2(9.63 mL, 11.1 g, 78.0 mmol, 2.00 eq) at -78 °C. Afterwards 1-(tert-Butyldimethylsilyloxy)-1-methoxyethene (10.2 mL, 8.82 g, 46.8 mmol, 1.20 eq) was added dropwise and the reaction stirred at this temperature for 1 h. The reaction was quenched by addition of saturated aqueous solution of NaHCOs. The phases were separated, and the aqueous phase was extracted with CH2CI2. The combined organic phases were dried over Na2SO4 and the solvent removed under reduced pressure.

[0973] Purification via flash chromatography (cyclohexane / EtOAc 20:1— >1:1) yielded V4 as a mixture of anti- and syn-isomer (4:1) as colorless oil (8.16 g, 82%).

[0974] e) Synthesis of methyl (E)-2-(2-acetoxy-1-hydroxycyclooct-3-en-1-yl)acetate (V5)

[0975]

[0976] M / 65047-PCTVERAXA 98

[0977] A 2-neck 500 mL flask was connected to the continuous flow photoreactor. A 100 g Biotage SNAP cartridge was equipped with 10% AgNOs silica gel (58.4 g, 34.4 mmol, 1.30 eq) on top of normal silica. The photoisomerization system was equilibrated at a flow of 100 mL / min with the cartridge attached with Et2O / hexane (500 mL) for 20 min. Then V4 (6.78 g, 26.5 mmol, 1.00 eq) dissolved in a minimal amount of Et20 and methyl benzoate (6.67 mL, 7.20 g, 52.9 mmol, 2.00 eq) were added into the 2-neck flask and the system equilibrated for another 20 min while cooling at 0 °C. Afterwards the two 55 W UV-lamps were switched on and the photoisomerization conducted under continuous flow (100 mL / min) for 30 h while cooling at 0 °C. Afterwards the system was flushed with 500 mL Et20 and the cartridge was purged with air. The AgNOs silica was transferred into an Erlenmeyer flask and CH2CI2 and a 25% aqueous solution of NH4OH was added and the mixture stirred vigorously for 5 min.

[0978] The silica was filtered off and the filter cake washed with 25% NH4OH and CH2CI2 several times. The phases were separated, and the aqueous phase was extracted with CH2CI2. The combined organic phases were washed with water, dried over Na2SO4 and the solvent removed under reduced pressure.

[0979] Purification via flash chromatography (cyclohexane / EtOAc 20:1— >3:1) yielded V5 as a separable mixture of anti- and syn-isomer (anti-V5 and syn-V5) as colorless crystalline solid (syn-isomer) and colorless oil (anti-isomer) (3.02 g, 45%).

[0980] f) Synthesis of - (E)-2-(1,2-dihydroxycyclooct-3-en-1-yl)acetic acid (anti-V6, syn-V6)

[0981]

[0982] To a solution of anti-V5 (1.17 g, 4.57 mmol, 1.00 eq) in MeOH (45 mL) was added 1 M NaOHaq (45.7 mL, 45.7 mmol, 10.0 eq) and the mixture was stirred at rt for 1.5 h. It was acidified with 1 M HCIaqand extracted with EtOAc 2 times. The combined organic phases were dried over Na2SO4and the solvent removed under reduced pressure. Coevaporation

[0983] M / 65047-PCTVERAXA

[0984] with toluene was conducted to remove residual AcOH impurities to yield anti-V6 as a colorless crystalline solid (837 mg, 92%).

[0985]

[0986] syn-V6

[0987] To a solution of syn-V5 (150 mg, 585 pmol, 1.00 eq) in MeOH (5.85 mL) was added 1 M NaOHaq (5.85 mL, 5.85 mmol, 10.0 eq) and the mixture was stirred at rt for 1 h. It was acidified with 1 M HClaqand extracted with EtOAc 2 times. The combined organic phases were dried over Na2SO4and the solvent removed under reduced pressure. Coevaporation with toluene was conducted to remove residual AcOH impurities to yield syn-V6 as a colorless crystalline solid (79.0 mg, 67%).

[0988] g) Synthesis of 2,5-dioxopyrrolidin-1-yl (E)-2-(1,2-dihydroxycyclooct-3-en-1-yl)acetate (6)

[0989] OH HO O

[0990] O

[0991]

[0992] To a solution of anti-V6 (1.20 g, 5.99 mmol, 1.00 eq) in DMF (8 mL) was added N, N, N', N'-tetramethyl-0-(N-succinimidyl)uronium tetrafluoroborate (2.17 g, 7.19 mmol, 1.20 eq) and DIPEA (1.3 mL, 968 mg, 7.49 mmol, 1.25 eq) and the reaction stirred at rt for 1 h. The reaction was quenched with H2O, extracted with CH2Cl2. The combined organic phases were dried over Na2SO4and the solvent removed under reduced pressure.

[0993] Purification via flash chromatography (cyclohexane / EtOAc 9:1— >1:1) yielded 6 as colorless solid (1.34 g, 75%).

[0994] M / 65047-PCTVERAXA 100

[0995] EXAMPLE 7 - Synthesis of 2,5-dioxopyrrolidin-1-yl (E)-2-(1-hydroxy-2-methoxycyclooct-3-en-1 -yl)acetate (7)

[0996] The compound was produced as previously described in PCT / EP2024 / 07114.

[0997] a) Synthesis of methyl (E)-2-(2-acetoxy-1-hydroxycyclooct-3-en-1-yl)acetate (anti-V5) Anti-V5 was prepared as described above in Example 6.

[0998] b) Synthesis of methyl 2-((1S, E)-1,2-dihydroxycyclooct-3-en-1-yl)acetate (V26):

[0999] O

[1000] OH

[1001]

[1002] To a solution of compound anti-V5 as prepared above, (4.78 g, 1.0 eq, 18.7 mmol) in methanol (15.0 mL) was added sodium methanolate (806 mg, 853 μL, 0.200 eq, 3.7 mmol) at 25 °C and the reaction mixture was stirred for 1 hour. The reaction mixture was diluted with CH2CI2 and washed with brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure to yield V26 (3.88 g, 18.1 mmol, 97%) as yellow oil.

[1003] c) Synthesis of methyl (E)-2-(1-hydroxy-2-methoxycyclooct-3-en-1-yl)acetate (V27):

[1004]

[1005] V27

[1006] To a solution of V26 (1.52 g, 1.0 eq, 7.1 mmol) in DMF (5.0 mL) was added iodomethane

[1007] M / 65047-PCTVERAXA 101

[1008] (5.02 g, 5.0 eq, 35.4 mmol) under N2atmosphere. The reaction mixture was cooled to 0 °C. A suspension of sodium hydride (311 mg, 1.1 eq, 7.8 mmol) in DMF (50.0 mL) was slowly added to the reaction mixture and left stirring at room temperature for 3 h. The reaction mixture was quenched with sat. NH4CI. The organic layer was extracted with ethyl acetate, dried over Na2SO4and concentrated under reduced pressure. The crude was purified by column chromatography on silica gel (ethyl acetate in cyclohexane: 2-20%). Fractions of product were combined and concentrated to yield V27 (0.77 g, 3.39 mmol, 48%) as colorless oil.

[1009] d) Synthesis of (E)-2-(1-hydroxy-2-methoxycyclooct-3-en-1-yl)acetic acid (V28):

[1010] O OH^-OH

[1011] I / / — ^OMe

[1012]

[1013] V28

[1014] To a solution of V27 (0.77 g, 1.0 eq, 3.4 mmol) in methanol (10.0 mL) was added 1 M NaOHaq (1.36 g, 10.0 eq, 33.9 mL, 33.9 mmol) and the reaction mixture was left stirring at room temperature for 2 h. Afterwards 1 M HCI solution was added until the mixture became acidic. The organic layer was extracted with ethyl acetate, dried over Na2SO4and concentrated under reduced pressure to yield V28 (0.72 g, 3.35 mmol, 99%) as colorless oil.

[1015] e) Synthesis of 2,5-dioxopyrrolidin-1-yl (E)-2-(1-hydroxy-2-methoxycyclooct-3-en-1-yl)acetate (7):

[1016]

[1017] 7

[1018] To a solution of V28 (0.72 g, 1.0 eq, 3.3 mmol) and 2-(2,5-dioxopyrrolidin-1-yl)-1, 1,3,3-tetramethylisouronium tetrafluoroborate (1.21 g, 1.2 eq, 4.0 mmol) in DMF (8.0 mL) was

[1019] M / 65047-PCTVERAXA 102

[1020] added DIPEA (541 mg, 1.3 eq, 720 pL, 4.2 mmol) and the reaction mixture was left stirring at room temperature for 2 h.

[1021] The reaction mixture was diluted with ethyl acetate and washed with brine. The organic layer was dried over Na2SO4and concentrated under reduced pressure. The crude was purified by column chromatography on silica gel (ethyl acetate in cyclohexane: 5-100%). Fractions of product were combined, concentrated and triturated with diethyl ether to get rid of remaining impurities. Product was dried in vacuo to yield 7 (0.88 g, 2.83 mmol, 85%) as white powder.

[1022] EXAMPLE 8 - Synthesis of 2,5-dioxopyrrolidin-1-yl (E)-2-(1,2-dimethoxycyclooct-3-en-1-yl)acetate (8)

[1023] The compound was produced as previously described in PCT / EP2024 / 07114.

[1024] a) Synthesis of (E)-2-(1-hydroxy-2-methoxycyclooct-3-en-1-yl)acetic acid (V28):

[1025] OMe

[1026]

[1027] V28

[1028] Compound V28 was prepared as described above in Example 7.

[1029] b) Synthesis of methyl (E)-2-(1,2-dimethoxycyclooct-3-en-1-yl)acetate (V30):

[1030]

[1031] OMe

[1032] V30

[1033] To a solution of V28 (100 mg, 1.0 eq, 471 pmol) in DMF (2.0 mL) was added iodomethane

[1034] M / 65047-PCTVERAXA 103

[1035] (335 mg, 5.0 eq, 147 pL, 2.4 mmol) under N2 atmosphere. This mixture was added dropwise to a cooled suspension of sodium hydride (226 mg, 12.0 eq, 5.7 mmol) in DMF (3.0 mL) at 0 °C under N2atmosphere. The reaction mixture was left stirring at room temperature for 3 h. The reaction mixture was quenched with sat. NH4CI. The organic layer was extracted with diethyl ether, dried over Na2SO4and concentrated under reduced pressure. The crude was purified by column chromatography on silica gel (ethyl acetate in cyclohexane: 5-60%). Fractions of product were combined and concentrated to yield V30 (5.3 mg, 218 pmol, 46%) as colorless oil.

[1036] c) Synthesis of (E)-2-(1,2-dimethoxycyclooct-3-en-1-yl)acetic acid (V31):

[1037]

[1038] V31

[1039] To a solution of V30 (70.0 mg, 1.0 eq, 289 pmol) in methanol (3.0 mL) was added 1 M NaOHaq (116 mg, 10.0 eq, 2.89 mL, 2.89 mmol) and the reaction mixture was left stirring at room temperature for 2 h. Afterwards 1 M HCI solution was added until the mixture became acidic. The organic layer was extracted with ethyl acetate, dried over Na2SO4and concentrated under reduced pressure to yield V31 (49.2 mg, 216 pmol, 75%) as colorless oil.

[1040] d) Synthesis of 2,5-dioxopyrrolidin-1-yl (E)-2-(1,2-dimethoxycyclooct-3-en-1-yl)acetate (8):

[1041] M / 65047-PCTVERAXA

[1042] 104

[1043]

[1044] To a solution of V31 (48.0 mg, 1.0 eq, 0.2 mmol) and 2-(2,5-dioxopyrrolidin-1 -yl)-1,1,3,3-tetramethylisouronium tetrafluoroborate (76.0 mg, 1.2 eq, 0.3 mmol) in DMF (2.0 mL) was added DI PEA (34.0 mg, 1.3 eq, 45.0 pL, 0.3 mmol) and the reaction mixture was left stirring at room temperature for 3 h. The reaction mixture was diluted with ethyl acetate and washed with brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude was purified by column chromatography on silica gel (ethyl acetate in cyclohexane: 5-100%). Fractions of product were combined, concentrated and trituarted with diethyl ether to get rid of remaining impurities. Product was dried in vacuo to yield 8 (34.2 mg, 0.1 mmol, 50%) as white powder.

[1045] EXAMPLE 9 - Synthesis of (2R,4S,5R,6S)-5-acetamido-2-(((((2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1 (2H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-6-((1R,2R)-3-(2-((E)-1,2-dihydroxycyclooct-3-en-1 -yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxytetrahydro-2H-pyran-2-carboxylic acid (9)

[1046]

[1047] M / 65047-PCTVERAXA 105

[1048] Compound 5 (4.00 mg, 6.53 pmol, 1.00 eq) was dissolved in H2O (200 pL). A solution of 6 (5.00 mg, 16.8 pmol, 2.58 eq) in DMF (400 pL) and DIPEA (2.50 pL, 15.0 pmol, 2.20 eq) were added. The reaction was incubated at 37 °C, shaking at 300 rpm for 1.5 h.

[1049] Purification via preparative HPLC (Amide column) yielded 9 as colorless solid (1 mg, 19%).

[1050] EXAMPLE 10 - Synthesis of (2R,4S,5R,6S)-5-acetamido-2-(((((2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-6-((1R,2R)-1,2-dihydroxy-3-(2-((E)-1-hydroxy-2-methoxycyclooct-3-en-1-yl)acetamido)propyl)-4-hydroxytetrahydro-2H-pyran-2-carboxylic acid (10)

[1051]

[1052] Compound 5 (4.00 mg, 6.53 pmol, 1.00 eq) was dissolved in H2O (200 pL). A solution of 7 (3.00 mg, 9.65 pmol, 1.48 eq) in DMF (200 pL) and DIPEA (2.50 pL, 15.0 pmol, 2.20 eq) were added. The reaction was incubated at 37 °C, shaking at 300 rpm for 30 min. Another solution of 7 (3.00 mg, 9.65 pmol, 1.48 eq) in DMF (200 pL) and DIPEA (2.50 pL, 15.0 pmol, 2.20 eq) were added, and the reaction was incubated at 37 °C, shaking at 300 rpm for another 30 min.

[1053] Purification via preparative HPLC (Amide column) yielded 10 as colorless solid (1 mg, 19%).

[1054] M / 65047-PCTVERAXA 106

[1055] EXAMPLE 11 - Synthesis of (2R,4S,5R,6S)-5-acetamido-2-(((((2R,3S,4R,5R)-5-(4-amino- 2-oxopyrimidin-1(2H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-6-((1R,2R)-3-(2-((E)-1,2-dimethoxycyclooct-3-en- 1 -yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxytetrahydro-2H-pyran-2-carboxylic acid (11)

[1056]

[1057] Compound 5 (4.00 mg, 6.53 pmol, 1.00 eq) was dissolved in H2O (200 pL). A solution of 8 (3.00 mg, 9.20 pmol, 1.41 eq) in DMF (200 pL) and DIPEA (2.50 pL, 15.0 pmol, 2.20 eq) were added. The reaction was incubated at 37 °C, shaking at 300 rpm for 30 min. Another solution of 8 (3.00 mg, 9.20 pmol, 1.41 eq) in DMF (200 pL) and DIPEA (2.50 pL, 15.0 pmol, 2.20 eq) were added, and the reaction was incubated at 37 °C, shaking at 300 rpm for another 30 min.

[1058] Purification via preparative HPLC (Amide column) yielded 11 as colorless solid (1 mg, 19%).

[1059] EXAMPLE 12 - Synthesis of 6-(4-((5S,8S,11 S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1 R,2 R)-3-(((1 S,2R)-1 -hydroxy-1 -phenylpropan-2-yl)amino)-1 -methoxy-2-methyl-3-oxopropyl)pyrrolidin-1 -yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-2-(3-(4-(6-(phosphonomethyl)-1,2,4,5-tetrazin-3-yl)benzamido)propanamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (12)

[1060] M / 65047-PCTVERAXA 107

[1061] OH

[1062]

[1063] 12

[1064] To a solution of ((6-(4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)phenyl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonic acid (27.8 mg, 2 eq, 70.8 pmol) (prepared in analogy to the description in WO2023 / 104941. Reference is made to Example 12 and compound 12 described therein) in DMF (1.00 mL) was added triethylamine (10.7 mg, 14.8 pL, 3 eq, 106 pmol) and Glucuronide-MMAE (40.0 mg, 1 eq, 35.4 pmol) (prepared in analogy to the description in WO2023 / 104941. Reference is made to Example 44 and compound 44 described therein) and the mixture was stirred at rt over night.

[1065] Purification via preparative HPLC (C18 column) yielded 12 (30.0 mg, 60%).

[1066] EXAMPLE 13a - Synthesis of (2S,3S,4S,5R,6S)-6-(4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1 R,2 R)-3-(((1 S,2R)-1 -hydroxy-1 -phenylpropan-2-yl)amino)-1 -methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-2-(3-(2-((6-(6-((diethoxyphosphoryl)methyl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)acetamido)propanamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (13a)

[1067] M / 65047-PCTVERAXA

[1068] 108

[1069]

[1070] 13a

[1071] To a solution of (6-(6-((diethoxyphosphoryl)methyl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)glycine (16.9 mg, 1 eq, 44.2 pmol) (prepared in analogy to the description in WO2023 / 104941. Reference is made to Example 38 and compound 38 described therein) in DMF (0.50 mL) was added HATU (16.8 mg, 1 eq, 44.2 pmol) and DIPEA (11.4 mg, 15.2 pL, 2 eq, 88.5 pmol) and the mixture stirred for 5 h at rt. Afterwards, Glucuronide-MMAE (50.0 mg, 1 eq, 44.2 pmol) (prepared in analogy to the description in WO2023 / 104941. Reference is made to Example 44 and compound 44 described therein) was added and the mixture was stirred at rt over night.

[1072] Purification via preparative HPLC (C18 column) yielded 13a (34.3 mg, 52%).

[1073] EXAMPLE 13 - Synthesis of 6-(4-((5S,8S,11 S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1 R,2R)-3-(((1 S,2R)-1 -hydroxy-1 -phenylpropan-2-yl)amino)-1 -methoxy-2-methyl-3-oxopropyl)pyrrolidin-1 -yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-2-(3-(2-((6-(6-(phosphonomethyl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)acetamido)propanamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (13)

[1074] M / 65047-PCTVERAXA

[1075] 109

[1076]

[1077] To a solution of 13a (16.9 mg, 1 Eq, 44.2 pmol) in in N, N-Dimethylformamide (0.50 mL) was added bromotrimethylsilane (35.2 mg, 30.2 pL, 10 Eq, 230 pmol) and the mixture stirred at rt for 3 d.

[1078] Purification via preparative HPLC (C18 column) yielded 13 (4.0 mg, 12%).

[1079] EXAMPLE 14 - Synthesis of 6-(4-((5S,8S,11 S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1 R,2R)-3-(((1 S,2R)-1 -hydroxy-1 -phenylpropan-2-yl)amino)-1 -methoxy-2-methyl-3-oxopropyl)pyrrolidin-1 -yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-2-(3-(3-(6-(4-phosphonophenyl)-1,2,4,5-tetrazin-3-yl)propanamido)propanamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (14)

[1080] M / 65047-PCTVERAXA

[1081] 110

[1082]

[1083] This compound was prepared in analogy to the description in WO2023 / 104941. Reference is made to Example 65 and compound 65 described therein

[1084] EXAMPLE 15 - Synthesis of 6-(4-((5S,8S,11 S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1 R,2R)-3-(((1 S,2R)-1 -hydroxy-1 -phenylpropan-2-yl)amino)-1 -methoxy-2-methyl-3-oxopropyl)pyrrolidin-1 -yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-2-(3-(3-(6-(phosphonomethyl)-1,2,4,5-tetrazin-3-yl)propanamido)propanamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (15)

[1085] M / 65047-PCTVERAXA

[1086] 111

[1087]

[1088] To a solution of 2,5-dioxopyrrolidin-1 -yl 3-(6-((diethoxyphosphoryl)methyl)-1,2,4,5-tetrazin-3-yl)propanoate (20.0 mg, 1.6 Eq, 49.8 pmol) (prepared in analogy to the description in WO2023 / 104941. Reference is made to Example 69 and compound 69 described therein) in Acetonitrile (1.00 mL), was added bromotrimethylsilane (153 mg, 131 pL, 20 Eq, 997 pmol) and the mixture stirred for 2 h at rt. The product was directly isolated via HPLC and product containing fractions were combined. Then, Glucuronide-MMAE (35.0 mg, 1 eq, 31.0 pmol) (prepared in analogy to the description in WO2023 / 104941. Reference is made to Example 44 and compound 44 described therein) and triethylamine (21.8 mg, 30.0 pL, 6.95 Eq, 215 pmol) were added immediately and the mixture stirred for 1 h at rt.

[1089] Purification via preparative HPLC (C18 column) yielded 15 (3.1 mg, 7%).

[1090] EXAMPLE 16a - Synthesis of 6,6'-((((10-((tert-butoxycarbonyl)amino)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecanedioyl)bis(azanediyl))bis(4-(((((1 S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1 H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-2,1-phenylene))bis(oxy))bis(3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid) (16a)

[1091] M / 65047-PCTVERAXA

[1092] 112

[1093]

[1094] 16a

[1095] To a solution of 6-(2-(3-aminopropanamido)-4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1 H,12H benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (279 mg, 2.2 Eq, 330 pmol) and triethylamine (45.5 mg, 62.6 pL, 3 Eq, 449 pmol)in THF (25.0 mL) was added a solution of 2- (t-butoxycarbonylamido)-1,3-bis(PFP-oxycarbonylethoxy)propane (100 mg, 1 Eq, 150 pmol) in N, N-Dimethylformamide (5.00 mL) dropwise. The mixture was stirred at rt over night.

[1096] Purification via reverse phase flash chromatography (10-100% MeCN in H2O) yielded 16a (250 mg, 84%).

[1097] EXAMPLE 16b - Synthesis of 6,6'-((((10-amino-5,15-dioxo-8,12-dioxa-4,16-diazanonadecanedioyl)bis(azanediyl))bis(4-(((((1 S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1 H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-2,1-phenylene))bis(oxy))bis(3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid) (16b)

[1098] M / 65047-PCTVERAXA

[1099] 113

[1100]

[1101] 16b

[1102] To a solution of 16a (279 mg, 2.2 Eq, 330 pmol) in DCM (0.50 mL) was added 4 N HCI in dioxane (0.50 mL). The mixture was stirred at rt for 5 h.

[1103] Purification via reverse phase flash chromatography (10-100% MeCN in H2O) yielded 16a (150 mg, 63%).

[1104] EXAMPLE 16 - Synthesis of 6,6'-((((5,15-dioxo-10-(4-(6-(phosphonomethyl)-1,2,4,5-tetrazin-3-yl)benzamido)-8,12-dioxa-4,16-diazanonadecanedioyl)bis(azanediyl))bis(4-(((((1 S,9S)-9-ethyl-5-f luoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1 H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-2,1 -phenylene))bis(oxy))bis(3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid) (16)

[1105] M / 65047-PCTVERAXA

[1106] 114

[1107]

[1108] To a solution of ((6-(4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)phenyl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonic acid (31.1 mg, 2 Eq, 79.2 pmol) (prepared in analogy to the description in WO2023 / 104941. Reference is made to Example 12 and compound 12 described therein) and triethylamin (8.01 mg, 11.0 pL, 2 Eq, 79.2 pmol) in DMF (0.50 mL) was added 16b (75.0 mg, 1 Eq, 39.6 pmol) and the mixture was stirred at rt over night. Purification via reverse phase flash chromatography (2-100% MeCN in H2O) yielded 16 (13.5 mg, 16%).

[1109] EXAMPLE 17 - Synthesis of (2R,4S,5R,6S)-5-acetamido-2-carboxy-6-((1 R,2R)-3-(((((E)-cyclooct-2-en-1-yl)oxy)carbonyl)amino)-1,2-dihydroxypropyl)-4-hydroxytetrahydro-2H-pyran-2-yl (((2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1 (2H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl) phosphate 17)

[1110]

[1111] M / 65047-PCTVERAXA 115

[1112] To a solution of 5 (100 mg, 0.163 mmol) in 1 mL H2O was added (E)-cyclooct-2-en-1-yl (2,5- dioxopyrrolidin-1 -yl) carbonate (113.5 mg, 0.425 mmol) in 2 mL DMF under nitrogen atmosphere. Afterwards DIPEA (46.34 mg, 0.359 mmol) was added to the reaction, and the resulting mixture was stirred at 25eC for 1.5 h. Upon completion, the reaction solution was filtered, and the filtrate was purified by HILIC-HPLC Prep-column (gradient 95%^55% ACN / 10 mmol NH4OAC in H2O over 13.5 min, flow rate: 25 mL / min). The product 17 (54 mg, 44%) was obtained as a white solid.

[1113] EXAMPLE 18 - Synthesis of (2R,4S,5R,6S)-5-acetamido-2-carboxy-6-((1 R,2R)-3-(((((E)- cyclooct-4-en-1-yl)oxy)carbonyl)amino)-1,2-dihydroxypropyl)-4-hydroxytetrahydro-2H- pyran-2-yl (((2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3,4- dihydroxytetrahydrofuran-2-yl)methyl) phosphate (18)

[1114]

[1115] To a solution of 5 (100 mg, 0.163 mmol) in 1 mL H2O was added (E)-cyclooct-4-en-1-yl (2,5- dioxopyrrolidin-1 -yl) carbonate (113.5 mg, 0.425 mmol) in 2 mL DMF under nitrogen atmosphere. Afterwards DIEPA (46.34 mg, 0.359 mmol) was added and the reaction was stirred at 25eC for 1.5 h. Upon completion, the reaction solution was filtered, and the resulting filtrate was purified by HILIC-HPLC Prep column (gradient 95%^60% MeCN / 10 mmol HCOONH4 in H2O over 19 min, flow rate: 30 mL / min). The product 18 was obtained (20 mg, 16%) as a white solid.

[1116]

[1117] of ADCs /

[1118] M / 65047-PCTVERAXA 116

[1119] PREPARATION EXAMPLE 1 - Preparation of Trastuzumab-(N297-p-AZ-GlcNAc- p-(1,4)-Gal)2(17)

[1120] Trastuzumab SEQ ID NO: 1 (for HC) and SEQ ID NO: 2 (for LC) (10 mg / mL; 100 mg) was incubated with EndoS2 (0.01 U / pg IgG), B4GalT1 (0.05 mg / mL; 0.5 mg), alkaline phosphatase (10 U / mL), UDP-Gal (2 mM; 20 mmol), and MnCl2(1 mM) in 25 mM Tris-HCI, pH 7.4, 150 mM NaCI (total volume: 10 mL) at 37°C, 250 rpm for 26 h. Afterwards, another portion of UDP-Gal (10 mmol) was added and the mixture was incubated at 37 °C, 250 rpm for another 21 h. Another portion of B4GalT1 (0.25 mg) and UDP-Gal (5 mmol) was added and the mixture was incubated at 37°C, 150 rpm for 14 h, after which another portion of UDP-Gal (5 mmol) was added, and the incubation was continued for further 20 h.

[1121] Purification via affinity chromatography (Cytiva HiTrap MabSelect SuRe column) and buffer exchange into 50 mM Tris-HCI, pH 8.0, yielded IgG 17 (78 mg, 78%).

[1122] PREPARATION EXAMPLE 2 - Preparation of Trastuzumab-(N297-p-AZ-GlcNAc-p-(1,4)-Gal-a-(2,6)-(9-deoxy-9-(2-(1,2-dihydroxycyclooct-3-en-1 -yl)acetamido)-Neu5Ac))2(18)

[1123] IgG 17 (10 mg / mL; 0.54 mg) was incubated with ST6Gal1 (0.1 mg / mL) and 9 (2 mM) in 100 mM Tris-HCI, pH 8.0 (total volume: 55 pL) at 37 °C, shaking at 300 rpm for 55 h.

[1124] Purification via affinity chromatography (Cytiva HiTrap Fibro PrismA column) and buffer exchange into PBS, pH 7.4, yielded IgG 18 (0.32 mg, 59%).

[1125] PREPARATION EXAMPLE 3 - Preparation of Trastuzumab-(N297-p-AZ-GlcNAc-p-(1,4)-Gal-a-(2,6)-(9-deoxy-9-(2-(1-hydroxy-2-methoxycyclooct-3-en-1-yl)acetamido)-Neu5Ac))2(19)

[1126] IgG 17 (10 mg / mL; 0.54 mg) was incubated with ST6Gal1 (0.1 mg / mL) and 10 (2 mM) in 100 mM Tris-HCI, pH 8.0 (total volume: 55 pL) at 37 °C, shaking at 300 rpm for 55 h.

[1127] M / 65047-PCTVERAXA 117

[1128] Purification via affinity chromatography (Cytiva HiTrap Fibro PrismA column) and buffer exchange into PBS, pH 7.4, yielded IgG 19 (0.35 mg, 65%).

[1129] PREPARATION EXAMPLE 4 - Preparation of Trastuzumab-(N297-p-AZ-GlcNAc-p-(1,4)-Gal-a-(2,6)-(9-deoxy-9-(2-(1,2-dimethoxycyclooct-3-en-1 -yl)acetamido)-Neu5Ac))2(20)

[1130] IgG 17 (10 mg / mL; 0.54 mg) was incubated with ST6Gal1 (0.1 mg / mL) and 11 (2 mM) in 100 mM Tris-HCI, pH 8.0 (total volume: 55 pL) at 37 °C, shaking at 300 rpm for 55 h.

[1131] Purification via affinity chromatography (Cytiva HiTrap Fibro PrismA column) and buffer exchange into PBS, pH 7.4, yielded IgG 20 (0.38 mg, 70%).

[1132] In the formulae depicted in the subsequent preparation examples for different ADCs of the invention the glycan linker is depicted in symbols

[1133] Square = GIcNac

[1134] Dot = Gal

[1135] Diamond ♦ = Neu5Ac

[1136] An identical glycan linked drug moiety is also present in the second heavy chain of the mAb molecule (but not depicted in said formulae).

[1137] PREPARATION EXAMPLE 5 - Preparation of Trastuzumab-(N297-p-AZ-GlcNAc-p-(1,4)-Gal-a-(2,6)-(9-deoxy-9-(2-(1,2-dihydroxycyclooct-3-en-1-yl)acetamido)-Neu5Ac)-12)2(21)

[1138] M / 65047-PCTVERAXA

[1139] 118

[1140]

[1141] IgG 18 (2 mg / mL; 0.2 mg, 1.3 nmol, 1.00 eq) was incubated with 12 (0.2 mM; 20 nmol, 15.0 eq) in PBS, pH 7.4 (total volume: 100 pL) at 37 °C, shaking at 300 rpm for 1.5 h.

[1142] Purification via size exclusion chromatography (Cytiva Superdex 200 column) yielded ADC 21 (0.16 mg, 80%).

[1143] PREPARATION EXAMPLE 6 - Preparation of Trastuzumab-(N297-p-AZ-GlcNAc-p-(1,4)-Gal-a-(2,6)-(9-deoxy-9-(2-(1,2-dihydroxycyclooct-3-en-1-yl)acetamido)-Neu5Ac)-13)2 (22)

[1144] M / 65047-PCTVERAXA

[1145] 119

[1146]

[1147] IgG 18 (2 mg / mL; 0.2 mg, 1.3 nmol, 1.00 eq) was incubated with 13 (0.2 mM; 20 nmol, 15.0 eq) in PBS, pH 7.4 (total volume: 100 pL) at 37 °C, shaking at 300 rpm for 1.5 h.

[1148] Purification via size exclusion chromatography (Cytiva Superdex 200 column) yielded ADC 22 (0.14 mg, 70%).

[1149] PREPARATION EXAMPLE 7 - Preparation of Trastuzumab-(N297-p-AZ-GlcNAc-p-(1,4)-Gal-a-(2,6)-(9-deoxy-9-(2-(1,2-dihydroxycyclooct-3-en-1-yl)acetamido)-Neu5Ac)-14)2(23)

[1150] M / 65047-PCTVERAXA

[1151] 120

[1152]

[1153] 23 IgG 18 (2 mg / mL; 0.2 mg, 1.3 nmol, 1.00 eq) was incubated with 14 (0.2 mM; 20 nmol, 15.0 eq) in PBS, pH 7.4 (total volume: 100 pL) at 37 °C, shaking at 300 rpm for 1.5 h.

[1154] Purification via size exclusion chromatography (Cytiva Superdex 200 column) yielded ADC 23 (0.15 mg, 75%).

[1155] PREPARATION EXAMPLE 8 - Preparation of Trastuzumab-(N297-p-AZ-GlcNAc-p-(1,4)-Gal-a-(2,6)-(9-deoxy-9-(2-(1,2-dihydroxycyclooct-3-en-1-yl)acetamido)-Neu5Ac)-15)2 (24)

[1156] M / 65047-PCTVERAXA 121

[1157] o

[1158]

[1159] IgG 18 (2 mg / mL; 0.2 mg, 1.3 nmol, 1.00 eq) was incubated with 15 (0.2 mM; 20 nmol, 15.0 eq) in PBS, pH 7.4 (total volume: 100 pL) at 37 °C, shaking at 300 rpm for 1.5 h.

[1160] Purification via size exclusion chromatography (Cytiva Superdex 200 column) yielded ADC 24 (0.16 mg, 80%).

[1161] PREPARATION EXAMPLE 9 - Preparation of Trastuzumab-(N297-p-AZ-GlcNAc-p-(1,4)-Gal-a-(2,6)-(9-deoxy-9-(2-(1-hydroxy-2-methoxycyclooct-3-en-1-yl)acetamido)-Neu5Ac)-12)2(25)

[1162] M / 65047-PCTVERAXA 122

[1163]

[1164] ’5

[1165] IgG 19 (2 mg / mL; 0.2 mg, 1.3 nmol, 1.00 eq) was incubated with 12 (0.2 mM; 20 nmol, 15.0 eq) in PBS, pH 7.4 (total volume: 100 pL) at 37 °C, shaking at 300 rpm for 1.5 h.

[1166] Purification via size exclusion chromatography (Cytiva Superdex 200 column) yielded ADC 25 (0.15 mg, 75%).

[1167] PREPARATION EXAMPLE 10 - Preparation of Trastuzumab-(N297-p-AZ-GlcNAc-p-(1,4)-Gal-a-(2,6)-(9-deoxy-9-(2-(1-hydroxy-2-methoxycyclooct-3-en-1-yl)acetamido)-Neu5Ac)-16)2(26)

[1168] M / 65047-PCTVERAXA 123

[1169]

[1170] 26

[1171] IgG 19 (2 mg / mL; 0.2 mg, 1.3 nmol, 1.00 eq) was incubated with 16 (0.2 mM; 20 nmol, 15.0 eq) in PBS, pH 7.4 (total volume: 100 pL) at 37 °C, shaking at 300 rpm for 1.5 h.

[1172] Purification via size exclusion chromatography (Cytiva Superdex 200 column) yielded ADC 26 (0.12 mg, 60%).

[1173] PREPARATION EXAMPLE 11 - Preparation of Trastuzumab-(N297-p-AZ-GlcNAc-p-(1,4)-Gal-a-(2,6)-(9-deoxy-9-(2-(1,2-dihydroxycyclooct-3-en-1-yl)acetamido)-Neu5Ac)-Cy5)2 (27)

[1174] M / 65047-PCTVERAXA

[1175] 124

[1176]

[1177] IgG 18 (2.9 mg / mL; 0.29 mg, 1.93 nmol, 1.00 eq) was incubated with Cy5-HTet (0.1 mM; 10 nmol, 5.18 eq) in PBS, pH 7.4 (total volume: 100 pL) at 25 °C for 1 h.

[1178] Purification via size exclusion chromatography (Cytiva Superdex 200 column) yielded ADC 27 (0.13 mg, 45%).

[1179] PREPARATION EXAMPLE 12 - Preparation of Trastuzumab-(N297-p-AZ-GlcNAc-p-(1,4)-Gal-a-(2,6)-(9-deoxy-9-(2-(1-hydroxy-2-methoxycyclooct-3-en-1-yl)acetamido)-Neu5Ac)-Cy5)2(28)

[1180]

[1181] M / 65047-PCTVERAXA 125

[1182] IgG 19 (3.0 mg / mL; 0.30 mg, 2.00 nmol, 1.00 eq) was incubated with Cy5-HTet (0.1 mM; 10 nmol, 5.00 eq) in PBS, pH 7.4 (total volume: 100 pL) at 25 °C for 1 h.

[1183] Purification via size exclusion chromatography (Cytiva Superdex 200 column) yielded ADC 28 (0.23 mg, 77%).

[1184] PREPARATION EXAMPLE 13 - Preparation of Trastuzumab-(N297-p-AZ-GlcNAc-p-(1,4)-Gal-a-(2,6)-(9-deoxy-9-(2-(1,2-dimethoxycyclooct-3-en-1-yl)acetamido)-Neu5Ac)-Cy5)2 (29)

[1185]

[1186] IgG 20 (3.2 mg / mL; 0.32 mg, 2.13 nmol, 1.00 eq) was incubated with Cy5-HTet (0.1 mM; 10 nmol, 4.69 eq) in PBS, pH 7.4 (total volume: 100 pL) at 25 °C for 1 h.

[1187] Purification via size exclusion chromatography (Cytiva Superdex 200 column) yielded ADC 29 (0.24 mg, 75%).

[1188] PREPARATION EXAMPLE 14 - Preparation of FLYSYN-(N297-p- GlcNAc)2(30)

[1189] To prepare a FLYSYN stock, mAb FLYSYN (Syn Immune) was buffer-exchanged into PBS, pH 7.4.

[1190] M / 65047-PCTVERAXA 126

[1191] mAb FLYSYN has the following amino acid sequences for HC and LC:

[1192] mAb FLYSYN HC: SEQ ID NO: 3

[1193] 1 QVQLQQPGAE LVKPGASLKL SCKSSGYTFT SYWMHWVRQR PGHGLEWIGE

[1194] 51 IDPSDSYKDY NQKFKDKATL TVDRSSNTAY MHLSSLTSDD SAVYYCARAI

[1195] 101 TTTPFDFWGQ GTTLTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY

[1196] 151 FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI

[1197] 201 CNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPD VFLFPPKPKD

[1198] 251 TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST

[1199] 301 YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APEEKTISKA KGQPREPQVY

[1200] 351 TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD

[1201] 401 SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGKSG

[1202] 451 KPTHVNVSVV MAEEQKLISE EDLLR

[1203] Potential glycosylated Asn residues are highlighted in bold letters mAb FLYSYN LC: SEQ ID NO: 4

[1204] 1 DIVLTQSPAT LSVTPGDSVS LSCRASQSIS NNLHWYQQKS HESPRLLIKY

[1205] 51 ASQSISGIPS RFSGSGSGTD FTLSINSVET EDFGVYFCQQ SNTWPYTFGG

[1206] 101 GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV

[1207] 151 DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG

[1208] 201 LSSPVTKSFN RGEC

[1209] FLYSYN (8.5 mg / mL; 8.5 mg) was deglycosylated employing immobilized EndoS2 (GlycINATOR microcolumn, Genovis) and converted according to the user manual.

[1210] Buffer exchange into 50 mM Tris-HCI, pH 8.0, yielded IgG 30 (6 mg, 71%).

[1211] PREPARATION EXAMPLE 15 - Preparation of FLYSYN-(N297-p-AZ-GlcNAc-p-(1,4)-Gal)2(31)

[1212] IgG 30 (10 mg / mL; 2 mg) was incubated with B4GalT1 (0.05 mg / mL; 0.01 mg), UDP-Gal (0.5 mM; 0.1 mmol), and MnCI2(10 mM) in 25 mM Tris-HCI, pH 8.0 (total volume: 200 pL) at 37°C, 300 rpm (thermomixer) for 15 h. To ensure a complete galactosylation, another portion of B4GalT1 (0.01 mg) and UDP-Gal (0.1 mmol) was added, and the mixture was incubated at 37 °C, 300 rpm (thermomixer) for another 72 h.

[1213] Purification via affinity chromatography (Cytiva HiTrap Fibro PrismA column) and buffer exchange into 50 mM Tris-HCI, pH 8.0, yielded IgG 31 (0.8 mg, 40%).

[1214] M / 65047-PCTVERAXA 127

[1215] PREPARATION EXAMPLE 16 - Preparation of FLYSYN-(N297-p- GlcNAc-p-(1,4)-Gal-a-(2,6)-(9-deoxy-9-(2-(1,2-dihydroxycyclooct-3-en-1 -yl)acetamido)-Neu5Ac))2(32)

[1216] IgG 31 (6 mg / mL; 0.59 mg) was incubated with ST6Gal1 (0.1 mg / mL) and 9 (2 mM) in 100 mM Tris-HCI, pH 8.0 (total volume: 58 pL) at 37 °C, 300 rpm (thermomixer) for 18 h. Purification via affinity chromatography (Cytiva HiTrap Fibro PrismA column) and buffer exchange into PBS, pH 7.4, yielded IgG 32 (0.27 mg, 46%).

[1217] PREPARATION EXAMPLE 17 - Preparation of FLYSYN-(N297-p- GlcNAc-p-(1,4)-Gal-a-(2,6)-(9-deoxy-9-(2-(1,2-dihydroxycyclooct-3-en-1 -yl)acetamido)-Neu5Ac)-12)2(33)

[1218]

[1219] 33

[1220] IgG 32 (2 mg / mL; 0.2 mg, 1.3 nmol, 1.00 eq) was incubated with 12 (0.4 mM; 40 nmol, 30.0 eq) in PBS, pH 7.4 (total volume: 100 pL) at 37 °C, 300 rpm (thermomixer) for 1.5 h. Purification via size exclusion chromatography (Cytiva Superdex 200 column) yielded ADC 33 (0.17 mg, 85%).

[1221] M / 65047-PCTVERAXA 128

[1222] PREPARATION EXAMPLE 18 - Preparation of 20D9-(N297-p-AZ-GlcNAc-p-(1,4)-Gal)2(34)

[1223] To prepare a 20D9 stock, mAb 20D9 (Proteogenix) was buffer-exchanged into 50 mM Tris-HCI, pH 7.4.

[1224] mAb 20D9 has the following amino acid sequences for HC and LC

[1225] mAb 20D9 HC: SEQ ID NO: 5 QVQLVESGGGVVQPGRSLRLSCAASGFTFNNYWMTWVRQAPGKGLEWIASITKTGGGTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCTTLQQLGVMDAWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1226] Potential glycosylated Asn residue is highlighted in bold letter.

[1227] mAb 20D9 LC: SEQ ID NO: 6 DIQMTQSPSVLSASVGDRVTINCRASQNINKELNWYQQKLGEAPKLLIYNTNNLQSGVPSRFSGSGSG TDYTLTISSLQPEDVATYFCFQHKSWPLTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC

[1228] was buffer-exchanged into 50 mM Tris-HCI, pH 7.4.

[1229] 20D9 (9 mg / mL; 0.92 mg) was incubated with EndoS2 (0.1 U / pg IgG), B4GalT1 (0.1 mg / mL; 0.01 mg), UDP-Gal (2 mM; 0.2 mmol), and MnCI2(10 mM) in 25 mM Tris-HCI, pH 7.4, 150 mM NaCI (total volume: 100 pL) at 37°C, 300 rpm (thermomixer) for 21 h. To ensure a complete galactosylation, another portion of B4GalT1 (0.005 mg) and UDP-Gal (0.2 mmol) was added, and the mixture was incubated at 37 °C, 300 rpm (thermomixer) for another 44 h. Removal of the released glycans and excess galactose by spin filtration (Amicon Ultra Centrifugal Unit, 30 kDa MWCO), and buffer exchange into 50 mM Tris-HCI, pH 8.0, yielded IgG 34 (0.61 mg, 66%).

[1230] M / 65047-PCTVERAXA 129

[1231] PREPARATION EXAMPLE 19 - Preparation of 20D9-(N297-p- GlcNAc-p-(1,4)-Gal-a-(2,6)-(9-deoxy-9-(2-(1,2-dihydroxycyclooct-3-en-1 -yl)acetamido)-Neu5Ac))2(35)

[1232] IgG 34 (3 mg / mL; 0.61 mg) was incubated with ST6Gal1 (0.15 mg / mL) and 9 (2 mM) in 100 mM Tris-HCI, pH 8.0 (total volume: 200 pL) at 37 °C, 300 rpm (thermomixer) for 19 h. Purification via affinity chromatography (Cytiva HiTrap Fibro PrismA column) and buffer exchange into PBS, pH 7.4, yielded IgG 35 (0.52 mg, 85%).

[1233] PREPARATION EXAMPLE 20 - Preparation of 20D9-(N297-p- GlcNAc-p-(1,4)-Gal-a-(2,6)-(9-deoxy-9-(2-(1,2-dihydroxycyclooct-3-en-1 -yl)acetamido)-Neu5Ac)-12)2(36)

[1234]

[1235] IgG 35 (2 mg / mL; 0.2 mg, 1.3 nmol, 1.00 eq) was incubated with 12 (0.2 mM; 20 nmol, 15.0 eq) in PBS, pH 7.4 (total volume: 100 pL) at 37 °C, 300 rpm (thermomixer) for 1.5 h. Purification via size exclusion chromatography (Cytiva Superdex 200 column) yielded ADC 36 (0.12 mg, 60%).

[1236] M / 65047-PCTVERAXA 130

[1237] Test Examples

[1238] Test Example 1 -In vivo study (conducted at Charles River Laboratories)

[1239] Female NSG mice were injected with 10 mio MAXHER BT-474 cells in a mixture of Matrigel / PBS (0.2 mL, 1:1) into the subcutaneous tissue.

[1240] BT-474-tumor-bearing animals with a suitable tumor volume of 50-250 mm3were randomized into 8 groups of 6 animals and treated as described in the table below. Tumor volume was measured twice weekly. An ADC of the invention (Compound 26; DAR = 3.94) prepared according to preparation example 10 was compared to a commercial ADC commercialized under the trade name Enhertu ® (Trastuzuman-Deruxtecan, drug attached via thioether linkage to the antibody molecule) (DAR = approx. 8) were compared as regards their anti-tumor effect at equal doses.

[1241] Total daily Dosing

[1242] Group Dosing

[1243] Compound dose volume Route Vehicle n=

[1244] ID days

[1245] [mg / kg / d] [ml / kg]

[1246] 1 Control Vehicle 5 ml / kg 5 QD*1 i.v. PBS 6

[1247] 2 ADC 26 1 5 QD*1 i.v. PBS 6

[1248] 3 ADC 26 3 5 QD*1 i.v. PBS 6

[1249] 4 ADC 26 10 5 QD*1 i.v. PBS 6

[1250] 5 Enhertu 1 5 QD*1 i.v. PBS 6

[1251] 6 Enhertu 3 5 QD*1 i.v. PBS 6

[1252] 7 Enhertu 10 5 QD*1 i.v. PBS 6

[1253]

[1254] QD*1 = single dose on day 1; * = based on last body weight,

[1255] The test results are shown in Figure 2. As can be seen ADC 26 (Trastuzumab-Exatecan) despite of a 50% lower DAR surprisingly performs very similar to Enhertu ® at equal doses over the entire test period.

[1256] Test Example 2 - in vitro cell modification with CMP-sialic acid derivative (9) in presence of neuraminidase

[1257] TROP2 hybridoma cellswere surface modified with the TCO-Neu5Ac-CMP-derivative of above Example 9 (100 pM) by the action of ST6Gal1 (42 pg / mL) in reaction mixture (100 pL)

[1258] M / 65047-PCTVERAXA 131

[1259] using serum free DMEM with 0,66 pL of BSA (2 mg / mL), 15 pL of 3 M sucrose, and Vibrio cholerae (VC) neuraminidase (50 mU / mL) for 2 h.for 2 h.

[1260] After cells were washed with DPBS, they were labeled with Cy5-tetrazine (30 pM) in 2% FBS containing DPBS for 1 h at room temperature and then were analyzed by microscopy. Here, the Cy5 dye is localized via click reation between its tetrazine moiety and the TCO moiety of the sialic acid attached on the cell surface of said TROP2 hybridoma cells.

[1261] The result is shown in Figure 6.

[1262] It was observed that an attachment of a TCO-type substituent of the invention at position 9 of Neu5Ac surprisingly results in a chemical entity which is not a substrate for neuraminidase, because neuraminidase is known to cleave terminal substituted sialic acids from glycans (Zhara Khedri, et al., Chem. Commun., 2012, 48, 3357-3359).

[1263] Hence, the pesent invention allows not only labeling with 9-TCO-Neu5Ac in presence of neuraminidase, but also the more efficient application of such chemical entities for in vivo click reactions.

[1264] M / 65047-PCTVERAXA

[1265] 132

[1266] Listing of Sequences

[1267] This listing is considered part of the general disclosure of the invention SEQ Designation Description / Source Type ID NO

[1268] 1 Trastuzumab IgHC Artrificial sequenc AA 2 Trastuzumab IgLC Artrificial sequenc AA 3 mAb FLYSYN IgHC Artrificial sequenc AA 4 mAb FLYSYN IgLC Artrificial sequenc AA 5 mAb 20D9 IgHC Artrificial sequenc AA 6 mAb 20D9 IgLC Artrificial sequenc AA

[1269]

[1270] Any document as cited herein above is incorporated by reference.

[1271] M / 65047-PCT

Claims

1. Claims1. / V-Acetyl neuraminic acid (Neu5Ac) derivatives of the general formula I:TCO-Neu5Ac-CMP(I)whereinNeu5Ac represents a moiety of the formula lao(la)CMP represents a residue of the formula lblinked via its phosphoryl group to the oxyl group in position 2 of the of Neu5Ac moiety;andTCO represents an optionally substituted mono- or bicyclic trans-cyclooctenyl- type residue linked to the terminal carbon atom in position 9 of the Neu5Ac moiety andrepresents a moiety of the general formula IcX1-X2a- X3- (lc)whereinM / 65047-PCTX1is selected from a residue of the general formula VII to XII:(VIII) (IX) (X)whereinR1, R2and R3may be bound to any ring position except for the carbon atoms of the C=C double bond of the cyclooctene ring, and are independently of each other selected from hydrogen, or substituents selected from halogen, linear or branched C1-C4- alkyl, linear or branched C2-C4-alkenyl, CF3, CN, OH, linear or branched C1-C4-alkoxy, linear or branched C2-C4-alkeneoxy, -O- CF3, linear or branched C2-C5-alkanoyloxy, linear or branched Ci-C4-alkylaminocarbonyloxy, or linear or branched C1-C4- alkylthio, -NR4R4a, carboxy, or linear or branched C1-C4-alkyl carboxylate groups,whereinR4and R4aindependently of each other represent H or linear or branched Ci-C4-alkyl;Cyc represents together with the ring atoms to which it is attached form a three- to six-membered carbocyclic or heterocyclic, particularly carbocyclic, nonaromatic or aromatic, saturated or unsaturated ring, optionally carrying at least one ring substituent, selected from OH, carboxylic acid groups, or linear or branched C1-C4 alkyl carboxylate groups, linear or branched C1-C4-alkoxy groups and keto groups;M / 65047-PCTfrepresents the chemical bond linking the terminal group X1to spacer group X2or to the linking group X3as defined below, and may be linked to any ring position within a X1moiety of any one of the formulae VII to XII except for the carbon atoms of the C=C double bond of the trans-cyclooctene ring; X2is a spacer group selected from lower alkylene, preferably methylene; a is 0 or 1;X3is a linking group selected from -O-, -S-,-NR8-, -N+R8R8a-, -C(O)-NR9-, - NR9-C(O)-, -NH-CH(NH2)-, -CH(NH2)-NH-, -NH-C(NH)-NH-, -NR9-C(O)-O-, -O-C(O)-NR9-, -C(O)-NH-CH(NH2)-, -C(O)-NH-C(NH)-NH-, NH-CH(NH2)- C(O)-, -NH-C(NH)-NH-C(O)-, =N-, =N-O-, =N-NR9;whereinR8, R8aand R9independently of each other represent H or linear or branched Ci-C4-alkyl or linear or branched C2-C4-alkenyl.

2. The Neu5Ac derivative of clam 1, which is a CMP-p-D- / V-acetylneuraminic acid (CMP-Neu5Ac) derivative of the general formula IIIwhereinTCO is as defined above.

3. The Neu5Ac derivative of claim 1 or 2, wherein X1is selected from residues of the general formulae XIII, XIV or XVM / 65047-PCT(XV) whereinR1, R2and R3are as defined above, in particular independently from each other H, OH or C1-C4-alkoxy; andis as defined above.

4. The Neu5Ac derivative of claim 3, wherein X1is selected from a residue of the general formula XVII(XVII)whereinR1and R2independently of each other are OH or C1-C4-alkoxy; andis as defined above.

5. A glycan-engineered immunoglobulin of the general formula XXM / 65047-PCTlg-(GlcNAc-Gal-Neu5Ac-TCO)n(FllC)m(XX)whereinn represents an integer of at least 1, preferably 2;m is 0 or 1Ig represents an immunoglobulin; more particularly a monoclonal antibodyTCO is as defined above,GlcNAc represents an N-acetyl-glucosamin moiety, in particular N- acetyl-B-D-glucosamin;Gal represents a galactosyl moiety, in particular p-D-galactosyl moiety;Neu5Ac represents an / V-acetylneuraminic acid moiety, in particular a-D- acetyl neuraminic acid moiety, andFuc represents a fucosyl moiety, in particular 6-desoxy-L-galactosyl moiety.

6. The glycan-engineered immunoglobulin of claim 5, which is of the general formula (XXI)lg-[GlcNAc(B1-4)-Gal(a2-6)-Neu5Ac-TCO]n(XXI)whereinn, Ig, TCO, GlcNAc, Gal and Neu5Ac are as defined above,and wherein TCO is covalently attached to carbon atom C-9 of Neu5Ac, and wherein GlcNAc in said formula XXI is optionally linked via an (a1-6)- glycosidic linkage to a Fuc moiety as defined above.M / 65047-PCT7. The glycan-engineered immunoglobulin of anyone of the claims 5 and 6, wherein said immunoglobulin is an antibody molecule or Fc fragment of an antibody; particularly, wherein the glycan moiety is N-linked to the side chain of amino acid Asn in position 297, according to Ell numbering of the C-domain of immunoglobulins, of the CH2 domain of the antibody heavy chain Ig-HC or the corresponding Fc fragment thereof.

8. The glycan-engineered immunoglobulin of claim 7, wherein the antibody represents a polyclonal or monoclonal antibody, particularly an IgG antibody.

9. A method of preparing a glycan-engineered immunoglobulin of anyone of the claims 5 to 8, which method comprisesa) the enzyme-catalyzed conversion of a natural glycan side chain of the immunoglobulin molecule carrying at least one glycan side chain, which is attached to the Ig-heavy chain of the molecule via an optionally fucosylated GlucNAc moiety into an optionally fucosylated -GalNAc-Gal side chain, thereby obtaining a first glycan-modified immunoglobulin; andb) enzymatically attaching an / V-acetyl neuraminic acid (Neu5Ac) derivative as defined in anyone of the claims 1 to 5, under conditions allowing the covalent binding of the TCO-Neu5Ac- moiety of said Neu5Ac derivative to the terminal Gal moiety of said first glycan-modified immunoglobulin, thereby obtaining a second glycan-engineered immunoglobulin which is as defined in anyone of the claims 5 to 8;particularly, wherein step a) is performed in the simultaneous presence of an enzyme having endoglycosidase activity, an enzyme having galactosyl transferase activity, and an enzyme having alkaline phosphatase activity, UDP Gal and MnCl in a liquid buffered medium; andstep b) is performed in the simultaneous presence of an enzyme having sialyltransferase activity and an Neu5Ac derivative as defined in anyone of the claims 1 to 4; andmore particularly, whereinsaid enzyme having endoglycosidase activity is selected from EndoS or EndoS2, in particular EndoS2,said enzyme having galactosyl transferase is B4GalT 1; andM / 65047-PCTsaid enzyme having sialyltransferase activity is a beta-galactoside alpha-2, 6- sialyltransf erase ST6Gal1.

10. A conjugate of a glycan-engineered immunoglobulin of anyone of the claims 5 to 8 and a payload molecule PM functionalized with a terminal docking group selected from tetrazines or triazines capable of covalently binding (i.e. being conjugated) in a copper-free strain promoted inverse-electron-demand Diels-Alder cycloaddition SPIEDAC to said TCO group of said glycan-engineered immunoglobulin anyone of the preceding claims 5 to 8.

11. The conjugate of claim 10, which is an antibody payload conjugate (APC), comprising an antibody molecule or Fc-fragment thereof as defined in anyone of the preceding claims 7 and 8, covalently bound via its TCO group in a copper-free strain promoted inverse-electron-demand Diels-Alder cycloaddition SPIEDAC to said payload molecule (P) which is functionalized with a terminal docking group selected from triazines and tetrazines capable of covalently binding to said TCO group.

12. The conjugate of anyone of the claims 10 and 11, wherein the payload molecule is selected from bio-active compounds, labeling agents, protein degraders, in particular payloads applicable in proteolysis targeting chimeras PROTACs, photosensitizers, and chelators.

13. A pharmaceutical composition comprising in a pharmaceutically acceptable carrier at least one conjugate as defied in anyone of the claim 10 to 12, or a diagnostic composition comprising in a diagnostically applicable carrier at least one conjugate as defined in anyone of the claim 10 to 1214. The conjugate as defined in anyone of the claims 10 to 12 for use in medicine, as in particular in diagnosis and / or therapy.in particular,said conjugate for use in the diagnosis and / or treatment of cancers, like in particular breast cancer, gastric cancer or other Her2 overexpressing tumors, as for example tumors of ovary, endometrium, bladder, lung, colon, and head and neck.

15. A diagnostic or analytical kit comprising at least one conjugate as defined in anyone of the claims 10 to 12.M / 65047-PCT

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