Antibody-linker-lipid conjugate

Abstract

An antibody-linker-lipid conjugate is provided wherein the linker includes a moiety which is obtainable by an inverse electron demand Diels-Alder reaction. The antibody-linker-lipid conjugate is suitable for micelles, liposomes or LNPs. A method for obtaining the antibody-linker-lipid conjugate is also provided.

Description

[0001] ANTIBODY-LINKER-LIPID CONJUGATE

[0002] Technical Field:

[0003] The present invention relates to an antibody-linker-lipid conjugate and a method for preparing the same. The linker of the antibody-linker-lipid conjugate according to the invention includes a moiety which is obtainable by an inverse electron demand Diels-Alder reaction (iEDDA).

[0004] Background of the Invention:

[0005] In past years lipid nanoparticles (LNPs) gained increasing attention as a novel approach for therapeutic applications especially due to the coronavirus disease 2019 (COVID-19) vaccine.

[0006] LNPs possess distinctive advantages, such as simple formulation, high stability, high encapsulation efficiency, efficient cellular internalization, endosomal escape capabilities, and low immunogenicity. Their efficiency as RNA delivery systems has been well-documented. Typically, LNP formulations consist of phospholipids, cholesterol, PEGylated lipids, and ionizable lipids. Phospholipids enhance the cellular uptake of LNPs, while cholesterol, a natural component of the cell membrane, improves LNP stability by filling intervals between lipids and promoting fusion with endosomal membranes upon uptake. PEGylated lipid components prevent LNP aggregation, boost stability, provide a stealth effect, and prolong systemic circulation. In addition, the proportion of PEGylated lipids may determine the size of LNPs, with approximately 1.5% PEGylated lipids being considered ideal. After intravenous administration many LNPs end up in the liver or spleen, which are known as natural targets.

[0007] For some applications, e.g. in vivo CAR-T generation, gene editing or protein replacement, it is necessary or beneficial to target other tissues or specific cells. In the prior art, it has been investigated whether this object can be achieved by altering the particles physiochemical properties (see e.g. T. Zhang, iScience 2024, 27, 109804). More recent publications aim for an active targeting approach. Here, antibodies or antibody fragments are anchored to the LNP surface to bind a desired antigen, enabling a more selective delivery of the cargo. At present, it has been attempted to attach the lipid to the antibody by chemical conjugation methods. However, these attempts resulted in heterogeneous conjugates (antibody lipid conjugates) which has a negative impact on biophysical properties, antibody function and orientation as well as final product quality and manufacturing consistency (see e.g. M. Bruckner, Nanoscale 2021, 13, 9816-9824).

[0008] Similar challenges have to be addressed in the production of antibody-drug conjugates (ADC). In this context, WO2023 / 170239 Al describes a microbial transglutaminase which enables the site-specific conjugation of drug-linker constructs to a glutamine in the antibody sequence, more specifically to position HC-Q295 of native, fully glycosylated IgG-type antibodies (see also Dickgiesser, S. et al., Bioconjugate chemistry 2020, 31, 1070-1076), even in the absence of the artificial introduction of a transglutaminase recognition motif.

[0009] W02024 / 102770 Al relates to a conjugate comprising a targeting moiety and a lipid nanoparticle (LNP) encapsulating a therapeutic agent, wherein the targeting moiety is conjugated to the lipid nanoparticle through a linker, and wherein the linker comprises a Click product formed from a Click reaction between a first Click handle on the targeting moiety and a second Click handle on the LNP, wherein the first Click handle comprises a tetrazine (Tz) ring and the second Click handle comprises a trans-cyclooctene (TCO) moiety.

[0010] WO2024 / 102772 Al relates to a conjugate comprising a targeting moiety and a lipid nanoparticle (LNP) encapsulating a therapeutic agent, wherein the targeting moiety is conjugated to the lipid nanoparticle through a linker, and wherein the linker comprises an enzyme recognition sequence, preferably wherein the enzyme recognition sequence comprises a sortase recognition motif.

[0011] WO2024 / 102769 Al relates to a conjugate comprising a targeting moiety and a lipid nanoparticle (LNP) encapsulating a therapeutic agent, wherein the targeting moiety is conjugated to the lipid nanoparticle through a linker, and wherein the linker comprises an enzyme recognition sequence and a Click product formed from a Click reaction between a first Click handle on the targeting moiety and a second Click handle on the LNP. Example 1 of WO2024 / 102769 Al relates to a conjugate where the antibody having a sortase enzyme recognition sequence is reacted with a Triglycine methyltetrazine, and where a TCO modified dye was reacted with the antibody-Tz. There remains a need to provide antibody-lipid conjugates of high quality and with high yields and purity, e.g., with a defined lipid to antibody ratio, which are useful for targeted LNPs. In particular, it would be desirable to attach a lipid to an antibody in a site-specific manner. A process for producing such antibody-lipid conjugates in a high yield and / or purity would also be desirable.

[0012] Summary of the Invention

[0013] The aforementioned problems are solved by the antibody-linker-lipid conjugates and the methods as defined in the claims and / or described in this specification. Herein the sitespecific particle optimization for targeting (SPOT) to specific cells is described.

[0014] The current invention deviates from the disclosure of W02024 / 102770 Al, WO2024 / 102772 Al and WO2024 / 102769 Al at least (i) in that the first Click handle on the targeting antibody is not a tetrazine but a dienophil selected from MeCyp-, Norbornen SCO-, TCO-, or BCN-, which may optionally be substituted, and (ii) in that the second Click handle on the linker is not a trans-cyclooctene moiety but a tetrazine which may optionally be substituted, and furthermore in that a transglutaminase recognition motif forms part of the antibody for attaching the MeCyp-, Norbornen SCO-, TCO-, or BCN- Click handle to the antibody.

[0015] Although W02024 / 102770 Al explicitly discourages the configuration of the current invention: "when LNP is modified with Tz ring and targeting moiety (e.g., antibody. Fab fragment orScFv) is modified with a TCO moiety, the conjugation efficiency is substantially lower than when the LNP is modified with a TCO moiety and the targeting moiety, e.g., antibody, Fab fragment or ScFv, is modified with a Tz ring", the inventors of the current invention have found that the specific configuration of tetrazine as first Click handle and any of MeCyp-, Norbornen, SCO-, TCO-, BCN- or a derivative thereof as second Click handle, combined with the use of the Transglutaminase recognition motiv, results in an improved lipid yield of over 90% of the antibody-lipid conjugate, while the inverted orientation resulted in substantially lower yields, if combined with the Transglutaminase setup.

[0016] Brief Description of the Figures

[0017] Fig. 1 shows a scheme of the method for preparing LNPs according to the present invention. Fig.2 shows the results of the HIC analysis of the antibody-linker conjugates obtained in Example 1.

[0018] Figs. 3 to 6 show the results of the SEC analysis of the antibody-linker-lipid conjugates obtained in Example 2.

[0019] Figs. 7 to 10 show the results of the reverse phase HPLC analysis of the antibody- linker-lipid conjugates obtained in Example 2.

[0020] Fig. 11 shows the results of SDS-PAGE analysis of the antibody-linker-lipid conjugates obtained in Example 2. oN: overnight (18h);

[0021] MeCyp: Methylcyclopropene-PEG3-amine; TCO3: TCO-PEG3-amine; TCO8: TCO-PEG8-amine; BCN3: BCN-PEG3-amine; BCN8: BCN-PEG8- amine; Tz4: Tetrazine-PEG4-amine; Tz6: Tetrazine-PEG6-amine; MeTz3: Metyhltetrazine-PEG3-amine; MeTz7: Methyltetrazine-PEG7- amine; Azide3: Azido-PEG3-amine; Azide8: Azido-PEG8-amine; DBCO4: DBCO-PEG4-amine; DBCO8: DBCO-PEG8-amine; NC: Negative control (unmodified antibody).

[0022] Fig. 12 shows the results of SDS-PAGE analysis of the antibody-linker-lipid conjugates obtained in Example 3. Tz: DSPE-PEG2K-Tetrazine; PE: 18:0 PEG2000 PE; mAb: Monoclonal Antibody.

[0023] Fig. 13 shows the results of the SEC analysis of the antibody-linker-lipid conjugates obtained in Example 3. mAb: monoclonal antibody; PE: 18:0 PEG2000 PE; Tz: DSPE-PEG2K-Tetrazine.

[0024] Figs. 14 and 15 show the results of the reverse phase HPLC analysis of the antibody- linker-lipid conjugates obtained in Example 3. mAb: monoclonal antibody; PE: 18:0 PEG2000 PE; Tz: DSPE-PEG2K-Tetrazine; Fig 14: reverse phase HPLC, non-reduced; Fig 15: reverse phase HPLC, reduced.

[0025] Fig. 16 shows the results of the SEC analysis (A) and SDS-PAGE (B) of the antibody-linker-lipid conjugates obtained in Example 4.

[0026] Fig. 17 shows the (A), (B) results of the SEC analysis of the antibody-linker- lipid conjugates, (C) SDS-PAGE for the antibody-linker-lipid conjugates, (D) HIC results for the antibody-linker-lipid conjugates, (E) PBMC transfection results obtained using the resulting tLNPs according to Example 5, (F) OVCAR-3 / SKOV-3 transfection results obtained using the resulting tLNPs according to Example 5. NC: Negative control (naked antibody); Con: Lipid conjugate; Upi: Upifitamab; Lifa: Lifastuzumab; Pert: Pertuzumab; Tras: Trastuzumab.

[0027] Fig. 18 shows the results of the HIC analysis of the antibody-linker conjugates obtained in Example 6.

[0028] Fig. 19 shows the results of the SEC analysis of the antibody-linker-lipid conjugates obtained in Example 6.

[0029] Fig.20 shows the mass spectra generated according to Example 6 with nonreduced antibody samples.

[0030] Fig.21 shows the mass spectra generated according to Example 6 with reduced antibody samples.

[0031] Fig.22 shows the results of the SDS-PAGE analysis of the LNP postmodification in Example 7. NC: Negative control (naked antibody only).

[0032] Fig.23 shows SEC analysis (A), DLS results (B) and RiboGreen data (C) of the tLNP conjugation kinetic experiment in Example 8.

[0033] Fig.24 shows the influence of mAb density on tLNPs upon PBMC transfection in Example 9. Ratios are provided as mAB: Tz ratio.

[0034] Fig.25 shows the results of the HIC analysis of the Fab-fragment-linker conjugates obtained in Example 10.

[0035] Fig.26 shows the results of the SEC analysis of the Fab-fragment-linker-lipid conjugates obtained in Example 10.

[0036] Fig.27 shows the results of the SDS-PAGE analysis of the Fab-fragment-linker- lipid conjugates obtained in Example 10. Pert: Pertuzumab; Rit: Rituximab; Tras: Trastuzumab; C: Control - unmodified Fab; M: Fab micelles.

[0037] Fig.28. shows the transfection results on PBMCs by using Fab-fragment-linker conjugates for tLNP generation.

[0038] Fig.29 shows a schematic representation of a preferred embodiment of the current invention, (upper pannel) where the antibody is either a conventional antibody comprising a Glutamine which is recognized by an enhanced microbial transglutaminase or (lower pannel) wherein the antibody is an antibody fragment having a transglutaminase recognition motif. In each case, TCO is attached to the antibody as a first click handle, while Tz is attached to the lipid of a LNP as a second click handle. The orientation of the first and second click handle defines the click product and thereby the final conjugate.

[0039] Detailed Description

[0040] This invention addresses the challenges associated with targeted drug delivery system, particularly in the context of antibody-linker-lipid conjugates. Existing methods often suffer from low efficiency and specificity, limiting their therapeutic potential and applications. The present invention provides a site-specific method for producing antibody-linker-lipid conjugates. These antibody-linker-lipid conjugates can form micelles and can be incorporated into liposomes and LNPs, thereby providing targeted micelles, liposomes or LNPs. A scheme which depicts the preparation of micelles and LNPs in accordance with the present invention is shown in Fig. 1 and Fig. 29.

[0041] By utilizing a unique configuration of Click handles, the method enhances the conjugation efficiency and lipid yield. This not only improves the performance of the conjugates but also opens avenues for their application in various therapeutic areas including cancer and immunology and rare diseases therapies. The ability to selectively target specific cells positions this technology as significant advancement in drug delivery systems.

[0042] Unless the context requires otherwise, throughout the present specification and claims, the word "comprise" and variations thereof, such as, "comprises" and "comprising" are to be construed in an open, inclusive sense, that is, as "including, but not limited to". However, in some embodiments the term "comprises" and variations thereof may refer to "consists of".

[0043] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0044] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

[0045] As used herein, a "domain" or "region" may be any region of a protein, generally defined on the basis of sequence homologies and often related to a specific structural or functional entity.

[0046] A "coding sequence" or a sequence "encoding" an expression product, such as a polypeptide, protein, or enzyme, is a nucleotide sequence that, when expressed, results in the production of that polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide, protein or enzyme. A coding sequence for a protein may include a start codon (usually ATG) and a stop codon.

[0047] As used herein, references to specific "proteins" (e.g. antibodies or enzymes) can include a polypeptide having a native amino acid sequence, as well as variants and modified forms regardless of their origin or mode of preparation. A protein which has a native amino acid sequence is a protein having the same amino acid sequence as obtained from nature. Such native sequence proteins can be isolated from nature or can be prepared using standard recombinant and / or synthetic methods. Native sequence proteins specifically encompass naturally occurring truncated or soluble forms, naturally occurring variant forms (e.g. alternatively spliced forms), naturally occurring allelic variants and forms including post-translational modifications. Native sequence proteins include proteins carrying post-translational modifications such as glycosylation, or phosphorylation, or other modifications of some amino acid residues.

[0048] The term "gene" means a DNA sequence that codes for, or corresponds to, a particular sequence of amino acids which comprises all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. Some genes, which are not structural genes, may be transcribed from DNA to RNA, but are not translated into an amino acid sequence. Other genes may function as regulators of structural genes or as regulators of DNA transcription. In particular, the term gene may be intended for the genomic sequence encoding a protein, i.e. a sequence comprising regulator, promoter, intron and exon sequences.

[0049] Herein, a sequence "at least 85% identical" to a reference sequence is a sequence having, over its entire length, 85% or more, for instance 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the entire length of the reference sequence. The percentage of "sequence identity" may thus be determined by comparing two such sequences over their entire length by global pairwise alignment using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443 (1970)), e.g. using the program Needle (EMBOSS) with the BLOSUM62 matrix and the following parameters: gap open=10, gap extend=0.5, end gap penalty=false, end gap open=10, end gap extend=0.5 (which are standard settings).

[0050] A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain with similar chemical properties (e.g., charge, size or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. Examples of groups of amino acids that have side chains with similar chemical properties include 1 ) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine. Conservative amino acid substitution groups can also be defined on the basis of amino acid size.

[0051] An "antibody" (also referred to as an "immunoglobulin") may e.g. be a natural or conventional type of antibody in which two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (I) and kappa (k). There are five main heavy chain classes (or isotypes) which determine aspects of the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each antibody chain contains distinct sequence domains (or regions). The light chain of a typical IgG antibody includes two regions, a variable region (VL) and a constant region (CL). The heavy chain of a typical IgG antibody includes four regions, namely a variable region (VH) and a constant region (CH), the latter being made up of three constant domains (CHI, CH2 and CH3). The variable regions of both light and heavy chains determine binding and specificity to the antigen. The constant regions of the light and heavy chains can confer important biological properties, such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an antibody and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the so-called hypervariable or complementarity determining regions (CDRs). Complementarity determining regions (CDRs) therefore refer to amino acid sequences which together define the binding affinity and specificity of the Fv region of an antibody. The light (L) and heavy (H) chains of an antibody each have three CDRs, designated CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. A conventional antibody's antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain variable region.

[0052] "Framework regions" (FRs) refer to amino acid sequences interposed between CDRs, i.e. to those portions of immunoglobulin light and heavy chain variable regions that are relatively conserved among different immunoglobulins in a single species. The light and heavy chains of an immunoglobulin each have four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L, and FR1-H, FR2-H, FR3-H, FR4-H, respectively. As used herein, a "human framework region" is a framework region that is substantially identical (about 85%, or more, for instance 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) to the framework region of a naturally occurring human antibody.

[0053] In the context of the invention, CDR / FR definition in an immunoglobulin light or heavy chain is determined based on the IMGT definition (Lefranc et al. Dev. Comp. Immunol., 2003, 27(l):55-77; www.imgt.org).

[0054] As used herein, the term "antibody" includes conventional antibodies and fragments thereof, as well as single domain antibodies and fragments thereof, such as variable heavy chain of single domain antibodies; the term "antibody" as used herein also includes chimeric, humanized, bispecific or multispecific antibodies, as well as other types of engineered antibodies. The term "antibody" includes monoclonal antibodies.

[0055] The term "monoclonal antibody" or "mAb" as used herein refers to an antibody molecule of a single amino acid sequence, which is directed against a specific antigen, and is not to be construed as requiring production of the antibody by any particular method. A monoclonal antibody may be produced e.g. by a single clone of B cells or hybridoma, but may also be recombinant, e.g. produced by methods involving genetic or protein engineering.

[0056] The term "chimeric antibody" refers to an engineered antibody which, in its broadest sense, contains one or more regions from one antibody and one or more regions from one or more other antibodies. In an embodiment, a chimeric antibody comprises a VH and a VL of an antibody derived from a non-human animal, in association with a CH and a CL of another antibody which is, in some embodiments, a human antibody. As the non-human animal, any animal such as mouse, rat, hamster, rabbit or the like can be used. A chimeric antibody may also denote a multispecific antibody having specificity for at least two different antigens.

[0057] The term "humanized antibody" refers to an antibody which is wholly or partially of non-human origin and which has been modified to replace certain amino acids, for instance in the framework regions of the VH and VL, in order to avoid or minimize an immune response in humans. The constant regions of a humanized antibody are typically human CH and CL regions.

[0058] "Fragments" of antibodies (e.g. of conventional antibodies) comprise a portion of an intact antibody such as an IgG, in particular an antigen binding region or variable region of the intact antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, as well as bispecific and multispecific antibodies formed from antibody fragments. A fragment of a conventional antibody may also be a single domain antibody, such as a heavy chain antibody or VHH.

[0059] The term "Fab" denotes an antibody fragment having a molecular weight of about 50,000 Da and antigen binding activity, in which about a half of the N-terminal side of the heavy chain and the entire light chain are bound together through a disulfide bond. It is usually obtained among fragments by treating IgG with a protease, papaine.

[0060] The term "F(ab')2" refers to an antibody fragment having a molecular weight of about 100,000 Da and antigen binding activity, which is slightly larger than 2 identical Fab fragments bound via a disulfide bond of the hinge region. It is usually obtained among fragments by treating IgG with a protease, pepsin. The term "Fab1" refers to an antibody fragment having a molecular weight of about 50,000 Da and antigen binding activity, which is obtained by cutting a disulfide bond of the hinge region of the F(ab')2.

[0061] A single chain Fv ("scFv") is a covalently linked VH:: VL heterodimer which is usually expressed from a gene fusion including VH and VL encoding genes linked by a peptide-encoding linker. The human scFv fragments of the invention include CDRs that are held in appropriate conformation, for instance by using gene recombination techniques. Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)2. "dsFv" is a VH:: VL heterodimer stabilised by a disulphide bond. "(dsFv)2" denotes two dsFv coupled by a peptide linker.

[0062] The term "bispecific antibody" or "BsAb" denotes an antibody which comprises two different antigen binding sites. Thus, BsAbs are able to e.g. bind two different antigens simultaneously. Genetic engineering has been used with increasing frequency to design, modify, and produce antibodies or antibody derivatives with a desired set of binding properties and effector functions as described for instance in EP 2050764 Al.

[0063] The term "multispecific antibody" denotes an antibody which comprises two or more different antigen binding sites.

[0064] The term "diabodies" refers to small antibody fragments with two antigen binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains of the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0065] A "Designed Ankyrin Repeat Protein" (DARPin) is an antibody variant having an N-terminal cap section, at least two Ankyrin Repeat (AR) module sections, and a C-terminal cap section.

[0066] The term "hybridoma" denotes a cell, which is obtained by subjecting a B cell prepared by immunizing a non-human mammal with an antigen to cell fusion with a myeloma cell derived from a mouse or the like which produces a desired monoclonal antibody having an antigen specificity. A "transglutaminase" denotes an enzyme which catalyzes the acyl transfer of a y-carboxyamide group from glutamine to a primary amine.

[0067] A "microbial transglutaminase" is a transglutaminase of micobial origin or a variant derived thereof. Useful microbial transglutaminases have been isolated from Streptomyces mobaraense, Streptomyces cinnamoneum, Streptomyces griseo-carneum, Streptomyces lavendulae, and Bacillus subtilis.

[0068] A transglutaminase according to the current invention can be a microbial transglutaminase as described in WO2023 / 170239, W02023 / 170240, W02016 / 100735, WO2016 / 096785, WO2019 / 057772, WO2022 / 058594 or CN102994469, or a functional fragment or variant thereof. A "functional fragment" of a transglutaminase is a fragment of a transglutaminase that can catalyze the acyl transfer of a y-carboxyamide group from glutamine to a primary amine. Preferably, the transglutaminase according to the current invention recognizes at least one of the following amino acid sequences as substrate(s): Q, GGTLQSPP (SEQ ID NO: 1), TLQSG (SEQ ID NO: 2), TLQSPP (SEQ ID NO: 3), GGTLQSG (SEQ ID NO: 4), TLQSA (SEQ ID NO: 5), RLQQP (SEQ ID NO: 6), YELQRPYHSELP (SEQ ID NO: 7), LLQG (SEQ ID NO: 8), GECTYFQAYGCTE (SEQ ID NO: 9), DIPIGQKMTG (SEQ ID NO: 10), DIPIGQGMTG (SEQ ID NO: 11), DIPIGQRMTG (SEQ ID NO: 12), GENTYFQAYGNTE (SEQ ID NO: 13) and TGTLQSVSY (SEQ ID NO: 14).

[0069] An amino acid sequences that is recognized by a transglutaminase is a "transglutaminase recognition motif".

[0070] By "purified" or "isolated" it is meant, when referring to a polypeptide (e.g. an antibody) or a nucleotide sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. The term "purified" as used herein means at least 75%, 85%, 95%, 96%, 97%, or 98% by weight, of biological macromolecules of the same type are present. An "isolated" nucleic acid molecule which encodes a particular polypeptide refers to a nucleic acid molecule which is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may include some additional bases or moieties which do not deleteriously affect the basic characteristics of the composition.

[0071] As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine, a primate or a human. In preferred embodiments of the invention, the subject (or patient) is a human. As used herein, the term "about" in connection with a numerical value means that the value can vary between + / -10% of said value.

[0072] As used herein, the term "Lipid" means any lipid that is suitable for micelles, liposomes or LNPs. For example, the Suitable Lipids include phosphoethanolamine, phosphocholines, dimyristoyl glycerol, sphingo, ditetradecylacetamide and n-tetamine-lipids with variable saturated and / or unsaturated carbon chains, in particular DSPE and DMG. For example, the saturated or unsaturated carbon chain can have a length of 12 to 22 C atoms.

[0073] These Lipids may be conjugated to a linker, e.g. a hydrophilic polymeric linker.

[0074] Suitable linker are or may comprise for example polyethylene glycol (e.g. PEGylated DMG and PEGylated DSPE), polysarcosine (pSAR) (e.g. DSPE-Polysarcosine), and polyoxazoline (pOx), or a combination of these. In this case, the polyethylene glycol, polysarcosine or polyoxazoline chain may be (part of) the linker within the meaning of the present invention, in particular it may be part of the moiety -L2- as defined herein.

[0075] "Halogen" refers to bromo, chloro, fluoro or iodo.

[0076] "Alkyl" refers to a saturated, straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, preferably having from one to twelve carbon atoms (C1-C12 alkyl), more preferably one to eight carbon atoms (C1-C8 alkyl) and even more preferably one to six carbon atoms (C1-C6 alkyl), which is attached to the rest of the molecule by a single bond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso propyl), n-butyl, 1,1-dimethylethyl (t-butyl ), n-pentyl, 2,2,2-trimethylethyl, n-hexyl, 3-methylhexyl, 2 methylhexyl, and the like.

[0077] "Alkenyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms and including one or more double bonds, preferably having from two to twelve carbon atoms (C2-C12 alkenyl), more preferably two to eight carbon atoms (C2-C8 alkenyl) and even more preferably two to six carbon atoms (C2-C6 alkenyl), which is attached to the rest of the molecule by a single bond, e.g., ethenyl, prop-l-enyl, but-l-enyl, pent-l-enyl, penta-1, 4-dienyl, hex-l-enyl, and the like.

[0078] "Alkynyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms and including one or more triple bonds, preferably having from two to twelve carbon atoms (C2-C12 alkynyl), more preferably two to eight carbon atoms (C2-C8 alkynyl) and even more preferably two to six carbon atoms (C2-C6 alkynyl), which is attached to the rest of the molecule by a single bond, e.g., ethynyl, prop-1 ynyl, but-1 ynyl, pent-l-ynyl, hex-l-ynyl, and the like.

[0079] "Alkylene" or "alkylene chain" refers to a saturated, straight or branched divalent hydrocarbon chain, consisting solely of carbon and hydrogen, preferably having from one to twelve carbon atoms (C1-C12 alkylene), more preferably one to eight carbon atoms (Cl-C8 alkylene) and even more preferably one to six carbon atoms (C1-C6 alkylene), which is attached to the rest of the molecule by single bonds, e.g., methylene, ethylene, propylene, n-butylene, and the like.

[0080] "Alkenylene" and "alkynylene" refer to a divalent group derived from the alkenyl group and alkynyl group as defined above, respectively.

[0081] "Aryl" refers to a carbocyclic ring system radical comprising 6 to 18 carbon atoms and at least one aromatic ring. For purposes of embodiments of this invention, the aryl radical is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems. Aryl includes, but is not limited to, aryl radicals derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene, such as phenyl, indenyl, naphthalenyl and the like

[0082] "Arylene" refers to a carbocyclic ring system that is derived from an aromatic hydrocarbon (arene) and is bivalent. Arylene includes, but is not limited to, arylene diradicals derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene, such as phenylene. "Carbocyclic" or "carbocycle" refers to a ring system, wherein each of the ring atoms are carbon.

[0083] "Cycloalkyl" refers to a stable non aromatic monocyclic or polycyclic carbocyclic radical consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems, having from three to fifteen carbon atoms, preferably having from three to ten carbon atoms, which is saturated and attached to the rest of the molecule by a single bond. Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic radicals include, for example, adamantyl, norbornyl, decalinyl, 7,7 dimethyl bicyclo[2.2.1]heptanyl, and the like. "Cycloalkylene" refers to a divalent group derived from the cycloalkyl group as defined above.

[0084] "Cycloalkenyl" refers to a carbocyclic ring comprising one or more carbon-carbon double bonds within the ring, such as, cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl.

[0085] "Cycloalkenylene" refers to a divalent group derived from the cycloalkenyl group as defined above.

[0086] "Heterocyclyl" or "heterocyclic ring" refers to a stable 3 to 18-membered nonaromatic ring radical having one to twelve ring carbon atoms and from one to six ring heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. Unless stated otherwise specifically in the specification, the heterocyclyl radical is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused, spirocyclic ("spiro-heterocyclyl") and / or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclyl radical is optionally oxidized; the nitrogen atom is optionally quaternized; and the heterocyclyl radical is partially or fully saturated. Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl [l,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxothiomorpholinyl, and 1,1-dioxothiomorpholinyl.

[0087] "Heterocyclylene" refers to a divalent group derived from the heterocyclyl group as defined above.

[0088] "Heteroaryl" refers to a 5 to 14-membered ring system radical comprising hydrogen atoms, one to thirteen ring carbon atoms, one to six ring heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, and at least one aromatic ring comprising a heteroatom. For purposes of embodiments of this invention, the heteroaryl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heteroaryl radical may be optionally oxidized; the nitrogen atom may be optionally quaternized. Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][l,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[l,2a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl, 1-phenyl-l-Hpyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e., thienyl).

[0089] "Heteroarylene" refers to a divalent group derived from the heteroaryl group as defined above.

[0090] Unless stated otherwise specifically in the specification, each of the above groups may optionally be substituted. The term "substituted" as used herein means any of the above groups (e.g., alkyl, alkylene, alkynyl, cycloalkyl, cycloalkenyl heterocyclyl, aryl, heteroaryl) wherein at least one hydrogen atom (e.g., 1, 2, 3 or all hydrogen atoms) is replaced by a bond to a non-hydrogen atom such as, but not limited to: a halogen atom such as F, Cl, Br, and I; an oxygen atom in groups such as hydroxyl groups, alkoxy groups, and ester groups; a sulfur atom in groups such as thiol groups, thioalkyl groups, sulfone groups, sulfonyl groups, and sulfoxide groups; a nitrogen atom in groups such as amines, amides, alkylamines, dialkylamines; and other heteroatoms in various other groups.

[0091] As used here, in unless otherwise specified, the concentration is given in % (v / v).

[0092] The following abbreviations are used herein:

[0093] DBCO dibenzocyclooctyne / dibenzocyclooctynyl

[0094] BCN bicyclo[6.1.0]non-4-yne / bicyclo[6.1.0]non-4-ynyl

[0095] SCO strained cyclooctyne / strained cyclooctynyl

[0096] MeCyp methyl cyclopropene / methyl cyclopropenyl

[0097] TCO trans-cyclooctene / trans-cyclooctenyl

[0098] Norbornen: bicyclo[2.2.1]hept-2-en / bicyclo[2.2.1]hept-2-enyl MeTz 3-methyl-6-phenyl-l,2,4,5-tetrazine / 3-methyl-6-phenyl-l,2,4,5-tetrazinyl Tz 6-phenyl-l,2,4,5-tetrazine / 6-phenyl-l,2,4,5-tetrazinyl

[0099] DMG l,2-dimyristoyl-rac-glycero-3-methoxy

[0100] DSPE l,2-distearoyl-sn-glycero-3-phosphoethanolamine

[0101] PEG polyethylene glycol

[0102] Antibodies

[0103] Any antibody can be used for the antibody-linker-lipid conjugate of this invention.

[0104] The antibody of the invention may preferably serve as substrate for the transglutaminase or microbial transglutaminase (herein also "mTG" or "MTG"). Preferred antibodies include antibodies, in particular monoclonal antibodies, that comprise a transglutaminase recognition motif selected from the group consisting of Q, GGTLQSPP (SEQ ID NO: 1), TLQSG (SEQ ID NO: 2), TLQSPP (SEQ ID NO: 3), GGTLQSG (SEQ ID NO: 4), TLQSA (SEQ ID NO: 5), RLQQP (SEQ ID NO: 6), YELQRPYHSELP (SEQ ID NO: 7), LLQG (SEQ ID NO: 8), GECTYFQAYGCTE (SEQ ID NO: 9), DIPIGQKMTG (SEQ ID NO: 10), DIPIGQGMTG (SEQ ID NO: 11), DIPIGQRMTG (SEQ ID NO: 12), GENTYFQAYGNTE (SEQ ID NO: 13) and TGTLQSVSY (SEQ ID NO: 14), more preferably the transglutaminase recognition motif TLQSPP or GGTLQSPP (most preferably the transglutaminase recognition motif GGTLQSPP).

[0105] For example one or more of these transglutaminase recognition motifs may be located at the C-Terminus or N-Terminus of the polypeptide chain of the antibody.

[0106] For example one or more of these transglutaminase recognition motifs may be located in at least one and preferably both of its light chain constant regions (CL) and / or in at least one and preferably both of its heavy chain constant regions (CH).

[0107] For example, the glutamine may be located at position 295, at position 253 and / or at position 297 (EU numbering).

[0108] These amino acid sequences have been found to be suitable to serve as substrate for the mTG, even when incorporated into an antibody light chain constant regions (CL) and / or heavy chain constant regions (CH).

[0109] In a particularly preferred embodiment of the invention, the substrate for the mTG is a glutamine at position 295.

[0110] The EU numbering system is well known (cf. Edelman et al., Proc. Natl. Acad. Sci. USA 1969, 63, 78-85 and Kabat, E. A. et al., National Institutes of Health (U. S.) Office of the Director. Sequences of Proteins of Immunological Interest, 5th ed.; DIANE Publishing: Collingdale, PA, USA, 1991) and the positions of the amino acid that are indicated follow this numbering system. The amino acids are specified using the single letter amino acid code.

[0111] In some embodiments of the method of the invention, the antibody that is used in the method of the invention is a conventional antibody, such as a conventional monoclonal antibody, or an antibody fragment, a bispecific or multispecific antibody.

[0112] In some embodiments, the antibody comprises or consists of an IgG, or a fragment thereof. For example, the antibody can be an IgG antibody, such as an IgGl, lgG2, lgG3, lgG4 or any animal equivalent thereof. The majority of IgGs comprises a conserved glutamine at position 295 according to EU numbering.

[0113] In some embodiments, the antibody that is used in the conjugation method of the invention may be e.g. a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody. Numerous methods for humanization of an antibody sequence are known in the art; see e.g. the review by Almagro & Fransson (2008) Front Biosci. 13: 1619-1633. One commonly used method is CDR grafting, or antibody reshaping, which involves grafting of the CDR sequences of a donor antibody, generally a mouse antibody, into the framework scaffold of a human antibody of different specificity. Since CDR grafting may reduce the binding specificity and affinity, and thus the biological activity, of a CDR grafted non-human antibody, back mutations may be introduced at selected positions of the CDR grafted antibody in order to retain the binding specificity and affinity of the parent antibody. Identification of positions for possible back mutations can be performed using information available in the literature and in antibody databases. Amino acid residues that are candidates for back mutations are typically those that are located at the surface of an antibody molecule, while residues that are buried or that have a low degree of surface exposure will not normally be altered. An alternative humanization technique to CDR grafting and back mutation is resurfacing, in which non-surface exposed residues of non-human origin are retained, while surface residues are altered to human residues. Another alternative technique is known as "guided selection" (Jespers et al. (1994) Biotechnology 12, 899) and can be used to derive from a murine antibody a fully human antibody conserving the epitope and binding characteristics of the parental antibody.

[0114] For chimeric antibodies, humanization typically involves modification of the framework regions of the variable region sequences. Amino acid residues that are part of a CDR will typically not be substantially altered in connection with humanization, although in certain cases it may be desirable to alter individual CDR amino acid residues, for example to remove a glycosylation site, a deamidation site or an undesired cysteine residue. N-linked glycosylation occurs by attachment of an oligosaccharide chain to an asparagine residue in the tripeptide sequence Asn-X-Ser or Asn-X-Thr, where X may be any amino acid except Pro. Removal of an N-glycosylation site may be achieved by mutating either the Asn or the Ser / Thr residue to a different residue, for instance by way of conservative substitution. Deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation, primarily when present in the sequence Asn-Gly, and to a lesser extent in other dipeptide sequences such as Asn-Ala. When such a deamidation site, for instance Asn-Gly, is present in a CDR sequence, it may therefore be desirable to remove the site, typically by conservative substitution to remove one of the implicated residues. Substitution in a CDR sequence to remove one of the implicated residues is also intended to be encompassed by the present invention.

[0115] In a humanized antibody or fragment thereof, the variable domains of heavy and light chains may comprise human acceptor framework regions. A humanized antibody may further comprise human constant heavy and light chain domains, where present.

[0116] In some embodiments, the antibody used in the conjugation methods of the invention may be an antibody fragment (for instance a humanized antibody fragment) selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, and diabodies.

[0117] In some embodiments, the antibody may be a bispecific or multispecific antibody formed from antibody fragments, at least one antibody fragment being a fragment of an antibody according to the present invention. Multispecific antibodies are polyvalent protein complexes as described for instance in EP2050764 Al or US2005 / 0003403 Al.

[0118] The antibodies useful in the conjugation methods of the invention can be produced by any technique known in the art. Antibodies according to the invention can be used e.g. in an isolated (e.g. purified) form or contained in a vector, such as a membrane or lipid vesicle (e.g. a liposome).

[0119] If an antibody is used in the conjugation method of the invention that is not an IgG antibody, then it is preferred that the antibody comprises an transglutaminase recognition motif selected from the group consisting of GGTLQSPP, TLQSG, TLQSPP, GGTLQSG, TLQSA, RLQQP, YELQRPYHSELP, LLQG, GECTYFQAYGCTE, DIPIGQKMTG, DIPIGQGMTG, DIPIGQRMTG, GENTYFQAYGNTE and TGTLQSVSY.

[0120] In some embodiments of the conjugation method of the invention, the antibody that is used in the method of the invention is a conventional antibody, such as a conventional monoclonal antibody, or an antibody fragment, a bispecific or multispecific antibody. In some embodiments, the antibody comprises or consists of an IgG, or a fragment thereof.

[0121] Antibody-linker-lipid conjugates

[0122] The present invention relates to antibody-linker-lipid conjugates represented by

[0123]

[0124] wherein

[0125] Ab represents an antibody;

[0126] LI and L2 each independently represent a linker;

[0127] X represents a moiety which is obtainable by an inverse electron demand Diels-Alder reaction; and

[0128] n represents an integer of 1 to 8, preferably an integer of 1 to 4, more preferably 1 or 2, even more preferably 2.

[0129] As used herein, the term "inverse electron demand Diels-Alder reaction" (iEDDA) refers to a chemical reaction wherein a dienophile reacts with an electron deficient tetrazine. This reaction results in the formation of a (fused) ring system which comprises a six-membered ring including two adjacent nitrogen atoms.

[0130] In one embodiment of the present invention, X represents a ring system which comprises a six-membered ring including two adjacent nitrogen atoms and which is obtainable by an inverse electron demand Diels-Alder reaction. The ring system may optionally be substituted as defined above.

[0131] In another embodiment, X may be obtained by reacting a moiety X1with a moiety X2in an inverse electron demand Diels-Alder reaction, wherein the moiety X1is capable of reacting with the moiety X2in an inverse electron demand Diels-Alder reaction to result in the moiety X. Preferably, one of X1and X2is selected from MeCyp-, TCO-, BCN-, SCO- and Norbornene-, each of which may optionally be substituted; and the other isTz-, which may optionally be substituted.

[0132] In a preferred embodiment, the antibody-linker-lipid conjugate is obtained by an inverse electron demand Diels-Alder reaction, of an antibody-linker conjugate represented by

[0133]

[0134] and a lipid-linker conjugate represented by X2L2Lipid.

[0135] In this case, it is preferred that X1is selected from MeCyp-, TCO-, BCN-, SCO- and Norbornene-, more preferably TCO- and BCN-, each of which may optionally be substituted; and X2is Tz-which may optionally be substituted.

[0136] In another preferred embodiment of the present invention, X represents one of the following structures:

[0137]

[0138] Wherein

[0139] R1, R2, R3and R4each independently represent H, halogen, cyano, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl,

[0140] -CF3, -CF2-R', -NO2, -SO2, -OR', -SR', -CO-R', -C(=S)-R', -O-CO-R',

[0141] -S-CO-R', -O-C(=S)-R', -S-C(=S)-R', -S(=O)-R‘, -SO2-R‘, -SO2-NR'R", -CO-O-R', -CO-S- R', -C(=S)-O-R', -C(=S)-S-R', -NR'-CO- R", -NR'R", -O-CO-NR'R", -NR'-CO-NR"R"', -CO-NR'R", -C(=S)-NR'R", -NR'- C(=S)-R", -NR'-CO-OR", -NR'-C(=S)-OR", -NR'-CO-SR", -NR'-C(=S)-S-R", -S-CO-NR'R", -O-C(=S )-R'R'", -S-C(=S)R'R" and -NR'-C(=S)-NR"R"';

[0142] preferably H, halogen, cyano, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, -CF3, -CF2-R', -NO2, -SO2, -OR', -SR', -CO-R', -O-CO-R',

[0143] -CO-O-R', -NR' -CO-R", -NR'R", -O-CO-NR'R", -NR'-CO-NR"R'", -CO-NR'R", -NR'-CO-OR"; more preferably H, C1-C6 alkyl, -OH, -O-C1-C6 alkyl, Cl, -CO-OH, -CO-CI-6 alkyl, -O-CO-OH, -O-CO-C1-6 alkyl, NH2, -NH(Cl-C6alkyl),-N(Cl-C6alkyl)2, -CO-NH2,

[0144] -CO-NH(Cl-C6alkyl), -CO-N(Cl-C6alkyl)2,-NH-CO-OH, -NH-CO-C1-C6 alkyl,

[0145] -O-CO-NH2, -O-CO-NH-C1-C6 alkyl, -O-CO-N(C1-C6 alkyl)2,

[0146] -NH-CO-NH2;-NH-CO-NH(C1-C6 alkyl), -NH-CO-N(C1-C6 alkyl)2;

[0147] even more preferably H or C1-C6 alkyl;

[0148] especially R1, R2, R3and R4are all H;

[0149] R5represents a bond, alkylene, alkenylene, alkynylene, arylene, heteroarylene, heterocyclylene, cycloalkylene, cycloalkenylene;

[0150] preferably C6-18 arylene or C5-C14 heteroarylene;

[0151] more preferably pyrimidinylene, pyridinylene or phenylene;

[0152] most preferably phenylene;

[0153] R6represents H, halogen, cyano, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, -CF3, -CF2-R', -NO2, -SO2, -OR', -SR', -CO-R', -C(=S)-R', -O-CO-R', -S-CO-R', -O-C(=S)-R', -S-C(=S)-R', -S(=O)-R‘,

[0154] -SO2-R', -SO2-NR'R", -CO-O-R', -CO-S-R', -C(=S)-O-R', -C(=S)-S-R', -NR'-CO- R", -NR'R", -O-CO-NR'R", -NR'-CO-NR"R'", -CO-NR'R", -C(=S)-NR'R",

[0155] -NR'-C(=S)-R", -NR'-CO-OR", -NR'-C(=S)-OR", -NR'-CO-SR",

[0156] -NR'-C(=S)-S-R", -S-CO-NR'R", -O-C(=S)-R'R'", -S-C(=S)R'R" and

[0157] -NR'-C(=S)-NR"R"';

[0158] preferably H, halogen, cyano, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, -CF3, -CF2-R', -NO2, -SO2, -OR', -SR', -CO-R', -O-CO-R',

[0159] -CO-O-R', -NR' -CO-R", -NR'R", -O-CO-NR'R", -NR'-CO-NR"R'", -CO-NR'R", -NR'-CO-OR"; more preferably H, C1-C6 alkyl, -OH, -O-C1-C6 alkyl, Cl, -CO-OH, -CO-CI-6 alkyl, -O-CO-OH -O-CO-C1-6 alkyl, NH2, -NH(Cl-C6alkyl),-N(Cl-C6alkyl)2, -CO-NH2,

[0160] -CO-NH(Cl-C6alkyl), -CO-N(Cl-C6alkyl)2,-NH-CO-OH, -NH-CO-C1-C6 alkyl, -O-CO-NH2, -0-C0-NH-C1-C6 alkyl, -O-CO-N(C1-C6 alkyl )2,

[0161] -NH-CO-NH2;-NH-CO-NH(C1-C6 alkyl), -NH-CO-N(C1-C6 alkyl)2;

[0162] even more preferably hydrogen, -C1-C6 alkyl, pyrimidinyl, pyridinyl or phenyl; particular preferably hydrogen or methyl;

[0163] especially hydrogen;

[0164] R', R" and R'" each independently represent H, alkyl or cycloalkyl; preferably H or C1-C6 alkyl; and

[0165] one of the wavy lines represents the bond to L1and the other represents the bond to L2; preferably the wavy line attached to R5represents the bond to L2and the other represents the bond to L1.

[0166] In a more preferred embodiment, X represents one of the following structures:

[0167]

[0168] wherein

[0169] R1, R2, R3, R4, R5, R6and the wavy lines are as defined above.

[0170] In another preferred embodiment, L1is attached to a glutamine of the antibody; preferably (i) to a glutamine at position 295, 253 and / or 297 of the heavy chain of the antibody, whereby the antibody is a monoclonal antibody and wherein Eu numbering is used for defining the position of said glutamine, and / or (ii) to a glutamine in an amino acid sequence selected from the group consisting of GGTLQSPP, TLQSG, TLQSPP, GGTLQSG, TLQSA, RLQQP, YELQRPYHSELP, LLQG, GECTYFQAYGCTE, DIPIGQKMTG, DIPIGQGMTG, DIPIGQRMTG, GENTYFQAYGNTE and TGTLQSVSY, located preferably at the C-terminus or N-terminus of the polypeptide chain of the antibody, e.g. of the heavy- and / or light chain of the antibody; more preferably to a glutamine at position 295 of the heavy chain of the antibody, whereby the antibody is a monoclonal antibody and wherein Eu numbering is used for defining the position of said glutamine.

[0171] In a particularly preferred embodiment, the antibody-linker-lipid conjugate according to the present invention is represented by formula (1), (2) or (3), preferably formula (1):

[0172]

[0173] wherein

[0174] Ab represents an antibody;

[0175] -Glu-CO-NH- represents a glutamine side chain of said antibody, preferably (i) of a glutamine at position 295 of the heavy chain of the antibody, and / or of a glutamine at position 253 of the heavy chain of the antibody, and / or of a glutamine at position 297 of the heavy chain of the antibody, whereby the antibody is a monoclonal antibody and wherein Eu numbering is used for defining the position of said glutamine, and / or (ii) of a glutamine in an transglutaminase recognition motif selected from the group consisting of Q, GGTLQSPP, TLQSG, TLQSPP, GGTLQSG, TLQSA, RLQQP, YELQRPYHSELP, LLQG, GECTYFQAYGCTE, DIPIGQKMTG, DIPIGQGMTG, DIPIGQRMTG, GENTYFQAYGNTE and TGTLQSVSY, preferably located at the C-terminus or N-terminus of the polypeptide chain of the antibody, e.g. of the heavy- and / or light chain of the antibody;

[0176] V represents a linker, preferably a linker having a chain length of 1 to 20 atoms; more preferably C1-C6 alkylene; especially -CH2- or -CH2-CH2-;

[0177] Z, Y and W represent a bond or a linker,

[0178] Z preferably represents a bond, C1-C6 alkylene, (C1-C6 alkylene)NHCOO,

[0179] (C1-C6 alkylene)NHCOO(Cl-C6 alkylene),

[0180] (C1-C6 alkylene)NHCO(Cl-C6 alkylene)CO,

[0181] (C1-C6 alkylene)CONH-(Cl-C6 alkylene)-, (C1-C6 alkylene)CO or

[0182] (C1-C6 alkylene)CO(Cl-C6 alkylene); more preferably a bond, -CH2-, -CH2-CH2-, -CH2-CH2-NH-CO-O-,

[0183] -CH2-CH2-NH-CO-O-CH2-, -CH2-CH2-NH-CO-CH2-CH2-CO-or -CH2-CH2-CO-NH-CH2-; Y preferably represents a bond, C1-C6 alkylene, -(C1-C6 alkylene)-CO-,

[0184] -NH-CO-(C1-C6 alkylene)-CO-, -NH-CO-(C1-C6 alkylene)-, -NH-CO-O-,

[0185] -NH-CO-O-(C1-C6 alkylene)-, -(C1-C6 alkylene)-NH-CO-(Cl-C6 alkylene)-O-,

[0186] -O-(C1-C6 alkylene)-CO-NH2-(Cl-C6 alkylene)-;

[0187] more preferably a bond, -CH2-NH-CO-CH2-O-, -CH2-CO-; -NH-CO-CH2-CH2-CO-, -NH- CO-CH2-CH2-, -NH-CO-O-CH2-, -NH-CO-O-, -O-CH2-CO-NH2-CH2-;

[0188] W preferably represents a bond, -NH-(C1-C6 alkylene)-, -(C1-C6 alkylene)-CO- or -CO-;

[0189] more preferably a bond, -NH-CH2-, -CH2-CO- or -CO-;

[0190] R represents H, alkyl, cycloalkyl, aryl, heterocyclyl or heteroaryl;

[0191] n represents an integer of 1 to 6, preferably an integer of 1 to 4, more preferably 1 or 2, even more preferably 2;

[0192] q is an integer from 1 to 10, preferably an integer from 3 to 8;

[0193] m, p and r each independently represent an integer from 10 to 100, preferably an integer from 20 to 50; and more preferably and integer from 40 to 50; and

[0194] X is a moiety which is obtainable by an inverse electron demand Diels-Alder reaction, preferably X is as defined above.

[0195] Method for Preparing an Antibody-Linker Conjugate

[0196] The antibody-linker-lipid conjugates according to the invention are obtainable by the methods described below. In this manner, antibody-linker-lipid conjugates can be provided in a site-specific manner in a high yield and purity.

[0197] When preparing the antibody-linker-lipid conjugate according to the invention, it is preferable to first provide an antibody-linker conjugate represented by

[0198]

[0199] This antibody-linker conjugate may be prepared by a method comprising the following steps:

[0200] (1) providing an antibody Ab;

[0201] (2) mixing together in a reaction buffer at least the following components:

[0202] (a) said antibody provided in step (1);

[0203] (b) an enzyme; and (c) a compound represented by X^ -R, wherein the R-moiety is capable of reacting, in the presence of said enzyme, with the antibody from step (1) in a site-specific manner, and the moiety X1is capable of reacting in an inverse electron demand Diels-Alder reaction;

[0204] thereby obtaining an antibody-linker conjugate represented by

[0205]

[0206] n

[0207] If necessary, the antibody-linker conjugate can be isolated from the mixture and purified by methods known to the skilled person. For example, the antibody-linker conjugate can be isolated from the mixture by chromatographic methods such as size exclusion chromatography.

[0208] In a preferred embodiment of the present invention, the antibody-linker conjugate is prepared in accordance with the method disclosed in WO2023 / 170239 Al which is herein incorporated by reference. This method uses a microbial transglutaminase which attaches X1-!1- to an transglutaminase recognition motif, and thus allows for site-specific conjugation to the antibody.

[0209] By this preferred method, the side chain of a glutamine of the aforementioned transglutaminase recognition motif, in particular the side chain of a glutamine at position 295 of the heavy chain, can react with the primary amino group of a compound represented by X1-L1-NH2, in the presence of an mTG enzyme:

[0210] AcyLdonor Acyl acceptor

[0211] 09

[0212] Antibody-^^^ +H2N-RmTG * Antibody^^ -RGlutamine Amine Isopeptide bond

[0213] In the above shown reaction scheme the acyl acceptor is X^L^NHz, i.e., R- represents X1-!1-. In some preferred embodiments, this method for preparing the antibody-linker conjugate comprises the following steps:

[0214] (1) providing an antibody, preferably an antibody that comprises (i) a glutamine at position 295 of the heavy chain of the antibody, and / or a glutamine at position 253 of the heavy chain of the antibody and / or of a glutamine at position 297 of the heavy chain of the antibody, whereby the antibody is a monoclonal antibody and wherein EU numbering is used for defining the position of said glutamine, and / or (ii) a glutamine in a tranglutaminase recognition motif selected from the group consisting of Q, GGTLQSPP, TLQSG, TLQSPP, GGTLQSG, TLQSA, RLQQP, YELQRPYHSELP, LLQG, GECTYFQAYGCTE, DIPIGQKMTG, DIPIGQGMTG, DIPIGQRMTG, GENTYFQAYGNTE and TGTLQSVSY, preferably located at the C-terminus or N-terminus of the polypeptide chain of the antibody, e.g., of the heavy- and / or light chain of the antibody;

[0215] (2) mixing together in a reaction buffer at least the following components:

[0216] (a) said antibody provided in step (1);

[0217] (b) a (microbial) transglutaminase; and

[0218] (c) a compound represented by X1-L1-NH2 wherein the H2N-moiety is capable of reacting with the antibody from step (1) in the presence of said transglutaminase, and the moiety X1is capable of reacting in an inverse electron demand Diels-Alder reaction;

[0219] thereby obtaining an antibody-linker conjugate represented by:

[0220]

[0221] ; and

[0222] (3) optionally separating the antibody-linker-conjugate produced in step (2) from unreacted linker and from said transglutaminase, preferably by subjecting said mixture from step (2) to a size-exclusion chromatography.

[0223] In a preferred embodiment, the reaction buffer comprises one or more of the following buffers adjusted to a pH of between 6 and pH 8.8: BICINE, BICINE / Tris, Tris-HCI, HEPES and / or Tricine. In a particularly preferred embodiment, the reaction buffer comprises between 1% and 15% DMSO, between 5 and 100 mM HEPES, and has a pH of between 6 and 8; preferably between 7 and 7.5.

[0224] In a further embodiment of the method of the invention, the mixture in step (2) comprises a molar excess of the compound represented by X1L1NH2, preferably at least 10 molar, and more preferably at least 50 molar, per conjugation site, wherein the conjugation site is the transglutaminase recognition motif.

[0225] To optimize the turnover, the reaction is preferably conducted at a temperature between 0 and 50°C, more preferably 20 and 40°C, especially 37°C. Preferably, the reaction time is between 0.5 to 3 hours, such as 1 hour or 2 hours. In another preferred embodiment of the method of the invention, the (microbial) transglutaminase is comprised in the mixture in step (2) of the method at an activity of between 10 U and 40 U per mg of the antibody. The compound represented by X^ -NHj is preferably used in an amount between 10 to 200, preferably between 80 to 120, particularly preferably 50 or 100, molar equivalents per glutamine.

[0226] The compound represented by X^L^NHz acts as a substrate for the (microbial) transglutaminase. It carries a primary amine for attachment to a glutamine side chain of the antibody and a moiety X1which is capable of reacting in an inverse electron demand Diels-Alder reaction.

[0227] In a preferred embodiment of the present invention, the compound X1-L1-NH2 is selected from the following compounds, wherein -L1- corresponds to -Z-L3-V-:

[0228]

[0229]

[0230] wherein R1, R2, R3, R4, R5, R6, V and Z are as defined above; and

[0231] L3represents linker, preferably a linker having chain length of 1 to 100 atoms, more preferably a linker having chain length of 1 to 50 atoms, most preferably -[O-CH2-CH2]q-O- wherein q represents an integer from 1 to 10, preferably an integer from 3 to 8.

[0232] In a more preferred embodiment of the invention, X1-L1-NH2 is selected from the following compounds:

[0233]

[0234] wherein R1, R2, R3, R4, V, L3and Z are as defined above.

[0235] In a particularly preferred embodiment of the invention, X1-L1-NH2 is selected from the following compounds:

[0236] TCO-L1-NH2;

[0237] TCO-PEG8-Amine

[0238]

[0239] TCO-PEG3-Amine

[0240]

[0241] Tz- -NHz;

[0242] Tz-PEG6-Amine

[0243]

[0244] Norbornene- -NHj;

[0245] Norbornene-PEG8-Amine

[0246]

[0247] Especially, X1-L1-NH2 is selected from TCO-PEGg-Amine, TCO-PEGg-Amine, BCN-PEGg-Amine, BCN-PEGg-Amine and MeCyp-PEGg-Amine. In particular, X^L^NHz is TCO-PEGg-Amine, TCO-PEG8-Amine, BCN-PEGg-Amine or BCN-PEG8-amine.

[0248] Compounds represented by X1-L1-NH2 are commercially available, e.g. from BroadPharm, ConjuProbe, Iris Biotech and Lumiprobe.

[0249] Method for Preparing the Antibody-Linker-Lipid Conjugate

[0250] Subsequently, the antibody-linker conjugate represented by

[0251]

[0252] and the lipid-linker conjugate represented by X2-L2-Lipid are subjected to an inverse electron demand Diels-Alder reaction (iEDDA), thereby obtaining the antibody-linker-lipid conjugate represented by

[0253]

[0254] In a preferred embodiment of the present invention, the lipid-linker conjugate represented by X2-L2-Lipid is selected from the following compounds, wherein -L2- corresponds to -Y-L4-W-: D-L2-Lipid; where D is tetrazine or a derivative thereof;

[0255]

[0256] wherein R1, R2, R3, R4, R5, R6, Y and W are as defined above; and

[0257] L4represents linker, preferably a linker having chain length of 1 to 500 atoms, most preferably -[CHJ-CHJ-O]™-, -[N(CH3)-CH2-CO]P- or -[N(CO-R)-CH2-CH2]r; wherein m, p and r each independently represent an integer from 10 to 100, preferably an integer from 20 to 50 more preferably 40 to 50; and R is H, alkyl, cycloalkyl, aryl, heterocyclyl or heteroaryl. In a particularly preferred embodiment, the lipid-linker conjugate represented by X2-L2-Lipid is

[0258]

[0259] wherein R5, R6, Y, L4 and W are as defined above.

[0260] According to the present invention, the Lipid can be any lipid that is suitable for the preparation of micelles, liposomes or LNPs, e.g. a lipid having a saturated or unsaturated carbon chain with a length of 12 to 22 C atoms. Preferably, the lipid is selected from phosphoethanolamine, phosphocholines, dimyristoyl glycerol, sphingo, ditetradecylacetamide and n-tetamine-lipids with variable saturated and / or unsaturated carbon chains. More preferably, the lipid is selected from DMG and DSPE. Most preferably, the lipid is DSPE.

[0261] According to the present invention, the Lipid can be a lipid having a saturated or unsaturated carbon chain with a length of 12 to 22 C atoms, and L2can be a hydrophilic polymeric linker. For example, L2can be selected from polyethylene glycol (PEG), polysarcosine (pSAR), polyoxazoline (pOX) or a combination thereof.

[0262] According to the present invention, the Lipid-L2moiety can be DMG-W-(PEG)m-Y-, DMG-W- (pSAR)m-Y-, DMG-W-(pOX)m-Y-, DSPE-W-(PEG)m-Y-, DSPE-W-(pSAR)m-Y-, DSPE-W- (pOX)m-Y-, Dil8:0N-W-(pEG)m-Y, Dil8:0N-W-(pSar)m-Y, Dil8: ON-W-(pOX)m-Y, wherein m is an integer from 10 to 100, preferably an integer from 40 to 50, and whereinW and Y represent a bond or a linker.

[0263] In a particularly preferred embodiment, Lipid-L2-X2represents DSPE-PEG2K-Tetrazine (n=43), Dil8:0N-pSar-Tetrazine (n=44) or DSPE-pOX-Tetrazine (n=45):

[0264]

[0265]

[0266] Compounds which are suitable as the lipid-linker conjugate represented by X2-L2-Lipid are commercially available e.g. from Avanti and BroadPharm.

[0267] Inverse Electron Demand Diels-Alder Reaction

[0268] The term "inverse electron demand Diels-Alder reaction" (iEDDA) refers to a [4+2] cycloaddition of electron deficient 1,2,4,5-tetrazines (in particular Tz and MeTz) and various dienophiles. It is known to the skilled person as one of the so-called "click chemistry" reactions (see B. Oliveira, Chem. Soc. Rev. 2017, 46, 4895-4950; A. Knall, Chem. Soc. Rev. 2013, 42, 5131-5142; M. Pagel, J Pep Sci. 2019, 25:e3141). The following scheme depicts the reaction between trans-cyclooctene (TCO) with Tz or MeTz (i.e., R is H or methyl) which represents an especially preferred embodiment of the present invention:

[0269]

[0270] In addition to trans-cyclooctene (TCO), suitable dienophiles include MeCyp, BCN, SCO and Norbornene. Preferably, the dienophile is TCO or BCN. In an alternative embodiment, the dienophile can be attached to -L2-Lipid, and Tz / MeTz can be attached to -L1-Ab. However, it is preferred that the dienophile is attached to -L1-Ab as shown above, because this results in a higher yield.

[0271] The inverse electron demand Diels-Alder reaction results in a moiety X which is selected from:

[0272]

[0273] wherein R1, R2, R3, R4and R5are as defined above, and

[0274] wherein one of the wavy lines represents the bond to L1and the other represents the bond to L2; preferably the wavy line attached to R5represents the bond to L2and the other represents the bond to L1.

[0275] Preferably the inverse electron demand Diels-Alder reaction results in a moiety X which is selected from:

[0276]

[0277] wherein R1, R2, R3, R4and R5are as defined above, and

[0278] wherein Ab represents an antibody or fragment thereof, and

[0279] wherein Lipid represents a lipid or an LNP.

[0280] The inverse electron demand Diels-Alder reaction may be conducted in a manner known to the skilled person. A suitable method for conducting the reaction is disclosed in US8, 236,949 which is hereinafter incorporated by reference. For example, the reaction may be carried out in a reaction buffer, such as PBS (phosphate buffered saline) adjusted to a pH of 6 to 8, preferably 7.4.

[0281] In a preferred embodiment of the method of the invention, the mixture in step (5) comprises a molar excess of the lipid-linker conjugate represented by X2-L2-Li pid compared to the antibody-linker conjugate. Preferably, the antibody-linker conjugate represented by

[0282]

[0283] and the lipid-linker conjugate represented by X2-L2-Lipid are subjected to the reaction in a molar ratio from 1:1 to 1:5, preferably 1:2 to 1:4, more preferably 1:3.

[0284] To optimize the turnover, the reaction is preferably conducted at a temperature between 0 and 50°C, more preferably 20 and 40°C, especially 37°C. Preferably, the reaction time is between 0.5 to 5 hours, such as 2 hours.

[0285] Micelles, Liposomes and LNPs The antibody-1 inker-1 ipid conjugates according to the invention can be used for obtaining micelles, liposomes and LNPs as shown in Fig. 1. Methods for producing such micelles, liposomes and LNPs are known to the skilled person.

[0286] In one embodiment of the invention, the lipid-linker conjugate represented by X2-L2-Lipid described above can be incorporated into micelles, liposomes and LNPs. Subsequently, an antibody-linker conjugate represented by

[0287]

[0288] n

[0289] can be attached to these micelles, liposomes or LNPs by way of an inverse electron demand Diels-Alder reaction.

[0290] In another embodiment, targeted LNPs may be generated by post-modification. For example, an antibody-linker conjugate is prepared as described before, in particular by reacting the antibody with a compound represented by X1L1NH2 in the presence of a (microbial) transglutaminase. In parallel, LNPs comprising the lipid-linker conjugate represented by X2-L2-Lipid are prepared in a manner known to the skilled person, in particular the lipid-linker conjugate can be added to a mixture which is suitable for the preparation of LNPs. Subsequently, the antibody-linker conjugate is added to the functionalized LNP, and the mixture is incubated, for example at 37°C for 2 hours, thereby subjecting the moieties X1and X2to an inverse electron demand Diels-Alder reaction. In another embodiment, targeted LNPs may be generated by post-insertion. For example, an antibody-linker-lipid conjugate according to the present invention is prepared as described above and incorporated into micelles. Subsequently, the micelles are added to the unmodified LNP in an amount from 0.0001 up to 0.1 mol%, and the mixture is incubated at 37°C and 400 rpm for 2 hours.

[0291] Examples

[0292] Size-exclusion chromatography (SEC)

[0293] Analytical size exclusion chromatography was performed on an Agilent HPLC System Serie 1260 Infinity II system using a TSKgel SuperSW3000 column (Tosho) equipped with SuperSW3000 Guard Column (Tosho) by applying an isocratic flow (0.35 ml / min) of lxPBS pH 7.4 (Gibco) for 20 min.

[0294] Reversed phase HPLC Reversed phase HPLC was performed on an Agilent HPLC System Serie 1260 Infinity II system using a PLRP-S 4000 A column (Agilent) by applying a gradient from 0 to 90% eluent B in 11 min (0.6 ml / min). H2O + 0.1% TFA (A) and ACN + 0.1% TFA (B) were used as eluents. Column compartment was heated to 80°C. For reduction of antibodies, the protein was mixed by with 10 mM TCEP or lx NuPAGE reducing agent (Thermo Fisher).

[0295] Hydrophobic interaction chromatography (HIC)

[0296] Hydrophobic interaction chromatography was performed on an Agilent HPLC System Serie 1260 Infinity II system using a TSKgel Butyl-NPR column (Tosho) by applying a gradient from 0 to 100% eluent B in 20 min (1 ml / min). 1.5 M Ammonium sulfate + 50 mM TRIS pH 7.2 (A) and 50 mM TRIS pH 7.2 (B) were used as eluents.

[0297] SDS-PAGE

[0298] For SDS-PAGE a XCell SureLock Mini-Cell Electrophoresis system was used (Invitrogen). Sample was mixed with NuPAGE LDS-sample buffer (Invitrogen), NuPAGE reducing agent (Invitrogen) and heated to 95°C. Typically, 0.5 pg protein were applied to the gel (4-12 % NuPAGE Bis-TRIS; Invitrogen), separation was done in NuPAGE MES SDS-running buffer (Invitrogen) and according to the manufactures protocol (200 V, 3000 mA, 350 W for 30 min).

[0299] PBMC transfection

[0300] To assess the in vitro transfection efficiency of targeted lipid nanoparticles, experiments were conducted using human peripheral blood mononuclear cells (PBMCs) isolated from whole blood. PBMCs were thawed, washed once with AIMV medium supplemented with 10% fetal bovine serum (FBS) centrifuged at 800 x g for 3 minutes, and resuspended in AIM-V medium supplemented with 10% FBS. Cells were counted and the required volume was taken to achieve a final cell density of 5 x 105cells / mL. The cell suspension was centrifuged again at 800 x g for 5 minutes, the supernatant was aspirated, and the cells were resuspended in the appropriate volume of medium. Subsequently, 50 pL of the cell suspension was seeded per well in a 384-well flat-bottom plate (non-cell culture treated). Cells were allowed to recover for 1-2 hours at 37°C in a humidified incubator with 5% CO2and >95% relative humidity. LNPs were diluted in AIM-V + 10% FBS using a 1:2 serial dilution, starting at a concentration of 6 pg / mL. Fifty microliters of each LNP dilution was added to the wells, resulting in an effective starting concentration of 300 ng LNP per well. All border wells were filled with PBS. Plates were incubated overnight at 37°C, 5% CO2, and >95% relative humidity.

[0301] Following incubation, plates were analyzed using an LSR Fortessa flow cytometer. Data were gated for singlets and lymphocytes by forward and side scatter, and mCherry expression was measured in the 610 / 20 channel with a 561 nm laser. Transfection efficiency was evaluated by determining the median mCherry fluorescence intensity and the percentage of mCherry-positive cells among all lymphocytes.

[0302] SKOV-3 / OVCAR-3 transfection

[0303] For transfection, 20000 OVCAR-3 and SKOV-3 cells / well were placed in a 96-well tissue culture plate with 200 pl of growth medium and were incubated for 24 h. Medium was removed, cells were treated with 60ng / well mRNA containg LNPs, and transfection was performed in technical duplicates. In order to detect a targeting effect rather than a passive uptake over time cells were washed once with IX PBS after 1 hour to remove LNPs. Cells were incubated overnight in fresh medium, detached from the culture plate with accutase and were analyzed by using a LSR Fortessa flow cytometer. Data were gated for singlets by forward and side scatter, and eGFP expression was measured in the 530 / 30 channel with a 488 nm laser. Transfection efficiency was evaluated by determining the mean eGFP fluorescence intensity.

[0304] LC-MS

[0305] For analysis of the TCO activated antibody and the lipid conjugates a sciex eksigent M5 MicroLC system (Framingham, MA, USA) connected to a Sciex X500B Q. TOF mass spectrometer was used. Intact antibody samples were injected on a nanoEase M / Z Protein BEH C4, 300A, 5 pm, 300 pm x 50 mm Trap Column (Waters) at room temperature with 5% buffer A (0.1% FA, 0.025% DFA in LC-MS grade water) for 1 min at a flow rate of 30 pL / min. A second aforementioned column was used to perform protein separation by applying a linear gradient from 5 to 95% buffer B (0.1% FA, 0.025% DFA in ACN) in 1.6 min with a flow of 10 pl / min. For reduction of the antibodies samples were incubated with 20 mM TCEP for 30 min at 37°C. For the mass spectrometry ESI turbo v ion source ionspray voltage was set to 5 kV and the temperature 350°C. Ion source gas 1 and 2 was set to 30 and 40 psi, respectively. The TOF MS mode was positive with intact protein mode on. MS scans were acquired over a m / z range of 1000-3000 with an accumulation time of 1 and a declustering potential of 150 V.

[0306] Abbreviations:

[0307] mAb monoclonal antibody

[0308] MeCyp MeCyp-PEG3-amine

[0309] TCO3 TCO-PEG3-amine

[0310] TCO8 TCO-PEG8-amine

[0311] BCN3 BCN-PEG3-amine

[0312] BCN8 BCN-PEG8-amine

[0313] Tz4 Tetrazine-PEG4-amine

[0314] Tz6 Tetrazine-PEG6-amine

[0315] MeTz3 Methyltetrazine-PEG3-amine

[0316] MeTz7 Methyltetrazine-PEG7-amine

[0317] Azide3 Azido-PEG3-amine

[0318]

[0319] Tz DSPE-PEG2K-Tetrazine

[0320] BCN DSPE-PEG2K-BCN

[0321] TCO DSPE-PEG2K-TCO

[0322] DBCO DSPE-PEG2K-DBCO

[0323] Azide DSPE-PEG2K-azide PE 18:0-PEG2K-PE

[0324] TCO-PEG4-m TCO-PEG4-Metyhl

[0325] mTG Enhanced Microbial Transglutaminase SAE0217 (Merck)

[0326] NC negative control (unmodified antibody)

[0327] oN overnight (18h)

[0328] In Examples 1 -4 and 6 - 9, an antibody having the following sequence was used: a) Heavy Chain (SEQ ID NO: 15)

[0329] b) Light Chain (SEQ ID NO: 16).

[0330] Example 1: Preparation of the antibody-linker conjugate

[0331] 1.5 mg (5 mg / ml) antibody were conjugated with 50 molar-equivalents per Q295 of different compounds represented by X1-L1-NH2 in the presence of 15 U of mTG (10 U per mg antibody) in 24 mM HEPES pH 7.0 for 2 h at 37°C. The antibody-linker conjugate was purified via size-exclusion chromatography using a YMC Eco column filled with Superdex 200 pg (Cytiva) on an Akta pure system (Cytiva). Eluted antibody conjugate containing fractions were united and concentrated via Vivaspin 20 MWCO 50kDa (Sartorius) filters. Purified antibody-linker conjugates were analyzed via HIC by injecting 50 pg of sample. The results are shown in Fig. 2.

[0332] To verify enzymatic turnover of the transglutaminase substrates, the purified and conjugated mAb was analyzed via hydrophobic-interaction chromatography (HIC). It was observed that the unmodified antibody showed one main elution peak with a smaller shoulder peak, which eluted earlier. This signal might relate to a subpopulation in the mAb possibly due to a different glycosylation or post-translational modification.

[0333] All tested antibody conjugates showed a delayed retention time compared to the unmodified counterpart (Fig. 2) which indicates turnover by the enzyme. For the azide conjugates the shift appeared not that significant, but, due to its more hydrophilic nature, a minor retention shift, if any at all, would be expected. Although a mixture of peaks was observed for the DBCO substrates, HIC data indicated that conjugation occurred.

[0334] Example 2: Preparation of the antibody-linker-lipid conjugate For iEDDA and SPAAC (Strain-promoted alkyne azide cycloaddition), 200 pg of antibodylinker conjugate (1.9 mg / ml) were mixed with 3 molar-equivalents of lipid-linker conjugate (38.8 pM) in PBS and were incubated at 37°C and 1000 rpm. After 2 h and 18 h samples were taken, and the reaction was stopped by addition of 20 molar-equivalents of m-PEG4-TCO (iEDDA) or Azido-PEG3-amine (SPAAC).

[0335] Products were analyzed via analytical size-exclusion chromatography and reversed phase HPLC by injecting 40 and 35 pg, respectively. The products were also analyzed by SDS-PAGE. The results of SEC, reversed phase HPLC and SDS-PAGE are shown in Figs. 3 to 11.

[0336] SDS-PAGE showed that conjugation by iEDDA or SPAAC was successful for many of the analysed combinations. Since DSPE-PEG2K compounds have a MW of ~ 3000 Da a size increase of the conjugated antibody chain can be detected. No conjugation of the lipid occurred to the light chain of the antibody. This observation confirms that the reaction with transglutaminase results in an attachment to Q295 on the heavy chain, only.

[0337] For iEDDA reactions, lipid conjugation to the heavy (chain) was observed with yields up to 80% or even more within 2h. Especially, TCO8 and BCN8 showed a high degree of conjugation. SDS-PAGE and reversed phase samples indicate that there was nearly no unmodified heavy chain remaining.

[0338] SEC data reflect the results which were obtained by SDS-PAGE. Unconjugated mAb showed one single elution peak at 8.6 min. Antibodies being conjugated to micelles elute earlier on SEC, since they are larger in size. Those antibody-micelles were detected at 6.2 min. All micelles, which are not conjugated with an antibody do not show absorbance at 280 nm and therefore cannot be detected. Comparable to SDS-PAGE, TCO8 and BCN8 showed the highest turnover with 93.8 / 96.6 % (2h and 18h) and 92.1 / 94.8 %, respectively.

[0339] SEC results indicate that orientation of the partners affects the efficiency of the reaction. For example, conjugation of TCO to the mAb via transglutaminase with subsequent iEDDA to a tetrazine modified lipid provided a much higher yield than inversion of the pairs (Tetrazine-mAb and TCO-lipid), see Table 2.1 below.

[0340] For example, the reaction between mAb-TCO3 or mAb-TCO8 with Lipid-Tz yielded 92 - 97 % product after 18 hours, while the inverse orientation of mAb-Tz4 or mAb-Tz6 with Lipid-TCO yielded only 63 - 76 % product after 18 hours. In another example, the reaction between mAb-BCN3 or mAb-BCN8 with Lipid-Tz yielded 82 - 95 % product after 18 hours, while the inverse orientation of mAb-Tz4 or mAb-Tz6 with Lipid-BCN yielded only 65 - 78 % product.

[0341] This trend was also observed for other reaction pairs.

[0342] Table 2.1. Yield of antibody-linker-lipid conjugate calculated based on the SEC analysis.

[0343]

[0344] Table 2.2. Yield of antibody-linker-lipid conjugate calculated based on the SEC analysis.

[0345]

[0346]

[0347] This data demonstrates that, using iEDDA instead of SPAAC, a linker between the antibody and the lipid can be provided in a higher yield. Combining iEDDA and an enzymatic transglutaminase reaction, therefore, provides a beneficial process for obtaining antibody-linker-lipid conjugates in a site-specific manner and resulting conjugates can be synthesized with low effort, high yield and high purity.

[0348] Example 3: Conjugation matrix with TC0-PEG8-amine

[0349] To optimize the process, TCO-Tz reaction was investigated with different ratios of tetrazinelipid to antibody. To assess the impact of sterical hindrance, DSPE-PEG2K-Tetrazine was mixed with 18:0-PEG2K to increase space between the tetrazine molecules.

[0350] 3.5 mg (6.86 mg / ml) antibody were conjugated with 50 molar-equivalents per Q295 of TCO-PEG8-amine (4.7 mM) and 105 U of mTG (29 U per mg antibody) in 47 mM HEPES pH 7.0 for 2 h at 37°C. The antibody was purified via size-exclusion chromatography using a Superdex® 200 Increase 10 / 300 GL on an Akta pure system (Cytiva).

[0351] Before iEDDA, DSPE-PEG2K-Tetrazine and 18:0-PEG2K-PE were mixed in different ratios (1:0; 1:1; 1:2), the lipid preparation was added to 100 pg activated antibody (1.42 mg / ml) in molar ratios of 1:2, 1:3 and 1:5 (mAb: Tz) and incubated at 37°C and 1000 rpm for 2h. Reaction was quenched by addition of TCO-PEG8-amine (632 pM).

[0352] Products were analyzed via SDS-PAGE, analytical size-exclusion chromatography, and reversed phase HPLC by injecting ~ 25 pg. The results are shown in Figs. 12 to 15.

[0353] Comparing the RP chromatograms with those of a reduced unmodified mAb revealed, that modification of the light chain was not occurring. This shows that both mTG reaction and iEDDA were site-specific (Fig. 15).

[0354] Analysis via SEC, RP and SDS-PAGE showed comparable results for all tested conjugations. Significant amounts of unconjugated mAb could only be observed when TCO and tetrazine were mixed in equimolar ratios (mAb: Tz - 1:2) without any 18:0-PEG2K lipid. For all other reactions, free mAb was hardly detectable. This implies that increasing tetrazine concentrations or decreasing sterically demand improves the reaction. RP data of reduced samples showed that conjugation only occurs at the heavy chain of the mAb.

[0355] Example 4: Attachment of different lipid

[0356] To verify possible attachment of other lipids using the presented chemoenzymatic approach, Dil8:0N-pSar2k-Tz was utilized in the second-step click reaction. DSPE-PEG2k-Tz served as a reference. Hereby, 200 pg TCO-PEG8-amine activated antibody (prepared as before mentioned) were mixed with 3 molar-equivalents of the respective lipid and incubated for 2 h at 37°C and 1000 rpm. Reaction was quenched with a TCO carrying molecule. Analysis of the product was performed with analytical size-exclusion chromatography by measuring absorbance at 280 nm and SDS-PAGE (Fig. 16).

[0357] Example 5: Conjugation of Lipid to Different Antibodies

[0358] A. Upifitamab:

[0359] 3 mg antibody were conjugated with 50 molar-equivalents per Q295 of TCO8 and 7 U of mTG for 2 h at 37°C. The activated mAb was purified via size-exclusion chromatography and concentrated. 2 mg activated antibody were mixed with 3 molar-equivalents of DSPE-PEG2K-Tetrazine and incubated for 2h at 37°C and 1000 rpm. Reaction was quenched with TCO-PEG4-m.

[0360] B. Lifastuzumab:

[0361] 6 mg antibody were conjugated with 50 molar-equivalents per Q295 of TCO8 and 240 U of mTG for 2 h at 37°C. The activated mAb was purified via size-exclusion chromatography and concentrated. 4.2 mg activated antibody were mixed with 3 molar-equivalents of DSPE-PEG2K-Tetrazine and incubated for 2h at 37°C and 1000 rpm. Reaction was quenched with TCO-PEG4-m.

[0362] C. Pertuzumab & Trastuzumab:

[0363] 3 mg antibody were conjugated with 50 molar-equivalents per Q295 of TCO8 and 120 U of mTG for 2 h at 37°C. The activated mAb was purified via size-exclusion chromatography and concentrated. 1.3 mg activated antibody were mixed with 3 molar-equivalents of DSPE-PEG2K-Tetrazine and incubated for 2h at 37°C and 1000 rpm. Reaction was quenched with TCO carrying molecule. Analysis of the products was performed with analytical size-exclusion chromatography by measuring absorbance at 280 nm and SDS-PAGE. The results are shown in Figs. 16 and 17.

[0364] It was observed that conjugation was successful for all antibodies with some variations in the degree of labelling.

[0365] D. Cetuximab:

[0366] 1.6 mg Cetuximab were conjugated with 50 molar-equivalents per Q295 of TCO8 and 16 U of mTG for 1 h at 37°C. Purification of the activated mAb was carried out via size-exclusion chromatography. Subsequently, the concentrated antibody was mixed with DSPE-PEG2K-Tetrazine carrying LNPs in an molar ratio of 0.5:1 (mAb: Tz) and incubated for 2h at 37°C (see also Example 6: Generation of tLNPs (post-modification)). tLNPs were then tested in vitro for transfection efficiency.

[0367] tLNPs generated with TCO-activated Cetuximab showed improved transfection on EGFR high and EGFR medium expressing cells compared to the naked LNP (Fig. 17F).

[0368] Table 5.1. Dynamic light scattering analysis of generated LNPs

[0369]

[0370] Table 5.2. RiboGreen analysis of generated LNPs

[0371]

[0372] E. Teplizumab & Siplizumab:

[0373] 3 mg of each antibody were conjugated in presence of 30U mTG and 50 molar-equivalents per Q295 of TCO8 for lh at 37°C and 400 rpm. After purification via size-exclusion chromatography, concentration with VivaSpin 2050kDa, and analysis via HIC, the activated mAbs were mixed with DSPE-PEG2K-Tetrazine carrying LNPs in an molar ratio of 0.5:1 (mAb: Tz) and incubated for 2h at 37°C. LNPs were analyzed via RiboGreen-assay and DLS. In vitro testing of the tLNPs was performed afterwards.

[0374] Table 5.3. Dynamic light scattering analysis of generated LNPs

[0375]

[0376] Table 5.4.: RiboGreen analysis of generated LNPs

[0377]

[0378] tLNPs generated with TCO-activated Siplizumab or Teplizumab showed improved transfection on PBMC compared to unmodified LNPs (Fig. 17E).

[0379] Example 6: Scale-Up Experiment

[0380] 30 mg antibody were conjugated with 50 molar-equivalents per Q295 of TCO8 in the presence of 300 U of mTG for lh at 37°C. The activated mAb was purified via size-exclusion chromatography and subsequently concentrated.

[0381] DSPE-PEG2K-Tetrazine was added in 3-fold molar excess and incubated for 2h at 37°C and 1000 rpm. Reaction was quenched with TCO-PEG4-m. Generated micelles were purified via size-exclusion chromatography. Different product intermediates were analyzed via HIC, SEC and mass spectra. The results are shown in Figs. 18, 19, 20 and 21.

[0382] The experiment yielded 21 mg of antibody-lipid conjugate with no detectable loss in conjugation efficiency. The additional mass analysis revealed complete turnover during the enzymatic conjugation as well as for the click reaction.

[0383] Example 7: Generation of tLNPs (post-modification)

[0384] In order to generate targeted LNPs (tLNPs) via post-modification, the antibody was activated with TCO-PEG8-amine and mTG in a manner as described before and was purified via SEC.

[0385] LNPs were generated with Nanoassemblr Ignite (Cytiva) by using two different formulations (SM-102, Cholesterol, DSPC, DMG-PEG2000, DSPE-PEG2K-Tetrazine; 50 / 38.5 / 10 / 1.41 / 0.09% and 50 / 38.5 / 10 / 1.46 / 0.04%). After buffer exchange of the LNPs via ZebaSpin columns into lxPBS, the LNPs were mixed in different ratios with the activated mAb to generate targeted LNPs. Ratios were chosen based on the tetrazine molecules on the LNP surface assuming no lipid loss or lipid composition changes during the process (3.18 mM concentration of total lipid after buffer exchange). In detail, lipid concentration was set to 2 mM final concentration and the antibody was added in an excess of 0.5 / 1 / 2 to DSPE-PEG2K-Tetrazine. Samples were filled up with lxPBS to get desired concentrations and incubated at 37°C for 2h. Reaction was quenched by addition of TCO-PEG4-m SDS-PAGE showed that conjugation to the antibody occurred, and a size increase related to the attached lipid was observed for all tested samples (Fig. 22). DLS measurements showed an increase in size for the particles compared to the unmodified buffer exchanged counterparts. PDI before and after conjugation stayed in the same order of magnitude.

[0386] Table 7.1.: Dynamic light scattering analysis of generated LNPs

[0387]

[0388] Example 8: Post-mod kinetics

[0389] In order to track the progress of post-modification a conjugation kinetic study was conducted. Therefore, anti-CD45 antibody was conjugated with TCO-PEG8-amine and mTG in a manner as described before. In parallel LNPs were generated with Nanoassemblr Ignite (Cytiva) by using two different formulations (SM-102, Cholesterol, DSPC, DMG-PEG2000, DSPE-PEG2K-Tetrazine; 50 / 38.5 / 10 / 1.41 / 0.09% and 50 / 38.5 / 10 / 1.46 / 0.04%) and buffer exchanged in lxPBS via PD10 desalting columns. Assuming no lipid loss or change in composition, antibody was mixed with LNPs at a ratio of 1:3 mAb: Tetrazine and a total final lipid concentration of 1.5 mM. After given timepoint samples were taken and the reaction was quenched with a TCO-carrying molecule. For O min sample, the LNP was quenched before addition of the TCO-activated mAb. All sample were analyzed via DLS for size determination and RiboGreen assay for encapsulated mRNA and EE%. Conjugation progress was analyzed via analytical SEC by using a GTxResolve Premier SEC 1000 Å (Waters) colum and 2x PBS, 5% Isopropanol and 0.001% Poloxamer 188 as eluent. Three individual experiments were conducted, and means were calculated for each analysis method. Integration of mAb peak showed an area count decrease over time signifying conjugation to the LNP. Most mAb was conjugated by incubating the reaction >= 2 h. Throughout the entire process, size and encapsulation efficiency remained inconspicuous.

[0390] Example 9: Influence of antibody density on tLNPs on In Vitro transfection efficiency in human PBMCs

[0391] Anti-CD45 antibody was conjugated with TCO-PEG8-amine and mTG in a manner as described before and was purified via SEC.

[0392] LNPs were generated with Nanoassemblr Ignite (Cytiva) by using 50% SM-102, 38.5% Cholesterol, 10% DSPC, 1.46% DMG-PEG2000, 0.04% DSPE-PEG2K-Tetrazine and mRNA coding for mCherry. After buffer exchange into lxPBs using PD10 columns conjugations with different mAb: Tetrazine ratios were conducted by decreasing the total added amount of mAb to the LNP solution. Reaction was carried out at 37°C for 2h and reaction was quenched by addition of m-PEG4-TCO. LNPs were characterized via RiboGreen Assay and DLS-measurement. Followed by transfection of human PBMCs.

[0393] Table 9.1.: RiboGreen analysis of generated LNPs

[0394]

[0395] Table 9.2.: Dynamic light scattering analysis of generated LNPs

[0396]

[0397]

[0398] Fig.24 shows a targeting effect that can already be observed for a 1:300 conjugation ratio compared to the "no mAb" LNP (untargeted) control, demonstrating flexibility in conjugation degree while maintaining effective targeting.

[0399] Example 10: Fab-fragment: Lipid conjugation and post-modification

[0400] 4 mg of an anti CD45 antibody (aCD45) (Heavy Chain: SEQ ID No: 17; Light Chain: SEQ ID No: 18), Pertuzumab (Heavy Chain: SEQ ID No: 19; Light Chain: SEQ ID No: 20), Rituximab (Heavy Chain: SEQ ID No: 21; Light Chain: SEQ ID No: 22) or Trastuzumab (Heavy Chain: SEQ ID No: 23; Light Chain: SEQ ID No: 24) Fab fragment were conjugated with 50 molarequivalents per transglutaminase recognition motif of TCO-PEG8-Amine and 62.5 U of mTG for 1 h at 37°C. The activated Fab was purified via cation exchange chromatography, buffer exchanged via Zeba Spin column, concentrated and analyzed via HIC measuring absorbance at 280 nm. 2000 pg TCO activated Fab were mixed with 1.5 molar-equivalents of DSPE-PEG2K-Tetrazine and incubated for 2h at 37°C and 1000 rpm. Reaction was quenched with a TCO carrying molecule. Micelles were purified using size-exclusion chromatography. Unpurified intermediate (crude) and purified micelles were analyzed using analytical sizeexclusion chromatography measuring absorbance at 280 nm.

[0401] In addition aCD45 and Rituximab TCO-activated Fab fragments were used to generate tLNPs via post-modification as described elsewhere herein. Fab: Tz ratio was set to 1:1.5 at a total lipid concentration of 2 mM.

[0402] HIC reveals a shift to higher retention time for all the used Fab-fragments, with little remaining umodified Fab, which implies attachment of TCO-PEG8-amine (Fig. 25).

[0403] Fig. 26 shows SEC data with a single elution peak for the purified micelles indicating successful purificiation. The crude reaction mixture revealed that most TCO activated Fab-fragments reacted with a lipid, however there was a small propotion, with unconjugated fragments. This was expected since not all Fab-fragments carry a TCO, which was determined in HIC. General conversion yield was > 81%.

[0404] SDS-PAGE proved attachment to the Fab A heavy chain (AHC), since size increase was only obtained for the respective band (Fig. 27).

[0405] Transfection of PBMC with the respective Fab-tLNPs showed improved transfection compared to the naked LNP treated cells. (Fig. 28).

[0406] Table 10.1: Antibody-linker-lipid yield determined by SEC-analysis

[0407]

[0408] Table 10.2: Dynamic light scattering analysis of generated LNPs

[0409]

[0410] Table 10.3: RiboGreen analysis of generated LNPs

[0411]

Claims

CLAIMS1. An antibody-1 inker-lipid conjugate represented by:whereinAb represents an antibody;L1and L2each independently represent a linker;X represents a moiety which is obtainable by an inverse electron demand Diels-Alder reaction; andn represents an integer of 1 to 8, preferably an integer of 1 to 4, more preferably 1 or 1, even more preferably 1,preferably the lipid is selected from phosphoethanolamine, phosphocholines, dimyristoyl glycerol, sphingo, ditetradecylacetamide and n-tetamine-lipids with variable saturated and / or unsaturated carbon chains, more preferably the lipid is selected from DMG and DSPE,wherein the antibody-linker-lipid conjugate is obtainable by an inverse electron demand Diels-Alder reaction, of an antibody-linker conjugate represented byand a lipid-linker conjugate represented by X2-I_2-I_ipid;wherein the moiety X1is capable of reacting with the moiety X2in the inverse electron demand Diels-Alder reaction, to result in the moiety X;characterized in that X1is selected from MeCyp-, TCO-, BCN-, SCO- and Norbornen-, each of which may optionally be substituted; andX2is Tz, which may optionally be substituted;wherein L1is covalently bound to a glutamine of the antibody, wherein said glutamine is part of a transglutaminase recognition motif, preferably of a transglutaminase recognition motif of an enhanced microbial transglutaminase.

2. The antibody-linker-lipid conjugate according to claim 1 wherein the glutamine is located at the C-terminus of the heavy- and / or light chain of the antibody, more preferably wherein the glutamine is located at position 295, 297 or 253 of the heavy chain of the antibody, wherein the antibody is a monoclonal antibody and wherein Eu numbering is used for defining the position of said glutamine.

3. The antibody-linker-lipid conjugate according to any of claims 1 or 2, wherein the transglutaminase recognition motif is an amino acid sequence selected from the group consisting of Q, GGTLQSPP (SEQ ID NO: 1), TLQSG (SEQ ID NO: 2), TLQSPP (SEQ ID NO: 3), GGTLQSG (SEQ ID NO: 4), TLQSA (SEQ ID NO: 5), RLQQP (SEQ ID NO: 6), YELQRPYHSELP (SEQ ID NO: 7), LLQG (SEQ ID NO: 8), GECTYFQAYGCTE (SEQ ID NO: 9), DIPIGQKMTG (SEQ ID NO: 10), DIPIGQGMTG (SEQ ID NO: 11), DIPIGQRMTG (SEQ ID NO: 12), GENTYFQAYGNTE (SEQ ID NO: 13) and TGTLQSVSY (SEQ ID NO: 14), preferably wherein the transglutaminase recognition motif is at the C-terminus or N-terminus of the polypeptide chain of the antibody.

4. The antibody-linker-lipid conjugate according to any one of claims 1 to 3, wherein the Lipid is a lipid having a saturated or unsaturated carbon chain with a length of 12 to 22 C atoms, and wherein L2is a hydrophilic polymeric linker.

5. The antibody-linker-lipid conjugate according to any one of claims 1 to 4, wherein L2is selected from polyethylene glycol (PEG), polysarcosine (pSAR), polyoxazoline (pOX) or a combination thereof,preferably wherein the Lipid-L2moiety is DMG-W-(PEG)m-Y-, DMG-W-(pSAR)m-Y-, DMG-W-(pOX)m-Y-, DSPE-W-(PEG)m-Y-, DSPE-W-(pSAR)m-Y-, DSPE-W-(pOX)m-Y-, Dil8:0N-W-(pEG)m-Y, Dil8:0N-W-(pSar)m-Y, Dil8: ON-W-(pOX)m-Y,wherein m is an integer from 10 to 100, preferably an integer from 40 to 50, and wherein W and Y represent a bond or a linker.

6. The antibody-linker-lipid conjugate according to any one of claims 1 to 5, wherein the moiety Lipid-L2-X2is selected from DSPE-PEG2K-MeTz and DSPE-PEG2K-Tz, wherein Tz may optionally be substituted.

7. The antibody-linker-lipid conjugate according to claim 1 to 6, wherein X represents one of the following structures:Wherein R1, R2, R3and R4each independently represent H, halogen, cyano, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl,cycloalkenyl, -CF3, -CF2-R', -NO2, -SO2, -OR', -SR', -CO-R', -C(=S)-R',-O-CO-R', -S-CO-R', -O-C(=S)-R', -S-C(=S)-R', -S(=O)-R‘,-SO2-R‘, -SO2-NR'R", -CO-O-R', -CO-S-R', -C(=S)-O-R', -C(=S)-S-R',-NR' -CO-R", -NR'R", -O-CO-NR'R", -NR'-CO-NR"R'", -CO-NR'R1',-C(=S)-NR'R", -NR'-C(=S)-R", -NR'-CO-OR", -NR'-C(=S)-OR", -NR'-CO-SR",-NR'-C(=S)-S-R", -S-CO-NR'R", -O-C(=S)-R'R'", -S-C(=S)R'R" and-NR'-C(=S)-NR"R"';R5represents a bond, alkylene, alkenylene, alkynylene, arylene, heteroarylene heterocyclylene, cycloalkylene, cycloalkenylene;R6represents H, halogen, cyano, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, -CF3, -CF2-R', -NO2, -SO2, -OR',-SR', -CO-R', -C(=S)-R', -O-CO-R', -S-CO-R', -O-C(=S)-R',-S-C(=S)-R', -S(=O)-R‘, -SO2-R‘, -SO2-NR'R",-CO-O-R', -CO-S-R', -C(=S)-O-R', -C(=S)-S-R‘, -NR'-CO-R",-NR'R", -O-CO-NR'R", -NR'-CO-NR"R"', -CO-NR'R"-C(=S)-NR'R", -NR'-C(=S)-R", -NR'-CO-OR", -NR'-C(=S)-OR", -NR'-CO-SR",-NR'-C(=S)-S-R", -S-CO-NR'R", -O-C(=S)-R'R'", -S-C(=S)R'R" and -NR'-C(=S)-NR"R"'; R', R" and R'" each independently represent H, alkyl or cycloalkyl; andthe wavy line attached to R5represents the bond to L2and the other wavy line represents the bond to L1.

8. The antibody-linker-lipid conjugate according to any previous claim, which is represented bywherein Ab represents an antibody;-Glu-CO-NH- represents a glutamine side chain of said antibody,preferablyi. of a glutamine at position 295, 297 and / or 253 of the heavy chain of the antibody, whereby the antibody is a monoclonal antibody and wherein Eu numbering is used for defining the position of said glutamine, and / or ii. of a glutamine in a transglutaminase recognition motif selected from the group consisting of GGTLQSPP (SEQ ID NO: 1), TLQSG (SEQ ID NO: 2),TLQSPP (SEQ ID NO: 3), GGTLQSG (SEQ ID NO: 4), TLQSA (SEQ ID NO: 5), RLQQP (SEQ ID NO: 6), YELQRPYHSELP (SEQ ID NO: 7), LLQG (SEQ ID NO: 8), GECTYFQAYGCTE (SEQ ID NO: 9), DIPIGQKMTG (SEQ ID NO: 10), DIPIGQGMTG (SEQ ID NO: 11), DIPIGQRMTG (SEQ ID NO: 12), GENTYFQAYGNTE (SEQ ID NO: 13) and TGTLQSVSY (SEQ ID NO: 14), wherein the amino acid sequence is preferably at the C-terminus or N-terminus of the polypeptide chain of the antibody;X is a moiety which is obtainable by an inverse electron demand Diels-Alder reaction, preferably X is as defined in claim 7;V represents a linker, preferably a linker having a chain length of 1 to 20 atoms; more preferably C1-C6 alkylene; especially -CH2- or -CH2-CH2-;Z, Y and W represent a bond or a linker,preferablyZ represents a bond, C1-C6 alkylene, -(C1-C6 alkylene)-NH-CO-O-,-(C1-C6 alkylene)-NH-CO-O-(Cl-C6 alkylene)-,-(C1-C6 alkylene)-NH-CO-(Cl-C6 alkylene)-CO-,-(C1-C6 alkylene)-CO-NH-(Cl-C6 alkylene), -(C1-C6 alkylene)-CO- or-(C1-C6 alkylene)-CO-(Cl-C6 alkylene)-;more preferably a bond, -CH2-, -CH2-CH2-, -CH2-CH2-NH-CO-O-,-CH2-CH2-NH-CO-O-CH2-, -CH2-CH2-NH-CO-CH2-CH2-CO-or -CH2-CH2-CO-NH-CH2-. Y represents a bond, C1-C6 alkylene, -(C1-C6 alkylene)-CO-,-NH-CO-(C1-C6 alkylene)-CO-, -NH-CO-(C1-C6 alkylene)-, -NH-CO-O-,-NH-CO-O-(C1-C6 alkylene)-, -(C1-C6 alkylene)-NH-CO-(Cl-C6 alkylene)-O-, -O-(C1-C6 alkylene)-CO-NH2-(Cl-C6 alkylene)-;more preferably a bond, -CH2-NH-CO-CH2-O-, -CH2-CO-, andW represents a bond or -NH-(C1-C6 alkylene)-, -(C1-C6 alkylene)-CO- or -CO-; more preferably a bond, -NH-CH2-, -CH2-CO- or -CO-;n represents an integer of 1 to 6, preferably an integer of 1 to 4, more preferably 1 or 2, even more preferably 2;q is an integer from 1 to 10, preferably an integer from 3 to 8;m is an integer from 10 to 100, preferably an integer from 40 to 50; andp is an integer from 10 to 100, preferably an integer from 20 to 50.

9. The antibody-linker-l ipid conjugate according to any one of claims 1 to 8, wherein the antibody is a human IgG, a Fv, a Fab, a F(ab')2, a Fab', a dsFv, a (dsFv)2, a scFv, a sc(Fv)2, a diabody, a bispecific antibody, a multispecific antibody, a single domain antibody, a VHH or a DARPin.

10. A method for preparing an antibody-linker-lipid conjugate according to any one of claims 1 to 9, which comprises:(1) providing an antibody Ab;(2) mixing together in a reaction buffer at least the following components:(a) said antibody provided in step (1);(b) an enzyme; and(c) a compound represented by X^ -R, wherein the R-moiety is capable of reacting, in the presence of said enzyme, with the antibody from step (1) in a site-specific manner, and the moiety X1is capable of reacting in an inverse electron demand Diels-Alder reaction;thereby obtaining an antibody-linker conjugate represented bywherein n is an integer of 1 to 8, preferably an integer of 1 to 4, more preferably 1 or 2, even more preferably 2, and L1is preferably attached to the C-terminus or N-terminus of the polypeptide chain of the antibody Ab;(3) optionally isolating the antibody-linker conjugate from the mixture and purifying the same;(4) providing a lipid-linker conjugate represented by X2-I_2-I_ipid wherein the moiety X2is capable of reacting with the moiety X1in an inverse electron demand Diels- Alder reaction;(5) subjecting the antibody-linker conjugate represented byand the lipid-linker conjugate represented by X2-I_2-I_ipid to an inverse electron demand Diels-Alder reaction, thereby obtaining the antibody-linker-lipid conjugate represented by11. A method according to claim 10, which comprises:(1) providing an antibody,preferably an antibody that comprises(i) a glutamine at position 295, 297 and / or 253 of the heavy chain of the antibody, whereby the antibody is a monoclonal antibody and wherein Eu numbering is used for defining the position of said glutamine, and / or (ii) a glutamine in an transglutaminase recognition motif selected from the group consisting of GGTLQSPP (SEQ ID NO: 1), TLQSG (SEQ ID NO: 2), TLQSPP (SEQ ID NO: 3), GGTLQSG (SEQ ID NO: 4), TLQSA (SEQ ID NO: 5), RLQQP (SEQ ID NO: 6), YELQRPYHSELP (SEQ ID NO: 7), LLQG (SEQ ID NO: 8), GECTYFQAYGCTE (SEQ ID NO: 9), DIPIGQKMTG (SEQ ID NO: 10), DIPIGQGMTG (SEQ ID NO: 11), DIPIGQRMTG (SEQ ID NO: 12), GENTYFQAYGNTE (SEQ ID NO: 13) and TGTLQSVSY (SEQ ID NO: 14), wherein the transglutaminase recognition motif is preferably located at the C-terminus or N-terminus of the polypeptide chain of the antibody; (2) mixing together in a reaction buffer at least the following components:(a) said antibody provided in step (1);(b) a transglutaminase; and(c) a compound represented by X^L^NHz wherein the HjN-moiety is capable of reacting with the antibody from step (1) in the presence of said transglutaminase, and the moiety X1is capable of reacting in an inverse electron demand Diels-Alder reaction;thereby obtaining an antibody-linker conjugate represented byn(3) optionally separating the antibody-linker-conjugate produced in step (2) from unreacted linker and from said transglutaminase, preferably by subjecting said mixture from step (2) to a size-exclusion chromatography;(4) providing a lipid-linker conjugate represented by X2-I_2-I_ipid wherein the moiety X2is capable of reacting with the moiety X1in an inverse electron demand Diels- Alder reaction;(5) subjecting the antibody-linker conjugate represented byand the lipid-linker conjugate represented by X2-I_2-I_ipid to an an inverse electron demand Diels-Alder reaction, thereby obtaining the antibody-linker-lipid conjugate represented by12. The method according to claim 11, wherein said antibody comprises a glutamine at position 295 of the heavy chain of the antibody, whereby the antibody is a monoclonal antibody and wherein Eu numbering is used for defining the position of said glutamine.

13. The method according to any of claims 10 to 12, wherein the mixture in step (2) comprises a molar excess of the compound represented by X1-L1-NHz, preferably at least 2 molar, and more preferably at least 5 molar, per conjugation site, preferably wherein the conjugation site isa glutamine at position 295, 297 and / or 253 of the heavy chain of the antibody, whereby the antibody is a monoclonal antibody and wherein Eu numbering is used for defining the position of said glutamine, and / or(ii) a glutamine in an transglutaminase recognition motif selected from the group consisting of GGTLQSPP (SEQ ID NO: 1), TLQSG (SEQ ID NO: 2), TLQSPP (SEQ ID NO: 3), GGTLQSG (SEQ ID NO: 4), TLQSA (SEQ ID NO: 5), RLQQP (SEQ ID NO: 6), YELQRPYHSELP (SEQ ID NO: 7), LLQG (SEQ ID NO: 8), GECTYFQAYGCTE (SEQ ID NO: 9), DIPIGQKMTG (SEQ ID NO: 10), DIPIGQGMTG (SEQ ID NO: 11), DIPIGQRMTG (SEQ ID NO: 12), GENTYFQAYGNTE (SEQ ID NO: 13) and TGTLQSVSY (SEQ ID NO: 14),wherein the transglutaminase recognition motif is preferably located at the C-terminus or N-terminus of the polypeptide chain of the antibody.

14. The method according to any one of claims 10 to 13, wherein the compound represented by X1-L1-NH2 is selected from TCO-Z-PEGq-V-Amine, BCN-Z-PEGq-V-Amine, MeCyp-Z-PEGq-V-Amine, preferably TCO-Z-PEGq-V-Amine and BCN-Z-PEGq-V-Amine; wherein q is an integer from 1 to 10, preferably an integer from 3 to 8; andV and Z each independently represent a bond or a linker, preferably V and Z are each as defined in claim 8;more preferably the compound represented by X^L^NHz is selected fromTCO-PEGg-Amine, TCO-PEGg-Amine, BCN-PEGg-Amine, BCN-PEGg-Amine, MeCyp-PEGg-Amine, especially TCO-PEGg-Amine, TCO-PEGg-Amine, BCN-PEGg-Amine, BCN-PEGg-Amine.

15. The method according to any one of claims 10 to 14, wherein the antibody-linker conjugate represented byand the lipid-linker conjugate represented by X2-I_2-I_ipid are subjected to the reaction in a molar ratio of X1to X2a. from 1:1 to 1:500, preferably 1:1 to 1:5, more preferably 1:1 to 1:2, most preferably 1:1.5, orb. from 500:1 to 1:1.

16. A process for preparing micelles, liposomes or lipid nanoparticles comprising an antibody-linker-lipid conjugate, which comprises the method according to any one of claims 10 to 15.

17. An antibody-linker-lipid conjugate which is obtainable by a method according to any one of claims 10 to 15.

18. Micelles, liposomes or lipid nanoparticles comprising the antibody linker lipid conjugate according to any one of claims 1 to 9 or 17.

19. A composition comprising the micelles, liposomes or lipid nanoparticles according to claim 18.

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