Patent Description:
Currently, the predominant methods to label and/or to conjugate molecules to proteins, especially, when small-molecule payloads or labels are concerned, involve the chemical conjugation with specific linker molecules that covalently attach the payload to free lysine and/or cysteine amino acids of the proteins.

However, many proteins, like e.g. antibodies that are of particular interest for immunotargeting strategies, are fairly large proteins, that may contain several lysine and cysteine residues. Because linker-mediated, chemical conjugation is a stochastic process, linker-mediated chemical ligation of payloads leads to heterogeneous mixtures of conjugated proteins that may differ in their therapeutic efficacy and/or diagnostic utility. Obviously, mixtures of protein-payload conjugates also represent a significant challenge in the regulatory approval process for therapeutic conjugates, as batch-to-batch variation and/or variations in the active pharmaceutical ingredient (API) are negatively viewed by regulatory authorities due to potential safety concerns.

In addition, if a defined ratio of payload to protein is desired, it is often necessary to purify the conjugate with the desired conjugation stoichiometry. This is not only tedious, but can significantly add to the cost-of-goods in the manufacturing process, as often only a fraction of the linker-mediated conjugated protein represents the desired ratio of payload conjugation. This is particularly true for therapeutically relevant antibody/drug conjugates (ADCs), where depending on the toxin employed, <NUM> to <NUM> toxin molecules appear to be advantageous, but antibodies with no toxin coupled to up to <NUM> toxins per antibody coupled are found in typical linker-mediated chemical conjugation reactions (Panowski et al.

Despite of the limitations described above, all antibody/drug conjugates currently in clinical trials, or approved by the health authorities for the therapy of disease, have been generated by linker-mediated chemical conjugation of toxic small-molecule drugs to antibodies (Lambert (<NUM>) or Mullard (<NUM>)).

It is widely acknowledged in the industry and by scientific experts in the field, that site-specific and stoichiometric conjugation of molecular payloads, including toxin or label molecules to immunoligands would have significant advantages in comparison to chemical, linker-mediated conjugation. This is evidenced by attempts to target the chemical conjugation to specific amino acids in the protein structure (Panowski et al.

On one hand, this is attempted by mutating certain positions in the protein structure to delete unwanted and/or to provide desired conjugation sites (i.e. lysine and/or cysteine residues) to which the linker-ligation can be targeted (McDonagh et al. (<NUM>) or Junutula et al.

On the other hand, control of chemical conjugation to proteins is attempted by incorporation of unnatural amino acids at certain positions, like selenocysteine, p-azidophenylalanine, or acetylphenylalanine (Hofer et al. (<NUM>), Axup et al. (<NUM>), or Lemke (<NUM>)).

However, all of these approaches change the primary amino acid sequence of the protein to be conjugated, and may result in undesired functional properties. Furthermore, the incorporation of unnatural amino acids, as described above, is often low efficient, and does not allow for a quantitative incorporation of specific labeling sites to proteins.

Therefore, there is an urgent need in the industry to overcome the known issues of stochastic conjugation methods in particular for the generation of therapeutically relevant immunoconjugates, including, but not limited to ADCs.

It is thus one object of the present invention to provide an efficient method for conjugating immunoligands and payloads, e.g., drugs, toxins, cytokines, markers, or the like, preferably full-length monoclonal antibodies to small-molecular weight toxins, for the generation of site-specifically conjugated antibody drug conjugates (ADCs).

It is another object of the present invention to create immunoligand/payload conjugates, which have better efficacy and/or can be produced with higher reproducibility.

It is another object of the present invention to allow the conjugation of payloads to immunoligands in a site-specific and/or sequence specific manner.

It is another object of the present invention to create immunoligand/payload conjugates which preserve the characteristic features of its components, e.g., target affinity, target specificity, target sensitivity, solubility, pharmacological function and the like.

These objects are achieved by the subject matter of the independent claims, while the dependent claims as well as the specification disclose further preferred embodiments.

As used herein, the term "immunoligand" is meant to define an entity, an agent or a molecule that has affinity to a given target, e.g., a receptor, a cell surface protein, a cytokine or the like. Such immunoligand may optionally block or dampen agonist-mediated responses, or inhibit receptor-agonist interaction. Most importantly, however, the immonoligand may serve as a shuttle to deliver a payload to a specific site, which is defined by the target recognized by said immunoligand. Thus, an immunoligand targeting, for instance, but not limited to a receptor, delivers its payload to a site which is characterized by abundance of said receptor. Immunoligands include, but are not limited to, antibodies, antibody fragments, antibody-based binding proteins, antibody mimetics, receptors, soluble decoy receptors, scaffold proteins with affinity for a given target and ligands of receptors.

"Antibodies", also synonymously called "immunoglobulins" (Ig), are generally comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, and are therefore multimeric proteins, or an equivalent Ig homologue thereof (e.g., a camelid nanobody, which comprises only a heavy chain, single domain antibodies (dAbs) which can be either be derived from a heavy or light chain); including full length functional mutants, variants, or derivatives thereof (including, but not limited to, murine, chimeric, humanized and fully human antibodies, which retain the essential epitope binding features of an Ig molecule, and including dual specific, bispecific, multispecific, and dual variable domain immunoglobulins; Immunoglobulin molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) and allotype.

An "antibody-based binding protein", as used herein, may represent any protein that contains at least one antibody-derived VH, VL, or CH immunoglobulin domain in the context of other non-immunoglobulin, or non-antibody derived components. Such antibody-based proteins include, but are not limited to (i) Fc-fusion proteins of binding proteins, including receptors or receptor components with all or parts of the immunoglobulin CH domains, (ii) binding proteins, in which VH and or VL domains are coupled to alternative molecular scaffolds, or (iii) molecules, in which immunoglobulin VH, and/or VL, and/or CH domains are combined and/or assembled in a fashion not normally found in naturally occuring antibodies or antibody fragments.

An "antibody drug conjugate" (ADC), as used herein, relates to either an antibody, or an antibody fragment, or and antibody-based binding protein, coupled to a small molecular weight active pharmaceutical ingredient (API), including, but not limited to a toxin (including e.g., but not limited to, tubulin inhibitors, actin binders, RNA polymerase inhibitors, DNA-intercalating and modifying/damaging drugs), a kinase inhibitor, or any API that interferes with a particular cellular pathway that is essential for the survival of a cell and/or essential for a particular physiologic cellular pathway.

An "antibody derivative or fragment", as used herein, relates to a molecule comprising at least one polypeptide chain derived from an antibody that is not full length, including, but not limited to (i) a Fab fragment, which is a monovalent fragment consisting of the variable light (VL), variable heavy (VH), constant light (CL) and constant heavy <NUM> (CH<NUM>) domains; (ii) a F(ab')<NUM> fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a heavy chain portion of a Fab (Fd) fragment, which consists of the VH and CH<NUM> domains; (iv) a variable fragment (Fv) fragment, which consists of the VL and VH domains of a single arm of an antibody, (v) a domain antibody (dAb) fragment, which comprises a single variable domain; (vi) an isolated complementarity determining region (CDR); (vii) a single chain Fv Fragment (scFv); (viii) a diabody, which is a bivalent, bispecific antibody in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementarity domains of another chain and creating two antigen binding sites; and (ix) a linear antibody, which comprises a pair of tandem Fv segments (VH-CH<NUM>-VH-CH<NUM>) which, together with complementarity light chain polypeptides, form a pair of antigen binding regions.

The term "modified antibody format", as used herein, encompasses antibody-drug-conjugates, Polyalkylene oxide-modified scFv, Monobodies, Diabodies, Camelid Antibodies, Domain Antibodies, bi- or trispecific antibodies, IgA, or two IgG structures joined by a J chain and a secretory component, shark antibodies, new world primate framework + non-new world primate CDR, IgG4 antibodies with hinge region removed, IgG with two additional binding sites engineered into the CH3 domains, antibodies with altered Fc region to enhance affinity for Fc gamma receptors, dimerised constructs comprising CH3+VL+VH, and the like.

The term "antibody mimetic", as used herein, refers to proteins not belonging to the immunoglobulin family, and even non-proteins such as aptamers, or synthetic polymers. Some types have an antibody-like beta-sheet structure. Potential advantages of "antibody mimetics" or "alternative scaffolds" over antibodies are better solubility, higher tissue penetration, higher stability towards heat and enzymes, and comparatively low production costs.

Some antibody mimetics can be provided in large libraries, which offer specific binding candidates against every conceivable target. Just like with antibodies, target specific antibody mimetics can be developed by use of High Throughput Screening (HTS) technologies as well as with established display technologies, just like phage display, bacterial display, yeast or mammalian display. Currently developed antibody mimetics encompass, for example, ankyrin repeat proteins (called DARPins), C-type lectins, A-domain proteins of S. aureus, transferrins, lipocalins, 10th type III domains of fibronectin, Kunitz domain protease inhibitors, ubiquitin derived binders (called affilins), gamma crystallin derived binders, cysteine knots or knottins, thioredoxin A scaffold based binders, nucleic acid aptamers, artificial antibodies produced by molecular imprinting of polymers, peptide libraries from bacterial genomes, SH-<NUM> domains, stradobodies, "A domains" of membrane receptors stabilised by disulfide bonds and Ca2+, CTLA4-based compounds, Fyn SH3, and aptamers (oligonucleic acid or peptide molecules that bind to a specific target molecules).

In case the immunoligand is not a protein or a peptide, e.g., if its an aptamer, it should preferably be provided with a peptide tag in order to provide a suitable substrate for the enzymatic conjugation disclosed further herein.

"Conjugation", as used herein, relates to the covalent association of a molecule to another molecule by formation of a covalent bond.

An "immunotoxin", as used herein, relates to an immunoligand conjugated to a protein or polypeptide representing a toxin, including, but not limited to bacterial toxins, e.g. diphteria-toxin A, Pseudomonas exotoxin, botulinum toxin, or e.g. proteinaceous venoms from invertebrates (e.g. but not limited spiders, scorpions, molluscs, jelly-fish), or vetrebrates (e.g., but not limited to snakes), or functional fragments thereof.

The term "low molecular-weight payload" as used herein, represents a payload with a molecular weight not exceeding <NUM>'<NUM> Dalton.

The term "payload", as used herein, represents any naturally occuring or synthetically generated molecule, including small-molecular weight molecules or chemical entities that can chemically be synthesized, and larger molecules or biological entities that need to be produced by fermentation of host cells and that confer a novel functionality to an immunoligand specific for binding to targets or antigens.

The term "small molecular weight toxin", as used herein, means a cytotoxic compound of small molecular weight not exceeding a molecular weight of <NUM>'<NUM> Dalton that is cytotoxic to mammalian cells.

A "transpeptidase", as used herein, is an enzyme or a catalytic domain of an enzyme or a protein that is able to catalyze the breakage of peptide bonds and subsequently either directly, or by way of several reaction intermediates, the formation of novel peptide bonds, such that the energy of the first peptide bond is preserved during the reaction and transfered to a new peptide bond. Preferably, said transpeptidases preferably connect the C-terminus of one peptide or protein with the N-terminus of another peptide or protein. Due to the formation of a new peptide bond, these enzymes or functional domains are also refered to as "protein ligases", "peptide ligases", or nicknamed "protein or peptide staplers". Such protein ligases comprise, but are not limited to sortase enzymes.

As used herein, the term "sequence-specific transpepeptidase" is meant to define a transpeptidase which needs at least one substrate peptide or protein with a given peptide sequence as recognistion sequence (N-terminally and/or C-terminally) to connect said substrate peptide or protein to another peptide or protein, or a small-molecular weight compound containing a peptide or protein component.

As used herein, the term "site-specific transpepeptidase" is meant to define a transpeptidase which has a specific site in at least one substrate peptide or protein which it uses to conjugate to another peptide or protein, or a small-molecular weight compound containing a peptide or protein component.

The invention discloses methods that utilize site-specific transpeptidases, e.g., sortase enzymes, to site-specifically and selectively conjugate payloads, preferably small molecular weight toxins to immunoligands, preferably antibodies, for the generation of immunoligand payloads, preferably antibody drug conjugates (ADCs). The preferred payloads are small molecular weight toxins modified with short, preferably less than <NUM> (thirteen) amino acid long synthetic amino acid sequence, which renders them as substrates for sortase enzymes or split intein mediated covalent conjugation either at the N- or C-terminus of the immunoligands (<FIG> & <FIG>). This conjugation is achieved in a site-specific manner and with defined stoichiometry, which is a distinguishing feature to conventional chemical conjugation of payloads to immunoligands, where the conjugation is a stochastic process, as disclosed further above.

The invention further discloses site specific transpeptidase, e.g., sortase mediated conjugation of multimeric immunoligands, preferably antibodies specifically with two different toxin molecules or other labels using different modifications of the subunits of the multimeric protein, e.g., antibody heavy and light chains, and different payloads modified with different, short amino acid stretches specific for different transpeptidases, in order to conjugate at least two different functional payloads to the multimeric immunoligand (<FIG>).

The invention further discloses methods to add affinity purification and/or detection tags to the N- or C-termini of the immunoligands, which undergo enzyme-mediated transpeptidation. such that the removal of the affinity purification and/or detection tag can be utilized to select for immunologands with complete (<NUM>%) conjugation of the payload to the modified binding protein, by means of affinity resins that retain immunoligands that have not been completely conjugated, and therefore still retain the additional affinity purification and/or detection tag (<FIG>).

The invention further discloses immunoligands in which a catalytic transpeptidase domain is directly fused to the N- or C-terminus of the protein to be conjugated, such that the transpeptidation activity is integral part of immunoligand to be conjugated, and no additional soluble sortase enzyme needs to be provided in the course of the transpeptidase-mediated conjugation reaction (<FIG>).

All of these embodiments mentioned above allow the site-specific and stoichiometrically controlled conjugation of any payload, including small molecule toxins (chemical entities), toxic proteins, or fluorescent labels, preferably small molecular weight toxins to immunoligands, including preferably antibodies, which is superior to standard chemical conjugation of payloads to proteins by chemical linker chemistry methods, which cannot be controlled for conjugation ratio and site. Therefore, for the generation of antibody drug conjugates (ADCs) conjugation of toxic payloads by transpeptidases, preferably sortase enzymes and split inteins to antibodies will lead to more homogeneous products with expected improved therapeutic properties for cancer therapy (<FIG>).

The enzymatic conjugation of payloads to immunoligands by sortase enzymes allows site-specific and stoichiometric payload conjugation to proteins and immunoligands, lowering cost-of-goods and providing homogeneous immunoligand-payload conjugates, especially as the selectivity of the transpeptidases allows the conjugation of payloads to immunoligands in crude cell culture supernatant, and does not require purified components as in traditional linker-mediated chemical conjugation. Therefore, the use of sequence-specific transpeptidases for conjugation of payloads to immunoligands could significantly lower the cost of goods in immunoligand-payload, and particularly ADC manufacturing.

The type of transpeptidase disclosed herein, the sortase enzymes, has been identified in a variety of gram-positive bacteria, like Staphylococcus, Streptococcus and Pneumococcus species, and catalyse the coupling of virulence factors to cell wall proteoglycans, in order to change the surface signature of the bacteria for evading an efficient immune response by the infected host (Mazmanian et al. Sortase A enzyme of the gram-positive bacterium Staphylococcus aureus has been characterized first (Ton-That et al. (<NUM>)) and has subsequently been characterized further as a tool for many protein modifications (Tsukiji (<NUM>)). The attraction of sortase enzymes is that the two molecules to be conjugated only require to be modified or expressed on one hand with a short <NUM> amino-acid long peptide tag (sortase tag, LPXTG in case of Staphylococcus aureus sortase A, X being any of the <NUM> naturally occuring aminoacids), and a short, preferably <NUM> to <NUM> amino acid long glycine stretch (Antos et al. (2009a)) (<FIG>), which can easily be added to each of the molecules to achieve either N-terminal or C-terminal conjugation of proteins. This allows to utilize the system on one hand for the coupling or conjugation of two proteins, but also for the conjugation of smaller molecules to proteins.

Sortase enzymes have been widely described in the prior art for protein-protein or protein-peptide ligations (Mao et al. (<NUM>), Parthasarathy et al. (<NUM>) or <CIT>), even including circularization of proteins (Antos et al. The applications of sortase protein or peptide ligation also included protein or peptide ligation using antibody fragments, like Fab- and scFv-fragments with protein- or peptide labels (Möhlmann et al. (<NUM>), Madej et al. (<NUM>), or <CIT> and <CIT>). Even two prior art documents were published, in which full-length antibodies have been sortase-ligated to proteins (Levary et al. (<NUM>), e.g. EGFP, albumin, gelonin were conjugated to the light chain of an antibody), or in which full-length antibodies have been sortase-ligated to short peptides (Swee et al. However, no prior art document could be identified demonstrating the sortase-mediated conjugation of small-molecular weight toxins, like e.g. auristatins or maytansins and the like, to full-length antibodies or antibody fragments. In particular no prior art documents could be identified, in which generation of ADCs with small molecular weight toxins has been been disclosed resulting in ADCs with small molecular weight toxins homogeneously conjugated to either IgH or IgL chains (drug-to-antibody ratio <NUM>), or to IgH and IgL chains (drug to antibody ratio <NUM>), as disclosed herein.

While the prior art also discloses the modification of non-protein substrates with glycine residues such that they could be used for sortase modification of simple, single-subunit proteins or peptides (Tsukiji (<NUM>), or <CIT>, respectively), the more challenging homogeneous conjugation of non-protein substrates, preferably small molecular weight toxins, to multimeric proteins, preferrably antibodies, has not been described before, despite the fact that sortase enzyme mediated protein or peptide ligation has been in the prior art for many years.

Moreover, the conjugation of multimeric proteins, particularly full-length monoclonal antibodies with two different payloads, preferrably two different small molecular weight toxins as disclosed herein, has not been described in the prior art before, despite the fact that sortase enzyme mediated protein or peptide ligation has been in the prior art for many years (Panowski et al.

It is known from the prior art that sortase enzymes may accept substrates that contain a minimum of <NUM> glycine amino acids (Parthasarathy et al. (<NUM>), therefore the invention may include payloads that contain at least three (<NUM>) glycine amino acid residues added to the payload molecule of interest, although even one or two glycine residues may be sufficient, and should be comprised by the method disclosed herein. In case of small molecular weight payloads the addition of few glycine amino acid residues can be achieved by conventional synthetic peptide chemistry, as described herein. In case of proteins glycine residues can be added either by adding codons for a number of glycine residues, preferably at least three glycine residues, in-frame to the open reading frame of the protein, or by conventional synthetic peptide chemistry such that the recombinant protein contains at least three N-terminal glycine amino acid residues.

It is known from the literature that different Sortase enzymes, e.g. Sortase B from Staphylococcus aureus, or Sortases from other gram-positive bacteria recognize different pentapeptide motifs, which differ from the LPXTG sortase A recognition motif (X = any amino acid) from Staphylococcus aureus (Spirig et al. Therefore, the invention shall also include the concept of adding other sortase recognition motifs to proteins and immunoligands, including preferably antibodies, that differ from the Staphylococcus aureus sortase A recognition motif LPXTG, in order to prepare them for sortase conjugation with different cognate sortase enzyme of different gram-positive bacterial species. Therefore, proteins and immunoligands, preferably antibodies, can also be expressed with a different sortase recognition motif, e.g. a NPQTN pentapeptide motif specific for sortase B from Staphylococcus aureus which can then be conjugated to glycine modified payloads.

In another aspect of the invention, multimeric immunoligands, preferably but not limited to antibodies, which are composed of immunoglobulin heavy and light chains, allow the utilization of said different sortase recognition sequences added to the different polypeptides of such multimeric proteins (in case of antibodies adding different sortase recognition sequences to the antibody heavy and light chains), in order to allow conjugation of different payloads to said different polypeptides by performing sequential conjugations with Glyn-tagged payloads (n ><NUM>) in the presence of the respective sortase enzyme (<FIG>). For this, an antibody needs to be expressed with different C-terminal modifications at heavy and light chains comprising different sortase recognition motifs for different sortase enzymes. Such an antibody can then sequentially be conjugated to two different payloads containing a glycine modification as described further above.

This format may have the advantage that ADCs specifically be loaded with two different toxins, preferably interfering with a different cellular pathway will be more potent in cancer cell killing, because it is more difficult for a targeted cancer cell to evade the attack of two toxins comprised in the ADCs.

It is clear to a person skilled in the art, that a sortase pentapeptide recognition motif, like the Staphylococcus aureus sortase A LPXTG motif, can be added selectively to individual polypeptides of multimeric immunoligands, in order to provide desired conjugation sites. For instance, in the case of antibodies, this allows the generation of modified antibodies, either only containing sortase recognition motifs added to the heavy chains (resulting in two payloads per antibody conjugation), or only containing sortase recognition motifs added to the light chains (resulting in two payloads per antibody conjugation), or containing sortase recognition motifs added to the heavy and the light chains (resulting in four payloads per antibody conjugation). These designed variations will allow specific conjugation of payloads to antibodies by sortase enzymes either to the heavy chains alone (generating ADCs with drug to antibody ratio of <NUM>, i.e., DAR2), or to the light chains alone (generating ADCs with drug to antibody ratio of <NUM>, i.e., DAR2), or simultaneously to the heavy and the light chains (generating ADCs with drug to antibody ratio of <NUM>, i.e., DAR4). This way, the conjugation sites and stoichiometries for antibodies can be varied in a controlled fashion, either generating two payload conjugations per antibody heavy or light chain, or generating four payload conjugations per antibody by addition of the payload to the heavy and the light chains.

Similar to the above-described variations in conjugation sites and stoichiometries using different sortase recognition motifs and sortase enzymes in multimeric proteins or immunoligands, it is a further aspect of the invention to conjugate different payloads to different polpeptide chains of multimeric proteins combining sortase-mediated and split-intein mediated conjugation. This concept allows the simultaneous conjugation of different payloads to different polypeptide chains of multimeric proteins and immunoconjugates in one step, because different transpeptidases and substrates are being employed (<FIG>).

It is to be understood that the above-mentioned conjugation of two different payloads to a multimeric protein, preferably an antibody, which is composed of each two disulphide linked heavy and lights chains, can be accomplished by sortase enzyme mediated conjugation, but that it is also possible to conjugate two different payloads to a multimeric protein, preferably an antibody, by utilizing two different sortase enzymes, recognizing different sortase peptide motifs, for instance sortase A and sortase B from Staphylococcus aureus, as mentioned further above (<FIG>). However, this may also include sortase enzymes of other sortase classes (e.g. sortases C, D, E, F), or sortase enzymes from other bacterial species, differing in their sortase motif specificity.

Sortase-mediated conjugation of payloads to proteins and immunoligands can be achieved either by providing sortase recognition motif tagged proteins and at least tri-glycine tagged payloads and adding enzymatically active sortase enzyme or a functional fragment thereof as a soluble enzmye. In another aspect of the invention the enzymatically active domain of sortase enzyme can also be provided as a domain fused to either the N- or C-terminus of the protein. In this variation, is is advantageous, but not mandatory, to add the sortase enzymatic domain either N-terminal to an N-terminal sortase recognition motif, or C-terminal to a C-terminal sortase recognition motif. Both possibilities ensure that the after the reaction with a glycine-tagged payload, that the enzymatic sortase domain is removed from the protein in the course of the reaction (<FIG>).

Sortase-mediated conjugation of payloads can be performed at either the N- or the C-termini of proteins and immunoligands. This is only dependent on how the sortase-motif/glycine stretch are positioned at protein and payload (<FIG>).

In the case of antibodies, which are the preferred immunoligands, it is preferred to conjugate the payloads to the C-termini of the antibodies, because this positions the payloads most distally to the antigen-binding sites of the antibody. However, this preference shall not be interpreted by way of limitation, and it may be advantageous to conjugate payloads to the N-terminus of other immunoligand molecules, like e.g. antibody mimetics, in which the functional binding domains are not located at the N-terminus of the molecule.

Another aspect of the invention is to improve the efficiency of sortase conjugation of payloads to proteins and immunoligands by adding affinity purification or detection tags, like e.g., but not limited to small peptide tags (e.g. histidine tags, strep-tag, MYC-tag or HA-tag) or larger protein affinity purification tags (e.g. maltose-binding protein (MBP) tag, Glutathione-S-transerase (GST) tag, or Chitin-binding tag) distal to the sortase recognition motif.

With this aspect of the invention the affinity purification tag will be removed from the immunoligand to be conjugated as part of the transpeptidation reaction. This can be exploited to enrich fully payload conjugated immunoligands, as unreacted proteins and immunoligands, that still contain the affinity purification tag, can be removed by binding to a suitable affinity resin, while completely payload conjugated proteins and immunoligands will no longer contain the affinity purification tag, and can thus be specifically separated from the unreacted immunoligand substrates. This aspect of the invention is particularly powerful in the context of multimeric proteins and immunoligands, like the preferred antibodies, in which several payloads need to be conjugated. The use of affinity purification tags located distal to the sortase transpeptidase conjugation site ensures that one can remove proteins and immunoligands in which the affinity purification tag is still present due to incomplete payload conjugation (<FIG>).

In comparison to chemical conjugation, this provides a significant advantage in the process to obtain homogeneous immunoligand/payload conjugates, and preferably ADCs in which small molecular weight toxins are site specifically conjugated to the C-termini of antibody heavy and/or light chains.

Generally, the disclosed method provides a novel and efficient method to site-specifically and stoichiometrically conjugate payloads, preferably small molecular weight toxins to immunoligands, preferably antibodies, by which defined immunoligand/payload conjugates, preferably ADCs are generated, that are useful for the therapy of diseases, preferably of cancer. The method may also be utilized for the generation of immunoligand/payload conjugates useful for the diagnosis of diseases, preferably oncology diseases. The novel method allows generation covalent immunoligand/payload conjugates by utilization of peptide-bond breaking and forming enzymes (transpeptidases), including sortase enzymes, or catalytically active fragments thereof. Said enzymes can catalyze the covalent and site-specific conjugation of payloads containing short amino acid stretches (preferably shorter than <NUM> amino acids) either to the N- or C-termini of immunoligands which are suitably modified allowing sortase to break and to form peptide bonds in the course of the reaction. Immunoligands are preferably antibodies, for the site-specific conjugation of small molecular weight toxins, in order to generate antibody drug conjugates (ADCs) with defined antibody payload, or drug to antibody ratios.

According to the invention, a method of producing an immunoligand/payload conjugate is disclosed, which method encompasses conjugating at least one payload to an immunoligand by means of a sequence-specific transpeptidase, or a catalytic domain thereof, wherein the immunoligand comprised in the immunoligand/payload conjugate is at least one selected from the group consisting of an antibody, antibody fragment, antibody-based binding protein, or an antibody mimetic, wherein.

Disclosed herein, but not as part of the invention, it is provided that the payload and/or the immunoligand either.

and, further, the protein or peptide or domain comprises, preferably, an amino acid sequence that can be detected by the sequence-specific transpeptidase, or a catalytic domain thereof.

This means, for example, that, in case the payload and/or the immunoligand is a protein, it means that said protein comprises, at its N- or C-terminus, an amino acid sequence which can be detected by the sequence-specific transpeptidase. If such amino acid sequence is lacking to the naive protein, it can be fused to the N- or C-terminus of said protein by recombinant methods known in the art.

In case the payload and/or the immunoligand is not a protein, such amino acid sequence which can be detected by the sequence-specific transpeptidase, is to be conjugated to the former by conventional chemical crosslinking methods known in the art.

Additional functionalities may be incorprated between the recognition sequence for a specific transpeptidase and the payload. This can be realized by chemical structures either being categorized by being cleavable (e.g. containing hydrazone, or disulfide chemistry, or specific peptide sequences for intracellular proteases) or being non-cleavable (e.g. containing thioether chemistry) following internalization into cells.

Chemical structures containing hydrazone chemistry can selectively be cleaved within the intracellular compartment of lysosomes (lower pH compared to the systemic blood circulation).

Peptide linkers have the potential to be selectively cleaved by lysosomal proteases (e.g. cathepsin-B) and have demonstrated increased serum stability and improved anti-tumor effects compared to hydrazone linkers. Valine-citruline (Val-Cit) pairs are the most commonly used peptide linkers and are ideally suited to work with the auristatin family of drugs such as monomethyl auristatin E (MMAE).

Non-cleavable linkers have long been overlooked as researchers were convinced the cleaving of the linker was the most reasonable way to free the drug. However, conjugates can, upon binding to a membrane receptor, get rapidly internalized and once internalized, the immunoligand can be degraded to the point where the payload, e.g., the drug is exposed. As one prominent example, thioether linkers, use the SMCC (N-succinimidyl-<NUM>-(N-maleimidomethyl)- cyclohexane-<NUM>-carboxylate) linker (See <FIG>, structure <NUM>).

All of these approaches have in common that there is no true site-specificty of the coupling reaction. Because linker-mediated, chemical conjugation is a stochastic process, linker-mediated chemical ligation of payloads leads to heterogeneous mixtures of conjugated proteins that may differ in their therapeutic efficacy and/or diagnostic potential. Obviously, mixtures of protein-payload conjugates also represent a significant challenge in the regulatory approval process for therapeutic conjuagtes, as batch-to-batch variation and/or variations in the active pharmaceutical ingredient (API) are negatively viewed by regulatory authorities due to potential safety concerns.

Preferably, in this embodiment, a small molecular payload is rendered as substrate for the sequence-specific transpeptidase by coupling of a peptide of less than <NUM> amino acids to the small molecular payload, such that it can be conjugated by a transpeptidase to the C-termini of a monoclonal antibody containing C-terminal modifications recognized by said transpeptidases. Such C-terminal modifications may be contained on either both heavy chains, or both light chains, or of heavy and light chains of a full-length antibody, thereby allowing generation of a site-specifically conjugated ADC with either drug-to-antibody ratio of <NUM> or <NUM> (DAR2 or DAR4).

Disclosed herein, but not as part of the invention, it is provided that the immunoligand binds at least one entity selected from the group consisting of.

As used herein, the term "receptor" means a cell surface molecule, preferably a cell surface molecule that (i) binds specific, or groups of specific, signalling molecules (i.e. a receptor, like, e.g., the VEGF receptor), and/or (ii) has no known ligand (i.e. an orphan receptor, like, e.g. HER2/neu). The natural receptors are expressed on the surface of a population of cells, or they merely represent the extracellular domain of such a molecule (whether such a form exists naturally or not), or a soluble molecule performing natural binding function in the plasma, or within a cell or organ. Preferably, such receptor is a member of a signalling cascade that is involved in a particular pathogenic process (e.g., a receptor that belongs to a signalling cascade of a growth factor), or is expressed on the surface of a cell or particle that is involved in a pathological process, e.g., a cancer cell.

As used herein, the term "antigen" means a substance that has the ability to induce a specific immune response, and may include surface proteins or protein complexes (e.g. ion channels). Often times, antigens are associated to pathogenic entities, e.g., a cancer cell.

As used herein, the term "cytokine" refers to small cell-signaling protein molecules that are secreted by numerous cells and are a category of signaling molecules used extensively in intercellular communication. Cytokines can be classified as proteins, peptides, or glycoproteins; the term "cytokine" encompasses a large and diverse family of regulators produced throughout the body by cells of diverse embryological origin.

As used herein, the term "growth factor" relates to naturally occurring substances capable of stimulating cellular growth, proliferation and cellular differentiation. Usually a growth factor is a protein or a steroid hormone. Growth factors are important for regulating a variety of cellular processes.

As used herein, the term "hormone" relates to a chemical released by a cell, a gland, or an organ in one part of the body that sends out messages that affect cells in other parts of the organism. The term encompasses peptide hormones, lipid and phospholipid-derived hormones including steroid hormones, and monoamines.

In case the immunoligand binds a receptor or an antigen, the immunoligand-payload conjugate can for example be directed to a specific site, e.g., to a pathogenic entity, e.g., a cancer cell, where the payload, e.g. a toxin or a chemotherapeutic agent, is delivered. Thus, the systemic toxicity of the toxin or the chemotherapeutic agent is reduced, while the local concentration of the latter at the site of action is increased, thus provding a better efficacy while side effects are reduced. Furthermore, a respective signalling cascade can be inhibited by the binding of the immunoligand. In case the payload is a marker the latter can thus be used to mark a specific site, e.g., a cancer cell charcterized by a given surface antigen detected by the immunoligand, for diagnosis.

In case the immunoligand binds a growth factor, a cytokine, and/or a hormone, the immunologand/payload conjugate can for example be directed to the site the growth factor cytokine or hormone usuall binds to, in oder to deliver the payload in a site-specific manner. Further, a respective signalling cascade can be inhibited by the binding of the immunoligand.

As used herein, the term "to bind" means the well-understood interaction or other nonrandom association between immunoligands, e.g., antibodies, or antibody fragments, and their targets. Preferably, such binding reaction is characterized by high specifity and/or sensitivity to the target. Preferably, the binding reaction is characterized by a dissociation constant (Kd) ≤ <NUM>-<NUM> M, preferably ≤ <NUM>-<NUM> M, ≤ <NUM>-<NUM> M, ≤ <NUM>-<NUM> M, ≤ <NUM>-<NUM> M, ≤ <NUM>-<NUM> M, ≤ <NUM>-<NUM> M, and most preferred ≤ <NUM>-<NUM>.

Acccording to a preferred embodiment of the invention, it is provided that at least one catalytic domain of the sequence-specific transpeptidase is fused to the C-terminus of the immunoligand.

Such fusion may take place by recombinant engineering, or by chemical coupling. In this embodiment, the enzymatic activity leading to the site-specific conjugation of the immunoligand to the payload does not need to be added to the reaction as a separate recombinant enzyme, but is rather part of protein substrate to be conjugated.

Disclosed herein, but not as part of the invention, it is provided that where the transpeptidase is a sortase, the payload, e.g., a toxin, is preferably rendered as substrate for sortase conjugation by addition of a small number of glycine amino acid residues, preferably <NUM> or <NUM> glycine residues.

The use of transpeptidases, preferably sortase enzymes for the generation of antibody drug conjugates, in which small molecular weight toxins are conjugated to full-length antibodies, has not yet been described in the prior art (Panowski et al.

Sortase enzymes have been identified in a variety of gram-positive bacteria, like Staphylococcus, Streptococcus and Pneumococcus species, and catalyze, in vivo, the coupling of virulence factors to cell wall proteoglycans, in order to change the surface signature of the bacteria for evading an efficient immune response by the infected host (Mazmanian et al.

The sortase A enzyme of the gram-positive bacterium Staphylococcus aureus has been characterized first (Ton-That et al. (<NUM>)) and has subsequently been characterized further as a tool for many protein modifications (Tsukiji (<NUM>)).

One beneficial feature of sortase enzymes is that the two molecules to be conjugated only require short peptide tags ("sortase tags"), which in case of Staphylococcus aureus sortase A is for example LPXTG at the C-terminus of one molecule (e.g., the payload), and a short <NUM> to <NUM> amino acid glycine stretch at the N-terminus of the other molecule (e.g., the immunoligand, see <FIG>). These peptide tags can either be fused to the molecules, or conjugated thereto by means of conventional crosslinking chemistry. This allows to utilize the system on one hand for the ligation of two proteins, but also for the conjugation of small molecular weight compounds, preferably small molecular weight toxins to proteins. In case of Staphylococcus aureus sortase B, the respective sortase motif is NPQTN.

In case of sortase enzymes addition of a short glycine stretch (> <NUM> glycine residues) to a molecule of choice is sufficient to allow the molecule to be conjugated to immunoligands containing a penta-peptide sortase recognition motif, like e.g. LPXTG in case of sortase A of S.

Other than chemical conjugation, the transpeptidase-mediated conjugation occurs under physiologic aqueous buffer conditions and physiologic temperatures, thereby minimally affecting the protein or antibody integrity in the conjugation reaction. This feature ensures optimal functionality of the resulting conjugate.

Disclosed herein, but not as part of the invention, it is provided that the payload comprised in the immunoligand/ payload conjugate is at least one selected from the group consisting of.

The term "marker" (also called "detection tag"), as used herein, may refer to any molecule or moiety that comprises one or more appropriate chemical substances or enzymes, which directly or indirectly generate a detectable compound or signal in a chemical, physical or enzymatic reaction.

The term "processing tag" as used herein, may encompass affinity tags, solubilization tags, chromatography tags and epitope tags. Affinity tags (also used as purification tags) are appended to proteins so that they allow purifification of the tagged molecule from their crude biological source using an affinity technique. These include chitin-binding protein (CBP), maltose binding protein (MBP), and glutathione-S-transferase (GST). The poly(His) tag, preferably a 6xHis tag, is a widely-used processing tag; it binds to metal matrices. Solubilization tags are used, especially for recombinant proteins expressed in chaperone-deficient species such as E. coli, to assist in the proper folding in proteins and keep them from precipitating. These include thioredoxin (TRX) and poly(NANP). Some affinity tags have a dual role as a solubilization agent, such as MBP, and GST.

Chromatography tags are used to alter chromatographic properties of the protein to afford different resolution across a particular separation technique. Often, these consist of polyanionic amino acids, such as FLAG-tag.

Epitope tags are short peptide sequences which are chosen because high-affinity antibodies can be reliably produced in many different species. Epitope tags are usually derived from viral genes, which explain their high immunoreactivity. Epitope tags include e.g. the V5-tag, MYC-tag, and HA-tag. These tags are particularly useful for western blotting, immunofluorescence and immunoprecipitation experiments, although they also find use in protein purification.

Processing tags find many other usages, such as specific enzymatic modification (such as biotin ligase tags) and chemical modification (FlAsH) tag. Often tags are combined to produce multifunctional modifications of the protein.

Disclosed herein, but not as part of the invention, it is provided that said marker is at least one selected from the group consisting of.

This enumeration of potential marker payloads is by no means restrictive. Disclosed herein, but not as part of the invention, it is provided that, said drug is at least one selected from the group consisting of.

As used herein, the term "cytokine" refers to small cell-signaling protein molecules that are secreted by numerous cells and are a category of signaling molecules used extensively in intercellular communication. Cytokines can be classified as proteins, peptides, or glycoproteins; the term "cytokine" encompasses a large and diverse family of regulators produced throughout the body by cells of diverse embryological origin. In the present context, cytokines are for example meant to impair, or even kill, pathogenic entity, e.g., a cancer cell.

As used herein, the term "radioactive agent" relates to an entity which has at least one atom with an unstable nucleus, and which is thus prone to undergo radioactive decay, resulting in the emission of gamma rays and/or subatomic particles such as alpha or beta particles, which have a cell killing effect. In the present context, radioactive agents are meant to impair, or even kill, pathogenic entity, e.g., a cancer cell.

As used herein, the term "anti-inflammatory drug" relates to compounds that reduce inflammation. This can be, e.g., steroids, just like specific glucocorticoids (often referred to as corticosteroids), which reduce inflammation or swelling by binding to glucocorticoid receptors. The term further encompasses non-steroidal anti-inflammatory drugs (NSAIDs), which counteract the cyclooxygenase (COX) enzyme. On its own, COX enzyme synthesizes prostaglandins, creating inflammation. In whole, the NSAIDs prevent the prostaglandins from ever being synthesized, reducing or eliminating the pain. The term further encompasses Immune Selective Anti-Inflammatory Derivatives (ImSAIDs), which are a class of peptides that alter the activation and migration of inflammatory cells, which are immune cells responsible for amplifying the inflammatory response.

As used herein, the term "toxin" relates to a molecule which is toxic to a living cell or organism. Toxins may be peptides, or proteins or preferably small molecular weight compounds, that are meant to impair, or even kill, pathogenic entity, e.g., a cancer cell. Toxins, as meant herein, encompass, in particular, cellular toxins. Preferably, said toxin is a small molecular toxin, i.e., having a molecular weight of ≤ <NUM> Da.

As used herein, the term "chemotherapeutic agent" relates to molecules that have the functional property of inhibiting a development or progression of a neoplasm, particularly a malignant (cancerous) lesion, such as a carcinoma, sarcoma, lymphoma, or leukemia. Inhibition of metastasis or angiogenesis is frequently a property of anti-cancer or chemotherapeutic agents. A chemotherapeutic agent may be a cytotoxic or chemotherapeutic agent. Preferably, said chemotherapeuic agent is a small molecular weight cytostatic agent, which inhibits or suppresses growth and/or multiplication of cancer cells.

Conjugating cytokines, radioactive agents, toxins or chemotherapeutic agents to an immunologand can help to reduce side effects and risks related to their administration, because.

The following table is a non restrictive list of potential targets/antigens (<NUM>st column) and examples for existing immunoligands targeting the former (<NUM>nd column). The <NUM>rd column shows a non restrictive list of potential toxins, cytokines or chemotherapeutic agents. Note that the examples from the <NUM>st and the <NUM>rd column can be combined with one another ad libitum, while hundereds of further targets and payloads exist. Respective target/payload combinations not explicitly mentioned in the table are encompassed by the scope of the present invention.

Acccording to a preferred embodiment of the invention, it is provided that said immunoligand comprises at least two subunits each being conjugated to a cytotoxic compound not exceeding a molecular weight of <NUM>'<NUM> Daltons that is cytotoxic to mammalian cells and impairs and kills cancer cells.

Preferably, at least two different payloads can be conjugated to the at least two subunits. This option provides a versatile toolbox with which a large variety of different immunoligand-payload constructs can be created. For example, a bispecific dual-domain immunoligand can be conjugated with two different payloads, for example one marker and one toxin.

Preferably the at least two different payloads are toxic payloads interfering with one or more cellular pathways.

Such embodiment can be accomplished, e.g., by conjugating the two different payloads to each the <NUM> light chains of a full-length antibody, and to the <NUM> heavy chains of a full length antibody, respectively, by utilizing two different sortase enzymes, recognizing different sortase recognition motifs, plus an antibody that contains different C-terminal modifications at heavy and light chains comprising the respective recognition motifs for said different sortase enzymes.

In such way, an Antibody Drug Conjugate can be created which is composed of each two full-length Ig light chains and Ig heavy chains, containing different payloads covalently attached to said heavy and light chains.

Such embodiment results, preferably, in the synchronous conjugation of the at least two subunits for the generation of immunoligand payloads with equal payload conjugation to each of said subunits.

Disclosed herein, but not as part of the invention, it is provided that said immunoligand with at least two subunits is being conjugated with at least <NUM>% efficiency per conjugation site.

The present invention further relates to said immunoligand with at least two subunits which contains a peptide spacer sequence of at least two amino acids, preferably <NUM>-<NUM> amino acids, appended to the C-termini of at least one of the two subunits.

This approach results, advantageously, in synchronous conjugation of the at least two subunits for the generation of immunoligand payloads with equal payload conjugation to each of said subunits.

Acccording to a preferred embodiment of the invention, the method allows a stoichiometrically defined relationship between immunoligand and payload.

Disclosed herein, but not as part of the invention, it is provided that a strict quantitative relationship between immunoligand and payload can be provided, thus improving the reproducibility and the overall performance of the respective immunoligand/payload conjugate particularly for clinical and/or therapeutic applications. This is accounted for by the sequence- and/or site specificity of the transpeptidase used.

According to a particularly prefered embodiment, said stoichiometrically defined relationship between immunoligand and payload is achieved by removal of partially reacted C-terminally modified immunoligand substrate.

Such removal can, for example, be carried out via affinity purification. Said approach results, preferably, in a homogeneous drug to immunoligand ratio.

According to a particularly prefered embodiment of the invention, the immunoligand has an affinity purification tag distal to the sortase recognition motif fused to the protein or immunoligand, preferably wherein the affinity purification tag is removed from the immunoligand as part of the transpeptidase reaction.

Preferably, said removal is carried out by affinity purification using an affinity tag positioned C-terminal to the transpeptidase recognition motif or domain. Standard methods known to the skilled person can be used for this purpose, e.g., HIS tag, CBP tag, CYD (covalent yet dissociable NorpD peptide) tag, Strep II tag, FLAG tag, HPC (heavy chain of protein C) tag, and the GST and MBP protein fusion tags.

According to another embodiment of the present invention, the method allows a site-specific conjugation of a payload to the immunoligand.

Disclosed herein, but not part of the invention, it is provided that the conjugation process does not interfere with the activity of the immunoligand, or the payload, itself, thus improving the reproducibility and the overall performance of the respective immunoligand/payload conjugate particularly for clinical and/or therapeutic applications. This is accounted for by the sequence- and/or site specificity of the transpeptidase used. Other than with conventional binding chemistry, which is not site specific in most cases, or has limited site specificity (e. g, when the payload is conjugated to a free amino group, like in Arg, Lys, Asn or Gln), the binding site can thus be exactly determined, so that the characterizing features of the immunoligand (e.g., target specificity) or the payload (e.g., toxicity) are not affected.

The present invention further relates to an immunoligand/payload conjugate which is obtainable with said method as described above, wherein said conjugate comprises an antibody, antibody fragment, antibody-based binding protein, or an antibody mimetic, wherein.

The present invention further relates to an immunoligand/payload conjugate which is obtainable with said method for use in the treatment of a human or animal subject suffering from a neoplastic disease.

In all these cases, the immunoligand/payload conjugate according to the invention can have beneficial effects, e. g, by directing the latter to a specific site, e.g., a cancer cell, a site of neuropathology, or a site of an autoimmune reaction.

The payload, e.g., a toxin, a chemotherapeutic agent, a cytokine or a drug is delivered at said site, e.g., to deplete a cancer cell, to act anti-proliferatively on a cancer cell, and the like.

In all these cases, the immunoligand/payload conjugate according to the invention can have beneficial effects, e. g, by directing the latter to a specific site, e.g., a cancer cell, where the payload, e.g. a toxin or a chemotherapeutic agent, is delivered, e.g., to deplete a cancer cell, to act anti-proliferatively on a cancer cell.

Thus, the systemic toxicity of the toxin or the chemotherapeutic agent is reduced, while the local of the latter at the site of action is increased, thus provding a better efficacy while side effects are reduced. Further, a respective signalling cascade can be inhibited by the binding of the immunoligand. In case the payload is a marker the latter can thus be used to mark a specific site, e.g., a cancer cell charcterized by a given surface antigen detected by the immunoligand, for diagnosis.

The site-specifity of the conjugating process ensures a high reproducibility and overall performance of the respective immunoligand/payload conjugate particularly for clinical and/or therapeutic applications.

The term "neoplastic disease", as used herein, refers to an abnormal state or condition of cells or tissue characterized by rapidly proliferating cell growth or neoplasm. In a more specific meaning, the term relates to cancerous processes, e.g., tumors and/or leukemias.

The term "neuropathological diseases" encompasses, among others, neurodegenerative diseases, neuroinflammatory diseases or seizure disorders.

Neurodegenerative diseases are characterized by progressive loss of structure or function of neurons, including death of neurons. Many neurodegenerative diseases including Parkinson's, Alzheimer's, Huntington's, Amyotrophic lateral sclerosis and Multiple Sclerosis occur as a result of neurodegenerative processes. There are many parallels between different neurodegenerative disorders including atypical protein assemblies as well as induced cell death. Neurodegeneration can further be found in many different levels of neuronal circuitry ranging from molecular to systemic.

The terms "Neurodegenerative diseases" and "Neuroinflammatory diseases" have a partially overlapping scope. Inflammatory responses are a hallmark of neurodegenerative disease and participate, or contribute, through different mechanisms in the neuronal cell death. The tryptophan catabolism along the Kynurenine pathway (KP) represents one of these mechanisms.

Seizure disorders are brain disorders which are characterized by abnormal signaling between brain cells. Seizure disorders can affect part of the brain (Partial seizures) or the entire brain (Generalized seizures). The most prominent Seizure disorder is epilepsy.

The term "Autoimmune disease", as used herein, encompasses organ-specific autoimmune diseases, in which an autoimmune response is directed against a single tissue, such as Crohn's disease and ulcerative colitis, Type I diabetes mellitus, myasthenia gravis, vitiligo, Graves' disease, Hashimoto's disease, Addison' s disease and autoimmune gastritis and autoimmune hepatitis. The term also encompasses non-organ specific autoimmune diseases, in which an autoimmune response is directed against a component present in several or many organs throughout the body.

Such autoimmune diseases include, for example, rheumatoid arthritis, disease, systemic lupus erythematosus, progressive systemic sclerosis and variants, polymyositis and dermatomyositis.

Additional autoimmune diseases include pernicious anemia including some of autoimmune gastritis, primary biliary cirrhosis, autoimmune thrombocytopenia, Sjögren's syndrome, multiple sclerosis and psoriasis. One skilled in the art understands that the methods of the invention can be applied to these or other autoimmune diseases, as desired.

The term "infectious disease" as used herein, includes, but is not limited to any disease that is caused by an infectious organism. Infectious organisms may comprise viruses, (e.g., single stranded RNA viruses, single stranded DNA viruses, human immunodeficiency virus (HIV), hepatitis A, B, and C virus, herpes simplex virus (HSV), cytomegalovirus (CMV) Epstein-Barr virus (EBV), human papilloma virus (HPV)), parasites (e.g., protozoan and metazoan pathogens such as Plasmodia species, Leishmania species, Schistosoma species, Trypanosoma species), bacteria (e.g., Mycobacteria, in particular, M. tuberculosis, Salmonella, Streptococci, E. coli, Staphylococci), fungi (e.g., Candida species, Aspergillus species), Pneumocystis carinii, and prions.

The present invention further relates to a use of a low molecular-weight cytotoxic compound not exceeding a molecular weight of <NUM>'<NUM> Daltons for conjugation thereof by means of a sortase enzyme to an immunoligand selected from the group consisting of an antibody, antibody fragment, antibody-based binding protein, or an antibody mimetic, wherein.

The present invention also further relates to said method of producing an immunoligand/payload conjugate as described above, wherein the immunoligand-payload conjugation is performed in crude cell culture supernatant.

As used herein, the term "Glyn-modification" means that an oligo- or polypepide consisting of n Glycin residues has been added to said payload. As used herein, the term "low molecular-weight payload compound" shall encompass payloads that have a molecular weight of <NUM> Da or less.

Said payload is, preferably, at least one selected from the group consisting of.

Said marker is at least one selected from the group consisting of.

Said drug is at least one selected from the group consisting of.

As disussed above already, said toxin is preferably a small molecular toxin, i.e., having a molecular weight of ≤ <NUM> Da. Preferably, said toxin is at least one selected from the group constisting of.

or derivatives of the former. Examples for such Glyn-modified toxions are shown in structures <NUM> to <NUM> of <FIG>.

Preferably, and as mentioned above, the conjugation is a transpeptidease-mediated conjugation, preferably with a sortase. Likewise preferably, the immunologand is an antibody.

Preferably, said immunoligand is an antibody. In such way, an antibody drug conjugate (ADC) can be provided.

Preferably, the immunologand-payload conjugation reaction is performed in crude cell culture supernatant. This means that, preferably, the conjugation reaction may take place with unpurified or only partially purified components.

The invention has been illustrated and described in detail in the drawings and foregoing description.

All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown <NUM>'-><NUM>'.

In order to perform the C-terminal conjugation of a payload to an antibody, first a recombinant antibody needs to be expressed that contains C-terminal modifications, including a recognition motif, e.g. for sortase A of Staphylococcus aureus.

For this, first ORFs for heavy and light chains of an anti-human CD19 specific antibody can be gene synthesized, e.g. at contract research organizations (CROs) offering such gene synthesis services, like e.g. Genscript (www. com, Piscataway, NJ, USA). As an example, the heavy and light chain sequences of a humanized anti-human CD19 antibody hBU12 can be found in patent <CIT> under Seq <NUM> (variant HF) and Seq <NUM> (variant LG). The VH and VL regions of this anti-human CD19 antibody are as follows:.

These sequences can be fused to human IgG<NUM> constant heavy and constant light chain regions containing additional C-terminal tags, in order to realize the method disclosed herein.

In order to realize the invention, the human constant IgG1 heavy chain region can be synthesized with additional <NUM>'-codons, encoding an LPETG Staphylococcus aureus sortase A recognition tag, followed by a 6xHis tag (HHHHHH), a MYC-tag (EQKLISEEDL) and a strep II tag (WSHPQFEK) resulting in a sequence, which is as follows:.

Furthermore, the human constant IgG1 kappa light chain region can be synthesized with additional <NUM>'-codons, encoding an LPETG Staphylococcus aureus sortase A recognition tag, followed by a 6xHis tag and a strep II tag (WSHPQFEK) resulting in a sequence, which is as follows:.

The complete coding regions for LPETG sortase tag, 6xHis and strepII tagged heavy and light chains of the humanized anti-human CD19 antibody hBU12 are then as follows:.

The coding regions for the heavy and light chains of the anti-human CD19 specific antibody as disclosed in SEQ ID NOs <NUM> and <NUM>, respectively, can then be synthesized with flanking restriction enzyme sites (e.g. HindIII and NotI) such that they can be cloned into a standard mammalian expression vector, such as pCDNA3. <NUM>-hygro (+) (Invitrogen), by standard molecular biology methods known in the art.

The complete DNA sequence of pCDNA3. <NUM>-hygro (+)-IgH chain expression vector for the tagged hBU12 anti-human CD19 antibody will be as follows:
SEQ ID NO <NUM> (coding region of human IgG1 VH-CH heavy chain for hBU12 with C-terminal LPETG sortase tag, 6xHis tag and a strepII tag underlined, and HindIII and NotI cloning sites shaded):
<IMG>
<IMG>.

The complete DNA sequence of pCDNA3. <NUM>-hygro (+)-IgL chain expression vector for the tagged hBU12 anti-human CD19 antibody will be as follows:
SEQ ID NO <NUM> (coding region of human IgG1 VL-CL kappa light chain for hBU12 with C-terminal LPETG sortase tag, 6xHis tag, Myc tag, and a strepII tag underlined, and HindIII and NotI cloning sites shaded):
<IMG>
<IMG>.

These constructs allow upon transfection into mammalian cells, like e.g. - but not limited to -CHO cells, that are typically used for recombinant antibody expression, the expression of the anti-human CD19 specific humanized antibody hBU12 with C-terminal additions of a sortase A tag, a 6xHis tag, a Myc tag, and a strepII tag at both the IgH and IgL chains.

Similar to the design of expression cassettes and vectors of Staphylococcus aureus sortase A tagged IgG1 heavy and light chains, the coding regions for a C-terminal fusion of N-intein domain of Ssp GyrB <NUM> split-intein to either the IgH and IgL chain can be designed as follows, in order to gene synthesize the genes by a qualified CRO (e.g. Genscript (www. com, Piscataway, NJ, USA), with the same elements for the anti-human CD19 antibody as disclosed further above.

The <NUM> amino acid sequence of the N-intein domain of Ssp GyrB <NUM> split-intein can be found in a publication by Appleby et al. (<NUM>), and is as follows:
<IMG>.

Reverse translation of that amino acid sequence with mammalian codon usage will result in the coding sequence for the N-intein domain of Ssp GyrB <NUM> split-intein as follows:
<IMG>.

With this sequence information at hand, the complete IgG1 heavy chain coding region for anti-human CD19 antibody hBU12 with C-terminal extension, comprising the N-intein domain of Ssp GyrB <NUM> split-intein, followed by a 6xHis-tag and a strepII tag can be designed as disclosed in SEQ ID NO <NUM> below:
<IMG>
<IMG>.

This translates to amino acid sequence SEQ ID NO <NUM> (amino acids of the N-intein domain are underlined, 6xHis tag and strepII tag are shaded):
<IMG>.

Likewise, a complete IgG1 kappa light chain coding region for anti-human CD19 antibody hBU12 with C-terminal extension, comprising the N-intein domain of Ssp GyrB <NUM> split-intein, followed by a 6xHis-tag and a strepII tag can be designed as disclosed in SEQ ID NO <NUM> below:
<IMG>.

The coding regions for the N-intein modified heavy and light chains of the anti-human CD19 specific antibody as disclosed in SEQ ID NOs <NUM> and <NUM>, respectively, can then be synthesized with flanking restriction enzyme sites (e.g. HindIII and NotI) such that they can be cloned into a standard mammalian expression vector, such as pCDNA3. <NUM>-hygro (+) (Invitrogen), by standard molecular biology methods known in the art.

The complete DNA sequence of pCDNA3. <NUM>-hygro (+)-IgH chain expression vector for the N-intein tagged hBU12 anti-human CD19 antibody is then as follows:
SEQ ID NO <NUM> (coding region of human IgG1 VH-CH heavy chain for hBU12 with C-terminal N-intein domain of Ssp GyrB S11 split intein, followed by 6xHis tag strepII tag (underlined), and HindIII and NotI cloning sites (shaded)):
<IMG>
<IMG>
<IMG>.

The complete DNA sequence of pCDNA3. <NUM>-hygro (+)-IgL chain expression vector for the Ssp GyrB S11 N-intein domain tagged hBU12 anti-human CD19 antibody will be as follows:
SEQ ID NO <NUM> (coding region of human IgG1 VL-CL kappa light chain for hBU12 with C-terminal Ssp GyrB S11 N-intein domain, 6xHis tag and a strepII tag underlined, and HindIII and NotI cloning sites shaded):
<IMG>
<IMG>.

These pcDNA3. <NUM>-hygro(+) based expression vectors disclosed in SEQ ID NOs <NUM> and <NUM> allow upon transfection into mammalian cells, like e.g. but not limited to CHO cells, that are typically used for recombinant antibody expression, the expression of the anti-human CD19 specific humanized antibody hBU12 with C-terminal N-intein domain fused, followed by a 6xHis tag and a strepII tag at both the IgH and IgL chains.

The ORF of Sortase A from Staphylococcus aureus is published in Genbank and can be found under entry: AF162687. The aa-sequence in that record reads is shown as SEQ ID NO <NUM> (amino acid sequence of sortase A from Staphylococcus aureus):
<IMG>.

The corresponding nucleotide sequence in this Genbank entry is provided as SEQ ID NO <NUM>:
<IMG>.

Technical information with respect to the expression of an enzymatically active fragment of recombinant sortase A in E. coli, comprising amino acids <NUM>-<NUM> with 6xHis tag are disclosed in reference <CIT>. The coding region for a 6xHis tagged version of Staphylococcus aureus sortase A (aa60-<NUM>) is provided below as SEQ ID NO <NUM>:
<IMG>.

This translates to amino acid sequence SEQ ID NO <NUM>:
<IMG>.

The coding region for the 6xHis tagged sortase A fragment of Staphylococcus aureus, as provided in SEQ ID NO <NUM>, can be cloned into a standard bacterial expression vector, like e.g. pET29 (Novagen), in order to transform E. coli strain BL21(DE3) (Novagen) and to generate an E. coli clone that can be used for the bacterial production of recombinant sortase A according to standard methods known in the art. In short, E. coli BL21(DE3) transformed with pET29 expression plasmids for sortase A can be cultured at <NUM> in LB mediom with <NUM>µg/mL kanamycin until until an OD<NUM> = <NUM>-<NUM> is reached. IPTG can then be added to a final concentration of <NUM> and protein expression can be induced for three hours at <NUM>. The cells can then be harvested by centrifugation and resuspended in lysis buffer (<NUM> Tris pH <NUM>, <NUM> NaCl supplemented with <NUM> MgCl2, <NUM> units/mL DNAseI (NEB), <NUM> aprotinin, <NUM> leupeptin, and <NUM> PMSF). Cells can then be lysed by sonication and clarified supernatant can then be purified on Ni-NTA agarose following the manufacturer's instructions.

Fractions that are of ><NUM>% purity, as judged by SDS-PAGE, can then be consolidated and dialyzed against Tris-buffered saline (<NUM> Tris pH <NUM>, <NUM> NaCl), and the enzyme concentration can be calculated from the measured A<NUM> using the published extinction coefficient of <NUM>,<NUM>-<NUM> cm-<NUM>. The above-mentioned protocol has been followed and ca. <NUM> of > <NUM>% pure recombinant enzymatically active fragment (of ca. <NUM> kD) sortase A of Staphylococcus aureus has been produced and the analysis of the recombinant protein by SDS-PAGE and Western blotting is disclosed in <FIG>.

In brief, cleared cell culture supernatant is run over a protein A column of appropriate size and capacity equilibrated with PBS. Residual medium is washed with PBS and eventually bound IgG can be eluted with low pH buffer, like <NUM> citric acid-NaOH, pH <NUM>. Eluted IgG should be neutralized immediately with <NUM>/10th volume of <NUM> Tris/Cl, pH7. Combined fractions containing IgG can then be dialized against PBS over night at <NUM>.

The protocols provided in Example <NUM> provide the skilled person in the art with the instruction to produce sufficient quantities of purified, recombinant antibodies from the constructs disclosed in Examples <NUM> and <NUM>.

Monomethyl Auristatin A toxin coupled to a <NUM> amino acid glycine stretch and a <NUM> amino acid SSp GyrB S11 C-int split intein peptide according to the formulas provided below, can be custom ordered from qualified chemistry CROs. <CHM>
<CHM>
<CHM>.

Conjugation of <NUM> glycine amino acid modified MMAE toxic payload to LPETG sortase A tagged IgG1 antibody (that can be produced by following Examples <NUM> and <NUM>) can be achieved by mixing appropriate ratios of LPETG tagged IgG1 antibody with the glycine-modified MMAE toxin disclosed in Formula <NUM> (e.g. at <NUM>:<NUM> ratio and <NUM> concentration) and with recombinant sortase A (production described in Example <NUM>) (e.g. at <NUM> concentration), and using physiologic incubation buffer, like e.g.; <NUM> Tris/Cl, <NUM> NaCl, <NUM> CaCl<NUM>, pH <NUM>, and incubating at <NUM> to <NUM> for a minimum of <NUM> hours.

Efficiency of the conjugation can be monitored by analyzing the absence of the 6xHis tag and/or the strepII tag after stopping the reaction, e.g. by western-blot analysis or ELISA with anti-His-tag and/or anti strepII tag antibodies.

Completely conjugated product can be enriched by Nickel-NTA columns, or streptactin column binding, which bind to the 6xHis tag or strepII tag, respectively, which can only be present in incompletely reacted IgG1 substrate. Final IgG-payload conjugate can eventually be purified using protein A purification as described above.

Conjugation of Ssp GyrB S11 C-intein amino acid modified MMAE toxic payload to N-intein tagged IgG1 antibody (that can be produced by following Examples <NUM> and <NUM>) can be achieved by mixing appropriate ratios of N-intein tagged IgG1 antibody with the C-intein amino acid-modified MMAE toxin disclosed in Formula <NUM> (e.g. at <NUM>:<NUM> or <NUM>:<NUM> ratio at <NUM> concentration of the IgG antibody) using physiologic incubation buffer, like e.g.; <NUM> Tris/Cl, <NUM> NaCl, <NUM> EDTA, pH <NUM>, and incubating at room temperature or at <NUM> a minimum of <NUM> hours.

In summary, the Examples <NUM>-<NUM> disclosed above allow a person skilled in the art to practice the invention of enzymatically conjugating a toxic payload site-specifically to the C-terminus either using sortase A mediated or split-intein mediated transpeptidation.

Antibody expression constructs encoding monoclonal antibody Trastuzumab (Tras) heavy and light chains, either untagged (SEQ ID NOs: <NUM> - <NUM>) or C-terminally tagged with GS (glycine-serine) linker, LPETG Sortase tag, 6xHis tag, and Strep II tag (SEQ ID NOs: <NUM> - <NUM>) were generated essentially as described in Example <NUM>. Using these expression constructs, Tras-HC-GS-LHS and Tras-LC-GS-LHS (HC=heavy chain, LC=light chain, GS=glycine-serine, LHS=LPETG-tag+6xHis-tag+strepII-tag) were produced in CHO cells by co-transfection of the corresponding expression constructs. Tras-HC-GS-LHS is a Trastuzumab variant with an unmodified light chain (SEQ ID NOs: <NUM> - <NUM>), and a heavy chain C-terminally tagged with GS (glycine-serine) linker, LPETG Sortase motif, 6xHis-tag, and strepII-tag (SEQ ID NOs: <NUM> - <NUM>). Tras-LC-GS-LHS is a Trastuzumab variant with an unmodified heavy chain (SEQ ID NOs: <NUM> - <NUM>), and a light chain C-terminally tagged with GS linker, LPETG Sortase motif, 6xHis-tag, and strepII-tag (SEQ ID NOs: <NUM> - <NUM>). CHO cell transfection and affinity purification of antibodies by proteinA-sepharose chromatography was done essentially as described in Example <NUM>.

Conjugation reactions containing Gly<NUM>-modified DM1 toxin (ordered from Concortis, San Diego, CA, U. , structure see <FIG> a. ) and a 17kD recombinant sortase A fragment from Staphylococcus aureus (see Example <NUM>) were carried out with <NUM> of each monoclonal antibody (mAb) (see Example <NUM>) in 1x Sortase buffer (<NUM> Tris-HCl, pH8. <NUM>; <NUM> NaCl; <NUM> CaCl<NUM>), as shown in Table II, below. The Tras-HC-GS-LHS conjugation reaction was incubated at <NUM> for <NUM>; the Tras-LC-GS-LHS conjugation reaction was incubated at <NUM> for <NUM>. Each reaction mixture was then passed over a Strep-Tactin® Sepharose columns (IBA Life-Sciences, Göttingen, Germany). For this, <NUM> of Strep-Tactin Agarose was packed under gravity into a fritted column and equilibrated with <NUM> column volumes of equilibration buffer (<NUM> Tris-HCl, pH <NUM>; <NUM> NaCl; <NUM> EDTA). Each conjugation mixture was passed twice down the same column using gravity flow (to increase residence time on the resin). The resin was washed with an additional column volume of equilibration buffer to maximize conjugate yield and the pool then applied immediately to a protein A column. For this, a <NUM> Protein A HiTrap column was equilibrated with <NUM> column volumes of buffer (<NUM> sodium phosphate pH <NUM>). Each conjugation reaction was then applied to an equilibrated column and the column washed with a further <NUM> column volumes of buffer. Bound conjugate was eluted with <NUM> column volumes of elution buffer (<NUM> succinic acid, pH <NUM>) with <NUM> column volume fractions collected (into tubes containing <NUM>% v/v <NUM> Tris Base to neutralise the acid) and analysed for protein content. Protein containing fractions were pooled and formulated by G25 column chromatography. For this, NAP <NUM> columns of an appropriate size for each scale of manufacture were used to formulate the conjugates for long term storage. The columns were equilibrated, loaded and eluted with <NUM> Sodium Succinate pH <NUM>, <NUM>/mL Trehalose, <NUM>% % w/v Polysorbate <NUM> (Formulation Buffer for Kadcyla® (T-DM1), marketed by Roche/Genentech) according to the manufacturer's instructions.

The Tras-HC-GS-LHS and Tras-LC-GS-LHS DM1-conjugate yields were, respectively, <NUM> (<NUM>%) and <NUM> (<NUM>%). The major process losses occurred during Protein A and G25 purification, most probably as a result of peak cutting to ensure maximal concentration of the product for each subsequent step or storage.

The drug loading was assessed by Hydrophobic Interaction Chromatography (HIC), and was performed on a TOSOH Butyl-NPR <NUM> x <NUM>, <NUM> column run at <NUM>/min with a <NUM> minute linear gradient between A - <NUM> (NH<NUM>)<NUM>SO<NUM>, <NUM> NaPi, pH=<NUM>±<NUM> and B - <NUM>% <NUM> NaPi, pH=<NUM>±<NUM>, <NUM>% IPA. The HIC profiles revealed that for both, Tras-HC-GS-LHS and Tras-LC-GS-LHS, there was no detectable unconjugated mAb left, and a major fraction of each mAb was loaded with <NUM> drugs (see <FIG>).

Cytotoxicity of DM1-sortaseA-conjugated Tras-HC-GS-LHS and DM1-sortaseA-conjugated Tras-LC-GS-LHS was investigated and compared to Kadcyla® (Roche/Genentech) using SKBR3 cells, a human breast cancer cell line overexpressing the cognate antigen of trastuzumab (Tras) HER-<NUM>/neu, and T47D-5R cells, a breast cancer cell line naturally expressing low levels of HER-<NUM>/neu, engineered to be devoid of cell surface HER-<NUM>/neu (Graus-Porta et al. Cells were plated on <NUM> well plates in 100µl complete DMEM (<NUM>'<NUM> cells per well). After one day incubation, 50µl medium was carefully removed from each well and replaced by 50µl of <NUM>-fold serial dilutions of each ADC in complete DMEM, resulting in ADC concentrations ranging from 20µg/ml to <NUM>. Each dilution was done in duplicates or triplicates. After <NUM> additional days incubation at <NUM> in a humidified incubator at <NUM>% CO<NUM> atmosphere, plates were removed from the incubator and equilibrated to room temperature. After approximately <NUM> minutes, 100µl CellTiter-Glo® Luminescent Solution (Promega, Cat. No G7570) was added to each well and, after shaking the plates at 450rpm for <NUM> followed by a <NUM> incubation without shaking, luminescence was measured on a Tecan Infinity F200 with an integration time of <NUM> second per well. All three ADCs were highly cytotoxic for the HER-<NUM>/neu overexpressing SKBR3 breast cancer cell line, but not for the HER-<NUM>/neu-negative T47D-5R breast cancer cell line (see <FIG>). The EC<NUM> values for Her-<NUM>/neu positive breast cancer cell line SKBR3 were: Kadcyla®, <NUM>. 4ng/ml; DM1-conjugated Tras-HC-GS-LHS, <NUM>. 6ng/ml; Tras-LC-GS-LHS, <NUM>. 4ng/ml, and thus are within similar range of potency in the in vitro tumor cell killing experiment. Conversely, no specific cellular toxicity was detectable with the Her-<NUM>/neu negative breast cancer cell line T47D-5R, demonstrating the functional equivalence of sortaseA, enzymatically conjugated ADC versus traditional, chemically conjugated ADC, when the comparison entails the same targeting antibody and the same toxin (DM1) (<FIG>). However, it appears that the lower drug-to antibody ratio of ca. <NUM> (deducted from intergration of the DAR1 and DAR2 peaks in <FIG>) for the Tras-HC-GS-LHS and Tras-LC-GS-LHS sortase A-conjugated ADCs, as compared to the DAR of ca. <NUM>-<NUM>, reported for Kadcyla® does not translate into a proportionally different cellular cytotoxicity in the in vitro tumor cell killing assays (<FIG>). This unexpected finding may be the result of a more defined and site-specific toxin-antibody conjugation mediated by sortase A in comparison to the less defined, stochastically, chemically conjugated Kadcyla®.

The influence of peptide-spacer length positioned between the C-terminus of antibody heavy or light chain and LPETG sortase A recognition motif was investigated. For this, antibody heavy chain and light chain expression constructs encoding chimeric CD30-specific mAb Ac10 heavy and light chains (HC sequence derived from <CIT>, Seq1, LC sequence derived from <CIT>, Seq9), C-terminally modified with sequences comprising or not comprising a <NUM> amino acid GS (glycine-serine) spacer, and comprising a LPETG sortaseA recognition motif, and a strep-II purification tag (SEQ ID NOs: <NUM> - <NUM>), have been cloned essentially according to instructions disclosed in Example <NUM>. Using these expression constructs, mAbs Ac10-HC-GS-LHS/LC-GS-LHS and Ac10-HC-LS/LC-LS were produced in CHO cells by co-transfection of the corresponding plasmids. Ac10-HC-GS-LHS/LC-GS-LHS is an Ac10 variant with heavy and light chains modified at the C-termini of each HC and LC with a GS peptide spacer, a LPETG sortaseA motif, a 6xHis tag, and a strep-II tag (SEQ ID NOs:<NUM> - <NUM>; Table <NUM>). Ac10-HC-LS/LC-LS is an Ac10 variant with heavy and light chains modified at the C-termini with LPETG Sortase motif and strep-II tag without the <NUM>-peptide GS linker (SEQ ID NOs: <NUM> - <NUM>; Table <NUM>). CHO cell transfection and affinity purification of antibodies by protein A-sepharose chromatography was done essentially as described in Example <NUM>.

To investigate efficiency of conjugation, serial dilutions of Sortase A were used to conjugate penta-glycine-modified FITC (Gly<NUM>-FITC, see Formula <NUM> below). <CHM>
<CHM>.

For this, Gly<NUM>-FITC was sortaseA conjugated to two Ac10 variants in 1x Sortase buffer (<NUM> Tris-HCl, pH8. <NUM>; <NUM> NaCl; <NUM> CaCl<NUM>), as shown in Table <NUM>. After <NUM> at <NUM>, reaction products were analyzed by denaturing, reducing SDS-PAGE gel electrophoresis, and FITC was visualized by placing the gels on a UV box (<FIG>). Conjugation to the heavy chain was found to be highly efficient irrespective of the presence absence of the GS-linker between heavy chain C-terminus and LPETG Sortase recognition motif. Unexpectedly, sortaseA mediated conjugation to the light chain was significantly less efficient in comparison to sortaseA mediated heavy chain conjugation. Furthermore, it was surprisingly found that coupling efficiency was dramatically affected by the presence or absence of the <NUM> peptide GS (glycine-serine) spacer positioned between the C-terminus of the antibody light chains and the LPETG sortaseA recognition motif. Whereas in the presence of the GS-linker, conjugation to the light chain took place with about <NUM>-10x lower efficiency than to the heavy chain, it was about <NUM>-100x less efficient in the absence of a linker. Therefore, it was concluded that increasing the peptide spacer length between the light chain and the LPETG Sortase recognition motif might further improve conjugation efficiency.

Therefore, the influence of increasing the length of the peptide spacer between light chain and LPETG Sortase A recognition motif on conjugation efficacy was investigated next. Expression constructs encoding mAb Ac10 light chains, C-terminally tagged with LPETG Sortase recognition motif and strep-II purification tag, with a <NUM> to <NUM> amino acid linker (SEQ ID NOs: <NUM> - <NUM>), were generated essentially as described in Example <NUM>. Using these expression constructs, mAbs Ac10-HC-LS/LC-GS-LS, Ac10-HC-LS/LC-GGS-LS, Ac10-HC-LS/LC-GGGS-LS and Ac10-HC-LS/LC-GGGGS-LS were produced in CHO cells by co-transfection of the corresponding expression constructs. In each of these antibodies, the heavy chain is C-terminally modified with an LPETG Sortase recognition motif and a strep-II purification tag (SEQ ID NOs: <NUM> - <NUM>; Table <NUM>). The light chain is C-terminally modified with an LPETG Sortase tag and strep-II tag containing either a GS, GGS, GGGS, or a GGGGS peptide spacer (SEQ ID NOs: <NUM> - <NUM>; Table <NUM>) in front of the LPETG motif. CHO cell transfection and affinity purification of antibodies by protein A-sepharose chromatography was done essentially as described in Example <NUM>.

To investigate conjugation efficiency, serial dilutions of Sortase A were used to conjugate penta-glycine-modified FITC (Gly<NUM>-FITC, see Formula <NUM>, above) to the four different Ac10 mAb variants in 1x Sortase buffer (<NUM> Tris-HCl, pH8. <NUM>; <NUM> NaCl; <NUM> CaCl<NUM>), as shown in Table <NUM>. After <NUM> at <NUM>, reaction products were analyzed by denaturing, reducing SDS-PAGE gel electrophoresis, and FITC was visualized by placing the gels on a UV box (<FIG>). As expected, conjugation to the heavy chain was equally efficient in all four antibody variants. In contrast, conjugation to the light chain was improved significantly by increasing peptide-spacer length. Significantly, with the longest peptide-spacer analyzed (GGGGS), light chain conjugation efficiency was equally efficient in comparison to conjugation of the heavy chain, thereby allowing synchronous conjugation of heavy and light chains of an antibody C-terminally modified at both heavy and light chain. It is concluded that this antibody format will facilitate Sortase A-mediated production of homogeneous ADCs loaded with <NUM> drugs per antibody (DAR4).

Sortase A mediated conjugation with Gly<NUM>-labeled vc-PAB-MMAE (see Formula <NUM>, Example <NUM>) was performed with anti-CD30 antibody Ac10 modified at the C-termini of either the heavy chains, or the light chains with sequences comprising an LPETG sortase A motif and a strepII-affinity purification tag as provided in Table <NUM> below:.

The expression vectors encoding the Ac10 heavy or light chain sequences of Table <NUM> have been constructed essentially as disclosed in Example <NUM>. CHO cell transfection and affinity purification of antibodies by protein A-sepharose chromatography was done essentially as described in Example <NUM>.

Sortase A mediated conjugation of heavy or light chaing sortase motif tagged anti-CD30 antibodies with Gly<NUM>-labeled vc-PAB-MMAE (see Formula <NUM>, Example <NUM>) was performed essentially according to the protocol provided in Example <NUM>.

As described further above in the detailed description of the invention, unreacted antibody will retain the C-terminal strep-II affinity purification tag, which can be exploited to enrich fully reacted ADC with DAR2. Analysis of the heavy chain sortase A conjugation with vc-PAB-MMAE toxin via hydrophobicity interaction chromatography (HIC) (<FIG>, panel A), shows that the majority of the sortase-motif modified heavy chains have been conjugated, but a certain percentage of unreacted substrate (DAR0 = drug to antibody ratio = zero), or partially reacted substrate (DAR1 = drug to antibody ratio = <NUM>) was still detectable by HIC (<FIG>, Panel A).

Therefore, the protein A purified vc-PAB-MMAE conjugate was passed <NUM> times times over a StrepTactin® affinity column (IBA Sciences, Göttingen, Germany), essentially as described in Example <NUM>, in order to remove unreacted or partially reacted sortase A-modified antibody. <FIG>, Panel B shows that upon several passages of the heterogeneous vc-PAB-MMAE antibody drug conjugate, completely reacted DAR2 ADCs (DAR2 = drug to antibody ratio = <NUM>) could be highly enriched. This experiment demonstrates the feasibility to utilize additional affinity purification tags added C-terminally to the sortase A LPETG recognition motif to generate homogeneous ADC with a defined drugs per antibody ratio (here DAR2).

In order to allow conjugation of two different payloads, preferably toxic payloads to a single antibody, modified with different sortase motifs at heavy and light chain C-termini, it is required to modify two different toxins with glycine residues, preferably toxins with different mode of actions, such that a cancer cell targeted with a dual payload conjugated ADC, is attacked with via two different, potentially synergistic routes. The synthesis of two different glycine-modified toxic payloads (here maytansine and alpha-amanitin) satisfying this requirement has been performed and is described herein.

<NUM> alpha-amanitin (Structure <NUM>) (Sigma-Aldrich, order # A2263) was dissolved in <NUM> anhydrous DMSO. To this solution <NUM> NH-Boc-amino-hexylbromide were added, followed by potassium tert-butoxide (<NUM> solution in THF, <NUM>µl). The reaction mixture was stirred at room temperature for <NUM> and more potassium tert-butoxide (<NUM> solution in THF, <NUM>µl) was added. The reaction was kept at room temperature for <NUM>. Acetic acid (<NUM>µl) was added and the crude mixture was purified by RP-HPLC directly (Sunfire C18 5µ <NUM> x <NUM> column, <NUM>/min, <NUM>-<NUM>% acetonitrile/water <NUM> gradient). The desired fraction was collected and lyophilized to give Structure <NUM> as a white powder (<NUM>), which was treated with TFA/DCM solution (<NUM>/<NUM>, v/v, <NUM>) for <NUM> minutes at room temperature. The volatiles were removed under reduced pressure to give Structure <NUM> as a slightly yellowish gum, which was used in the next step without further purification.

Fmoc-Gly5-OH (<NUM>) was dissolved in anhydrous DMF (<NUM>). HATU (Sigma-Aldrich, order # <NUM>) (<NUM>) was added, followed by DIEA (<NUM>) (Sigma-Aldrich, order #<NUM>). The mixture was agitated gently at room temperature for <NUM> and then transferred to a solution of compound <NUM> in DMF (<NUM>). After <NUM> mins, LC/MS analysis showed that all of compound <NUM> was consumed. Piperidine (<NUM>µl) was added and the progress of the reaction was monitored by LC/MS. Acetic acid was added to neutralize the reaction after <NUM> and the mixture was purified by RP-HPLC (Sunfire C18 5µ <NUM> x <NUM> column, <NUM>/min, <NUM>-<NUM>% acetonitrile/water <NUM> gradient). The fractions were pooled and lyophilized to give structure <NUM> as a white powder (<NUM>). Analytical data for compound <NUM> is provided in <FIG>, panel A).

Maytansinol (<NUM>, <NUM> mmol) (Clearsynth Labs, Mumbai, India) was dissolved in anhydrous THF (<NUM>) and anhydrous DMF (<NUM>) after which <NUM>,<NUM> DIEA (Sigma-Aldrich, order #<NUM>) was added. The solution was placed under argon atmosphere. Zinc triflate (<NUM>) and NMeAla NCA (<NUM>) were added in one portion. The mixture was sonicated until the solid was dissolved. The reaction mixture was stirred at room temperature for <NUM> days and then diluted with ethyl acetate (<NUM>). It was washed with saturated NaHCO<NUM> (aq. solution, <NUM> x <NUM>) and brine (<NUM>). The organic layer was dried (over MgSO<NUM>) and concentrated to give the crude maytansinol <NUM>-(S)-alpha-N-methylaminopropionate (<NUM>) which was used directly in the next step without further purification.

Fmoc-Gly5-OH (<NUM>) was dissolved in anhydrous DMF (<NUM>). HATU (Sigma-Aldrich, order # <NUM>) (<NUM>) was added, followed by DIEA (<NUM>µL). The mixture was agitated gently at room temperature for <NUM> and then transferred to a solution of compound <NUM> in THF (<NUM>). After <NUM> mins, LC/MS analysis showed that all compound <NUM> was consumed. Piperidine (<NUM>µl) was added and the progress of the reaction was monitored by LC/MS. Ether (<NUM>) was added to the reaction after <NUM> and the precipitated solid was collected and washed with ether. The crude compound was purified by RP-HPLC (Sunfire C18 5µ <NUM> x <NUM> column, <NUM>/min, <NUM>-<NUM>% acetonitrile/water <NUM> gradient). The fractions were pooled and lyophilized to give compound <NUM> as a white powder (<NUM>). Analytical data for compound <NUM> is provided in <FIG>, Panel B.

Importantly, it is to be noted that in principle, any toxin can be functionalized for sortase mediated enzymatic conjugation, if either <NUM> glycines (as shown here), or any number of glycine residues greater or equal than one glycine, are attached to the toxins (see <FIG> ).

5x10<NUM> SKOV3 tumor cells in 200µl PBS/Matrigel (<NUM>:<NUM> ratio) were implanted subcutaneously into the left flanks of <NUM>-<NUM> weeks old female NMRI nude mice. Primary tumor volumes were monitored by calipering. After a mean tumor volume of <NUM>-<NUM><NUM> was reached, tumor-bearing animals were randomized into <NUM> Groups according to tumor sizes (<NUM> animals per group). On the day of randomization (day <NUM>) and on day <NUM>, animals of Groups <NUM>, <NUM> and <NUM> were injected intravenously with, respectively, <NUM>/kg PBS, <NUM>/kg Kadcyla®, or <NUM>/kg sortase A-conjugated Trastuzumab-DM1. Tumor volumes were measured bi-weekly by calipering (<FIG>). The study was terminated after <NUM> days and animals were euthanized according to accepted animal experimentation guidelines.

In the course of the study, tumors in control animals mock-injected with PBS grew steadily to a volume of approximately <NUM><NUM>. In contrast, tumors in Kadcyla®-treated animals shrank and were essentially undetectable on day <NUM>. Anti-tumor activity of Sortase A-conjugated Trastuzumab-DM1 did not differ significantly from that of commercially available Kadcyla®, despite the fact that the sortase-conjugated T-DM1 exhibited a lower drug to antibody ratio of approximately <NUM>, in comparison of a reported DAR of <NUM> of Kadcyla®. In combination with the data from Example <NUM>, the results demonstrate that sortase conjugated ADCs, using identical antibody and toxin moiety, have comparable tumor killing activity in comparison to commercially available chemically conjugated Kadcyla® in vitro and in vivo, albeit at lower drug to antibody ratio.

Claim 1:
A method of producing an immunoligand/payload conjugate, which method encompasses conjugating at least one payload to an immunoligand by means of a sequence-specific transpeptidase, or a catalytic domain thereof,
wherein the immunoligand comprised in the immunoligand/payload conjugate is at least one selected from the group consisting of an antibody, antibody fragment, antibody-based binding protein, or an antibody mimetic, wherein
a) the antibody fragment is selected from the group consisting of (i) a Fab fragment, (ii) a F(ab')<NUM> fragment; (iii) a heavy chain portion of a Fab(Fd) fragment, which consists of the VH and CH1 domains; (iv) a variable fragment (Fv) fragment; (v) a domain antibody (dAb) fragment, which comprises a single variable domain; (vi) an isolated complementarity determining region (CDR); (vii) a single chain FvFragment (scFv); (viii) a diabody; and (ix) a linear antibody, which comprises a pair of tandem Fvsegments (VH-CH1-VH-CH1) which, together with complementarity light chain polypeptides, form a pair of antigen binding regions;
b) the antibody based binding protein is a protein that contains at least one antibody-derived VH, VL, or CH immunoglobulin domain,
c) the antibody mimetic is selected from the group consisting of Ankyrin Repeat Proteins, C-Type Lectins, Transferrins, Lipocalins, 10th type III domains of fibronectin, Cysteine knots or knottins, SH-<NUM> domains, and Fyn SH3,
wherein the sequence-specific transpeptidase is a sortase enzyme which recognizes a pentapeptide recognition motif, added to a C-terminal end of the immunoligand,
wherein the payload is a cytotoxic compound not exceeding a molecular weight of <NUM>'<NUM> Daltons that is cytotoxic to mammalian cells and impairs and kills cancer cells,
and wherein the immunoligand comprises a sortase recognition motif and the cytotoxic compound is modified with a Glyn-modification, wherein n><NUM>, and
wherein the sortase recognition motif is a pentapeptide linked to the C-terminus of the immunoligand.