Improved antibody-payload conjugates (APCs) prepared by site-specific conjugation using genetic code expansion

Abstract

The present invention relates to novel site-specifically modified immunoglobulin molecules capable of binding to human epidermal growth factor receptor 2 (HER2) bearing non-canonical amino acid residues (ncAA) at predefined positions, the respective nucleic acid sequences encoding such modified immunoglobulin molecules, recombinant organisms useful for preparing such modified immunoglobulin molecules and adapted to express the respective coding nucleic acid sequences, methods for preparing said site-specifically modified immunoglobulin molecules, conjugates formed between said site-specifically modified immunoglobulin molecules and binding partners bearing functional groups reactive with said ncAA residues of the immunoglobulin molecules, more particularly antibody drug conjugates (ADCs). The present invention also relates to specific binding partners and their preparation. The present invention also relates to pharmaceutical compositions comprising such conjugates, as well as the use of such conjugates in medicine, in particular in the treatment of cancers overexpressing HER2.

Description

[Technical field]

[0001] The present invention relates to novel site-specifically modified immunoglobulin molecules capable of binding to human epidermal growth factor receptor 2 (HER2) carrying non-canonical amino acid residues (ncAA) at predefined positions, the respective nucleic acid sequences encoding such modified immunoglobulin molecules, recombinant organisms useful for the preparation of such modified immunoglobulin molecules and adapted to express the respective coding nucleic acid sequences, methods for the preparation of said site-specifically modified immunoglobulin molecules, conjugates formed between said site-specifically modified immunoglobulin molecules and binding partners carrying functional groups reactive with said ncAA residues of the immunoglobulin molecules, more particularly antibody drug conjugates (ADCs). The present invention also relates to specific binding partners and their preparation. The present invention also relates to pharmaceutical compositions comprising such conjugates, as well as medical uses of such conjugates, in particular in the treatment of cancers overexpressing HER2. [Background technology]

[0002] Antibody-drug conjugates (ADCs) combine the two main therapies currently applied in the treatment of cancer: chemotherapy and antibody therapy. Antibodies are important biologics, which bind to their specific antigens, e.g., receptors on cells that are overexpressed on cancer cells compared to healthy cells. Antibodies activate the compatibility system, so that the cancer cells are destroyed by killer cells. Chemotherapy, on the other hand, is a treatment with cytotoxic moieties, which are absorbed by cells and can kill them by various routes. Active cells, such as cancer cells, can take up more cytotoxic drugs than healthy cells. Nevertheless, this treatment shows significant side effects. Combining the killing effects of antibodies and cytotoxic drugs allows for directed and efficient cancer treatment. Therefore, the choice of the conjugation method, the method of labeling the antibody with the drug, is very important. The first commercially available ADCs were randomly conjugated by utilizing cysteines or lysines in the antibody sequence to attach a toxic payload. This results in heterogeneous species with different drug-to-antibody ratios (DAR), which negatively impacts the pharmacokinetics and safety profile of the ADC (Senter, PD & Sievers, EL The discovery and development of brentuximab vedotin for use in relapsed Hodgkin lymphoma and systemic anaplastic large cell lymphoma. Nat. Biotechnol. 30, 631-637 (2012);Junutula, JR et al. Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat. Biotechnol. 26, 925-932 (2008)).

[0003] Site-specific conjugation methods utilizing antibody glycosylation or enzymatic coupling emerged subsequently (Van Geel, R. et al. Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved N-Glycan of Native mAbs Provides Homogeneous and Highly Efficacious Antibody-Drug Conjugates. Bioconjug. Chem. 26, 2233-2242 (2015); Dennler, P. et al. Transglutaminase-based chemo-enzymatic conjugation approach yields homogeneous antibody-drug conjugates. Bioconjug. Chem. 25, 569-578 (2014)). These methods are limited to specific sites and cannot be transferred to other positions on the antibody sequence. One site-specific and position-unlimited conjugation method is the use of genetic code expansion technology. Therefore, non-standard amino acids (ncAAs) are introduced into the antibody sequence at the translational level in response to a stop codon previously inserted into the antibody gene (e.g., the amber stop codon TAG). An orthogonal aminoacyl-tRNA synthetase (aaRS) / tRNA pair capable of binding and introducing the ncAA into the growing antibody protein sequence must be introduced into the antibody expression host (Lemke, EA The exploding genetic code. ChemBioChem 15, 1691-1694 (2014);de la Torre, D. & Chin, JW Reprogramming the genetic code. Nat. Rev. Genet. 22, 169-184 (2021)). The ncAAs can be freely positioned within the antibody sequence and can be used for conjugation with toxic payloads depending on their chemical properties. Different ncAAs exist based on various endogenous amino acids such as lysine and tryptophan.They can have different head groups, which affect their chemical properties and which chemical reactions they can undergo. Tian et al. showed that multiple antibodies expressed in CHO cells were incorporated with ncAAs containing ketone head groups and subsequently coupled to cytotoxic payloads via copper-free click reactions. The reaction of alkoxyamine functional groups with ketones could only be performed at pH 4 and required additives otherwise (Tian, ​​F. et al. A general approach to site-specific antibody drug conjugates. Proc. Natl. Acad. Sci. USA 111, 1766-1771 (2014)).

[0004] Currently, the fastest bioorthogonal chemical reaction that can be performed even at neutral pH is the strain-promoted inverse electron demand Diels-Alder cycloaddition (SPIEDAC) between strained alkenes or alkynes and tetrazine groups (Nikic, I. & Lemke, EA Genetic code expansion enabled site-specific dual-color protein labeling: superresolution microscopy and beyond. Curr. Opin. Chem. Biol. 28, 164-173 (2015)). One special case of the SPIEDAC reaction is the conjugation of cyclooctenyl lysine (SCO) with 1,2,4,5-tetrazine. As can be shown by in vivo measurements, this may not be an inverse electron demand reaction and does not show the same reaction rate as other strained alkenes or alkynes (Figure 1). Therefore, SCOs and the resulting reaction products show the highest stability in the cellular environment compared to other strained alkenes / alkynes tested (Wagner, JA, Mercadante, D., Nikic, I., Lemke, EA & Grater, F. Origin of Orthogonality of Strain-Promoted Click Reactions. Chem. - A Eur. J. 21, 12431-12435 (2015);Reinkemeier, CD et al. Synthesis and Evaluation of Novel Ring-Strained Noncanonical Amino Acids for Residue-Specific Bioorthogonal Reactions in Living Cells. Chem. - A Eur. J. 27, chem.202100322 (2021)). Toxic payloads can be divided into linkers and cytotoxic drugs. Currently, many linker technologies exist, ranging from non-cleavable to enzymatic, acidic and glutathione cleavable linkers.The linker directly impacts the pharmacokinetics and pharmacodynamics of the ADC (Hafeez, U., Parakh, S., Gan, HK & Scott, AM Antibody-drug conjugates for cancer therapy. Molecules 25, 4764 (2020); Khongorzul, P., Ling, CJ, Khan, FU, Ihsan, AU & Zhang, J. Antibody-Drug Conjugates: A Comprehensive Review. Mol. Cancer Res. 18, 3-19 (2020)).

[0005] Only a handful of different cytotoxic drug families, such as the auristatins, maytansinoids, calicheamicins, and duocarmycins, are currently used as chemical warheads in ADCs. They damage either DNA or microtubules (Chau, CH, Steeg, PS & Figg, WD Antibody-drug conjugates for cancer. Lancet 394, 793-804 (2019);Sievers, EL & Senter, PD Antibody-drug conjugates in cancer therapy. Annu. Rev. Med. 64, 15-29 (2013)).

[0006] In this regard, there are 31 ongoing clinical trials, such as 177-lutetium rilotomab tetraxetane (Betalutin) for the treatment of non-Hodgkin's lymphoma (Kolstad, A et al Study of 177 Lu-Lilotomab Satetraxetan in Relapsed / Refractory Indolent Non-Hodgkin Lymphoma. Blood Adv 4 (17), 4091-4101 (2020), The use of radioimmunoconjugates (RIC) is also of growing interest in cancer treatment.

[0007] More specifically, RICs represent another interesting subclass of ADCs and can be generated by labeling monoclonal antibodies with a radioactive payload, which is then delivered to the tumor site, resulting in the shrinkage and / or growth inhibition of tumor cells.

[0008] The isotope most commonly used for therapeutic purposes has a half-life of 6.7 days. 177 Beta-emitting isotopes such as Lu.

[0009] Also, conjugating radioisotopes to mAbs opens the opportunity to replace therapeutic radioisotopes, often beta or alpha emitters, with diagnostic isotopes, often positron emitters for positron emission tomography (PET) to detect disease-related targets of interest.

[0010] The concept of combining therapeutic and diagnostic capabilities with one targeted molecule has become a highly dynamic field of nuclear medicine called theranostics. The beneficial impact of site-specific labeling on the properties of RICs, such as improved stability, immunoreactivity and biodistribution, has been previously shown (Kristensen, L et al Site-Specifically Labeled 89 Zr-DFO-Trastuzumab Improves Immuno-Reactivity and Tumor Uptake for Immuno-PET in a Subcutaneous HER2-Positive Xenograft Mouse Model. Theranostics 2019, 9 (15), 4409-4420). Compared to this prior art, our technique has several advantages. First, there is no need to use additional enzymes to modify our conjugation sites. Second, the position of conjugation can be freely selected and is not limited to glycosylation sites. In addition, our conjugation chemistry is much faster than the SPAAC used by Kristensen et al., and therefore, the click reaction is possible not only directly in vivo but also with pre-radiolabeled chelators.

[0011] The use of radioisotopes for therapeutic research has particularly high prerequisites for purity and stability in biological systems over hours to days.

[0012] In this context, the use of chelators that simultaneously exhibit high stability and reaction yield, such as the dodecanetetraacetic acid (DOTA) chelator (Figure 11), plays a key role and is directly relevant for the successful implementation of RIC in cancer treatment or diagnosis.

[0013] Here, reaction temperatures are typically higher and it is desirable to perform radiolabeling without heat-sensitive proteins such as monoclonal antibodies.

[0014] In that sense, currently available therapeutic antibody-drug conjugates and conjugation methods still exhibit shortcomings, especially with regard to their pharmacological activity.

[0015] Therefore, the problem to be solved by the present invention is the provision of improved therapeutically valuable ADCs. Summary of the Invention

[0016] The above problems could surprisingly be solved by the provision of improved therapeutic ADCs based on specific site-specifically modified antibody components of the conjugate. More specifically, improved ADCs of value for the treatment of breast cancer are provided.

[0017] In particular, a novel site-specific conjugation of trastuzumab with a toxic payload is provided that utilizes the reaction between SCO and a tetrazine group containing a cleavable linker coupled to a cytotoxic drug (Figure 1A). Two different payloads are illustrated, both containing monomethyl auristatin E (MMAE) as the cytotoxic drug, but with different linker technologies. Both linkers are shown in detail in Figure 1B. The first payload (P1) contains a PEG linker attached to a valine-alanine cleavable linker, followed by ρ-aminobenzyl (PAB). For the second payload (P2), a glucuronide linker connects the tetrazine group to the MMAE.

[0018] Furthermore, we have surprisingly improved the potency of our ADC in in vitro cytotoxicity assays compared to the commercially available ADC Kadcyla® (Genentech, Roche; INN name trastuzumab emtansine). Kadcyla® is an antibody / cytostatic conjugate that combines the humanized anti-HER2 IgG1 antibody trastuzumab with the microtubule inhibitor maytansinoid DM1. The component trastuzumab binds to HER2, where it exerts its antitumor effect. The conjugate is then internalized and degraded, releasing DM1 intracellularly, where it binds to tubulin and leads to apoptotic cell death. This is believed to enhance the therapeutic effect. Covalent conjugation of trastuzumab and DM1 via a thioether linker is believed to reduce the systemic release of DM1 and enhance the release of DM1 to the target.

[0019] The present invention provides the first study showing the effect of coupling site on ADC potency as well as the impact of DAR in in vitro cytotoxicity assays. Different variants of trastuzumab, either non-glycosylated or glycosylated, containing one ncAA at various sites in its heavy chain (IgH) or light chain (LgL) or two ncAAs at various sites in its heavy or light chain or in both heavy and light chains, were coupled with their respective toxic payloads. First, sites showing preferential behavior in in vitro cytotoxicity studies were investigated. Seven amber mutations in the heavy chain and five amber mutations in the light chain were generated and tested for their potency against cancer cell lines such as SK-BR-3 and BT-474 cells (Figure 2). Two of the mutations were also prepared in glycosylated form and tested for their activity against cancer cells (Figure 9).

[0020] The various mutation sites were selected based on the structural knowledge of trastuzumab and the human IgG1 Fc fragment (Trastuzumab Fab fragment PDB: 4HKZ, IgG1 Fc fragment PDB: 3DNK). Some sites, such as position K249 of the heavy chain of trastuzumab, are already known based on the literature. However, this position was only used for coupling to cyclic peptides without the use of genetic code expansion techniques (Shi, W. et al. Manipulating the Click Reactivity of Dibenzoazacyclooctynes: From Azide Click Component to Caged Acylation Reagent by Silver Catalysis. Angew. Chemie 132, 20112-20116 (2020)). Position K320 has been mentioned in the context of C1q binding, but has not been used for site-specific labeling so far.

[0021] In particular, the newly characterized mutation sites in the light chain have not previously been exploited for site-specific coupling, since Applicant's technology only allows for site-specific modification of discrete sites in the light chain as well.

[0022] The various trastuzumab-derived ADCs exemplified herein are summarized in the table below.

[0023] Table: Trastuzumab-derived ADCs [Table 1]

[0024] The above problems have also been surprisingly solved by providing improved trastuzumab-derived radiolabeled ADCs, in particular site-specifically labeled trastuzumab variants carrying an ncAA moiety that can be selectively coupled to, for example, tetrazine-modified chelators (Figure 10), in particular DOTA-based chelators (Figure 11), via a click reaction under mild conditions.

[0025] Table: Radiolabeled ADCs derived from trastuzumab [Table 2]

[0026] The findings of the present invention obtained using site-specifically modified trastuzumab (see FIG. 4a) can be transferred to other structurally and functionally related antibody molecules based on the teachings of the present invention, including, but not limited to, the anti-HER2 antibody pertuzumab (see FIG. 4b). [Brief description of the drawings]

[0027] [Figure 1A] Schematic diagram of our ADC platform. An antibody with an ncAA site-specifically introduced into its protein sequence reacts with a tetrazine moiety attached to a linker (oval) and a cytotoxic drug (dotted circle). [Figure 1B] Detailed structure of H-Tet-PEG9-Val-Ala-PAB-MMAE (Payload 1 = P1) is shown. The linker is enclosed in an oval and the cytotoxic drug is enclosed in a dotted oval. [Figure 1C] The structure of Payload 2 (P2, H-Tet-glucuronide-MMAE) is shown. The glucuronide linker is enclosed in an oval and the MMAE is enclosed in a dotted oval. [Figure 2A] In vitro cytotoxicity assays. Cytotoxicity assays representing cell viability measured after addition of different ADCs to cancer cells. Data points were measured in triplicate and error bars indicate the standard error of each data point. A) Cytotoxicity assay of different heavy chain variants of trastuzumab conjugated to payload 1 (P1) against SK-BR-3 cells. [Figure 2B] Two heavy chain mutant ADCs and Kadcyla® against BT-474 cells. [Figure 2C] The three light chain variant ADCs compared to ADC-H1-P2 and Kadcyla® on SK-BR-3 cells. [Figure 2D] Cavity mutant ADCs compared to ADC-H1-P2 and Kadcyla®. [Figure 2E] Different double mutant ADCs compared to Kadcyla®. [Figure 2F] Different double mutant ADCs compared to Kadcyla®. [Diagram 3] Best ADC on three different cell lines. Cytotoxicity assay of single mutant ADC-H1-P2 and double mutant ADC-H6L4-P2 compared to Kadcyla® on three different cell lines. Top row: SK-BR-3, middle row: BT-747, bottom row: MCF-7 cells. [Figure 4A] Trastuzumab heavy and light chain protein sequences and locations of the investigated mutation sites. [Figure 4B] Pertuzumab heavy and light chain protein sequences and locations of the investigated mutation sites. [Figure 4C] 1 shows the sequence alignment of the heavy and light chains of the monoclonal antibodies Pertuzumab (top row) and Trastuzumab (bottom row). [Figure 4D]1 shows the nucleic acid sequence of the HSA-trastuzumab heavy chain and the locations of the mutation sites and signal sequence investigated. [Figure 4E] The protein sequence of the HSA-trastuzumab heavy chain and the locations of the mutation sites and signal sequence investigated are shown. [Figure 4F] 1 shows the nucleic acid sequence of the HSA-trastuzumab light chain and the location of the signal sequence. [Figure 4G] The protein sequence of the HSA-trastuzumab light chain and the location of the signal sequence are shown. [Diagram 5] A three-dimensional model of the binding of different mutant positions in the heavy and light chains of Trastuzumab is shown. Candidate mutant positions in the light chain are highlighted and named (ALA-51, PRO-59, GLY-42, LYS-169, ALA-111). ALA-51 is located within the CDR-L2 motif. The side chains of the mutant positions can be seen to protrude outward. Each of the candidate positions is located in a section of the amino acid sequence that does not form significant secondary structure. [Figure 6] 1 shows the plasmid map of the expression plasmid pAceBacDUAL (SEQ ID NO:1). [Figure 7] FIG. 2 shows the plasmid map of the expression plasmid pAceBacDUAL-Trastuzumab heavy chain 6His-light chain (SEQ ID NO: 22), useful for preparing site-specifically modified trastuzumab immunoglobulins of the present invention. [Figure 8] FIG. 4 shows the plasmid map of the expression plasmid pCK-HSA-TrastuzumabHC-LC (SEQ ID NO:49), useful for preparing site-specifically modified glycosylated Trastuzumab immunoglobulins of the present invention. [Figure 9A]In vitro cytotoxicity assay. Cytotoxicity assay showing cell viability measured after addition of different ADCs to BT-474 cancer cells. Site-selectively mutated trastuzumab antibody moieties of ADCs were expressed in either Sf21 insect cells or HEK293F human cancer cells (ADCs named with the prefix "gly"). Data points were measured in triplicate and the error bars show the standard error of each data point. Cytotoxicity assay of different heavy chain variants of ADC-H1-P2, glyADC-H1-P2 and Kadcyla® against BT-474 cells. [Figure 9B] In vitro cytotoxicity assay of different heavy chain variants of ADC-H4-P2, glyADC-H4-P2 and Kadcyla® against BT-474 cells. [Figure 10] Schematic showing site-specific labeling using ncAA via click reaction with tetrazine-modified chelators. [Figure 11] Here is a specific example of a chelating agent, called H-Tet-PEG9-DOTA. [Figure 12] Ex vivo biodistribution of 177Lu-Trastuzumab A121 PEG9-DOTA in BT-474 xenograft mice was measured after 48 hours and is shown by dotted bars compared to the control group (black bars). [Figure 13] Figure 1 shows subcutaneous tumor size in BT-474 xenograft mice approximately 30 days after radiopharmaceutical injection. A comparison of the change in tumor volume (mm3) between test groups (squares) and untreated growth control groups (circles) is shown. [Figure 14] Ex vivo tumor weights 30 days after injection are shown, with a comparison between a group of four animals that received subcutaneous tumor implants and treatment (left column) and five animals that received only subcutaneous tumor implants without treatment (growth control). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] A. Abbreviation ADC = antibody-drug conjugate APC = antibody payload complex ATCC = American Type Culture Collection DOTA = dodecanetetraacetic acid EDC = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (coupling reagent) FBS = fetal bovine serum GCE = genetic code expansion HER2 = human epidermal growth factor receptor 2 HSA = human serum albumin H-Tet = 1,2,4,5-tetrazine-3-yl IgH = immunoglobulin heavy chain. IgL = immunoglobulin light chain. kDa = kilodaltons MMAE = monomethylauristatin E ncAA = nonstandard amino acid NES = nuclear export signal NLS = nuclear localization signal O-tRNA = orthogonal tRNA O-RS = Orthogonal RS PAB = para-aminobenzyl PBS = phosphate-buffered saline POI = Protein of Interest Aminoacyl RS = aminoacyl-tRNA synthetase PylRS = pyrrolysyl-tRNA synthetase rcf = relative centrifugal force RS = aminoacyl-tRNA synthetase RT = room temperature

[0029] SCO = 2-amino-6-(cyclooct-2-yn-1-yloxycarbonylamino)hexanoic acid [ka]

[0030] SPAAC = (copper-free) strain-promoted alkyne azide cycloaddition SPIEDAC = (copper-free) strain-promoted inverse electron demand Diels-Alder cycloaddition TCO = transcyclooctene

[0031] TCO-E-Lys = N6-((((R,E)-cyclooct-4-en-1-yl)oxy)carbonyl)-L-lysine [ka]

[0032] TCO*A-Lys = N6-((((S,E)-cyclooct-2-en-1-yl)oxy)carbonyl)-L-lysine [ka]

[0033] tRNA Pyl = a tRNA that can be acylated with pyrrolysine by a wild-type or modified PylRS, the anticodon of which is preferably the reverse complement of the selector codon for site-specific incorporation of an ncAA into the POI. tRNA アミノアシル = a tRNA that can be acylated with an aminoacyl residue by a wild-type or modified PyIRS, the anticodon of which is preferably the reverse complement of the selector codon for site-specific incorporation of an ncAA into the POI. UNAA = unnatural amino acid, synonymous with ncAA

[0034] B. Definition 1.General Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. The meaning and scope of the terms should be clear, but in the event of potential ambiguity, the definitions provided herein shall take precedence over any dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0035] Generally, the nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed herein, unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as commonly practiced in the art or as described herein. The nomenclature used in connection with, and experimental procedures and techniques of, analytical chemistry, synthetic organic chemistry, medicinal chemistry, and drug discovery chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0036] In the context of this description and the appended claims, the use of "or" means "and / or" unless stated otherwise.

[0037] Similarly, the words "comprise," "comprises," "comprising," "include," "includes," and "including" are not intended to be limiting and are interchangeable.

[0038] Further, although the term "comprising" is used in describing various embodiments, it should be understood that in some specific cases, a person of ordinary skill in the art would understand that an embodiment could alternatively be described using the phrase "consisting essentially of" or "consisting of."

[0039] The term "one or more" or the similar term "at least one" refers to, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

[0040] Where a lower and upper limit of a numerical range is disclosed, any numerical value falling within that range and any inclusive range is specifically disclosed, including both the upper and lower values. In particular, each range of values ​​disclosed herein should be understood to include all values ​​falling within that broader range as well as the narrower range.

[0041] The term "about" indicates a potential variation of ±25%, particularly ±15%, ±10%, and more particularly ±5%, ±2% or ±1% of the stated value.

[0042] The term "substantially" denotes a range of values ​​from about 80 to 100%, such as for example 85 to 99.9%, in particular 90 to 99.9%, more particularly 95 to 99.9%, or 98 to 99.9%, in particular 99 to 99.9%.

[0043] "Mainly" refers to a percentage in the range of more than 50%, for example, in the range of 51 to 100%, particularly 75 to 99.9%, more specifically 85 to 98.5%, 95 to 99%, etc.

[0044] Where the present disclosure refers to features, parameters and ranges thereof having different degrees of preference (including general features, parameters and ranges thereof that are not expressly preferred), unless otherwise stated, combinations of any two or more of such features, parameters and ranges thereof are included in the disclosure herein, regardless of their respective degrees of preference.

[0045] As used herein, the terms "purified," "substantially purified," and "isolated" refer to a state in which the compound of the invention is free of other distinct compounds with which it is normally associated in its natural state, whereby "purified," "substantially purified," and "isolated" refer to a subject that comprises at least 0.5%, 1%, 5%, 10%, or 20%, or at least 50% or 75%, by weight, of the mass of a given sample. In one embodiment, these terms refer to a subject in which the compound of the invention comprises at least 95, 96, 97, 98, 99, or 100%, by weight, of the mass of a given sample. As used herein, "purified," "substantially purified," and "isolated," when referring to a nucleic acid or protein, also refer to a state of purification or enrichment that is different from that which naturally occurs, for example, in a prokaryotic or eukaryotic environment, such as a bacterial or fungal cell, or in a mammalian organism, particularly a human. Any degree of purification or enrichment greater than that which occurs in nature is within the meaning of "isolated," including (1) purification from other structures or compounds with which they are associated, or (2) association with structures or compounds with which they are not normally associated in said prokaryotic or eukaryotic environment. The nucleic acids or proteins or classes of nucleic acids or proteins described herein can be isolated or otherwise associated with structures or compounds with which they are not normally associated in nature, according to a variety of methods and processes known to those of skill in the art.

[0046] The compounds described herein may contain one or more asymmetric elements, such as stereogenic centers, stereogenic axes, etc., such as asymmetric carbon atoms, so that the compounds may exist in different stereoisomeric forms.These compounds may be, for example, in the form of racemates or optically active forms.All stereoisomers, diastereomers, Z and E forms, purified forms and mixture forms are included.Therefore, when a compound is specifically named or a class of compounds is described, it is intended that all these forms are included.

[0047] The compounds described herein may also exist in two or more forms of structural isomers, also called constitutional isomers or positional isomers, which are molecules that have the same overall molecular formula but differ only in the different arrangement of their atoms or groups of atoms.

[0048] Thus, unless otherwise indicated for each of the compounds, biomolecules, and complexes described herein, any such possible stereoisomeric or regioisomeric form, or a mixture of two or more stereoisomeric and / or regioisomeric forms, is within the scope of the invention.

[0049] In addition to the ADC of the invention, a "pharmaceutical composition" comprises, for example, one or more substances selected from the group consisting of a pharma- ceutically acceptable preservative, a pharma- ceutically acceptable colorant, a pharma- ceutically acceptable protective colloid, a pharma- ceutically acceptable pH adjusting agent, and a pharma- ceutically acceptable osmolality adjusting agent. Such substances are described in the art. A more detailed description of pharmaceutical compositions of the invention is provided below.

[0050] As used herein, the term "effective amount" refers to the amount of a treatment sufficient to reduce or ameliorate the severity and / or duration of a disorder or one or more symptoms thereof, sufficient to prevent progression of a disorder, sufficient to cause regression of a disorder, sufficient to prevent the recurrence, development, onset or progression of one or more symptoms associated with a disorder, sufficient to detect a disorder, or sufficient to enhance or improve the prophylactic or therapeutic effects of another treatment (e.g., a prophylactic or therapeutic agent).

[0051] 2. Immunology The term "antibody" as used herein broadly refers to any immunoglobulin (Ig) molecule composed of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivative thereof that retains the essential epitope binding properties of an Ig molecule. Such functional fragment, mutant, variant, or derivative antibody formats are known in the art. Non-limiting embodiments thereof are discussed below. As used herein, a "full-length antibody" refers to an Ig molecule that comprises four polypeptide chains, two heavy chains and two light chains. The chains are usually linked to each other via disulfide bonds. Each heavy chain is composed of a heavy chain variable region (also referred to herein as "variable heavy chain" or abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (also referred to herein as "variable light chain" or abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0052] As used herein, the term "antigen-binding portion of an antibody" (or simply "antibody portion"), "antigen-binding moiety" (or simply "antibody moiety") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (i.e., an immunogenic product of the invention), i.e., functional fragments of an antibody. It has been shown that the antigen-binding function of an antibody can be performed by one or more fragments of a full-length antibody. Such antibody embodiments may also be bispecific, dual specific or multispecific, specifically binding to two or more different antigens. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL and CH1 domains, (ii) a F(ab') fragment, which is a bivalent fragment comprising two Fab fragments linked by disulfide bridges in the hinge region, and (iii) a Fab fragment, which is a bivalent fragment comprising two Fab fragments linked by disulfide bridges in the hinge region. 2(iii) Fd fragments consisting of the VH and CH1 domains, (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody, (v) dAb fragments comprising a single variable domain (Ward et al., Nature 341:544-546, 1989; Winter et al., WO90 / 05144 A1, herein incorporated by reference), and (vi) isolated complementarity determining regions (CDRs). Furthermore, the two domains VL and VH of the Fv fragment are encoded by separate genes, but can be joined together using recombinant methods by a synthetic linker that allows them to be made into a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as single-chain Fv (scFv), see, for example, Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). Such single-chain antibodies are also included in the term "antigen-binding portion" of an antibody. Other forms of single-chain antibodies, such as diabodies, are also included. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing of the two domains on the same chain, the domains pair with complementary domains on another chain, creating two antigen-binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993; Poljak et al., Structure 2:1121-1123, 1994). Such antibody binding moieties are known in the art (Kontermann and Dubel eds., Antibody Engineering, Springer-Verlag. New York. 790 pp., 2001, ISBN 3-540-41354-5).

[0053] The term "antibody" as used herein also includes antibody constructs. The term "antibody construct" as used herein refers to a polypeptide comprising one or more antigen-binding moieties of the present invention linked to a linker polypeptide or an immunoglobulin constant domain. A linker polypeptide comprises two or more amino acid residues joined together by peptide bonds and is used to link one or more antigen-binding moieties. Such linker polypeptides are well known in the art (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993; Poljak et al., Structure 2:1121-1123, 1994).

[0054] An immunoglobulin constant domain refers to a heavy or light chain constant domain. The amino acid sequences of the heavy and light chain constant domains of human IgG are known in the art.

[0055] Additionally, the binding proteins (e.g., antibodies) of the invention may be part of a larger immunoadhesion molecule formed by covalent or non-covalent association of the binding protein of the invention with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of a streptavidin core region to generate tetrameric scFv molecules (Kipriyanov et al., Human Antibodies and Hybridomas 6:93-101, 1995) and the use of cysteine ​​residues, a marker peptide and a C-terminal polyhistidine tag to generate bivalent biotinylated scFv molecules (Kipriyanov et al., Mol. Immunol. 31:1047-1058, 1994). Antibody moieties, such as Fab and F(ab') 2 Fragments can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Furthermore, antibodies, antibody portions, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.

[0056] As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities. However, an isolated antibody that specifically binds to an immunogenic product of the invention may have cross-reactivity to other antigens, such as Aβ globulomers, such as Aβ(20-42) globulomers or other Aβ forms. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals and / or any other targeted Aβ forms.

[0057] The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs, particularly CDR3. However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0058] As used herein, the term "recombinant human antibody" refers to any human antibody that is prepared, expressed, generated or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector transfected into a host cell (described further in section B below), antibodies isolated from a combinatorial recombinant human antibody library (Hoogenboom, TIB Tech. 15:62-70, 1997; Azzazy and Highsmith, Clin. Biochem. 35:425-445, 2002; Gavilondo JV and Larrick JW (2002) BioTechniques 29:128-145; Hoogenboom H. and Chames P. (2000) Immunology Today 21: 371-378), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (e.g., Taylor, LD, et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann SA. and Green LL (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al (2000) Immunology Today 21:364-370), or any other means involving splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies have been subjected to in vitro mutagenesis (or in vivo somatic mutagenesis, when animals transgenic for human Ig sequences are used) such that the amino acid sequences of the VH and VL regions of the recombinant antibody, while derived and related to human germline VH and VL sequences, are sequences that may not naturally exist within the in vivo human antibody germline repertoire.

[0059] The term "chimeric antibody" refers to an antibody that contains heavy and light chain variable region sequences derived from one species and constant region sequences derived from another species, e.g., an antibody in which murine heavy and light chain variable regions are linked to human constant regions.

[0060] The term "CDR-grafted antibody" refers to an antibody that comprises heavy and light chain variable region sequences from one species, but in which one or more sequences of the VH and / or VL CDR regions have been replaced with CDR sequences from another species, e.g., an antibody having a murine CDR (e.g. CDR3) in which one or more of the murine variable heavy and light chain regions have been replaced with human variable heavy and light chain sequences.

[0061] The terms "Kabat numbering," "Kabat definition," and "Kabat label" are used interchangeably herein. These terms are art-recognized and refer to a numbering system for amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody or antigen-binding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication NO: 91-3242). In the heavy chain variable region, the hypervariable region ranges from amino acid positions 31-35 for CDR1, amino acid positions 50-65 for CDR2, and amino acid positions 95-102 for CDR3. In the light chain variable region, the hypervariable region ranges from amino acid positions 24-34 for CDR1, amino acid positions 50-56 for CDR2, and amino acid positions 89-97 for CDR3.

[0062] As used herein, the terms "acceptor" and "acceptor antibody" refer to an antibody or nucleic acid sequence that provides or encodes at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% of the amino acid sequence of one or more of the framework regions. In some embodiments, the term "acceptor" refers to an antibody amino acid or nucleic acid sequence that encodes or provides the constant regions. In yet another embodiment, the term "acceptor" refers to an antibody amino acid or nucleic acid sequence that encodes or provides one or more of the framework regions and the constant regions. In certain embodiments, the term "acceptor" refers to a human antibody amino acid or nucleic acid sequence that provides or encodes at least 80%, e.g., at least 85%, at least 90%, at least 95%, at least 98%, or 100% of the amino acid sequence of one or more of the framework regions. In accordance with this embodiment, the acceptor may contain at least 1, at least 2, at least 3, at least 4, at least 5, or at least 10 amino acid residues that are not present in one or more specific positions of a human antibody. The acceptor framework regions and / or acceptor constant regions can be derived or obtained, for example, from germline antibody genes, mature antibody genes, functional antibodies (e.g., antibodies known in the art, antibodies in development, or commercially available antibodies).

[0063] The term "CDR" as used herein refers to the complementarity determining region in an antibody variable sequence. There are three CDRs in each of the heavy and light chain variable regions, which are called CDR1, CDR2 and CDR3 for each variable region. The term "CDR set" as used herein refers to a group of three CDRs present in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs are defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia et al. (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) found that, despite great diversity at the amino acid sequence level, certain subportions within the Kabat CDRs adopt nearly identical peptide backbone conformations. These subportions were designated L1, L2, and L3 or H1, H2, and H3, where "L" and "H" refer to the light and heavy chain regions, respectively. These regions were designated by Chothia. These may be referred to as CDRs that overlap with the Kabat CDRs. Other boundaries that define CDRs that overlap with the Kabat CDRs have been described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)). Still other CDR boundary definitions may not strictly follow one of the above systems, but would still overlap with the Kabat CDRs.However, they may be shortened or lengthened in light of predicted or experimental findings that particular residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, and in certain embodiments use CDRs defined by Kabat or Chothia.

[0064] The term "canonical" residues as used herein refers to residues within a CDR or framework that define a particular canonical CDR structure as defined by Chothia et al. (J. Mol. Biol. 196:901-907 (1987); Chothia et al., J. Mol. Biol. 227:799 (1992); both incorporated herein by reference). According to Chothia et al., a significant portion of the CDRs of many antibodies have nearly identical peptide backbone conformations, despite great diversity at the level of amino acid sequence. Each canonical structure primarily defines a set of peptide backbone torsion angles for a contiguous segment of amino acid residues that form a loop.

[0065] As used herein, the terms "donor" and "donor antibody" refer to an antibody providing one or more CDRs. In one embodiment, the donor antibody is an antibody of a different species than the antibody from which the framework region is obtained or derived. In the context of a humanized antibody, the term "donor antibody" refers to a non-human antibody providing one or more CDRs.

[0066] The term "framework" or "framework sequence" as used herein refers to the remaining sequence of the variable region excluding the CDRs. The exact definition of the CDR sequence may be determined by different systems, and therefore the meaning of the framework sequence is interpreted accordingly. The six CDRs (CDR-L1, -L2, and -L3 of the light chain, and CDR-H1, -H2, and -H3 of the heavy chain) also divide the framework region on the light and heavy chain into four subregions (FR1, FR2, FR3, and FR4) on each chain, in which CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. A certain framework region referred to by others without specifying a particular subregion as FR1, FR2, FR3, or FR4 represents the combination of FRs in the variable region of a single naturally occurring immunoglobulin chain. As used herein, a single FR represents one of the four subregions, and a plurality of FRs represents two or more of the four subregions that make up the framework region.

[0067] Human heavy and light chain acceptor sequences are known in the art.

[0068] As used herein, the term "germline antibody gene" or "gene fragment" refers to an immunoglobulin sequence encoded by a non-lymphoid cell that has not undergone the maturation process that leads to gene rearrangement and mutation for expression of a specific immunoglobulin (see, e.g., Shapiro et al., Crit. Rev. Immunol. 22(3):183-200 (2002); Marchalonis et al., Adv Exp Med Biol. 484:13-30 (2001)). One advantage provided by various embodiments of the present invention arises from the recognition that germline antibody genes are more likely than mature antibody genes to conserve essential amino acid sequence structures characteristic of individuals within a species, and therefore are less likely to be recognized as foreign when used therapeutically in that species.

[0069] The term "key residues" as used herein refers to certain residues in the variable region that have a major impact on the binding specificity and / or affinity of an antibody, particularly a humanized antibody. Key residues include, but are not limited to, one or more of the following: residues adjacent to the CDRs, potential glycosylation sites (whether N- or O-glycosylation sites), rare residues, residues that may interact with the antigen, residues that may interact with the CDRs, canonical residues, contact residues between the heavy and light chain variable regions, residues in the Vernier zone, and residues in the overlap region between the variable heavy chain CDR1 definition by Chothia and the first heavy chain framework definition by Kabat.

[0070] The term "humanized antibody" as used herein refers to an antibody or variant, derivative, analog or portion thereof that comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementarity determining region (CDR) having substantially the amino acid sequence of a non-human antibody, which immunospecifically binds to a target antigen. As used herein, the term "substantially", in the context of a CDR, refers to a CDR that has an amino acid sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of the non-human antibody CDR. A humanized antibody comprises at least one, and typically two, variable domains (Fab, Fab', F(ab') 2Humanized antibodies generally comprise substantially all of the CDR regions (FabC, Fv) of a non-human immunoglobulin (i.e., donor antibody) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are of a human immunoglobulin consensus sequence. According to one aspect, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In some embodiments, the humanized antibody contains both a light chain and at least a heavy chain variable domain. The antibody may also include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody contains only a humanized light chain. In some embodiments, the humanized antibody contains only a humanized heavy chain. In certain embodiments, the humanized antibody contains only humanized variable domains of the light chain and / or the heavy chain.

[0071] The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4. The humanized antibody may comprise sequences from more than one class or isotype, and particular constant domains can be selected to optimize desired effector functions using techniques well known in the art.

[0072] The framework and CDR regions of a humanized antibody need not correspond exactly to the parental sequences. For example, the donor antibody CDR or consensus framework may be mutated by substitution, insertion and / or deletion of at least one amino acid residue such that the CDR or framework residue at that site does not correspond to either the donor antibody or the consensus framework. In one embodiment, however, such mutations will not be extensive. Usually, at least 90%, at least 95%, at least 98%, or at least 99% of the humanized antibody residues will correspond to those of the parental FR and CDR sequences. The term "consensus framework" as used herein refers to the framework region in a consensus immunoglobulin sequence. The term "consensus immunoglobulin sequence" as used herein refers to a sequence formed from the amino acids (or nucleotides) that occur most frequently within a family of related immunoglobulin sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany, 1987)). In a family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid that occurs most frequently at that position within that family. If two amino acids occur equally frequently, either can be included in the consensus sequence.

[0073] As used herein, "Vernier" zone refers to a subset of framework residues that can adjust the CDR structure and fine-tune the fit to the antigen, as described by Foote and Winter (1992, J. Mol. Biol. 224:487-499, incorporated herein by reference). Vernier zone residues form a layer of support for the CDR and can affect the structure of the CDR and the affinity of the antibody.

[0074] The term "antibody" as used herein also includes multivalent binding proteins. As used herein, the term "multivalent binding protein" is used to refer to a binding protein that contains two or more antigen binding sites. A multivalent binding protein is engineered to have three or more antigen binding sites and is generally not a naturally occurring antibody. The term "multispecific binding protein" refers to a binding protein that can bind to two or more related or unrelated targets. A dual variable domain (DVD) binding protein as used herein is a binding protein that contains two or more antigen binding sites and is a tetravalent or multivalent binding protein. Such DVDs can be monospecific, i.e., capable of binding to one antigen, or multispecific, i.e., capable of binding to two or more antigens. A DVD binding protein that contains two heavy chain DVD polypeptides and two light chain DVD polypeptides is referred to as a DVD Ig. Each half of a DVD Ig contains a heavy chain DVD polypeptide and a light chain DVD polypeptide, and two antigen binding sites. Each binding site contains a heavy chain variable domain and a light chain variable domain, with a total of six CDRs involved in antigen binding per antigen binding site. DVD-binding proteins and methods for making DVD-binding proteins are disclosed in US patent application Ser. No. 11 / 507,050, which is incorporated herein by reference.

[0075] The term "labeled binding protein" as used herein refers to a binding protein that incorporates a label that allows for identification of the binding protein. Similarly, the term "labeled antibody" as used herein refers to an antibody that incorporates a label that allows for identification of the antibody. In one aspect, the label is a detectable marker, such as the incorporation of a radiolabeled amino acid, or the attachment of a biotinyl moiety to the polypeptide that is detectable by a marked avidin (e.g., streptavidin that contains a fluorescent marker or an enzymatic activity that is detectable by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g., 3 H, 14 C. 35 S, 90 Y, 99Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho, or 153 Sm), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), and magnetic agents such as gadolinium chelates.

[0076] As used herein, the term "antibody" also includes antibody conjugates, which refers to a binding protein, such as an antibody, chemically linked to a second chemical moiety, such as a therapeutic agent.

[0077] As used herein, "K D ” (“K d " or "KD") is intended to refer to the "equilibrium dissociation constant", which can be obtained by titration measurements at equilibrium, or the dissociation rate constant (k off ) to the association rate constant (k on The association rate constant (k on ), dissociation rate constant (k off ), and the equilibrium dissociation constant (K D ) is used to express the binding affinity of a binding protein (e.g., an antibody) to an antigen. Methods for determining association and dissociation rate constants are well known in the art. Fluorescence-based techniques allow for sensitive testing of samples in physiological buffer at equilibrium. Other experimental approaches and instruments such as BIAcore® (Biomolecular Interaction Analysis) assays can also be used (e.g., instruments available from BIAcore International AB, GE Healthcare, Uppsala, Sweden). In addition, KinExA® (KinExA) assays available from Sapidyne Instruments (Boise, Idaho) can also be used.

[0078] "Internalizing" or "internalization" of an immunoglobulin molecule refers to the ability of an immunoglobulin or ADC or APC described herein to bind to a cell surface receptor and induce receptor-mediated endocytosis upon binding.

[0079] "Deglycosylated" or "deglycosylation" refers to the partial, especially complete, removal of one or more glycosyl residues from a glycosylated species of a biomolecule, such as a glycosylated immunoglobulin molecule.

[0080] 3. Genetic Code Expansion "Aminoacyl-tRNA synthetase" (RS) is a tRNA アミノアシル with an amino acid or amino acid analogue.

[0081] "Pyrrolysyl-tRNA synthetase" (PylRS) is a tRNA (tRNA Pyl ) can be acylated with a specific amino acid or amino acid analog, preferably an ncAA or a salt thereof.

[0082] As used herein, the term "archaeal pyrrolysyl-tRNA synthetase" (abbreviated as "archaeal PylRS") refers to a PylRS in which at least a segment of the PylRS amino acid sequence or the entire PylRS amino acid sequence has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence of a naturally occurring PylRS from an archaea, or to the amino acid sequence of an enzymatically active fragment of such a naturally occurring PylRS.

[0083] The PylRS of the present invention may comprise a mutated archaeal PylRS, or an enzymatically active fragment thereof.

[0084] Generally, a "mutant PylRS" or "mutated PylRS" differs from the corresponding wild-type PylRS in that it contains one or more additions, substitutions and / or deletions of amino acid residues. Preferably, these are modifications that improve the stability of PylRS, modulate the substrate specificity of PylRS and / or enhance the enzymatic activity of PylRS. Particularly preferred "mutant archaeal PylRS" or "mutated archaeal PylRS" are described in more detail herein below.

[0085] The term "nuclear export signal" (abbreviated as "NES") refers to an amino acid sequence that can enable a polypeptide containing it (e.g., the NES-containing PylRS of the present invention) to be exported from the nucleus of a eukaryotic cell. The export is believed to be mediated primarily by Crm1 (chromosomal region maintenance 1, also known as karyopherin exportin 1). NESs are known in the art. For example, the ValidNESs database (http: / / validness.ym.edu.tw / ) provides sequence information for experimentally validated NES-containing proteins. Further, NES databases, such as NESbase 1.0 (see www.cbs.dtu.dk / databased / NESbase-1.0 / ; Le Cour et al., Nucl Acids Res 31(1), 2003), as well as NES prediction tools, such as NetNES (see www.cbs.dtu.dk / services / NetNES / ; La Cour et al., La Cour et al., Protein Eng Des Sel 17(6):527-536, 2004), NESpredictor (NetNES, http: / / www.cbs.dtu.dk / ; Fu et al., Nucl Acids Res 41:D338-D343, 2013; La Cour et al., Protein Eng Des Sel 17(6):527-536, 2004) and NESsential (a web interface in combination with ValidNESs) are publicly available. Hydrophobic leucine-rich NESs are the most common and represent the best characterized group of NESs to date. Hydrophobic leucine-rich NESs are non-conserved motifs with three or four hydrophobic residues. Many of these NESs contain the conserved amino acid sequence pattern LxxLxL (SEQ ID NO:111) or LxxxLxL (SEQ ID NO:112), where each L is independently selected from leucine, isoleucine, valine, phenylalanine and methionine amino acid residues and each x is independently selected from any amino acid (see La Cour et al., Protein Eng Des Sel 17(6):527-536, 2004).

[0086] The term "nuclear localization signal" (abbreviated "NLS" and also referred to in the art as "nuclear localization sequence") refers to an amino acid sequence that is capable of directing the import of a polypeptide containing it (e.g., wild-type archaeal PylRS) into the nucleus of a eukaryotic cell. The export is believed to be mediated by the NLS-containing polypeptide binding to importin (also known as karyopherin), forming a complex that passes through the nuclear pore. NLSs are known in the art. A number of NLS databases and NLS prediction tools are publicly available, such as NLSdb (see Nair et al., Nucl Acids Res 31(1), 2003), cNLS Mapper (see www.nls-mapper.aib.keio.ac.jp; see Kosugi et al., Proc Natl Acad Sci US A. 106(25):10171-10176, 2009; Kosugi et al., J Biol Chem 284(1):478-485, 2009), SeqNLS (see Lin et al., PLoS One 8(10):e76864, 2013), and NucPred (see www.sbc.su.se / ~maccallr / nucpred / ; see Branmeier et al., Bioinformatics 23(9):1159-60, 2007).

[0087] Unless otherwise indicated, as used herein, "tRNA アミノアシル. ", especially "tRNA Pyl " refers to a tRNA that can be acylated (substantially selectively, particularly selectively) by an aminoacyl RS, particularly a PylRS. The tRNAs described herein Pyl can be a wild-type tRNA that can be acylated with pyrrolysine by the PylRS, or a mutant of such a tRNA, e.g., a wild-type or mutant tRNA from Archaea, e.g., a species of Methanosarcina, e.g., M. mazei or M. barkeri. For site-specific incorporation of an ncAA into the POI, the "tRNA" used with the respective RS is アミノアシル" or tRNA Pyl The anticodon included in the "tRNA" is the reverse complement of the selector codon. アミノアシル ", especially tRNA Pyl The anticodon of is the reverse complement of the amber stop codon.

[0088] As used herein, the term "selector codon" refers to a codon that is selected from a tRNA アミノアシル " or tRNA Pyl The selector codon refers to a codon that is recognized (i.e., bound) by a tRNA and not recognized by endogenous tRNAs in a eukaryotic cell. The term is also used for the corresponding codon in a polypeptide-coding sequence of a polynucleotide that is not a messenger RNA (mRNA), such as a DNA plasmid. Preferably, the selector codon is a codon of low abundance in naturally occurring eukaryotic cells. "tRNA アミノアシル " or tRNA PylThe anticodon of binds to the selector codon in the mRNA, thereby site-specifically incorporating the ncAA into the growing chain of the polypeptide encoded by said mRNA. The 64 known genetic (triplet) codons code for 20 amino acids and three stop codons. Because only one stop codon is required for translation termination, the other two can in principle be used to code for non-proteinogenic amino acids. For example, the amber codon UAG has been successfully used in in vitro and in vivo translation systems as a selector codon to direct the incorporation of unnatural amino acids. The selector codons utilized in the methods of the invention extend the genetic codon framework of the protein biosynthetic machinery of the translation system used. Specifically, selector codons include, but are not limited to, nonsense codons, such as stop codons, e.g., amber (UAG), ochre (UAA), and opal (UGA) codons, codons consisting of more than three bases (e.g., four-base codons), and codons derived from natural or unnatural base pairs. For a given system, the selector codon can also include one of the natural three-base codons (i.e., a natural triplet), where the endogenous translation system does not (none or rarely) use said natural triplet, e.g., a system that lacks a tRNA that recognizes the natural triplet, or a system where the natural triplet is a rare codon.

[0089] Recombinant tRNAs that modulate the reading of mRNA in a given translation system (e.g., eukaryotic cells) to allow the read-through of, for example, a stop codon, a four-base codon, or a rare codon are called "suppressor tRNAs." The efficiency of suppression of a stop codon (e.g., an amber codon) that functions as a selector codon depends on the competition between the (aminoacylated) tRNA (acting as a suppressor tRNA) and a termination factor (e.g., RF1) that binds to the stop codon and initiates the release of the growing polypeptide chain from the ribosome. Thus, the efficiency of suppression of such stop codons can be increased by using a termination factor (e.g., RF1)-deficient strain.

[0090] The polynucleotide sequence encoding the "protein of interest" or "POI" is Pyl The polynucleotide sequence may include one or more, e.g., two or more, four or more, etc., codons (e.g., selector codons) that are the reverse complement of the anticodon contained in the POI. Conventional site-directed mutagenesis can be used to introduce the codons into the polynucleotide sequence at the site of interest to generate a polynucleotide sequence encoding the POI.

[0091] The RS (or a variant thereof) and the respective tRNA are preferably orthogonal.

[0092] The term "orthogonal" as used herein refers to a molecule (e.g., an orthogonal tRNA and / or an orthogonal RS) that is used with low efficiency by a translation system of interest (e.g., a eukaryotic cell used to express a POI described herein). "Orthogonal" refers to the inability or low efficiency of the orthogonal tRNA or orthogonal RS to function with the endogenous RS or endogenous tRNA, respectively, of the translation system of interest, e.g., less than 20% efficiency, less than 10% efficiency, less than 5% efficiency, or, for example, less than 1% efficiency.

[0093] Thus, in certain embodiments of the invention, any endogenous RS of a eukaryotic cell of the invention acylates an (orthogonal) tRNA with reduced or essentially zero efficiency compared to acylation of the endogenous tRNA by the endogenous RS. Pyl For example, with less than 20% efficiency, less than 10% efficiency, less than 5% efficiency, or less than 1% efficiency. Alternatively or in addition, the (orthogonal) PylRS of the present invention catalyzes the acylation of tRNA by a cell's endogenous RS. Pyl In one embodiment, the endogenous tRNA of a eukaryotic cell of the invention is acylated with low or substantially zero efficiency, e.g., less than 20% efficiency, less than 10% efficiency, less than 5% efficiency, or less than 1% efficiency, compared to the acylation of any endogenous tRNA of the eukaryotic cell of the invention.

[0094] Unless otherwise indicated, the terms "endogenous tRNA" and "endogenous aminoacyl-tRNA synthetase" ("endogenous RS") as used herein refer to PylRS and tRNA Pyl These terms refer to the tRNA and RS that were present in the cell that will ultimately be used as a translation system before the introduction of each of the tRNA and RS.

[0095] The term "translation system" generally refers to a set of components necessary to incorporate naturally occurring amino acids into a growing polypeptide chain (protein). Translation system components include, for example, ribosomes, tRNA, aminoacyl-tRNA synthetases (RS), mRNA, etc. Translation systems include artificial mixtures of the components, cell extracts, and living cells, e.g., living eukaryotic cells.

[0096] In the eukaryotic cells used to prepare the POI, tRNA アミノアシル The PylRS and tRNA used to prepare the POI according to the present invention are preferably acylated with an ncAA or a salt thereof (ncAA) by the PylRS of the present invention. アミノアシル The pair is preferably orthogonal. Advantageously, the orthogonal pair is capable of translating tRNA acylated with, for example, an ncAA. Pyl The ncAA residue functions in the eukaryotic cell to be incorporated into the growing polypeptide chain of the POI using, for example, a tRNA. Pyl recognizes a codon in the mRNA that encodes the POI (eg, a selector codon, such as an amber stop codon).

[0097] As used herein, the term "preferentially acylated" refers to a tRNA or amino acid that is preferentially acylated relative to the endogenous tRNA or amino acid of a eukaryotic cell, e.g., PylRS is a tRNA Pyl The ncAA is then acylated with a tRNA, for example, at about 50% efficiency, about 70% efficiency, about 75% efficiency, about 85% efficiency, about 90% efficiency, about 95% efficiency, or about 99% or more efficient. PylFor a given codon (e.g., a selector codon) that is the reverse complement of the anticodon contained in, is incorporated into the growing polypeptide chain with high fidelity, e.g., greater than 75%, greater than 80%, greater than 90%, greater than 95%, or greater than 99% efficiency or greater.

[0098] The term "non-standard amino acid" (abbreviated as "ncAA") as used herein refers to an amino acid that is not one of the 20 standard amino acids or selenocysteine ​​or pyrrolysine. The term also refers to amino acid analogs, e.g., compounds that differ from amino acids such that the α-amino group is replaced by a hydroxyl group and / or the carboxylic acid functional group forms an ester. When translationally incorporated into a polypeptide, said amino acid analogs result in an amino acid residue that differs from the corresponding amino acid residue of the 20 standard amino acids or selenocysteine ​​or pyrrolysine. When an ncAA, an amino acid analog in which the carboxylic acid functional group forms an ester of the formula -C(O)-OR, is used to prepare a polypeptide in a translation system (such as a eukaryotic cell), it is believed that R is removed in situ in the translation system, e.g., enzymatically, before being incorporated into the POI. Thus, R is advantageously selected to be compatible with the ability of the translation system to convert the ncAA or its salt into a form that is recognized and processed by the PylRS of the present invention. ncAA useful in the methods and kits of the present invention have been described in the prior art (for reviews, see, e.g., Liu et al., Annu Rev Biochem 83:379-408, 2010; Lemke, ChemBioChem 15:1691-1694, 2014).

[0099] The term "host cell" or "transformed cell" as used herein refers to a cell (or organism) that has been modified to carry at least one nucleic acid molecule, such as a recombinant gene, that encodes a desired protein or nucleic acid sequence, which upon transcription produces a polypeptide for use as described herein. The host cell may be a prokaryotic or eukaryotic cell, such as a bacterial cell, a fungal cell, a plant cell, an insect cell or a mammalian cell. The host cell may contain the recombinant gene integrated into the nuclear or organelle genome of the host cell. Alternatively, the host may contain the recombinant gene extrachromosomally.

[0100] A particular organism or cell is said to be "capable of producing a POI" if it naturally produces the POI, or if it does not naturally produce said POI but can be transformed to produce said POI.

[0101] C. Specific Aspects and Embodiments of the Invention The present invention relates to the following aspects and specific embodiments thereof.

[0102] A first aspect of the present invention relates to a site-selectively modified immunoglobulin molecule comprising at least one, more particularly one or two, immunoglobulin heavy chains (IgH) and at least one, more particularly one or two, immunoglobulin light chains (IgL), The IgH CDR-H1 selected from SEQ ID NOs: 9 and 10, CDR-H2 selected from SEQ ID NOs: 11 and 12, and A variable region V comprising a CDR-H3 selected from SEQ ID NOs: 13 and 14 H ,and Constant region C H Including, The IgL CDR-L1 selected from SEQ ID NOs: 15 and 16, CDR-L2 selected from SEQ ID NOs: 17 and 18, and Variable region V comprising CDR-L3 selected from SEQ ID NOs: 19 and 20L ,and Constant region C L Including, Where: a) at least one, more particularly one or two IgHs have at least one, e.g. one, two, three, four, or five, more particularly one or two, non-canonical amino acid (ncAA) residue in their amino acid sequence, in particular at at least one, e.g. one, two, three, four, or five, more particularly one or two positions, C H FR1, FR2, FR3 and FR4, more particularly one or two frameworks V selected from FR1, FR2, FR3 and FR4, more particularly one or two frameworks V selected from FR2 and FR3, and / or at least one, e.g. one, two, three, four or five, more particularly one or two positions. H or b) at least one, more particularly one or two IgLs, have at least one, e.g., 1, 2, 3, 4, or 5, more particularly one or two, non-canonical amino acid (ncAA) residues in their amino acid sequence, in particular L Framework V selected from FR1, FR2, FR3 and FR4, more specifically selected from FR2 and FR3, L and at a position selected from positions within the CDRs, in particular CDR-L1, CDR-L2 and CDR-L3, more particularly within CDR-L2; or c) at least one, more particularly one or two IgHs and at least one, more particularly one or two IgLs are simultaneously site-selectively modified by incorporating at least one, e.g. one, two, three, four, or five, more particularly one or two ncAA residues in their amino acid sequences, whereby the IgHs, in particular, contain at least one, e.g. one, two, three, four, or five, more particularly one or two positions, C HFR1, FR2, FR3 and FR4, more particularly one or two frameworks V selected from FR1, FR2, FR3 and FR4, more particularly one or two frameworks V selected from FR2 and FR3, and / or at least one, e.g. one, two, three, four or five, more particularly one or two positions. H In the region, it is mutated and IgL, in particular, L Framework V selected from FR1, FR2, FR3 and FR4, more specifically selected from FR2 and FR3, L and at a position selected from positions within the CDRs, in particular CDR-L1, CDR-L2 and CDR-L3, more particularly within CDR-L2, The site-selectively modified immunoglobulin molecule has the ability to bind to human epidermal growth factor receptor 2 (ERBB2 or HER2 / neu).

[0103] The inventors have found that site-specific labelling at appropriately selected amino acid positions of said immunoglobulins according to the present invention surprisingly results in an increase in the potency of the final ADC.

[0104] Without wishing to be bound by any theory, the inventors have found that substitution of any amino acid that is not involved in a specific secondary structure element such as an alpha helix or a beta sheet, but rather is within the linker region and is also in a surface accessible position, results in a highly efficient ADC.

[0105] In this respect, amino acid positions buried within functional pockets responsible for local / global structure stabilizing effects such as CDRs, antigen binding domains, alpha helices and beta sheets are preferably left intact, i.e. in their naturally occurring form, in order to preserve the overall structure of the IgG molecule.

[0106] More specifically, the modified immunoglobulin molecule may be an IgG antibody molecule.

[0107] More specifically, said modified immunoglobulin molecules exhibit the ability to be internalized by receptor-mediated endocytosis upon binding to a cell surface receptor.

[0108] According to another particular embodiment, the present invention also relates to each modified individual IgH or IgL polypeptide chain or fragment or derivative of such immunoglobulin molecule, wherein said polypeptide chain, fragment or derivative retains at least one site-specific modification as defined herein. For example, when a site-specifically modified immunoglobulin contains at least one site-specific modification within its Fab region, the Fab or (Fab) 2 For example, if the site-specifically modified immunoglobulin contains at least one site-specific modification within its Fv region, for example, an scFv fragment is also part of the present invention.

[0109] In another specific embodiment, the site-modified immunoglobulin molecules are produced with high affinity, e.g., 1×10 -6 K for affinity over M D Or 1×10 -7 of affinity above M, e.g. 1 x 10 -8 M to 1×10 -12 M, e.g. 1×10 -9 M to 1×10 -10 K in the M range D and binds to the target (ERBB2 or HER2 / neu).

[0110] The modified immunoglobulin molecule may be provided in a glycosylated form.

[0111] Alternatively, the modified immunoglobulin molecule may be provided in non-glycosylated or deglycosylated form.

[0112] In certain embodiments of the first aspect, a site-selectively modified immunoglobulin molecule is provided, comprising: wherein said at least one IgH has at least one ncAA residue in its amino acid sequence, H 2 and / or one or more, for example one, two, three, four, more particularly one or two, frameworks V selected from FR1, FR2, FR3 and FR4, more particularly selected from FR2 and FR3. H and / or has been site-selectively modified by incorporation at least one, e.g., one, two, three, four or five, more particularly one or two positions within the region; The at least one, more particularly one or two IgLs have at least one, e.g., one, two, three, four or five, more particularly one or two, non-canonical amino acid (ncAA) residues at the C L Framework V selected from FR1, FR2, FR3 and FR4, more specifically selected from FR2 and FR3, L and at positions selected from positions within the CDRs, in particular CDR-L1, CDR-L2 and CDR-L3, more particularly within CDR-L2, The site-selectively modified immunoglobulin molecule has the ability to bind to human epidermal growth factor receptor 2.

[0113] More specifically, in the site-selectively modified immunoglobulin molecule of this embodiment: Site-selectively modified IgH has been characterized by the modification of its constant region, especially C H 2, or in one or two positions within FR2 and FR3, more particularly in FR2, selected from framework V H The IgH is modified at one or two positions within the region, e.g., C H 2 or 1 modification in FR2, and / or The site-selectively modified IgL has a constant region C L or its variable region V L or its constant region CL and its variable region V L For example, IgL is modified once within FR2 and / or FR3.

[0114] In another particular embodiment of said first aspect, a site-selectively modified immunoglobulin molecule is provided, in which additionally or alternatively at least one, more particularly one or two IgHs have at least one, e.g. one, two, three, four or five, more particularly one or two non-canonical amino acid (ncAA) residues in their amino acid sequence, in particular in a V region selected from FR1, FR2, FR3 and FR4, more particularly in a V region selected from FR2 and FR3. H and / or at positions selected from positions within the CDRs, in particular CDR-H1, CDR-H2 and CDR-H3, The site-selectively modified immunoglobulin molecule has the ability to bind to human epidermal growth factor receptor 2.

[0115] In another particular embodiment of said first aspect of the invention there is provided a site-selectively modified immunoglobulin, wherein the single mutation is not at position A121 of the heavy chain of SEQ ID NO:2.

[0116] In another particular embodiment of said first aspect of the invention there is provided a site-selectively modified immunoglobulin, wherein the single mutation is not at position A132 of the heavy chain of SEQ ID NO:2.

[0117] In another particular embodiment of said first aspect of the invention there is provided a site-selectively modified immunoglobulin, wherein the double mutation is not at positions A121 and A132 of the heavy chain of SEQ ID NO:2.

[0118] Each of the above variants may be provided in a non-glycosylated, glycosylated or deglycosylated form.

[0119] In another particular embodiment of said first aspect of the invention, a site-selectively modified immunoglobulin is provided, wherein said site-selectively modified IgH comprises an ncAA at at least one, such as 1, 2, 3, 4 or 5, more particularly 1 or 2, amino acid sequence positions corresponding to positions selected from the following (referred to as Group 1): a) V H : S25, K43, R50, D62, K65, E89, D102 of SEQ ID NO: 2, b) C H Position 1: A121, E155, P156, S194, E219 of SEQ ID NO:2, c) C H Position 2: D252, E275, K277, D283, H288, K293, E296, R304, K323 of SEQ ID NO:2, and / or wherein the site-selectively modified IgL comprises an ncAA at at least one amino acid sequence position corresponding to a position selected from: d) V L Positions: K42, K45, R61, D70, E81 of SEQ ID NO:4, e) C L Positions: E143, D151, G157, G200 of sequence number 4.

[0120] In a particular embodiment thereof, a site-selectively modified immunoglobulin is provided, in which a single mutation at position A121 of the heavy chain of SEQ ID NO:2 is excluded.

[0121] Each of the above variants may be provided in a non-glycosylated, glycosylated or deglycosylated form.

[0122] More specifically, the two site-selective modifications are selected from: a) (Group 1) At least one single IgH modification at one of the amino acid sequence positions corresponding to a position selected from the following: i.V H S25, K43, R50, D62, K65, E89, D102 of SEQ ID NO: 2, ii. C H Position 1: A121, E155, P156, S194, E219 of SEQ ID NO:2, iii. C H Position 2: D252, E275, K277, D283, H288, K293, E296, R304, K323, and At least one single IgL modification in one of the amino acid sequence positions corresponding to iv. V L Positions: K42, K45, R61, D70, E81 of SEQ ID NO:4, v.C L Position: E143, D151, G157, G200 of SEQ ID NO:4, or b) Dual modifications in at least one IgH at two amino acid sequence positions corresponding to positions selected from: i.V H S25, K43, R50, D62, K65, E89, D102 of SEQ ID NO: 2, ii. C H Position 1: A121, E155, P156, S194, E219 of SEQ ID NO:2, iii. C H Position 2: D252, E275, K277, D283, H288, K293, E296, R304, K323 of SEQ ID NO:2, or c) Dual modifications in two amino acid sequence positions of at least one IgL corresponding to a position selected from: iv. V L Positions: K42, K45, R61, D70, E81 of SEQ ID NO:4, v.C L Positions: E143, D151, G157, G200 of sequence number 4.

[0123] Each of the above variants may be provided in a non-glycosylated, glycosylated or deglycosylated form.

[0124] According to another particular embodiment of the first aspect, a site-selectively modified immunoglobulin molecule is provided, wherein the site-selectively modified IgH chain, in particular C H , VH , or C H and V H both of which contain an ncAA at at least one, e.g., 1, 2, 3, 4 or 5, more particularly 1 or 2 amino acid sequence positions corresponding to positions selected from P41, G42, K291, K249, K251, K320 and K343 of SEQ ID NO:2; and / or wherein the site-selectively modified IgL comprises an ncAA at at least one, e.g., 1, 2, 3, 4 or 5, more particularly 1 or 2, amino acid sequence positions corresponding to positions selected from G41, A51, P59, A111 and K169 of SEQ ID NO:4.

[0125] In another particular embodiment of the first aspect of the invention, the site-selectively modified IgH comprises an ncAA at at least one, such as 1, 2, 3, 4 or 5, more particularly 1 or 2, amino acid sequence positions corresponding to positions selected from the following (referred to as Group 2): a) V H S25, P41, G42, K43, R50, D62, K65, E89, D102 of SEQ ID NO: 2, b) C H Position 1: A121, E155, P156, S194, E219 of SEQ ID NO:2, c) C H 2 positions: K249, K251, D252, E275, K277, D283, H288, K291, K293, E296, R304, K320, K323 and K343 of SEQ ID NO:2; and / or wherein said site-selectively modified IgL comprises an ncAA at at least one amino acid sequence position corresponding to a position selected from: d) V L Positions: G41, K42, K45, A51, P59, R61, D70, E81 of SEQ ID NO:4, e) C L Positions: A111, E143, D151, G157, K169, G200 of SEQ ID NO:4.

[0126] Each of the above variants may be provided in a non-glycosylated, glycosylated or deglycosylated form.

[0127] More specifically, the two site-selective modifications may be selected from: a) (referred to as Group 2) at least one single IgH modification at one of the amino acid sequence positions corresponding to a position selected from the following: vi. V H S25, P41, G42, K43, R50, D62, K65, E89, D102 of SEQ ID NO: 2, vii. C H Position 1: A121, E155, P156, S194, E219 of SEQ ID NO:2, viii. C H Position 2: K249, K251, D252, E275, K277, D283, H288, K291, K293, E296, R304, K320, K323 and K343 of SEQ ID NO:2, and At least one single IgL modification in one of the amino acid sequence positions corresponding to: ix. V L Positions: G41, K42, K45, A51, P59, R61, D70, E81 of SEQ ID NO:4, x.C L Position: A111, E143, D151, G157, K169, G200 of SEQ ID NO:4, or b) Dual modifications in at least one IgH at two amino acid sequence positions corresponding to positions selected from: xi. V H S25, P41, G42, K43, R50, D62, K65, E89, D102 of SEQ ID NO: 2, xii. C H Position 1: A121, E155, P156, S194, E219 of SEQ ID NO:2, xiii. C H Position 2: K249, K251, D252, E275, K277, D283, H288, K291, K293, E296, R304, K320, K323 and K343 of SEQ ID NO:2, or c) Dual modifications in two amino acid sequence positions of at least one IgL corresponding to a position selected from: xiv. V L Positions: G41, K42, K45, A51, P59, R61, D70, E81 of SEQ ID NO:4, xv. C L Positions: A111, E143, D151, G157, K169, G200 of SEQ ID NO:4.

[0128] Each of the above variants may be provided in a non-glycosylated, glycosylated or deglycosylated form.

[0129] In one embodiment, a site-selectively modified immunoglobulin molecule is provided that contains one ncAA in each of its IgH chains.

[0130] In one embodiment, a site-selectively modified immunoglobulin molecule is provided that contains one ncAA in each of its IgL chains.

[0131] In one embodiment, a site-selectively modified immunoglobulin molecule is provided that contains two ncAAs in each of its IgH chains.

[0132] In one embodiment, a site-selectively modified immunoglobulin molecule is provided that contains two ncAAs in each of its IgL chains.

[0133] In one embodiment, a site-selectively modified immunoglobulin molecule is provided that contains one ncAA in each of its IgH chains and one ncAA in each of its IgL chains.

[0134] In one embodiment, a site-selectively modified immunoglobulin molecule is provided that contains two ncAAs in each of its IgH chains and one ncAA in each of its IgL chains.

[0135] In one embodiment, a site-selectively modified immunoglobulin molecule is provided that contains one ncAA in each of its IgH chains and two ncAAs in each of its IgL chains.

[0136] In one embodiment, a site-selectively modified immunoglobulin molecule is provided that contains two ncAAs in each of its IgH chains and two ncAAs in each of its IgL chains.

[0137] Each of the above variants may be provided in a non-glycosylated, glycosylated or deglycosylated form.

[0138] In one embodiment, a site-selectively modified immunoglobulin molecule is provided that contains one ncAA in each constant region of its IgH chain.

[0139] In one embodiment, a site-selectively modified immunoglobulin molecule is provided that contains one ncAA in each constant region of its IgL chain.

[0140] In one embodiment, a site-selectively modified immunoglobulin molecule is provided that contains two ncAAs in each constant region of its IgH chain.

[0141] In one embodiment, a selectively modified immunoglobulin molecule is provided that contains two ncAAs in the constant region of each of its IgL chains.

[0142] In one embodiment, a site-selectively modified immunoglobulin molecule is provided that contains one ncAA in each of the constant regions of its IgH chains and one ncAA in each of the constant regions of its IgL chains.

[0143] In one embodiment, a site-selectively modified immunoglobulin molecule is provided that contains two ncAAs in each of the constant regions of its IgH chains and one ncAA in each of the constant regions of its IgL chains.

[0144] In one embodiment, a site-selectively modified immunoglobulin molecule is provided that contains one ncAA in each of the constant regions of its IgH chains and two ncAAs in each of the constant regions of its IgL chains.

[0145] In one embodiment, a site-selectively modified immunoglobulin molecule is provided that contains two ncAAs in each of the constant regions of its IgH chains and two ncAAs in each of the constant regions of its IgL chains.

[0146] Each of the above variants may be provided in a non-glycosylated, glycosylated or deglycosylated form.

[0147] In one embodiment, a site-selectively modified immunoglobulin molecule is provided that contains one ncAA in each variable region of its IgH chain.

[0148] In one embodiment, a site-selectively modified immunoglobulin molecule is provided that contains one ncAA in each variable region of its IgL chain.

[0149] In one embodiment, a site-selectively modified immunoglobulin molecule is provided that contains two ncAAs in the variable region of each of its IgH chains.

[0150] In one embodiment, a site-selectively modified immunoglobulin molecule is provided that contains two ncAAs in the variable region of each of its IgL chains.

[0151] In one embodiment, a site-selectively modified immunoglobulin molecule is provided that contains one ncAA in each variable region of its IgH chain and one ncAA in each variable region of its IgL chain.

[0152] In one embodiment, a site-selectively modified immunoglobulin molecule is provided that contains two ncAAs in each variable region of its IgH chain and one ncAA in each variable region of its IgL chain.

[0153] In one embodiment, a site-selectively modified immunoglobulin molecule is provided that contains one ncAA in each of the constant regions of its IgH chains and two ncAAs in each of the constant regions of its IgL chains.

[0154] In one embodiment, a site-selectively modified immunoglobulin molecule is provided that contains two ncAAs in each variable region of its IgH chain and two ncAAs in each variable region of its IgL chain.

[0155] Each of the above variants may be provided in a non-glycosylated, glycosylated or deglycosylated form.

[0156] In one embodiment, a site-selectively modified immunoglobulin molecule is provided that contains two ncAAs distributed on each of the constant and variable regions of its IgH chain.

[0157] In one embodiment, a site-selectively modified immunoglobulin molecule is provided that contains two ncAAs distributed on each of the constant and variable regions of its IgL chain.

[0158] In one embodiment, a site-selectively modified immunoglobulin molecule is provided that contains two ncAAs distributed on each of the constant and variable regions of its IgH chain and one ncAA in each of the constant regions of its IgL chain.

[0159] In one embodiment, a site-selectively modified immunoglobulin molecule is provided that contains one ncAA in each constant region of its IgH chain and two ncAAs distributed over each constant and variable region of its IgL chain.

[0160] In one embodiment, a site-selectively modified immunoglobulin molecule is provided that contains two ncAAs distributed on each of the constant and variable regions of its IgH chain and two ncAAs distributed on each of the constant and variable regions of its IgL chain.

[0161] Each of the above variants may be provided in a non-glycosylated, glycosylated or deglycosylated form.

[0162] According to another particular embodiment of the first aspect, there is provided a site-selectively modified immunoglobulin molecule comprising one single site-selective modification selected from: a) at least one IgH, more particularly each single IgH modification in one of the amino acid sequence positions corresponding to positions selected from P41, G42, K249, K251, K291, K320 and K343, particularly K249, of SEQ ID NO: 2, and b) at least one IgL, more particularly each single IgL modification in one of the amino acid sequence positions corresponding to positions G41, A51, P59, A111 or K169 of SEQ ID NO: 4, in particular K169.

[0163] Each of the above variants may be provided in a non-glycosylated, glycosylated or deglycosylated form.

[0164] According to another particular embodiment of the first aspect, there is provided a site-selectively modified immunoglobulin molecule comprising two site-selective modifications: a) each is located on at least one, more particularly on each IgH, and in particular on C H (More specifically, C H 2) and Framework V H or b) each located on at least one, more particularly on each IgL, and in particular on C L ,Framework V L and CDR-L2, or c) one of at least one IgH, more specifically, each C of that IgH H and the other is located on at least one IgL, more particularly on each of said IgLs, in particular at least one C L Above, and more specifically, that C L are located in each of the following areas:

[0165] Each of the above variants may be provided in a non-glycosylated, glycosylated or deglycosylated form.

[0166] More specifically, the two site-selective modifications are selected from: a) at least one IgH, more particularly each of said IgH, single IgH modification in one of the amino acid sequence positions corresponding to positions selected from P41, G42, K249, K251, K291, K320 and K343 of SEQ ID NO: 2, in particular K249, K320 or P41, and at least one IgL, more particularly each of said IgL, single IgL modification in one of the amino acid sequence positions corresponding to positions G41, A51, P59, A111 or K169 of SEQ ID NO: 4, in particular G41 or K169, b) at least one IgH, more specifically each of said IgH, double IgH modifications at two amino acid sequence positions corresponding to positions P41, G42, K249, K251, K291, K320 and K343 of SEQ ID NO: 2; c) A double IgL modification of at least one IgL, more particularly each of said IgLs, at two amino acid sequence positions corresponding to positions G41, A51, P59, A111 or K169 of SEQ ID NO:4.

[0167] Each of the above variants may be provided in a non-glycosylated, glycosylated or deglycosylated form.

[0168] According to another particular embodiment of the first aspect, the site-selectively modified immunoglobulin molecule is an IgG1 molecule or an antigen-binding fragment thereof.

[0169] According to another particular embodiment of the first aspect, the site-selectively modified immunoglobulin molecule is a monoclonal antibody or an antigen-binding fragment thereof.

[0170] According to another particular embodiment of the first aspect, the site-selectively modified immunoglobulin molecule is a site-selectively modified variant of trastuzumab (whose IgH and IgL chains are SEQ ID NOs: 2 and 4) or an antigen-binding fragment thereof, or a site-selectively modified variant of pertuzumab (whose IgH and IgL chains are SEQ ID NOs: 6 and 8) or an antigen-binding fragment thereof.

[0171] More particularly, said site-selectively modified immunoglobulin molecule is selected from a trastuzumab variant selected from: a) IgH single mutants of P41, G42, K249, K251, K291, K320 and K343 of SEQ ID NO: 2 in each of their IgHs, b) IgL single mutants G41, A51, P59, A111 and K169 of SEQ ID NO: 4 in each of their IgLs; c) IgH of (K249 / K320) and (K249 / K343) of SEQ ID NO: 2 in each of the IgHs (more specifically, C H 2) double mutants, d) (IgH / IgL) mixed double mutants (K249 / K169), (K249 / G41), (K320 / K169), (K320 / G41) and (P41 / G41) of SEQ ID NO: 2 and SEQ ID NO: 4, respectively, in each of their IgH / IgL pairs; e) or an antigen-binding fragment of any one of a) to d).

[0172] Each of the above variants may be provided in a non-glycosylated, glycosylated or deglycosylated form.

[0173] More particularly, said site-selectively modified immunoglobulin molecule is selected from a Pertuzumab variant selected from: a) IgH single mutants P41, G42, K248, K250, K291, K319 and K342 of SEQ ID NO: 6 in each of their IgHs; b) IgL single mutants G41, A51, P59, A111 and K169 of SEQ ID NO: 8 in each of their IgLs; c) IgH of (K248 / K319) and (K248 / K342) of SEQ ID NO: 6 in each of the IgHs (more specifically, C H 2) double mutants, d) (IgH / IgL) mixed double mutants (K248 / K169), (K248 / G41), (K319 / K169), (K319 / G41) and (P41 / G41) of SEQ ID NO: 6 and SEQ ID NO: 8, respectively, in each of their IgH / IgL pairs; e) or an antigen-binding fragment of any one of a) to d).

[0174] Each of the above variants may be provided in a non-glycosylated, glycosylated or deglycosylated form.

[0175] Sequences of specific examples of variants of the invention:

[0176] Particular examples of site-specifically modified immunoglobulin molecules include site-specifically modified trastuzumab immunoglobulins, non-limiting examples of which are selected from: a) an immunoglobulin comprising one, in particular two mutated IgH polypeptide chains according to SEQ ID NO: 2 containing an ncAA at position K249, and further comprising one, in particular two non-mutated IgL polypeptide chains according to SEQ ID NO: 4, b) an immunoglobulin comprising one, in particular two mutated IgH polypeptide chains according to SEQ ID NO: 2 containing an ncAA at position K251, and further comprising one, in particular two non-mutated IgL polypeptide chains according to SEQ ID NO: 4; c) an immunoglobulin comprising one, in particular two mutated IgH polypeptide chains according to SEQ ID NO: 2 containing an ncAA at position K320, and further comprising one, in particular two non-mutated IgL polypeptide chains according to SEQ ID NO: 4; d) an immunoglobulin comprising one, in particular two mutated IgH polypeptide chains according to SEQ ID NO: 2 containing an ncAA at position K343, and further comprising one, in particular two non-mutated IgL polypeptide chains according to SEQ ID NO: 4; e) an immunoglobulin comprising one, in particular two mutated IgH polypeptide chains according to SEQ ID NO: 2 containing an ncAA at position P41, and further comprising one, in particular two non-mutated IgL polypeptide chains according to SEQ ID NO: 4; f) an immunoglobulin comprising one, in particular two mutated IgH polypeptide chains according to SEQ ID NO: 2 containing an ncAA at position G42, and further comprising one, in particular two non-mutated IgL polypeptide chains according to SEQ ID NO: 4; g) an immunoglobulin comprising one, in particular two non-mutated IgH polypeptide chains according to SEQ ID NO: 2 and further comprising one, in particular two mutated IgL polypeptide chains according to SEQ ID NO: 4 containing an ncAA at position A51; h) an immunoglobulin comprising one, in particular two non-mutated IgH polypeptide chains according to SEQ ID NO: 2 and further comprising one, in particular two mutated IgL polypeptide chains according to SEQ ID NO: 4 containing an ncAA at position A111; i) an immunoglobulin comprising one, in particular two non-mutated IgH polypeptide chains according to SEQ ID NO: 2 and further comprising one, in particular two mutated IgL polypeptide chains according to SEQ ID NO: 4 containing an ncAA at position G41; j) an immunoglobulin comprising one, in particular two non-mutated IgH polypeptide chains according to SEQ ID NO: 2 and further comprising one, in particular two mutated IgL polypeptide chains according to SEQ ID NO: 4 containing an ncAA at position P59; k) an immunoglobulin comprising one, in particular two mutated IgH polypeptide chains according to SEQ ID NO: 2 containing an ncAA at positions K249 and K320, and further comprising one, in particular two non-mutated IgL polypeptide chains according to SEQ ID NO: 4; l) an immunoglobulin comprising one, in particular two mutated IgH polypeptide chains according to SEQ ID NO: 2 containing ncAAs at positions K249 and K343, and further comprising one, in particular two non-mutated IgL polypeptide chains according to SEQ ID NO: 4, m) an immunoglobulin comprising one, in particular two, mutant IgH polypeptide chains according to SEQ ID NO: 2 containing an ncAA at position K249 and further comprising one, in particular two, mutant IgL polypeptide chains according to SEQ ID NO: 4 containing an ncAA at position K169; n) an immunoglobulin comprising one, in particular two, mutant IgH polypeptide chains according to SEQ ID NO: 2 containing an ncAA at position K249 and further comprising one, in particular two, mutant IgL polypeptide chains according to SEQ ID NO: 4 containing an ncAA at position G41; o) an immunoglobulin comprising one, in particular two, mutant IgH polypeptide chains according to SEQ ID NO: 2, which contains an ncAA at position K320, and further comprising one, in particular two, mutant IgL polypeptide chains according to SEQ ID NO: 4, which contains an ncAA at position K169; p) an immunoglobulin comprising one, in particular two, mutant IgH polypeptide chains according to SEQ ID NO: 2 containing an ncAA at position K320, and further comprising one, in particular two, mutant IgL polypeptide chains according to SEQ ID NO: 4 containing an ncAA at position G41, and q) An immunoglobulin comprising one, in particular two mutated IgH polypeptide chains according to SEQ ID NO: 2 containing an ncAA at position P41 and further comprising one, in particular two mutated IgL polypeptide chains according to SEQ ID NO: 4 containing an ncAA at position G41.

[0177] Each of the above variants may be provided in a non-glycosylated, glycosylated or deglycosylated form.

[0178] The site-specifically modified trastuzumab immunoglobulins of the present invention may also comprise two site-selective modifications according to any of subgroups 1.1 to 1.10 within Group 1.

[0179] Group 1: a) Subgroup 1.1:V H V H (S25 / K43), (S25 / R50), (S25 / D62), (S25 / K65), (S25 / E89), (S25 / D102), (K43 / R50), (K43 / D62), (K43 / K65), (K43 / E89), (K43 / D1 02), (R50 / D62), (R50 / K65), (R50 / E89), (R50 / D102), (D62 / K65), (D62 / E89), (D62 / D102), (K65 / E89), (K65 / D102), (E89 / D102).

[0180] b) Subgroup 1.2 V H C H <h2 style=";text-align:left;direction:ltr">(S25 / A121), (S25 / E155), (S25 / P156), (S25 / S194), (S25 / E219), (S25 / D252), (S25 / E275), (S25 / K277), (S25 / D283), (S25 / H288), (S25 / K293), (S25 / E296), (S25 / R304), (S25 / K323), (K43 / A121), (K43 / E155), (K43 / P156), (K43 / S194), (K43 / E219), (K43 / D252), (K43 / E275), (K43 / K277), (K43 / D283), (K43 / H288), (K43 / K293), (K43 / E296), (K43 / R304), (K43 / K323), (R50 / A121), (R50 / E155), (R50 / P156), (R50 / S194), (R50 / E219), (R50 / D252), (R50 / E275), (R50 / K277), (R50 / D283), (R50 / H288), (R50 / K293), (R50 / E296), (R50 / R304), (R50 / K323), (D62 / A121), (D62 / E155), (D62 / P156), (D62 / S194), (D62 / E219), (D62 / D252), (D62 / E275), (D62 / K277), (D62 / D283), (D62 / H288), (D62 / K293), (D62 / E296), (D62 / R304), (D62 / K323), (K65 / A121), (K65 / E155), (K65 / P156), (K65 / S194), (K65 / E219), (K65 / D252), (K65 / E275), (K65 / K277), (K65 / D283), (K65 / H288), (K65 / K293), (K65 / E296), (K65 / R304), (K65 / K323), (E89 / A121), (E89 / E155), (E89 / P156), (E89 / S194), (E89 / E219), (E89 / D252), (E89 / E275), (E89 / K277), (E89 / D283), (E89 / H288), (E89 / K293), (E89 / E296), (E89 / R304),(E89 / K323), (D102 / A121), (D102 / E155), (D102 / P156), (D102 / S194), (D102 / E219), (D102 / D252), (D102 / E275), (D102 / K277), (D102 / D283), (D102 / H288), (D102 / K293), (D102 / E296), (D102 / R304), (D102 / K323). ,

[0181] c) Subgroup 1.3 V H V L (S25 / K42), (S25 / K45), (S25 / R61), (S25 / D70), (S25 / E81), (K43 / K42), (K43 / K45), (K43 / R61), (K43 / D70), (K43 / E81), (R50 / K42), (R50 / K45), (R50 / R61), (R50 / D70), (R50 / E81), (D62 / K42), (D62 / K45), (D62 / R61), (D62 / D70), (D62 / E81), (K65 / K42), (K65 / K45), (K65 / R61), (K65 / D70), (K65 / E81), (E89 / K42), (E89 / K45), (E89 / R61), (E89 / D70), (E89 / E81), (D102 / K42), (D102 / K45), (D102 / R61), (D102 / D70), (D102 / E81), (K42 / K45).

[0182] d) Subgroup 1.4 V H C L (S25 / E143), (S25 / D151), (S25 / G157), (S25 / G200), (K43 / E143), (K43 / D151), (K43 / G157), (K43 / G200), (R50 / E143), (R50 / D151), (R50 / G157), (R50 / G200), (D62 / E143), (D62 / D151), (D62 / G157), (D62 / G200), (K65 / E143), (K65 / D151), (K65 / G157), (K65 / G200), (E89 / E143), (E89 / D151), (E89 / G157), (E89 / G200), (D102 / E143), (D102 / D151), (D102 / G157), (D102 / G200).

[0183] e) Subgroup 1.5:C H C H <h2 style=";text-align:left;direction:ltr">(A121 / E155), (A121 / P156), (A121 / S194), (A121 / E219), (A121 / D252), (A121 / E275), (A121 / K277), (A121 / D283), (A121 / H288), (A121 / K293), (A121 / E296), (A121 / R304), (A121 / K323), (E155 / P156), (E155 / S194), (E155 / E219), (E155 / D252), (E155 / E275), (E155 / K277), (E155 / D283), (E155 / H288), (E155 / K293), (E155 / E296), (E155 / R304), (E155 / K323), (P156 / S194), (P156 / E219), (P156 / D252), (P156 / E275), (P156 / K277), (P156 / D283), (P156 / H288), (P156 / K293), (P156 / E296), (P156 / R304), (P156 / K323), (S194 / E219), (S194 / D252), (S194 / E275), (S194 / K277), (S194 / D283), (S194 / H288), (S194 / K293), (S194 / E296), (S194 / R304), (S194 / K323), (E219 / D252), (E219 / E275), (E219 / K277), (E219 / D283), (E219 / H288), (E219 / K293), (E219 / E296), (E219 / R304), (E219 / K323), (D252 / E275), (D252 / K277), (D252 / D283), (D252 / H288), (D252 / K293), (D252 / E296), (D252 / R304), (D252 / K323), (E275 / K277), (E275 / D283), (E275 / H288), (E275 / K293), (E275 / E296), (E275 / R304), (E275 / K323), (K277 / D283), (K277 / H288), (K277 / K293), (K277 / E296), (K277 / R304), (K277 / K323), (D283 / H288),(D283 / K293), (D283 / E296), (D283 / R304), (D283 / K323), (H288 / K293), (H288 / E296), (H288 / R304), (H288 / K323), (K293 / E296), (K293 / R304), (K293 / K323), (E296 / R304), (E296 / K323), (R304 / K323). ,

[0184] f) Subgroup 1.6 V L C H (A121 / K42), (A121 / K45), (A121 / R61), (A121 / D70), (A121 / E81), (E155 / K42), (E155 / K45), (E155 / R61), (E155 / D70), (E155 / E81), (P156 / K42), (P156 / K45), (P156 / R61), (P156 / D70), (P156 / E81), (S194 / K42), (S194 / K45), (S194 / R61), (S194 / D70), (S194 / E81), (E219 / K42), (E219 / K45), (E219 / R61), (E219 / D70), (E219 / E81), (D252 / K42), (D252 / K45), (D252 / R61), (D252 / D70), (D252 / E81), (E275 / K42), (E275 / K45), (E275 / R61), (E275 / D70), (E275 / E81), (K277 / K42), (K277 / K45), (K277 / R61), (K277 / D70), (K277 / E81), (D283 / K42), (D283 / K45), (D283 / R61), (D283 / D70), (D283 / E81), (H288 / K42), (H288 / K45), (H288 / R61), (H288 / D70), (H288 / E81), (K293 / K42), (K293 / K45), (K293 / R61), (K293 / D70), (K293 / E81), (E296 / K42), (E296 / K45), (E296 / R61), (E296 / D70), (E296 / E81), (R304 / K323), (R304 / K42), (R304 / K45), (R304 / R61), (R304 / D70), (R304 / E81), (K323 / K42), (K323 / K45), (K323 / R61), (K323 / D70), (K323 / E81).

[0185] g) Subgroup 1.7 C H C L (A121 / E143), (A121 / D151), (A121 / G157), (A121 / G200), (E155 / E143), (E155 / D151), (E155 / G157), (E155 / G200), (P156 / E143), (P156 / D151), (P156 / G157), (P156 / G200), (S194 / E143), (S194 / D151), (S194 / G157), (S194 / G200), (E219 / E143), (E219 / D151), (E219 / G157), (E219 / G200), (D252 / E143), (D252 / D151), (D252 / G157), (D252 / G200), (E275 / E143), (E275 / D151), (E275 / G157), (E275 / G200), (K277 / E143), (K277 / D151), (K277 / G157), (K277 / G200), (D283 / E143), (D283 / D151), (D283 / G157), (D283 / G200), (H288 / E143), (H288 / D151), (H288 / G157), (H288 / G200), (K293 / E143), (K293 / D151), (K293 / G157), (K293 / G200), (E296 / E143), (E296 / D151), (E296 / G157), (E296 / G200), (R304 / E143), (R304 / D151), (R304 / G157), (R304 / G200), (K323 / E143), (K323 / D151), (K323 / G157), (K323 / G200).

[0186] h) Subgroup 1.8 V L V L (K42 / K45), (K42 / R61), (K42 / D70), (K42 / E81), (K45 / R61), (K45 / D70), (K45 / E81), (R61 / D70), (R61 / E81), (D70 / E81).

[0187] i) Subgroup 1.9 V L CL (K42 / E143), (K42 / D151), (K42 / G157), (K42 / G200), (K45 / E143), (K45 / D151), (K45 / G157), (K45 / G200),), (R61 / E143), (R61 / D151), (R61 / G157), (R61 / G200), (D70 / E143), (D70 / D151), (D70 / G157), (D70 / G200), (E81 / E143), (E81 / D151), (E81 / G157), (E81 / G200).

[0188] j) Subgroup 1.10 C L C L (E143 / D151), (E143 / G157), (E143 / G200), (D151 / G157), (D151 / G200), (G157 / G200).

[0189] Alternatively, the site-specifically modified trastuzumab immunoglobulin of the present invention may also contain two modifications according to any of subgroups 2.1 to 2.10 within Group 2.

[0190] Group 2: a) Subgroup 2.1:V H V H (S25 / P41), (S25 / G42), (S25 / K43), (S25 / R50), (S25 / D62), (S25 / K65), (S25 / E89), (S25 / D102), (P41 / G42), (P41 / K43), (P41 / R50),(P41 / D62), (P41 / K65), (P41 / E89), (P41 / D102), (G42 / K43), (G42 / R50), (G42 / D62), (G42 / K65), (G42 / E89), (G42 / D102), (K43 / R50), (K43 / D62), (K43 / K65), (K43 / E89), (K43 / D102), (R50 / D62), (R50 / K65), (R50 / E89), (R50 / D102), (D62 / K65), (D62 / E89), (D62 / D102), (K65 / E89), (K65 / D102), (E89 / D102).

[0191] b) Subgroup 2.2 V H C H (S25 / A121), (S25 / E155), (S25 / P156), (S25 / S194), (S25 / E219), (S25 / K249), (S25 / K251), (S25 / D252), (S25 / E275), (S25 / K277), (S25 / D283), (S25 / H288), (S25 / K291), (S25 / K320), (S25 / K293), (S25 / E296), (S25 / R304), (S25 / K323), (S25 / K343), (P41 / A121), (P41 / E155), (P41 / P156), (P41 / S194), (P41 / E219), (P41 / K249), (P41 / K251), (P41 / D252), (P41 / E275), (P41 / K277), (P41 / D283), (P41 / H288), (P41 / K291), (P41 / K320), (P41 / K293), (P41 / E296), (P41 / R304), (P41 / K323), (P41 / K343), (G42 / A121), (G42 / E155), (G42 / P156), (G42 / S194), (G42 / E219), (G42 / K249), (G42 / K251), (G42 / D252), (G42 / E275), (G42 / K277), (G42 / D283), (G42 / H288), (G42 / K291), (G42 / K320), (G42 / K293), (G42 / E296), (G42 / R304), (G42 / K323), (G42 / K343), (K43 / A121), (K43 / E155), (K43 / P156), (K43 / S194), (K43 / E219), (K43 / K249), (K43 / K251), (K43 / D252), (K43 / E275), (K43 / K277), (K43 / D283), (K43 / H288), (K43 / K291), (K43 / K320), (K43 / K293), (K43 / E296), (K43 / R304), (K43 / K323), (K43 / K343), (R50 / A121), (R50 / E155), (R50 / P156), (R50 / S194), (R50 / E219), (R50 / K249), (R50 / K251),<h2 style=";text-align:left;direction:ltr">(R50 / D252), (R50 / E275), (R50 / K277), (R50 / D283), (R50 / H288), (R50 / K291), (R50 / K320), (R50 / K293), (R50 / E296), (R50 / R304), (R50 / K323), (R50 / K343), (D62 / A121), (D62 / E155), (D62 / P156), (D62 / S194), (D62 / E219), (D62 / K249), (D62 / K251), (D62 / D252), (D62 / E275), (D62 / K277), (D62 / D283), (D62 / H288), (D62 / K291), (D62 / K320), (D62 / K293), (D62 / E296), (D62 / R304), (D62 / K323), (D62 / K343), (K65 / A121), (K65 / E155), (K65 / P156), (K65 / S194), (K65 / E219), (K65 / K249), (K65 / K251), (K65 / D252), (K65 / E275), (K65 / K277), (K65 / D283), (K65 / H288), (K65 / K291), (K65 / K320), (K65 / K293), (K65 / E296), (K65 / R304), (K65 / K323), (K65 / K343), (E89 / A121), (E89 / E155), (E89 / P156), (E89 / S194), (E89 / E219), (E89 / K249), (E89 / K251), (E89 / D252), (E89 / E275), (E89 / K277), (E89 / D283), (E89 / H288), (E89 / K291), (E89 / K320), (E89 / K293), (E89 / E296), (E89 / R304), (E89 / K323), (E89 / K343), (D102 / A121), (D102 / E155), (D102 / P156), (D102 / S194), (D102 / E219), (D102 / K249), (D102 / K251), (D102 / D252), (D102 / E275), (D102 / K277), (D102 / D283), (D102 / H288), (D102 / K291),(D102 / K320), (D102 / K293), (D102 / E296), (D102 / R304), (D102 / K323), (D102 / K343). ,

[0192] c) Subgroup 2.3 V H V L (S25 / G41), (S25 / K42), (S25 / K45), (S25 / A51), (S25 / P59), (S25 / R61), (S25 / D70), (S25 / E81), (P41 / G41), (P41 / K42), (P41 / K45), (P41 / A51), (P41 / P59), (P41 / R61)(P41 / D70), (P41 / E81), (G42 / G41), (G42 / K42), (G42 / K45), (G42 / A51), (G42 / P59), (G42 / R61), (G42 / D70), (G42 / E81), (K43 / G41), (K43 / K42), (K43 / K45), (K43 / A51), (K43 / P59), (K43 / R61), (K43 / D70), (K43 / E81), (R50 / G41), (R50 / K42), (R50 / K45), (R50 / A51), (R50 / P59), (R50 / R61), (R50 / D70)(R50 / E81), (D62 / G41), (D62 / K42), (D62 / K45), (D62 / A51), (D62 / P59), (D62 / R61), (D62 / D70), (D62 / E81), (K65 / E89), (K65 / G41), (K65 / K42), (K65 / K45), (K65 / A51), (K65 / P59), (K65 / R61), (K65 / D70), (K65 / E81), (E89 / G41), (E89 / K42), (E89 / K45), (E89 / A51), (E89 / P59), (E89 / R61), (E89 / D70), (E89 / E81), (D102 / G41), (D102 / K42), (D102 / K45), (D102 / A51), (D102 / P59), (D102 / R61), (D102 / D70), (D102 / E81).

[0193] d) Subgroup 2.5:C H C H <h2 style=";text-align:left;direction:ltr">(A121 / E155), (A121 / P156), (A121 / S194), (A121 / E219), (A121 / K249), (A121 / K251), (A121 / D252), (A121 / E275), (A121 / K277), (A121 / D283), (A121 / H288), (A121 / K291), (A121 / K320), (A121 / K293), (A121 / E296), (A121 / R304), (A121 / K323), (A121 / K343), (E155 / P156), (E155 / S194), (E155 / E219), (E155 / K249), (E155 / K251), (E155 / D252), (E155 / E275), (E155 / K277), (E155 / D283), (E155 / H288), (E155 / K291), (E155 / K320), (E155 / K293), (E155 / E296), (E155 / R304), (E155 / K323), (E155 / K343), (P156 / S194), (P156 / E219), (P156 / K249), (P156 / K251), (P156 / D252), (P156 / E275), (P156 / K277), (P156 / D283), (P156 / H288), (P156 / K291), (P156 / K320), (P156 / K293), (P156 / E296), (P156 / R304), (P156 / K323), (P156 / K343), (S194 / E219), (S194 / K249), (S194 / K251), (S194 / D252), (S194 / E275), (S194 / K277), (S194 / D283), (S194 / H288), (S194 / K291), (S194 / K320), (S194 / K293), (S194 / E296), (S194 / R304), (S194 / K323), (S194 / K343), (E219 / K249), (E219 / K251), (E219 / D252), (E219 / E275), (E219 / K277), (E219 / D283), (E219 / H288), (E219 / K291), (E219 / K320), (E219 / K293), (E219 / E296),<h2 style=";text-align:left;direction:ltr">(E219 / R304), (E219 / K323), (E219 / K343), (K249 / K251), (K249 / D252), (K249 / E275), (K249 / K277), (K249 / D283), (K249 / H288), (K249 / K291), (K249 / K320), (K249 / K293), (K249 / E296), (K249 / R304), (K249 / K323), (K249 / K343), (K251 / D252), (K251 / E275), (K251 / K277), (K251 / D283), (K251 / H288), (K251 / K291), (K251 / K320), (K251 / K293), (K251 / E296), (K251 / R304), (K251 / K323), (K251 / K343), (D252 / E275), (D252 / K277), (D252 / D283), (D252 / H288), (D252 / K291), (D252 / K320), (D252 / K293), (D252 / E296), (D252 / R304), (D252 / K323), (D252 / K343), (E275 / K277), (E275 / D283), (E275 / H288), (E275 / K291), (E275 / K320), (E275 / K293), (E275 / E296), (E275 / R304), (E275 / K323), (E275 / K343), (K277 / D283), (K277 / H288), (K277 / K291), (K277 / K320), (K277 / K293), (K277 / E296), (K277 / R304), (K277 / K323), (K277 / K343), (D283 / H288), (D283 / K291), (D283 / K320), (D283 / K293), (D283 / E296), (D283 / R304), (D283 / K323), (D283 / K343), (H288 / K291), (H288 / K320), (H288 / K293), (H288 / E296), (H288 / R304), (H288 / K323), (H288 / K343), (K291 / K320), (K291 / K293), (K291 / E296), (K291 / R304),<h2 style=";text-align:left;direction:ltr">(K291 / K323), (K291 / K343), (K320 / K293), (K320 / E296), (K320 / R304), (K320 / K323), (K320 / K343), (K293 / E296), (K293 / R304), (K293 / K323), (K293 / K343), (E296 / R304), (E296 / K323), (E296 / K343), (R304 / K323), (R304 / K343), (K323 / K343).<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0194] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> e)サブグループ2.4V<h2 style=";text-align:left;direction:ltr"> H <h2 style=";text-align:left;direction:ltr"> C<h2 style=";text-align:left;direction:ltr"> L <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (S25 / A111), (S25 / E143), (S25 / D151), (S25 / G157), (S25 / K169), (S25 / G200), (P41 / A111), (P41 / E143), (P41 / D151), (P41 / G157), (P41 / K169), (P41 / G200), (G42 / A111), (G42 / E143), (G42 / D151), (G42 / G157), (G42 / K169), (G42 / G200), (K43 / A111), (K43 / E143), (K43 / D151), (K43 / G157), (K43 / K169), (K43 / G200), (R50 / A111), (R50 / E143), (R50 / D151), (R50 / G157), (R50 / K169), (R50 / G200), (D62 / A111), (D62 / E143), (D62 / D151), (D62 / G157), (D62 / K169), (D62 / G200), (K65 / E89), (K65 / A111), (K65 / E143), (K65 / D151), (K65 / G157), (K65 / K169), (K65 / G200), (E89 / A111), (E89 / E143), (E89 / D151), (E89 / G157), (E89 / K169), (E89 / G200), (D102 / A111), (D102 / E143), (D102 / D151), (D102 / G157), (D102 / K169), (D102 / G200)。<h2 style=";text-align:left;direction:ltr">

[0195] f) Subgroup 2.6 V L V L (G41 / K42), (G41 / K45), (G41 / A51), (G41 / P59), (G41 / R61), (G41 / D70), (G41 / E81), (K42 / K45), (K42 / A51), (K42 / P59), (K42 / R61), (K42 / D70), (K42 / E81), (K45 / A51), (K45 / P59), (K45 / R61), (K45 / D70), (K45 / E81), (A51 / P59), (A51 / R61), (A51 / D70), (A51 / E81), (P59 / R61), (P59 / D70), (P59 / E81), (R61 / D70), (R61 / E81), (D70 / E81).

[0196] g) Subgroup 2.7 V L C L (G41 / A111), (G41 / E143), (G41 / D151), (G41 / G157), (G41 / K169), (G41 / G200), (K42 / A111), (K42 / E143), (K42 / D151), (K42 / G157), (K42 / K169), (K42 / G200), (K45 / A111), (K45 / E143), (K45 / D151), (K45 / G157), (K45 / K169), (K45 / G200), (A51 / A111), (A51 / E143), (A51 / D151), (A51 / G157), (A51 / K169), (A51 / G200), (P59 / A111), (P59 / E143), (P59 / D151), (P59 / G157), (P59 / K169), (P59 / G200), (R61 / A111), (R61 / E143), (R61 / D151), (R61 / G157), (R61 / K169), (R61 / G200), (D70 / A111), (D70 / E143), (D70 / D151), (D70 / G157), (D70 / K169), (D70 / G200), (E81 / A111), (E81 / E143), (E81 / D151), (E81 / G157), (E81 / K169), (E81 / G200).

[0197] h) Subgroup 2.8 V L C H <h2 style=";text-align:left;direction:ltr">(G41 / A121), (G41 / E155), (G41 / P156), (G41 / S194), (G41 / E219), (G41 / K249), (G41 / K251), (G41 / D252), (G41 / E275), (G41 / K277), (G41 / D283), (G41 / H288), (G41 / K291), (G41 / K320), (G41 / K293), (G41 / E296), (G41 / R304), (G41 / K323), (G41 / K343), (K42 / A121), (K42 / E155), (K42 / P156), (K42 / S194), (K42 / E219), (K42 / K249), (K42 / K251), (K42 / D252), (K42 / E275), (K42 / K277), (K42 / D283), (K42 / H288), (K42 / K291), (K42 / K320), (K42 / K293), (K42 / E296), (K42 / R304), (K42 / K323), (K42 / K343), (K45 / A121), (K45 / E155), (K45 / P156), (K45 / S194), (K45 / E219), (K45 / K249), (K45 / K251), (K45 / D252), (K45 / E275), (K45 / K277), (K45 / D283), (K45 / H288), (K45 / K291), (K45 / K320), (K45 / K293), (K45 / E296), (K45 / R304), (K45 / K323), (K45 / K343), (A51 / A121), (A51 / E155), (A51 / P156), (A51 / S194), (A51 / E219), (A51 / K249), (A51 / K251), (A51 / D252), (A51 / E275), (A51 / K277), (A51 / D283), (A51 / H288), (A51 / K291), (A51 / K320), (A51 / K293), (A51 / E296), (A51 / R304), (A51 / K323), (A51 / K343), (P59 / A121), (P59 / E155), (P59 / P156), (P59 / S194), (P59 / E219), (P59 / K249), (P59 / K251),(P59 / D252), (P59 / E275), (P59 / K277), (P59 / D283), (P59 / H288), (P59 / K291), (P59 / K320), (P59 / K293), (P59 / E296), (P59 / R304), (P59 / K323), (P59 / K343), (R61 / A121), (R61 / E155), (R61 / P156), (R61 / S194), (R61 / E219), (R61 / K249), (R61 / K251), (R61 / D252), (R61 / E275), (R61 / K277), (R61 / D283), (R61 / H288), (R61 / K291), (R61 / K320), (R61 / K293), (R61 / E296), (R61 / R304), (R61 / K323), (R61 / K343), (D70 / A121), (D70 / E155), (D70 / P156), (D70 / S194), (D70 / E219), (D70 / K249), (D70 / K251), (D70 / D252), (D70 / E275), (D70 / K277), (D70 / D283), (D70 / H288), (D70 / K291), (D70 / K320), (D70 / K293), (D70 / E296), (D70 / R304), (D70 / K323), (D70 / K343), (E81 / A121), (E81 / E155), (E81 / P156), (E81 / S194), (E81 / E219), (E81 / K249), (E81 / K251), (E81 / D252), (E81 / E275), (E81 / K277), (E81 / D283), (E81 / H288), (E81 / K291), (E81 / K320), (E81 / K293), (E81 / E296), (E81 / R304), (E81 / K323), (E81 / K343). ,

[0198] i) Subgroup 2.9 C L C L (A111 / E143), (A111 / D151), (A111 / G157), (A111 / K169), (A111 / G200), (E143 / D151), (E143 / G157), (E143 / K169), (E143 / G200), (D151 / G157), (D151 / K169), (D151 / G200), (G157 / K169), (G157 / G200), (K169 / G200).

[0199] j) Subgroup 2.10 C H C L (A121 / A111), (A121 / E143), (A121 / D151), (A121 / G157), (A121 / K169), (A121 / G200), (E155 / A111), (E155 / E143), (E155 / D151), (E155 / G157), (E155 / K169), (E155 / G200), (P156 / A111), (P156 / E143), (P156 / D151), (P156 / G157), (P156 / K169), (P156 / G200), (S194 / A111), (S194 / E143), (S194 / D151), (S194 / G157), (S194 / K169), (S194 / G200), (E219 / A111), (E219 / E143), (E219 / D151), (E219 / G157), (E219 / K169), (E219 / G200), (K249 / A111), (K249 / E143), (K249 / D151), (K249 / G157), (K249 / K169), (K249 / G200), (K251 / A111), (K251 / E143), (K251 / D151), (K251 / G157), (K251 / K169), (K251 / G200), (D252 / A111), (D252 / E143), (D252 / D151), (D252 / G157), (D252 / K169), (D252 / G200), (E275 / A111), (E275 / E143), (E275 / D151), (E275 / G157), (E275 / K169), (E275 / G200), (K277 / A111), (K277 / E143), (K277 / D151), (K277 / G157), (K277 / K169), (K277 / G200), (D283 / A111), (D283 / E143), (D283 / D151), (D283 / G157), (D283 / K169), (D283 / G200), (H288 / A111), (H288 / E143), (H288 / D151), (H288 / G157), (H288 / K169), (H288 / G200), (K291 / A111), (K291 / E143), (K291 / D151), (K291 / G157), (K291 / K169),(K291 / G200), (K320 / A111), (K320 / E143), (K320 / D151), (K320 / G157), (K320 / K169), (K320 / G200), (K293 / A111), (K293 / E143), (K293 / D151), (K293 / G157), (K293 / K169), (K293 / G200), (E296 / A111), (E296 / E143), (E296 / D151), (E296 / G157), (E296 / K169), (E296 / G200), (R304 / A111), (R304 / E143), (R304 / D151), (R304 / G157), (R304 / K169), (R304 / G200), (K323 / A111), (K323 / E143), (K323 / D151), (K323 / G157), (K323 / K169), (K323 / G200), (K343 / A111), (K343 / E143), (K343 / D151), (K343 / G157), (K343 / K169), (K343 / G200). ,

[0200] According to another particular embodiment of said first aspect of said site-selectively modified immunoglobulin molecule, said ncAA carries a functional side chain, wherein said functional side chain is reactable via a Diels-Alder type cycloaddition reaction and is selected from: (i) a trans-cyclooctenyl dienophile group of the formula: [ka]

[0201] Where: R 1 is hydrogen, halogen, C 1 -C 4 Alkyl, (R a O) 2 P(O)OC 1 -C 4 Alkyl, (R b O) 2 P(O)-C 1 -C 4 Alkyl, CF3 , CN, hydroxyl, C 1 -C 4 Alkoxy, -O-CF 3 , C 2 -C 5 Alkenoxy, C 2 -C 5 Alkanoyloxy, C 1 -C 4 Alkylaminocarbonyloxy or C 1 -C 4 Alkylthio, C 1 -C 4 Alkylamino, di(C 1 -C 4 Alkyl)amino, C 2 -C 5 Alkenylamino, C 2 -C 5 Alkenyl-C 1 -C 4 Alkyl-amino or di(C 2 -C 5 alkenyl)amino, R a , R b are independently hydrogen or C 2 -C 5 alkanoyloxymethyl or (ii) a cyclooctynyl dienophile group of the formula: [ka]

[0202] Where: R 2 is hydrogen, halogen, C 1 -C 4 Alkyl, (R c O) 2 P(O)OC 1 -C 4 Alkyl, (R d O) 2 P(O)-C 1 -C 4 Alkyl, CF 3 , CN, hydroxyl, C 1 -C 4 Alkoxy, -O-CF3 , C 2 -C 5 Alkenoxy, C 2 -C 5 Alkanoyloxy, C 1 -C 4 Alkylaminocarbonyloxy or C 1 -C 4 Alkylthio, C 1 -C 4 Alkylamino, di(C 1 -C 4 Alkyl)amino, C 2 -C 5 Alkenylamino, C 2 -C 5 Alkenyl-C 1 -C 4 Alkyl-amino or di(C 2 -C 5 alkenyl)amino, R c , R d are independently hydrogen or C 2 -C 5 It is alkanoyloxymethyl.

[0203] More specifically, the ncAA is SCO (2-amino-6-(cyclooct-2-yn-1-yloxycarbonylamino)hexanoic acid), TCO-Lys (N-ε-((trans-cyclooct-4-en-1-yloxy)carbonyl)-L-lysine), TCO*-Lys (N-ε-((trans-cyclooct-2-en-1-yloxy)carbonyl)-L-lysine), TCO # -Lys(N-ε-((trans-cyclooct-3-en-1-yloxy)carbonyl)-L-lysine), TCO-E-Lys(N6-((((R,E)-cyclooct-4-en-1-yl)oxy)carbonyl)-L-lysine) and TCO*A-Lys(N6-((((S,E)-cyclooct-2-en-1-yl)oxy)carbonyl)-L-lysine).

[0204] According to a very particular embodiment, the ncAA applied for the preparation of said site-selectively modified immunoglobulin molecules is TCO*A-Lys (N6-((((S,E)-cyclooct-2-en-1-yl)oxy)carbonyl)-L-lysine).

[0205] A second aspect of the present invention relates to an antibody payload conjugate (APC), in particular an antibody drug conjugate (ADC), comprising at least one site-selectively modified immunoglobulin molecule according to the first aspect of the invention as identified above.

[0206] According to certain embodiments thereof, the payload molecule is selected from a pharma- ceutically active drug, a marker and a chelator as further defined herein below.

[0207] According to a more particular embodiment thereof, the payload molecule is a pharma- ceutically active drug, in particular chosen from cytotoxins, antiproliferative / antitumor agents.

[0208] According to a more particular embodiment thereof, the payload molecule is an anti-proliferative / anti-tumor drug.

[0209] According to very particular embodiments thereof, the payload molecule is an auristatin, such as dolastatin 10, monomethylauristatin E (MMAE), auristatin F, monomethylauristatin F (MMAF), auristatin F hydroxypropylamide (AF HPA), auristatin F phenylenediamine (AFP), monomethylauristatin D (MMAD), auristatin PE, auristatin EB, auristatin EFP, auristatin TP and auristatin AQ.

[0210] According to another particular embodiment thereof, the antibody moiety of the ADC is a site-specifically modified trastuzumab as defined herein above.

[0211] According to another particular embodiment thereof, the antibody moiety of the ADC is a site-specifically modified pertuzumab as defined herein above.

[0212] According to another particular embodiment thereof, the antibody moiety and the payload moiety are linked via a linker.

[0213] According to a more specific embodiment thereof, said linker comprises a cleavable moiety. More specifically, said cleavable moiety is cleavable under physiological conditions. More specifically, said linker is proteolytically cleavable or as a pH-sensitive cleavable linker.

[0214] In another particular embodiment of the second aspect of the invention, an ADC is provided that comprises at least one site-selectively modified immunoglobulin molecule comprising at least one immunoglobulin heavy chain (IgH) and at least one immunoglobulin light chain (IgL), The IgH CDR-H1 of SEQ ID NO: 9, CDR-H2 of SEQ ID NO: 11, and Variable region V comprising CDR-H3 of SEQ ID NO: 13 H ,and Constant region C H Including, The IgL CDR-L1 of SEQ ID NO: 15, CDR-L2 of SEQ ID NO: 17, and Variable region V including CDR-L3 of SEQ ID NO: 19 L ,and Constant region C L Including, Where: at least one IgH is site-selectively modified by incorporating one or two SCO residues within their amino acid sequence at sequence positions corresponding to positions selected from K249 and K320 of SEQ ID NO:2, respectively; the site-selectively modified immunoglobulin molecule has the ability to bind to human epidermal growth factor receptor 2 (ERBB2 or HER2 / neu); Each SCO is linked to an H-tetrazine functionalized payload moiety P that includes a drug moiety D selected from auristatins and maytansinoids.

[0215] In another particular embodiment of the second aspect of the invention, there is provided an ADC capable of binding to human epidermal growth factor receptor 2 (ERBB2 or HER2 / neu) and having the following general formula (1): [ka]

[0216] Where: n represents the (average) number of attached side chains, in particular selected from 1, 2, 3 or 4, more particularly 2 or 4, each chain comprising a payload moiety -LD, Where: D is selected from auristatins and maytansinoids; L is optionally a cleavable linker moiety, particularly an enzymatically or chemically cleavable linker, such as a proteolytically cleavable or pH-sensitive cleavable linker; A represents a site-selectively modified immunoglobulin molecule comprising at least one immunoglobulin heavy chain (IgH) and at least one immunoglobulin light chain (IgL); Where: The IgH is CDR-H1 of SEQ ID NO: 9, CDR-H2 of SEQ ID NO: 11, and A variable region VH comprising the CDR-H3 of SEQ ID NO: 13, and Contains the constant region CH, The IgL is CDR-L1 of SEQ ID NO: 15, CDR-L2 of SEQ ID NO: 17, and A variable region VL comprising the CDR-L3 of SEQ ID NO: 19, and Contains the constant region CL, at least one IgH is site-selectively linked to said payload moiety-LD at one or two sequence positions corresponding to positions selected from K249 and K320 of SEQ ID NO:2, respectively; Either in the form of any stereoisomer and / or positional isomer, or as a mixture of at least two different stereoisomers and positional isomers thereof, as well as in non-glycosylated, glycosylated, or deglycosylated form.

[0217] A non-limiting example of such a regioisomer includes a compound of formula 1a: [ka]

[0218] More generally, the linker group L is an enzymatically or chemically cleavable linker group selected from: a) peptidyl groups, in particular di-, tri- or tetrapeptidyl groups, b)-(CR 7 R 8 ) n -SS-(CR 7 R 8 ) n -X 5 - a disulfide group of the formula Where: residue R 7 and R 8 are independently selected from H or lower alkyl, in particular methyl, or two residues R 7 and R 8 together with the carbon atom to which they are attached form a ring C 4 Forming a -C8 alkyl group, Part X 5 is selected from -C(O)- and -O-; c)>C=NN(R 9 )- and -N(R 9 )-N=C<hydrazone group Where: R 9 is H or lower alkyl, and d) beta-glucuronidase-sensitive cleavable linker groups, particularly those bearing a beta-glucuronic acid derived trigger residue.

[0219] Non-limiting examples illustrating the types of linkages between the tetrazine moiety and the -LD moiety include residues of formulas 5, 6, 7, 11, 12 and 13 below. [ka]

[0220] The residues of formulae 5, 6, 7, 11, 12 and 13 may be linked directly to L or, in particular, to -((CH 2 ) x1 -O) y1 - or -(O-(CH 2 ) x1 ) y1 -, and their branched analogues. Where: x1 each independently represent an integer selected from 1, 2, 3 or 4, in particular 1 or 2, Each y1 independently represents an integer of 1 to 20, particularly 1 to 4.

[0221] In another particular embodiment of the second aspect of the invention, there is provided an APC, in particular an ADC, site-selectively linked to at least one payload moiety comprising the moiety -LP of formula 4.1 below. [ka]

[0222] Where: m is an integer from 1 to 8; M is a radioactive metal isotope selected from 111-indium, 64-copper, 67-copper, 227-thorium, 188-rhenium, 177-lutetium, 89-zirconium, 68-gallium, 99m-technetium, 225-actinium, 213-bismuth, 90-yttrium and 212-lead, preferably 177-lutetium.

[0223] In a further embodiment, the APC comprises at least one IgH, such as one or two IgHs, which is site-selectively modified by binding to the payload moiety LP at one sequence position corresponding to position A121 of SEQ ID NO:2.

[0224] More specifically, at least one site-selectively modified IgH of said APC is site-selectively modified by incorporating a TCO*A, e.g. a TCO*A-Lys residue, at a sequence position in their amino acid sequence corresponding to position A121 according to SEQ ID NO:2, and may be further characterized by: (i) the site-selectively modified immunoglobulin molecule has the ability to bind to human epidermal growth factor receptor 2 (ERBB2 or HER2 / neu); (ii) At least one, and in particular each TCO*A, such as TCO*A-Lys, is bound to an H-tetrazine functionalized payload moiety-LP.

[0225] In that regard, the H-tetrazine functionalized payload moiety-LP is of formula 5 below. [ka]

[0226] Where: m is an integer from 1 to 8; M is a radioactive metal isotope selected from 111-indium, 64-copper, 67-copper, 227-thorium, 188-rhenium, 177-lutetium, 89-zirconium, 68-gallium, 99m-technetium, 225-actinium, 213-bismuth, 90-yttrium and 212-lead, preferably 177-lutetium.

[0227] A third aspect of the present invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding at least one site-selectively modified immunoglobulin polypeptide chain as defined for the first aspect of the invention above, which comprises at least one codon, in particular a stop codon, allowing the incorporation of said ncAA into the encoded polypeptide sequence during protein expression.

[0228] In certain embodiments, nucleic acid sequences are provided that are derived from the nucleic acid sequences of SEQ ID NOs: 1, 3, 5 and 7, and nucleic acid sequences derived therefrom that have at least 50%, such as at least 55%, or 60%, particularly at least 75%, more particularly at least 80 or 85%, such as 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID NOs: 1, 3, 5 and 7, and that include one, two or more, more than three, etc. codons (e.g. selector codons) that are reverse complements of the anticodons contained in the tRNA required to incorporate an ncAA into the polypeptide sequence encoded by said nucleic acid sequence. The sequence deviations are selected such that the polypeptides encoded by the nucleic acid sequences derived from SEQ ID NOs: 1, 3, 5 and 7 essentially retain the CDR sequence motifs of the polypeptides of SEQ ID NOs: 2, 4, 6 and 8, respectively. By "essentially" it is meant that site-specific modifications within the sequences encoding the individual CDR motifs are permitted, so long as the binding specificity of the respective immunoglobulin molecule is partially or completely retained.

[0229] According to a particular embodiment of said third aspect, the nucleic acid molecule may be part of an expression construct or expression vector useful for the preparation of the site-selectively modified immunoglobulin molecules of the invention.

[0230] According to another particular embodiment of the third aspect, there is provided a recombinant prokaryotic or eukaryotic host comprising at least one of the encoding nucleic acid molecules identified above and / or at least one of the expression constructs and / or viral vectors identified above.

[0231] According to a more particular embodiment thereof, said eukaryotic organism is selected from a non-human organism or a cell or cell line.

[0232] A fourth aspect of the present invention relates to a method for preparing a site-selectively modified immunoglobulin molecule as defined for the first aspect of the invention above, comprising one or more non-natural amino acid residues (ncAA), wherein said method comprises the steps of: (a) providing a eukaryotic cell comprising: (i) a suitable aminoacyl-tRNA synthetase, in particular a pyrrolysyl-tRNA synthetase; (ii) a suitable tRNA アミノアシル , especially tRNA Pyl ), (iii) ncAA or a salt thereof, and (iv) a polynucleotide encoding the site-selectively modified immunoglobulin molecule; wherein any position of the site-selectively modified immunoglobulin molecule occupied by an ncAA residue is selected from the group consisting of tRNA, アミノアシル , especially tRNA Pyl is encoded by the reverse complement of the anticodon in Here, the aminoacyl-tRNA synthetase, particularly pyrrolysyl-tRNA synthetase (i), アミノアシル , especially tRNA Pyl (ii) can be acylated with the unnatural amino acid or salt (iii); and (b) allowing translation of said polynucleotide (iv) by said eukaryotic cell, thereby producing said site-selectively modified immunoglobulin molecule.

[0233] A fifth aspect of the present invention relates to a method for preparing a polypeptide complex comprising the steps of: (a) preparing a site-selectively modified immunoglobulin molecule comprising one or more ncAA residues using the method of the fourth aspect of the invention identified above, and (b) reacting the site-selectively modified immunoglobulin molecule of step a) with one or more binding partner molecules such that the binding partner molecules are covalently attached to the ncAA residues of the site-selectively modified immunoglobulin molecule.

[0234] According to a particular embodiment of said method, said complex is an APC, in particular an ADC.

[0235] According to another particular embodiment thereof, the binding partner comprises a moiety selected from a pharma- ceutically active drug, a marker and a chelator, as further defined herein below.

[0236] According to another particular embodiment, said binding partner represents a functionalized payload molecule capable of chemically reacting with at least one ncAA residue of a mutated immunoglobulin molecule of the invention.

[0237] More specifically, the functionalized payload molecule may have the following general formula 4: YLD (4) Where: X is selected from functional chemical groups reactive with ncAA residues as defined herein; D is selected from a pharma- ceutical active drug, a marker, and a chelator, as defined herein; L is an optionally cleavable linker moiety as defined herein.

[0238] More generally, the linker group L is an enzymatically or chemically cleavable linker group selected from: a) peptidyl groups, in particular di-, tri- or tetrapeptidyl groups, b)-(CR 7 R 8 ) n -SS-(CR 7 R 8 ) n -X 5 - a disulfide group of the formula Where: residue R 7 and R8 are independently selected from H or lower alkyl, in particular methyl, or two residues R 7 and R 8 together with the carbon atom to which they are attached form a ring C 4 Forming a -C8 alkyl group, Part X 5 is selected from -C(O)- and -O-; c)>C=NN(R 9 )- and -N(R 9 )-N=C<hydrazone group Where: R 9 is H or lower alkyl, and d) beta-glucuronidase-sensitive cleavable linker groups, particularly those bearing a beta-glucuronic acid derived trigger residue.

[0239] Non-limiting examples illustrating the types of linkages between the tetrazine moiety and the -LD moiety include residues of formulas 5, 6, 7, 11, 12 and 13 below. [ka]

[0240] The residues of formulae 5, 6, 7, 11, 12 and 13 may be linked directly to L or, in particular, to -((CH 2 ) x1 -O) y1 - or -(O-(CH 2 ) x1 ) y1 -, and their branched analogues. Where: x1 each independently represent an integer selected from 1, 2, 3 or 4, in particular 1 or 2, Each y1 independently represents an integer of 1 to 20, particularly 1 to 4.

[0241] According to a more particular embodiment thereof, the binding partner comprises a component D, which is a pharma- ceutically active drug, in particular chosen from cytotoxins, antiproliferative / antitumor agents.

[0242] According to a more particular embodiment thereof, the binding partner comprises a component D, which is an anti-proliferative / anti-tumor drug.

[0243] According to a very particular embodiment thereof, the binding partner comprises a component D, which is an auristatin, such as dolastatin 10, monomethylauristatin E (MMAE), auristatin F, monomethylauristatin F (MMAF), auristatin F hydroxypropylamide (AF HPA), auristatin F phenylenediamine (AFP), monomethylauristatin D (MMAD), auristatin PE, auristatin EB, auristatin EFP, auristatin TP and auristatin AQ.

[0244] According to another particular embodiment thereof, the antibody portion of the APC or ADC is a site-specifically modified trastuzumab as defined herein above.

[0245] According to another particular embodiment thereof, the antibody portion of the APC or ADC is a site-specifically modified pertuzumab as defined herein above.

[0246] According to another particular embodiment thereof, the antibody moiety and the payload moiety are linked via a linker L.

[0247] According to a more specific embodiment thereof, said linker comprises a cleavable moiety L. More specifically, said cleavable moiety is cleavable under physiological conditions. More specifically, said linker is proteolytically cleavable or as a pH-sensitive cleavable linker.

[0248] According to certain embodiments of the method, the binding partner carries at least one functional group X capable of reacting with the at least one ncAA side chain contained in the site-selectively modified immunoglobulin molecule.

[0249] More specifically, at least one functional group X includes a 1,2,4,5-tetrazine moiety.

[0250] According to a particular embodiment, the method relates to a method for preparing an ADC of general formula 1: [ka]

[0251] Where: n, L, D and A are as defined above; in any stereoisomeric and / or positional isomeric form, or as a mixture of at least two different stereoisomeric and positional isomeric forms thereof, as well as in non-glycosylated, glycosylated or deglycosylated form.

[0252] The method comprises: a) providing a SCO-functionalized immunoglobulin molecule of general formula 2: [ka]

[0253] where n and A are as defined above. b) reacting the compound of formula 2 with an H-tetrazine functionalized payload molecule of general formula 3: [ka]

[0254] where L and D are as defined above. Obtaining an ADC of general formula (1), and optionally c) isolating the product. A non-limiting example of such a regioisomer to be prepared includes a compound of formula 1a below: [ka]

[0255] Non-limiting examples illustrating the types of linkages between the tetrazine moiety and the -LD moiety include residues of formulas 5, 6, 7, 11, 12 and 13 below. [ka]

[0256] The residues of formulae 5, 6 and 7 may be linked directly to L or, in particular, to -((CH 2 ) x1 -O) y1 - or -(O-(CH 2 ) x1 ) y1 -, and their branched analogues. Where: x1 each independently represent an integer selected from 1, 2, 3 or 4, in particular 1 or 2, Each y1 independently represents an integer of 1 to 20, particularly 1 to 4.

[0257] A sixth aspect of the invention relates to a pharmaceutical composition comprising, in a pharma- ceutically acceptable carrier, at least one ADC according to the second aspect of the invention as specified above or at least one ADC prepared according to the fifth aspect of the invention as specified above, or to a diagnostic composition comprising, in a diagnostically applicable carrier, at least one APC according to the second aspect of the invention as specified above or at least one APC prepared according to the fifth aspect of the invention as specified above.

[0258] According to certain embodiments, the pharmaceutical composition comprises, in a pharma- ceutical acceptable carrier, at least one ADC selected from the following: 1. An ADC comprising at least one site-selectively modified immunoglobulin molecule comprising at least one immunoglobulin heavy chain (IgH) and at least one immunoglobulin light chain (IgL), The IgH CDR-H1 of SEQ ID NO: 9, CDR-H2 of SEQ ID NO: 11, and Variable region V comprising CDR-H3 of SEQ ID NO: 13 H ,and Constant region C H Including, The IgL CDR-L1 of SEQ ID NO: 15, CDR-L2 of SEQ ID NO: 17, and Variable region V including CDR-L3 of SEQ ID NO: 19 L ,and Constant region C L Including, Where: at least one IgH is site-selectively modified by incorporating one or two SCO residues within their amino acid sequence at sequence positions corresponding to positions selected from K249 and K320 of SEQ ID NO:2, respectively; the site-selectively modified immunoglobulin molecule has the ability to bind to human epidermal growth factor receptor 2 (ERBB2 or HER2 / neu); Each SCO is linked to an H-tetrazine functionalized payload moiety P that includes a drug moiety D selected from auristatins and maytansinoids. Alternatively, it is selected from ADCs that have the ability to bind to human epidermal growth factor receptor 2 (ERBB2 or HER2 / neu) and have the following general formula (1): [ka]

[0259] Where: n represents the (average) number of attached side chains, each chain containing a payload moiety -LD; Where: D is selected from auristatins and maytansinoids; L is an optionally cleavable linker moiety; A represents a site-selectively modified immunoglobulin molecule comprising at least one immunoglobulin heavy chain (IgH) and at least one immunoglobulin light chain (IgL); Where: The IgH is CDR-H1 of SEQ ID NO: 9, CDR-H2 of SEQ ID NO: 11, and A variable region VH comprising the CDR-H3 of SEQ ID NO: 13, and Contains the constant region CH, The IgL is CDR-L1 of SEQ ID NO: 15, CDR-L2 of SEQ ID NO: 17, and A variable region VL comprising the CDR-L3 of SEQ ID NO: 19, and Contains the constant region CL, at least one IgH is site-selectively linked to said payload moiety-LD at one or two sequence positions corresponding to positions selected from K249 and K320 of SEQ ID NO:2, respectively; Either in the form of any stereoisomer and / or positional isomer, or as a mixture of at least two different stereoisomers and positional isomers thereof, as well as in non-glycosylated, glycosylated, or deglycosylated form.

[0260] A non-limiting example of such a regioisomer includes a compound of formula 1a: [ka]

[0261] More generally, the linker group L is an enzymatically or chemically cleavable linker group selected from: a) peptidyl groups, in particular di-, tri- or tetrapeptidyl groups, b)-(CR 7 R 8 ) n -SS-(CR 7 R8 ) n -X 5 - a disulfide group of the formula Where: residue R 7 and R 8 are independently selected from H or lower alkyl, in particular methyl, or two residues R 7 and R 8 together with the carbon atom to which they are attached form a ring C 4 Forming a -C8 alkyl group, Part X 5 is selected from -C(O)- and -O-; c)>C=NN(R 9 )- and -N(R 9 )-N=C<hydrazone group Where: R 9 is H or lower alkyl, and d) beta-glucuronidase-sensitive cleavable linker groups, particularly those bearing a beta-glucuronic acid derived trigger residue.

[0262] Non-limiting examples illustrating the types of linkages between the tetrazine moiety and the -LD moiety include residues of formulas 5, 6, 7, 11, 12 and 13 below. [ka]

[0263] The residues of formulae 5, 6, 7, 11, 12 and 13 may be linked directly to L or, in particular, to -((CH 2 ) x1 -O) y1 - or -(O-(CH 2 ) x1 ) y1 -, and their branched analogues. Where: x1 each independently represent an integer selected from 1, 2, 3 or 4, in particular 1 or 2, Each y1 independently represents an integer of 1 to 20, particularly 1 to 4.

[0264] A seventh aspect of the invention relates to an APC or ADC according to the second aspect of the invention specified above, for medical use, for example in diagnosis and therapy.

[0265] An eighth aspect of the invention relates to an APC or ADC according to the second aspect of the invention identified above for use in the diagnosis or treatment of breast cancer, gastric cancer, or other Her2-overexpressing tumours, such as ovarian, endometrial, bladder, lung, colon, and head and neck tumours.

[0266] In particular embodiments for use in diagnosing or treating breast cancer, the following are provided:

[0267] 1. An ADC comprising at least one site-selectively modified immunoglobulin molecule comprising at least one immunoglobulin heavy chain (IgH) and at least one immunoglobulin light chain (IgL), The IgH CDR-H1 of SEQ ID NO: 9, CDR-H2 of SEQ ID NO: 11, and Variable region V comprising CDR-H3 of SEQ ID NO: 13 H ,and Constant region C H Including, The IgL CDR-L1 of SEQ ID NO: 15, CDR-L2 of SEQ ID NO: 17, and Variable region V including CDR-L3 of SEQ ID NO: 19 L ,and Constant region C L Including, Where: at least one IgH is site-selectively modified by incorporating one or two SCO residues within their amino acid sequence at sequence positions corresponding to positions selected from K249 and K320 of SEQ ID NO:2, respectively; the site-selectively modified immunoglobulin molecule has the ability to bind to human epidermal growth factor receptor 2 (ERBB2 or HER2 / neu); Each SCO is linked to an H-tetrazine functionalized payload moiety P that includes a drug moiety D selected from auristatins and maytansinoids. Alternatively, it is selected from ADCs that have the ability to bind to human epidermal growth factor receptor 2 (ERBB2 or HER2 / neu) and have the following general formula (1): [ka]

[0268] Where: n represents the (average) number of attached side chains, each chain containing a payload moiety -LD; Where: D is selected from auristatins and maytansinoids; L is an optionally cleavable linker moiety; A represents a site-selectively modified immunoglobulin molecule comprising at least one immunoglobulin heavy chain (IgH) and at least one immunoglobulin light chain (IgL); Where: The IgH is CDR-H1 of SEQ ID NO: 9, CDR-H2 of SEQ ID NO: 11, and A variable region VH comprising the CDR-H3 of SEQ ID NO: 13, and Contains the constant region CH, The IgL is CDR-L1 of SEQ ID NO: 15, CDR-L2 of SEQ ID NO: 17, and A variable region VL comprising the CDR-L3 of SEQ ID NO: 19, and Contains the constant region CL, at least one IgH is site-selectively linked to said payload moiety-LD at one or two sequence positions corresponding to positions selected from K249 and K320 of SEQ ID NO:2, respectively; Either in the form of any stereoisomer and / or positional isomer, or as a mixture of at least two different stereoisomers and positional isomers thereof, as well as in non-glycosylated, glycosylated, or deglycosylated form.

[0269] A non-limiting example of such a regioisomer includes a compound of formula 1a: [ka]

[0270] More generally, the linker group L is an enzymatically or chemically cleavable linker group selected from: a) peptidyl groups, in particular di-, tri- or tetrapeptidyl groups, b)-(CR 7 R 8 ) n -SS-(CR 7 R 8 ) n -X 5 - a disulfide group of the formula Where: residue R 7 and R 8 are independently selected from H or lower alkyl, in particular methyl, or two residues R 7 and R 8 together with the carbon atom to which they are attached form a ring C 4 Forming a -C8 alkyl group, Part X 5 is selected from -C(O)- and -O-; c)>C=NN(R 9 )- and -N(R 9 )-N=C<hydrazone group Where: R 9 is H or lower alkyl, and d) beta-glucuronidase-sensitive cleavable linker groups, particularly those bearing a beta-glucuronic acid derived trigger residue.

[0271] Non-limiting examples illustrating the types of linkages between the tetrazine moiety and the -LD moiety include residues of formulas 5, 6, 7, 11, 12 and 13 below. [ka]

[0272] The residues of formulae 5, 6, 7, 11, 12 and 13 may be linked directly to L or, in particular, to -((CH 2 ) x1 -O) y1 - or -(O-(CH 2 ) x1 ) y1 -, and their branched analogues. Where: x1 each independently represent an integer selected from 1, 2, 3 or 4, in particular 1 or 2, Each y1 independently represents an integer of 1 to 20, particularly 1 to 4.

[0273] In a particular embodiment there is provided an APC, in particular an ADC, according to the second embodiment site-selectively linked to at least one payload moiety comprising the moiety-LP of formula 4.1 below, for the diagnosis or treatment of breast cancer. [ka]

[0274] Where: m is an integer from 1 to 8; M is a radioactive metal isotope selected from 111-indium, 64-copper, 67-copper, 227-thorium, 188-rhenium, 177-lutetium, 89-zirconium, 68-gallium, 99m-technetium, 225-actinium, 213-bismuth, 90-yttrium and 212-lead, preferably 177-lutetium.

[0275] The APC may comprise at least one IgH, such as one or two IgH, which is site-selectively modified by binding to the payload moiety LP at one sequence position corresponding to position A121 of SEQ ID NO:2.

[0276] More specifically, said at least one site-selectively modified IgH of said APC is site-selectively modified by incorporating a TCO*A-Lys residue within their amino acid sequence at a sequence position corresponding to position A121 of SEQ ID NO:2, and may be further characterized by: (i) the site-selectively modified immunoglobulin molecule has the ability to bind to human epidermal growth factor receptor 2 (ERBB2 or HER2 / neu); (ii) Each TCO*A-Lys is coupled to an H-tetrazine functionalized payload moiety-LP.

[0277] In this regard, the H-tetrazine functionalized payload moiety-LP is of formula 5: [ka]

[0278] Where: m is an integer from 1 to 8; M is a radioactive metal isotope selected from 111-indium, 64-copper, 67-copper, 227-thorium, 188-rhenium, 177-lutetium, 89-zirconium, 68-gallium, 99m-technetium, 225-actinium, 213-bismuth, 90-yttrium and 212-lead, preferably 177-lutetium.

[0279] A ninth aspect of the present invention relates to specific novel binding partners, especially for site-selectively modified immunoglobulin molecules such as those described above. The binding partners of this aspect of the invention are H-tetrazine functionalized payload molecules of the following general formula 20: H-Tet-X 1 -GX 2 -D (20) wherein H-Tet represents a functionalizing group comprising a moiety of formula 21: [ka]

[0280] G represents a beta-glucuronidase sensitive cleavable group, in particular carrying a moiety derived from beta-glucuronic acid, D represents an auristatin-type drug moiety; X 1 represents a chemical bond or group connecting H-Tet and G, X 2 represents a chemical bond or group connecting G and D, where said linking group is -C(=O)-. or a salt thereof, optionally in stereomerically pure form or as a mixture of at least two stereoisomers.

[0281] According to a particular embodiment of this aspect of the invention, the residue H-Tet of formula 20 is selected from the residues of formulas 5, 6, 7, 11, 12 or 13. [ka]

[0282] According to another particular embodiment of this aspect of the invention, group G of formula 20 is a residue of formula 22: [ka]

[0283] According to yet another particular embodiment of this aspect of the invention, group D of formula 20 is selected from dolastatin 10, monomethylauristatin E (MMAE), auristatin F, monomethylauristatin F (MMAF), auristatin F hydroxypropylamide (AF HPA), auristatin F phenylenediamine (AFP), monomethylauristatin D (MMAD), auristatin PE, auristatin EB, auristatin EFP, auristatin TP and auristatin AQ, in particular MMAE.

[0284] According to yet another particular embodiment of this aspect of the invention, in formula 20, X 1 is a chemical bond, and X 2 is -C(=O)-.

[0285] More specifically, the binding partner of this aspect of the invention is of general formula 23: [ka]

[0286] Particular binding partners according to this aspect of the invention are selected from compounds of any one of formulas 24.1-24.6 below. [ka] [ka] [ka]

[0287] According to yet another particular embodiment of this aspect of the invention, there is provided an antibody payload conjugate (APC), in which an antibody molecule functionalized by incorporating at least one non-natural amino acid (ncAA) residue into at least one of its polypeptide chains is conjugated via the side chain of said ncAA residue to at least one H-tetrazine functionalized payload molecule of any one of the preceding embodiments of the ninth aspect of the invention. More particularly, said ncAA is a SCO as defined above.

[0288] According to yet another particular embodiment of this aspect of the invention, the antibody molecule is derived from trastuzumab.

[0289] In a further embodiment of this aspect of the invention there is provided an APC as defined in the ninth aspect of the invention, particularly for medical use in therapy, more particularly for use in the treatment of breast cancer, gastric cancer or other Her2 overexpressing tumours, such as ovarian, endometrial, bladder, lung, colon and head and neck tumours.

[0290] According to yet another particular embodiment of this aspect of the invention there is provided a pharmaceutical composition comprising at least an APC as defined in the ninth aspect of the invention in a pharma- ceutically acceptable carrier.

[0291] Finally, according to yet another particular embodiment of this ninth aspect of the invention there is provided a method of preparing compounds of formula 20, in particular formula 23, which is described in more detail in Section D below.

[0292] D. Further Embodiments 1. Polypeptides of the Invention

[0293] 1.1. General In this context the following definitions apply: "Functional variants" of the polypeptides described herein encompass "functional equivalents" of such polypeptides, as defined below.

[0294] An "enzyme", "protein" or "polypeptide" as described herein may be a naturally occurring or recombinantly produced enzyme, protein or polypeptide. It may be a wild-type enzyme, protein or polypeptide or may be genetically modified by suitable mutations or by C- and / or N-terminal amino acid sequence extensions, such as His-tag containing sequences. The enzyme, protein or polypeptide may essentially be mixed with cellular, e.g. protein, impurities, but in particular in pure form. Suitable detection methods are described, for example, in the experimental section below or are known from the literature.

[0295] In the present invention, a "pure form" or a "pure" or "substantially pure" enzyme, protein or polypeptide is to be understood as an enzyme, protein or polypeptide having a degree of purity of more than 80% by weight, in particular more than 90% by weight, in particular more than 95% by weight, in particular more than 99% by weight, based on the total protein content as determined by the usual methods for protein detection, such as the Biuret method or the protein detection according to Lowry et al. (see description in RK Scopes, Protein Purification, Springer Verlag, New York, Heidelberg, Berlin (1982)).

[0296] "Proteinogenic" amino acids include in particular the following (single letter code): G, A, V, L, I, F, P, M, W, S, T, C, Y, N, Q, D, E, K, R and H.

[0297] The general terms "polypeptide" or "peptide" can be used interchangeably and refer to any natural or synthetic linear chain or sequence of consecutive peptidically linked amino acid residues, containing from about 10 residues to over 1,000 residues. Shorter polypeptides of up to 30 residues are also called "oligopeptides."

[0298] The term "protein" refers to a macromolecular structure consisting of one or more polypeptides. The amino acid sequence of that polypeptide represents the "primary structure" of the protein. The amino acid sequence also predetermines the "secondary structure" of the protein by the formation of special structural elements such as alpha-helical and beta-sheet structures formed within the polypeptide chain. The arrangement of several such secondary structural elements defines the "tertiary structure" or spatial arrangement of the protein. When a protein comprises two or more polypeptide chains, said chains are spatially arranged to form the "quaternary structure" of the protein. Correct spatial arrangement or "folding" of a protein is a prerequisite for the function of the protein. Denaturation or unfolding destroys the function of the protein. If such destruction is reversible, the function of the protein can be restored by refolding.

[0299] A "polypeptide" said to have a particular "activity" herein therefore implicitly refers to a correctly folded protein that exhibits the specified activity.

[0300] Similarly, the term "polypeptide fragment" encompasses the term "protein fragment."

[0301] The term "isolated polypeptide" refers to an amino acid sequence that has been removed from its natural environment by any method or combination of methods known in the art, including recombinant, biochemical, and synthetic methods.

[0302] The present invention also relates to "functional equivalents" (also called "analogs" or "functional variants") of the polypeptides specifically described herein.

[0303] For example, a "functional equivalent" refers to a polypeptide that exhibits at least 1-10%, or at least 20%, or at least 50%, or at least 75%, or at least 90% greater or less activity than that of the polypeptides specifically described herein in an assay used to measure NHase enzymatic activity.

[0304] The "functional equivalents" of the present invention also cover specific variants, which have, at at least one sequence position of the amino acid sequences described herein, an amino acid different from that specifically described, but still have one of the aforementioned biological activities, e.g. enzymatic activity. Thus, "functional equivalents" include variants that can be obtained by one or more, e.g. 1-20, in particular 1-15 or 5-10 amino acid additions, substitutions, in particular conservative substitutions, deletions and / or inversions, said changes may occur at any sequence position, as long as they result in a variant having the profile of properties according to the invention. Also, a qualitative match of the activity patterns between the variant and the unchanged polypeptide, i.e. for example different kinetics (i.e. EC 50 Or IC 50Functional equivalence is provided in particular when a similar interaction is observed with the same agonist or antagonist or substrate, expressed by a similar or similar parameter (expressed by a similar or similar parameter, or any other parameter suitable in the art). Examples of suitable (conservative) amino acid substitutions are shown in Table 3 below.

[0305] Table 3: Examples of conservative amino acid substitutions [Table 3]

[0306] "Functional equivalents" in the above sense are also "precursors" of the polypeptides described herein, as well as "functional derivatives" and "salts" of the polypeptides.

[0307] A "precursor" in this case is a natural or synthetic precursor of a polypeptide, with or without the desired biological activity.

[0308] The expression "salts" refers to salts of carboxyl groups of the protein molecules of the present invention as well as acid addition salts of amino groups. Salts of carboxyl groups can be produced in known manner and include inorganic salts, such as sodium, calcium, ammonium, iron and zinc salts, as well as salts with organic bases, such as amines, for example triethanolamine, arginine, lysine, piperidine, etc. Acid addition salts, such as salts with inorganic acids, such as hydrochloric acid or sulfuric acid, as well as salts with organic acids, such as acetic acid and oxalic acid, are also covered by the present invention.

[0309] "Functional derivatives" of the polypeptides of the invention can also be produced on functional amino acid side groups or at their N- or C-termini using known techniques. Such derivatives include, for example, aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups obtainable by reaction with ammonia or primary or secondary amines, N-acyl derivatives of free amino groups formed by reaction with acyl groups, or O-acyl derivatives of free hydroxyl groups formed by reaction with acyl groups.

[0310] "Functional equivalents" also include "fragments" of the polypeptides of the invention, such as individual domains or sequence motifs, or N-terminally and / or C-terminally truncated forms, which may or may not exhibit a desired biological function. In particular, such "fragments" retain at least a qualitatively desired biological function.

[0311] A "functional equivalent" is also a fusion protein having one of the polypeptide sequences described herein or functional equivalents derived therefrom and at least one additional, functionally distinct, heterologous sequence functionally linked to the N-terminus or C-terminus (i.e., the parts of the fusion protein do not substantially impair the function of each other).

[0312] "Functional equivalents" also included according to the present invention are homologues to the specifically disclosed polypeptides. These have at least 50%, 55% or 60%, in particular at least 75%, more particularly at least 80 or 85%, for example 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% homology (or identity) to one of the specifically disclosed amino acid sequences, as calculated by the algorithm of Pearson and Lipman, Proc. Natl. Acad, Sci. (USA) 85(8), 1988, 2444-2448. The homology or identity expressed as a percentage of the homologous polypeptides of the present invention means in particular the identity expressed as a percentage of the amino acid residues based on the full length of one of the specifically described amino acid sequences herein.

[0313] Identity data expressed as a percentage can also be determined by BLAST alignment, applying the algorithm blastp (protein-protein BLAST) or Clustal settings as specified below.

[0314] In the case of possible protein glycosylation, the "functional equivalents" of the present invention include deglycosylated or glycosylated forms of the polypeptides described herein, as well as modified forms which can be obtained by modulating the glycosylation pattern.

[0315] Functional equivalents or homologues of the polypeptides of the invention can be produced by mutagenesis, for example by point mutations, lengthening or shortening of the protein, or as described in more detail below.

[0316] 1.2 Immunoglobulins In certain embodiments, the antibody comprises a heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. According to one aspect, the heavy chain constant region is an IgG1 heavy chain constant region or an IgG4 heavy chain constant region. According to a further aspect, the antibody comprises a light chain constant region, either a kappa light chain constant region or a lambda light chain constant region. According to one aspect, the antibody comprises a kappa light chain constant region. The antibody portion may be, for example, a Fab fragment or a single chain Fv fragment.

[0317] The replacement of amino acid residues in the Fc portion to modulate the effector functions of antibodies is known in the art (Winter et al., U.S. Patent Nos. 5,648,260 and 5,624,821). The Fc portion of an antibody mediates several important effector functions, such as cytokine induction, ADCC, phagocytosis, complement-dependent cytotoxicity (CDC), and half-life / clearance rate of the antibody and antigen-antibody complex. In some cases, these effector functions are desirable for therapeutic antibodies, but in other cases, they may be unnecessary or even harmful, depending on the therapeutic purpose. Certain human IgG isotypes, particularly IgG1 and IgG3, mediate ADCC and CDC via binding to FcγR and complement C1q, respectively. The fetal Fc receptor (FcRn) is an important component that determines the circulating half-life of an antibody. In yet another embodiment, at least one amino acid residue is replaced in the constant region of an antibody, such as the Fc region of an antibody, resulting in a modulation of the effector function of the antibody.

[0318] Another embodiment of the invention provides a glycosylated antibody, in which the antibody comprises one or more carbohydrate residues. Nascent protein production in vivo may undergo further processing known as post-translational modification. In particular, sugar (glycosyl) residues may be added enzymatically in a process known as glycosylation. The resulting proteins with covalently attached oligosaccharide side chains are known as glycosylated proteins or glycoproteins.

[0319] Antibodies are glycoproteins that have one or more carbohydrate residues in the Fc domain as well as in the variable domain. Carbohydrate residues in the Fc domain have a significant effect on the effector functions of the Fc domain with minimal effect on antigen binding or half-life of the antibody (R. Jefferis, Biotechnol. Prog. 21 (2005), pp. 11-16). In contrast, glycosylation of the variable domain may have an effect on the antigen-binding activity of the antibody. Glycosylation of the variable domain can have a negative effect on the binding affinity of the antibody, possibly due to steric hindrance (Co, MS, et al., Mol. Immunol. (1993) 30:1361-1367), or can result in an increased affinity for the antigen (Wallick, SC, et al., Exp. Med. (1988) 168:1099-1109; Wright, A., et al., EMBO J. (1991) 10:2717 2723).

[0320] One aspect of the present invention is directed to generating glycosylation site mutants in which the O-linked or N-linked glycosylation site of an antibody is mutated. Such mutants can be generated by one of skill in the art using standard well-known techniques. It is another object of the present invention to generate glycosylation site mutants with increased or decreased binding activity while retaining biological activity.

[0321] In yet another embodiment, the glycosylation of the antibody of the invention is altered. For example, an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be modulated, for example, to increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be accomplished, for example, by modulating one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the removal of one or more variable region glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. Such approaches are described in further detail in International Application Publication No. WO 03 / 016466 A2 and U.S. Patent Nos. 5,714,350 and 6,350,861, each of which is incorporated herein by reference in its entirety.

[0322] Additionally or alternatively, modified antibodies of the invention can be made with a modulated type of glycosylation, e.g., hypofucosylated antibodies with reduced amounts of fucosyl residues or increased bisecting GlcNAc structures. Such modulated glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with modulated glycosylation machinery. Cells with modulated glycosylation machinery have been described in the art and can be used as host cells for expressing recombinant antibodies of the invention to produce antibodies with modulated glycosylation. See, e.g., Shields, RL et al. (2002) J. Biol. Chem. 277:26733-26740, Umana et al. (1999) Nat. Biotech. 17:176-1, as well as European Patent No. EP 1,176,195, International Application Publication Nos. WO 03 / 035835 and WO 99 / 54342 80, each of which is incorporated herein by reference in its entirety.

[0323] Protein glycosylation depends on the amino acid sequence of the protein of interest and the host cell in which the protein is expressed. Different organisms produce different glycosylation enzymes (e.g., glycosyltransferases and glycosidases) and may have different substrates (nucleotide sugars) available. Due to such factors, the glycosylation pattern and composition of glycosyl residues of a protein may vary depending on the host system in which a particular protein is expressed. Glycosyl residues useful in the present invention include, but are not limited to, glucose, galactose, mannose, fucose, N-acetylglucosamine, and sialic acid. In one embodiment, the glycosylated antibody comprises glycosyl residues whose glycosylation pattern is human.

[0324] It is known to those skilled in the art that differences in glycosylation of proteins can result in differences in the properties of the protein. For example, the potency of a therapeutic protein produced in a microbial host such as yeast and glycosylated using the endogenous pathway of yeast may be reduced compared to that of the same protein expressed in mammalian cells such as CHO or HEK293 or HEK293F cell lines. Also, such glycoproteins may be immunogenic in humans and exhibit reduced in vivo half-life after administration. Specific receptors in humans and other animals may recognize specific glycosyl residues and promote rapid clearance of the protein from the bloodstream. Other adverse effects include changes in protein folding, solubility, susceptibility to proteases, trafficking, transport, compartmentalization, secretion, recognition by other proteins or factors, antigenicity, or allergenicity. Thus, practitioners would prefer therapeutic proteins with a particular composition and pattern of glycosylation, e.g., the same or at least similar composition and pattern of glycosylation as that produced in human cells or species-specific cells of the intended target animal.

[0325] Expressing a protein with a glycosylated form different from that of the host cell can be achieved by genetically modifying the host cell to express a heterologous glycosylation enzyme. A person skilled in the art can use techniques known in the art to generate antibodies that show the glycosylation of human proteins. For example, yeast strains have been genetically modified to express non-naturally occurring glycosylation enzymes. As a result, the glycosylated forms of proteins (glycoproteins) produced in these yeast strains show the same protein glycosylation as animal cells, particularly human cells (US Patent Publication Nos. 20040018590 and 20020137134, and WO05 / 100584).

[0326] Another embodiment is directed to anti-idiotypic (anti-Id) antibodies specific to such antibodies of the invention. Anti-Id antibodies are antibodies that recognize unique determinants generally associated with the antigen-binding region of another antibody. Anti-Ids can be prepared by immunizing an animal with an antibody or its CDR-containing region. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody, producing anti-Id antibodies. Anti-Id antibodies can also be used as "immunogens" to induce an immune response in yet another animal, producing so-called anti-anti-Id antibodies.

[0327] Further, one skilled in the art will appreciate that a protein of interest can be expressed using a library of host cells genetically engineered to express various glycosylation enzymes, such that the host cells of the library members produce proteins of interest with variant glycosylation patterns. A practitioner can then select and isolate proteins of interest with specific novel glycosylation patterns. According to a further aspect, proteins with specifically selected novel glycosylation patterns exhibit improved or modulated biological properties.

[0328] 2. Nucleic Acids and Constructs 2.1 Nucleic acids In this context, the following definitions apply:

[0329] The terms "nucleic acid sequence", "nucleic acid", "nucleic acid molecule" and "polynucleotide" are used interchangeably and refer to a sequence of nucleotides. A nucleic acid sequence may be a single- or double-stranded deoxyribonucleotide or ribonucleotide of any length and may include coding and non-coding sequences of genes, exons, introns, sense and antisense complementary sequences, genomic DNA, cDNA, miRNA, siRNA, mRNA, rRNA, tRNA, recombinant nucleic acid sequences, isolated and purified naturally occurring DNA and / or RNA sequences, synthetic DNA and RNA sequences, fragments, primers, and nucleic acid probes. Those skilled in the art recognize that a nucleic acid sequence of RNA is identical to a DNA sequence, except that thymine (T) is replaced by uracil (U). The term "nucleotide sequence" should also be understood to include a polynucleotide or oligonucleotide molecule in the form of a single fragment or as a component of a larger nucleic acid.

[0330] An "isolated nucleic acid" or "isolated nucleic acid sequence" refers to a nucleic acid or nucleic acid sequence that is in an environment other than that in which it naturally occurs, and can include one that is substantially free of contaminating endogenous material.

[0331] As used herein, the term "naturally-occurring" when applied to nucleic acids refers to nucleic acids that are found in the cells of an organism in nature and that have not been intentionally modified by man in the laboratory.

[0332] A "fragment" of a polynucleotide or nucleic acid sequence refers to a contiguous nucleotide of the polynucleotide of the embodiments herein, particularly at least 15 bp, at least 30 bp, at least 40 bp, at least 50 bp, and / or at least 60 bp in length. In particular, a fragment of a polynucleotide comprises at least 25, more particularly at least 50, more particularly at least 75, more particularly at least 100, more particularly at least 150, more particularly at least 200, more particularly at least 300, more particularly at least 400, more particularly at least 500, more particularly at least 600, more particularly at least 700, more particularly at least 800, more particularly at least 900, and more particularly at least 1000 contiguous nucleotides of the polynucleotide of the embodiments herein. Without being limited thereto, a fragment of a polynucleotide of the present invention can be used as a PCR primer and / or a probe, or for antisense gene silencing or RNAi.

[0333] The term "hybridization" or hybridizing under certain conditions as used herein is intended to refer to hybridization and washing conditions under which nucleotide sequences that are significantly identical or homologous to each other remain bound to each other. The conditions can be such that sequences that are at least about 70%, such as at least about 80%, such as at least about 85%, 90%, or 95% identical to each other remain bound to each other. Definitions of low stringency, medium, and high stringency hybridization conditions are provided herein below. Appropriate hybridization conditions can also be selected by those skilled in the art with minimal experimentation, as exemplified by Ausubel et al. (1995, Current Protocols in Molecular Biology, John Wiley & Sons, sections 2, 4, and 6). In addition, stringency conditions are described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, chapters 7, 9, and 11).

[0334] A "recombinant nucleic acid sequence" is a nucleic acid sequence that results from the use of laboratory methods (e.g., molecular cloning) to combine genetic material from two or more sources to create or modify a non-naturally occurring nucleic acid sequence that would not otherwise be found in an organism.

[0335] "Recombinant DNA techniques" refers to the methods of molecular biology for preparing recombinant nucleic acid sequences, e.g., as described in Laboratory Manuals, Weigel and Glazebrook eds., 2002, Cold Spring Harbor Lab Press and Sambrook et al., 1989, Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press.

[0336] The term "gene" refers to a DNA sequence comprising a region that is transcribed into an RNA molecule, e.g., an mRNA in a cell, and is operably linked to a suitable regulatory region, e.g., a promoter. Thus, a gene may comprise several operably linked sequences, e.g., a promoter, a 5' leader sequence including sequences involved in translation initiation, a coding region of cDNA or genomic DNA, introns, exons, and / or 3' untranslated sequences including transcription termination sites, etc.

[0337] "Polycistronic" refers to a nucleic acid molecule, particularly an mRNA, that can separately encode two or more polypeptides within the same nucleic acid molecule.

[0338] "Chimeric gene" refers to any gene that is not normally found in nature in a species, specifically, a gene in which there are one or more portions of a nucleic acid sequence that are not naturally associated with each other. For example, a promoter is not naturally associated with all or a portion of a transcriptional region or with another control region. The term "chimeric gene" is understood to include an expression construct in which a promoter or transcriptional control sequence is operably linked to one or more coding sequences, or antisense, i.e., the reverse complement of the sense strand, or inverted repeat sequences (sense and antisense, whereby the RNA transcript forms a double-stranded RNA upon transcription). The term "chimeric gene" also includes genes obtained by combining portions of one or more coding sequences to generate a new gene.

[0339] "3'UTR" or "3' untranslated sequence" (also referred to as "3' untranslated region" or "3' end") refers to nucleic acid sequences found downstream of the coding sequence of a gene, which includes, for example, the transcription termination site and (in most, but not all, eukaryotic mRNAs) a polyadenylation signal such as AAUAAA or a variant thereof. After termination of transcription, the mRNA transcript may be cleaved downstream of the polyadenylation signal and a poly(A) tail may be added, which is involved in the transport of the mRNA to the site of translation, e.g., the cytoplasm.

[0340] The term "primer" refers to a short nucleic acid sequence that hybridizes to a template nucleic acid sequence and is used to polymerize a nucleic acid sequence that is complementary to the template.

[0341] The term "selectable marker" refers to any gene whose expression can be used to select cells that contain the selectable marker. Examples of selectable markers are described below. Those skilled in the art will know that different antibiotic, fungicide, auxotrophic or herbicide selectable markers are applicable to different target species.

[0342] The present invention also relates to nucleic acid sequences which code for the polypeptides defined herein.

[0343] In particular, the present invention also relates to nucleic acid sequences (single- and double-stranded DNA and RNA sequences, e.g. cDNA, genomic DNA and mRNA) encoding one of the abovementioned polypeptides and their functional equivalents, which can be obtained, for example, using artificial nucleotide analogues.

[0344] The present invention relates to both isolated nucleic acid molecules that encode the polypeptides of the invention or biologically active segments thereof, as well as nucleic acid fragments that can be used, for example, as hybridization probes or primers to identify or amplify the encoding nucleic acids of the invention.

[0345] The present invention also relates to nucleic acids having a certain degree of "identity" to the sequences specifically disclosed herein. "Identity" between two nucleic acids means in each case nucleotide identity over the entire length of the nucleic acid.

[0346] The "identity" between two nucleotide sequences (the same is true for peptide or amino acid sequences) is a function of the number of nucleotide residues (or amino acid residues) that are identical in the two sequences when an alignment of these two sequences is generated. Identical residues are defined as residues that are identical in the two sequences at a given position of the alignment. The percentage of sequence identity as used herein is calculated by taking the number of residues that are identical between the two sequences from the optimal alignment, dividing it by the total number of residues in the shortest sequence, and multiplying by 100. The optimal alignment is the alignment that results in the highest possible percentage of identity. Gaps can be introduced in one or both sequences at one or more positions of the alignment to obtain an optimal alignment. These gaps are then taken into account as non-identical residues for the calculation of the percentage of sequence identity. Alignment for the purpose of determining the percentage of identity of amino acid or nucleic acid sequences can be achieved in a variety of ways using computer programs, for example computer programs publicly available on the World Wide Web.

[0347] In particular, the BLAST program (Tatiana et al, FEMS Microbiol Lett., 1999, 174:247-250, 1999), set to default parameters and available from the National Center for Biotechnology Information (NCBI) website at ncbi.nlm.nih.gov / BLAST / bl2seq / wblast2.cgi, can be used to obtain optimal alignments of protein or nucleic acid sequences and to calculate the percentage of sequence identity.

[0348] In another example, identity can be calculated by the Vector NTI suite 7.1 program from Informax (USA) using the Clustal method (Higgins DG, Sharp PM. ((1989))) with the following settings:

[0349] Multiple alignment parameters: Gap Open Penalty 10 Gap extension penalty 10 Gap Separation Penalty Range 8 Gap separation penalty off % identity for alignment delay 40 Residue-specific gap off Hydrophilic residue gap off Transition Weighting 0

[0350] Pairwise alignment parameters: FAST algorithm on K tuple size 1 Gap Penalty 3 Window Size 5 Number of best diagonals: 5

[0351] Alternatively, identity can be determined according to Chenna, et al. (2003), web page http: / / www.ebi.ac.uk / Tools / clustalw / index.html# and following settings. DNA Gap Open Penalty 15.0 DNA Gap Extension Penalty 6.66 DNA Matrix Identity Protein Gap Open Penalty 10.0 Protein gap extension penalty 0.2 Protein Matrix Gonnet Protein / DNA ENDGAP -1 Protein / DNA GAPDIST 4

[0352] All nucleic acid sequences (single-stranded and double-stranded DNA and RNA sequences, e.g., cDNA and mRNA) referred to herein can be produced by chemical synthesis from nucleotide building blocks, for example by fragment condensation of individual overlapping complementary nucleic acid building blocks of a double helix, in a known manner. Chemical synthesis of oligonucleotides can be carried out, for example, by the phosphoramidite method, in a known manner (Voet, Voet, 2nd edition, Wiley Press, New York, p. 896-897). The accumulation of synthetic oligonucleotides and gap filling by the Klenow fragment of DNA polymerase and ligation reactions, as well as general cloning techniques, are described in Sambrook et al. (1989). See below.

[0353] The nucleic acid molecules of the present invention may also additionally contain untranslated sequences from the 3' and / or 5' ends of the coding gene region.

[0354] The present invention also relates to nucleic acid molecules which are complementary to the specifically described nucleotide sequences or segments thereof.

[0355] The nucleotide sequences of the present invention allow the generation of probes and primers that can be used for the identification and / or cloning of homologous sequences in other cell types and organisms. Such probes or primers generally comprise a nucleotide sequence region that hybridizes under "stringent" conditions (as defined elsewhere herein) over at least about 12, in particular at least about 25, e.g., about 40, 50 or 75 consecutive nucleotides of the sense strand or the corresponding antisense strand of the nucleic acid sequence of the present invention.

[0356] "Homologous" sequences include orthologous or paralogous sequences. Methods for identifying orthologs or paralogs, including phylogenetic methods, sequence similarity, and hybridization methods, are known in the art and described herein.

[0357] "Paralogue" results from gene duplication, which results in two or more genes with similar sequence and similar function. Paralogs are typically clustered together and formed by gene duplication in related plant species. Paralogs are found using pairwise Blast analysis in groups of similar genes, or using programs such as CLUSTAL in phylogenetic analysis of gene families. In paralogs, consensus sequences can be identified that are characteristic of sequences between related genes and have similar functions of genes.

[0358] "Orthologs" or orthologous sequences are sequences that are similar to each other because they are found in species derived from a common ancestor. For example, plant species with a common ancestor are known to contain many enzymes with similar sequences and functions. A person skilled in the art can identify orthologous sequences and predict the function of the orthologs, for example, by constructing a phylogenetic tree of a gene family of a species using CLUSTAL or BLAST programs. A method for identifying or confirming similar functions between homologous sequences is by comparing transcript profiles in a host cell or organism, such as a plant or microorganism, overexpressing or lacking (in a knockout / knockdown) the relevant polypeptide. A person skilled in the art will understand that genes with similar transcript profiles, with more than 50% of regulated transcripts in common, or more than 70% of regulated transcripts in common, or more than 90% of regulated transcripts in common, will have similar functions. Homologs, paralogs, orthologs and any other variants of the sequences herein are expected to function in a similar manner by creating host cells, organisms, such as plants or microorganisms, that produce the enzymes of the invention.

[0359] The nucleic acid molecules of the invention can be recovered by using standard techniques of molecular biology and the sequence information provided by the invention. For example, cDNA can be isolated from a suitable cDNA library using one of the complete sequences specifically disclosed herein or a segment thereof as a hybridization probe and standard hybridization techniques (e.g., as described in Sambrook, (1989)).

[0360] Furthermore, the nucleic acid molecule comprising one of the disclosed sequences or a segment thereof can be isolated by polymerase chain reaction using oligonucleotide primers constructed based on this sequence. The nucleic acid thus amplified can be cloned into a suitable vector and characterized by DNA sequencing. The oligonucleotide of the present invention can also be produced by standard synthetic methods, for example using an automatic DNA synthesizer.

[0361] The nucleic acid sequences of the invention or their derivatives, homologues or parts of these sequences can be isolated from other bacteria, for example from genomic or cDNA libraries, for example by conventional hybridization or PCR techniques, which DNA sequences hybridize under standard conditions with the sequences of the invention.

[0362] "Hybridize" refers to the ability of a polynucleotide or oligonucleotide to bind to a nearly complementary sequence under standard conditions, where non-specific binding does not occur between non-complementary partners under these conditions. For this purpose, the sequences may be 90-100% complementary. The property that complementary sequences can bind specifically to each other is utilized, for example, in Northern or Southern blotting, or for primer binding in PCR or RT-PCR.

[0363] Short oligonucleotides of the conserved regions are advantageously used for hybridization. However, it is also possible to use longer fragments or complete sequences of the nucleic acids of the present invention for hybridization. These "standard conditions" vary depending on the nucleic acid (oligonucleotide, longer fragment or complete sequence) used, or on what kind of nucleic acid, DNA or RNA, is used for hybridization. For example, the melting temperature of DNA:DNA hybrids is about 10°C lower than that of DNA:RNA hybrids of the same length.

[0364] For example, depending on the specific nucleic acid, standard conditions refer to a temperature of 42-58° C. in an aqueous buffer with a concentration of 0.1-5× SSC (1×SSC=0.15 M NaCl, 15 mM sodium citrate, pH 7.2) or additionally in the presence of 50% formamide, for example 42° C. in 5×SSC, 50% formamide. Advantageously, hybridization conditions for DNA:DNA hybrids are 0.1×SSC and a temperature of about 20° C.-45° C., in particular about 30° C.-45° C. Advantageously, hybridization conditions for DNA:RNA hybrids are 0.1×SSC and a temperature of about 30° C.-55° C., in particular about 45° C.-55° C. These stated hybridization temperatures are examples of calculated melting temperature values ​​in the absence of formamide for a nucleic acid having a length of about 100 nucleotides and a G+C content of 50%. The experimental conditions for DNA hybridization are described in relevant genetics textbooks, e.g., Sambrook et al., 1989, and can be calculated using formulas known to those skilled in the art, e.g., depending on the length of the nucleic acid, the type of hybrid, or the G+C content. Those skilled in the art can also obtain further information on hybridization from the following textbooks: Ausubel et al. (eds), (1985), Brown (ed) (1991).

[0365] "Hybridization" can be carried out under particularly stringent conditions, such as those described in Sambrook (1989) or Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6.

[0366] The term hybridization or hybridizing under certain conditions as used herein is intended to refer to hybridization and washing conditions under which nucleotide sequences that are significantly identical or homologous to each other remain bound to each other. The conditions can be such that sequences that are at least about 70%, such as at least about 80%, such as at least about 85%, 90%, or 95% identical to each other remain bound to each other. Definitions of low stringency, medium, and high stringency hybridization conditions are provided herein.

[0367] Appropriate hybridization conditions can be selected by one of ordinary skill in the art with minimal experimentation, as exemplified by Ausubel et al. (1995, Current Protocols in Molecular Biology, John Wiley & Sons, sections 2, 4, and 6). In addition, stringency conditions are described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, chapters 7, 9, and 11).

[0368] The defined low stringency conditions used herein are as follows: Filters containing DNA are pretreated for 6 hours at 40°C in a solution containing 35% formamide, 5xSSC, 50mM Tris-HCl (pH 7.5), 5mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500μg / ml denatured salmon sperm DNA. Hybridization is performed in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100μg / ml salmon sperm DNA, 10% (w / v) dextran sulfate, and 5-20x106 P-labeled probes are used. Filters are incubated in hybridization mixture for 18-20 h at 40°C and then washed in a solution containing 2x SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS for 1.5 h at 55°C. The wash solution is replaced with fresh solution and incubated for an additional 1.5 h at 60°C. Filters are dried and subjected to autoradiography.

[0369] The defined medium stringency conditions used herein are as follows: Filters containing DNA are pretreated for 7 h at 50 °C in a solution containing 35% formamide, 5x SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg / ml denatured salmon sperm DNA. Hybridization is performed in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg / ml salmon sperm DNA, 10% (w / v) dextran sulfate, and 5-20 x 106 P-labeled probes are used. Filters are incubated in hybridization mixture for 30 hours at 50° C. and then washed in a solution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at 55° C. The wash solution is replaced with fresh solution and incubated for an additional 1.5 hours at 60° C. Filters are dried and subjected to autoradiography.

[0370] The defined high stringency conditions used herein are as follows: Prehybridization of DNA-containing filters is performed at 65°C for 8 hours to overnight in a buffer consisting of 6xSSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg / ml denatured salmon sperm DNA. Filters are hybridized at 65°C for 48 hours in a prehybridization mixture containing 100 μg / ml denatured salmon sperm DNA and 5-20x106 cpm of 32P-labeled probe. Washing of filters is performed at 37°C for 1 hour in a solution containing 2xSSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1xSSC at 50°C for 45 minutes.

[0371] Other low, medium, and high stringency conditions well known in the art (e.g., used for cross-species hybridizations) can be used when the above conditions are inappropriate (e.g., used for cross-species hybridizations).

[0372] A detection kit for a nucleic acid sequence encoding a polypeptide of the present invention may include primers and / or probes specific for the nucleic acid sequence encoding the polypeptide, and associated protocols for using the primers and / or probes to detect the nucleic acid sequence encoding the polypeptide in a sample. Such detection kits may be used to determine whether a plant, organism, microorganism, or cell has been modified or transformed with a sequence encoding the polypeptide.

[0373] To test the functionality of variant DNA sequences of embodiments herein, the sequence of interest is operably linked to a selectable or screenable marker gene and expression of the reporter gene is tested in transient expression assays, for example in microorganisms or in protoplasts, or in stably transformed plants.

[0374] The present invention also relates to derivatives of the nucleic acid sequences specifically disclosed or derivable herein.

[0375] Thus, further nucleic acid sequences of the invention may be derived from the sequences specifically disclosed herein and may differ therefrom by one or more, e.g. 1-20, in particular 1-15 or 5-10 additions, substitutions, insertions or deletions of one or more (e.g. 1-10) nucleotides and may further encode a polypeptide having a desired property profile.

[0376] The present invention also encompasses nucleic acid sequences which contain so-called silent mutations or which are modulated compared to the specifically mentioned sequences according to the codon usage of a particular organism of origin or host.

[0377] According to certain embodiments of the present invention, variant nucleic acids can be prepared to adapt their nucleotide sequences to specific expression systems. For example, bacterial expression systems are known to express polypeptides more efficiently when amino acids are encoded by specific codons. Due to the degeneracy of the genetic code, two or more codons can code for the same amino acid sequence, and multiple nucleic acid sequences can code for the same protein or polypeptide, and all of these DNA sequences are encompassed by the embodiments herein. Where appropriate, nucleic acid sequences encoding the polypeptides described herein can be optimized for increased expression in host cells. For example, the nucleic acids of the embodiments herein can be synthesized using special codons for the host for improved expression.

[0378] The present invention also encompasses naturally occurring variants of the sequences described herein, such as splice variants or allelic variants.

[0379] Allelic variants may have at least 60% homology, particularly at least 80% homology, very particularly at least 90% homology at the derived amino acid level over the entire sequence range (for homology at the amino acid level, see details above for polypeptides). Advantageously, the homology may be higher in partial regions of the sequence.

[0380] The present invention also relates to sequences which can be obtained by conservative nucleotide substitutions, ie, resulting in the replacement of a given amino acid with an amino acid of the same charge, size, polarity and / or solubility.

[0381] The present invention also relates to molecules derived from the specifically disclosed nucleic acids by sequence polymorphisms. Such genetic polymorphisms may be present in cells from or within different populations due to natural allelic variations. Allelic variants may also include functional equivalents. These natural variations usually result in 1-5% variation in the nucleotide sequence of a gene. Said polymorphisms may result in changes in the amino acid sequence of the polypeptides disclosed herein. Allelic variants may also include functional equivalents.

[0382] Furthermore, derivatives should also be understood to be homologues of the nucleic acid sequences of the invention, such as animal, plant, fungal or bacterial homologues of coding and non-coding DNA sequences, truncated sequences, single-stranded DNA or RNA, for example, homologues have, at the DNA level, at least 40%, particularly at least 60%, particularly at least 70%, very particularly at least 80% homology over the entire DNA region given in the sequences specifically disclosed herein.

[0383] Furthermore, derivatives should be understood to be, for example, fusions with promoters. The promoters added to the described nucleotide sequences can be modified by at least one nucleotide exchange, at least one insertion, inversion and / or deletion without impairing the function or efficacy of the promoter. Furthermore, the efficacy of promoters can be increased by modulating their sequences or completely replaced by more effective promoters, even those of organisms of different genera.

[0384] 2.2 Constructs for expressing the polypeptides of the invention In this context, the following definitions apply:

[0385] "Expression of a gene" encompasses "heterologous expression" and "overexpression" and involves transcription of a gene and translation of mRNA into protein. Overexpression refers to the production of a gene product, as measured by levels of mRNA, polypeptide and / or enzymatic activity, in a transgenic cell or organism that exceeds the level of production in a non-transformed cell or organism of a similar genetic background.

[0386] As used herein, "expression vector" refers to a nucleic acid molecule that has been engineered using molecular biology methods and recombinant DNA technology to deliver foreign or exogenous DNA to a host cell. Expression vectors typically include sequences necessary for proper transcription of a nucleotide sequence. The coding region usually codes for a protein of interest, but can also code for RNA, such as antisense RNA, siRNA, etc.

[0387] As used herein, "expression vector" includes any linear or circular recombinant vector, including but not limited to viral vectors, bacteriophage and plasmids. Those skilled in the art can select a suitable vector depending on the expression system. In one embodiment, the expression vector includes a nucleic acid of an embodiment herein operably linked to at least one "control sequence" that controls transcription, translation, initiation and termination, such as a transcription promoter, operator or enhancer, or an mRNA ribosome binding site, and optionally includes at least one selectable marker. A nucleotide sequence is "operably linked" when the control sequence is functionally related to the nucleic acid of an embodiment herein.

[0388] As used herein, an "expression system" encompasses any combination of nucleic acid molecules necessary for the expression of one polypeptide, or the co-expression of two or more polypeptides, either in vivo or in vitro in a given expression host. Each coding sequence may be located on a single nucleic acid molecule or vector, such as a vector containing multiple cloning sites, or on a polycistronic nucleic acid, or may be distributed on two or more physically separate vectors. Particular examples include operons, which include a promoter sequence, one or more operator sequences, and one or more structural genes, each encoding an enzyme, as described herein.

[0389] As used herein, the terms "amplifying" and "amplification" refer to the use of any suitable amplification methodology to generate or detect naturally expressed or recombinant nucleic acids, as described in detail below. For example, the invention provides methods and reagents (e.g., specific degenerate oligonucleotide primer pairs, oligo dT primers) for amplifying (e.g., by polymerase chain reaction PCR) naturally expressed (e.g., genomic DNA or mRNA) or recombinant (e.g., cDNA) nucleic acids of the invention in vivo, ex vivo, or in vitro.

[0390] "Control sequence" refers to a nucleic acid sequence that can control the rate of transcription of a nucleic acid sequence operably linked to it, and determines the expression level of the nucleic acid sequence of the embodiments herein. Control sequences include promoters, enhancers, transcription factors, promoter elements, and the like.

[0391] "Promoter", "nucleic acid with promoter activity" or "promoter sequence" are understood in the present invention to mean a nucleic acid that controls the transcription of a nucleic acid when functionally linked to the nucleic acid to be transcribed. "Promoter" refers in particular to a nucleic acid sequence that controls the expression of a coding sequence by providing binding sites for RNA polymerase and other factors necessary for proper transcription, including but not limited to transcription factor binding sites, repressor and activator protein binding sites. The meaning of the term promoter also includes the term "promoter control sequence". Promoter control sequences may include upstream and downstream elements that may affect the transcription, RNA processing or stability of the associated coding nucleic acid sequence. Promoters include naturally occurring and synthetic sequences. The coding nucleic acid sequence is usually located downstream of the promoter with respect to the direction of transcription starting at the transcription start site.

[0392] In this context, a "functional" or "operably" linkage is understood to mean, for example, a contiguous arrangement of one of the nucleic acids with a control sequence. For example, the sequence with promoter activity and the nucleic acid sequence to be transcribed, as well as optionally further control elements, such as a nucleic acid sequence ensuring transcription of the nucleic acid and, for example, a terminator, are linked in such a way that each of the control elements can perform its function during transcription of the nucleic acid sequence. This does not necessarily require a direct link in the chemical sense. Genetic control sequences, such as enhancer sequences, can exert their function on the target sequence from more remote positions or even from other DNA molecules. A preferred arrangement is one in which the nucleic acid sequence to be transcribed is located behind (i.e. at the 3' end) of the promoter sequence, such that the two sequences are covalently joined. The distance between the promoter sequence and the nucleic acid sequence to be recombinantly expressed can be less than 200 base pairs, or less than 100 base pairs, or less than 50 base pairs.

[0393] In addition to promoters and terminators, examples of other control elements include: targeting sequences, enhancers, polyadenylation signals, selectable markers, amplification signals, origins of replication, etc. Suitable control sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).

[0394] The term "constitutive promoter" refers to an unregulated promoter that allows for the constant transcription of a nucleic acid sequence to which it is operably linked.

[0395] The term "operably linked" as used herein refers to the linking of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or rather a transcription control sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operatively linked means that the DNA sequences that are linked are typically contiguous. The nucleotide sequence linked to the promoter sequence can be of homologous or heterologous origin with respect to the plant to be transformed. The sequence can also be fully or partially synthetic. Regardless of origin, the nucleic acid sequence linked to the promoter sequence will be expressed or silenced according to the promoter characteristics to which it is linked after binding to the polypeptide of the embodiments herein. The linked nucleic acid may code for a protein that is desired to be expressed or suppressed all the time in the whole organism, or alternatively at a specific time or in a specific tissue, cell, or cell compartment. Such nucleotide sequences in particular code for proteins that confer desired phenotypic traits to the host cell or organism modulated or transformed thereby. More specifically, the linked nucleotide sequence results in the production of a product of interest as defined herein in a cell or organism. In particular, the nucleotide sequence encodes a polypeptide having an enzymatic activity as defined herein.

[0396] The nucleotide sequences described herein above may be part of an "expression cassette". The terms "expression cassette" and "expression construct" are used interchangeably. A (particularly recombinant) expression construct contains a nucleotide sequence that encodes a polypeptide of the invention and is under the genetic control of a regulatory nucleic acid sequence.

[0397] In the process applied according to the present invention, the expression cassette may be part of an "expression vector", in particular a recombinant expression vector.

[0398] An "expression unit" is understood in the present invention to mean an expressionally active nucleic acid. It comprises a promoter as defined herein, which, after functional linkage with the nucleic acid or gene to be expressed, controls the expression, i.e. the transcription and translation, of said nucleic acid or said gene. It is therefore also referred to in this context as a "regulatory nucleic acid sequence". In addition to the promoter, other control elements such as enhancers may also be present.

[0399] "Expression cassette" or "expression construct" is understood in the present invention to mean an expression unit that is functionally linked to a nucleic acid to be expressed or to a gene to be expressed. Thus, in contrast to an expression unit, an expression cassette not only comprises nucleic acid sequences that control transcription and translation, but also nucleic acid sequences that are to be expressed as a protein as a result of transcription and translation.

[0400] The term "expression" or "overexpression" in the context of the present invention refers to the increase in the production or intracellular activity of one or more polypeptides in a microorganism that are encoded by the corresponding DNA. For this purpose, it is possible, for example, to introduce a gene into the organism, to replace an existing gene with another gene, to increase the copy number of the gene, to use a strong promoter, or to use a gene that codes for a corresponding polypeptide with high activity. Optionally, these measures can be combined.

[0401] In particular, such constructs according to the invention comprise a promoter sequence 5' upstream and a terminator sequence 3' downstream of the respective coding sequence, and optionally other conventional control elements in each case operably linked to the coding sequence.

[0402] The nucleic acid constructs of the present invention particularly include sequences encoding polypeptides, e.g., from the amino acid related SEQ ID NOs described therein, or reverse complements thereof, or derivatives and homologues thereof, and preferably operatively or functionally linked to one or more regulatory signals for the control, e.g., increase, of gene expression.

[0403] In addition to these control sequences, the natural control of these sequences can still be present in front of the actual structural gene. Optionally, the natural control can be switched off and genetically modified so that the expression of the gene is enhanced. However, the nucleic acid construct can also be of a simpler structure. That is, no additional control signal is inserted in front of the coding sequence, and the natural promoter is not removed together with its control. Instead, the natural control sequence is mutated so that it is no longer controlled and gene expression is increased.

[0404] A preferred nucleic acid construct also advantageously comprises one or more of the already mentioned "enhancer" sequences operably linked to the promoter, which allow enhanced expression of the nucleic acid sequence. Additional advantageous sequences, such as further control elements or terminators, can also be inserted at the 3' end of the DNA sequence. One or more copies of the nucleic acid of the invention can be present in the construct. Other markers, such as genes that complement auxotrophies or antibiotic resistance, can also be optionally present in the construct to select the construct.

[0405] Examples of suitable regulatory sequences are cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacI. q , T7, T5, T3, gal, trc, ara, rhaP(rhaP BAD )SP6, lambda-P R or lambda-P L promoters, which are advantageously used in gram-negative bacteria. Further advantageous regulatory sequences are found, for example, in the gram-positive promoters amy and SPO2, the yeast or fungal promoters ADC1, MFalpha, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH. Artificial promoters can also be used for regulation.

[0406] For expression in the host organism, the nucleic acid construct is advantageously inserted into a vector, such as a plasmid or a phage, which allows optimal expression of the gene in the host. Vector is also understood to mean, in addition to plasmids and phages, all other vectors known to those skilled in the art, such as viruses, such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, phasmids, cosmids, and linear or circular DNA or artificial chromosomes. These vectors can replicate autonomously or otherwise chromosomally in the host organism. These vectors are further developments of the present invention. Binary vectors or cpo-integration vectors are also applicable.

[0407] A specific example of a baculovirus vector system is the MultiBacTAG system described in Koehler, C., et al. Genetic code expansion for multiprotein complex engineering. Nat Methods 13, 997-1000 (2016).

[0408] Suitable plasmids include, for example, pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III in E. coli. 113 -B1, λgt11 or pBdCI, pIJ101, pIJ364, pIJ702 or pIJ361 in Streptomyces, pUB110, pC194 or pBD214 in Bacillus, pSA77 or pAJ667 in Corynebacterium, pALS1, pIL2 or pBB116 in fungi, 2alphaM, pAG-1, YEp6, YEp13 or pEMBLYe23 in yeast, or pLGV23, pGHlac in plants +, pBIN19, pAK2004 or pDH51. The above plasmids are a small selection of possible plasmids. Further plasmids are well known to those skilled in the art and can be found, for example, in the book Cloning Vectors (Eds. Pouwels PH et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).

[0409] In the context of expression of multiprotein complexes in eukaryotic cells, plasmid vectors that allow the simultaneous insertion of coding sequences for immunoglobulin light and heavy chains are of particular interest, see for example pAceBacDUAL or pBI (expression plasmids for mammalian cells with two promoters for the expression of two genes (Clontech)).

[0410] In a further development of the vector, the nucleic acid construct of the invention or the vector comprising the nucleic acid of the invention can advantageously also be introduced into the microorganism in the form of linear DNA and integrated into the genome of the host organism via non-homologous or homologous recombination. This linear DNA can consist of a linearized vector such as a plasmid, or solely of the nucleic acid construct or nucleic acid of the invention.

[0411] For optimal expression of a heterologous gene in an organism, it is advantageous to modify the nucleic acid sequence to conform to the particular "codon usage" used by the organism, which can be readily determined by computer evaluation of other known genes of the organism of interest.

[0412] The expression cassettes of the invention are constructed by fusing a suitable promoter to a suitable coding nucleotide sequence and a terminator or polyadenylation signal using conventional recombination and cloning techniques, e.g., as described in T. Maniatis, EF Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989), and TJ Silhavy, ML Berman and LW Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984), and Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987).

[0413] For expression in a suitable host organism, the recombinant nucleic acid construct or gene construct is advantageously inserted into a host-specific vector that allows optimal expression of the gene in the host. Vectors are well known to those skilled in the art and can be found, for example, in "Cloning vectors" (Pouwels PH et al., Ed., Elsevier, Amsterdam-New York-Oxford, 1985).

[0414] Alternative embodiments of the present disclosure provide methods of "modulating gene expression" in a host cell. For example, polynucleotides of the present disclosure may be transcriptionally enhanced, overexpressed, or induced in a host cell or host organism in a particular context (e.g., by exposure to particular temperatures or culture conditions).

[0415] Modulation of expression of the polynucleotides provided herein can also result in ectopic expression, which is an expression pattern that is different between the modulated organism and the control or wild-type organism. Modulation of expression can result from the interaction of the polypeptides of the embodiments herein with exogenous or endogenous regulatory factors, or as a result of chemical modification of the polypeptide. This term also refers to the modulated expression pattern of the polynucleotides of the embodiments herein being modulated below detection level, or the activity being completely suppressed.

[0416] Also provided herein, in one embodiment, are isolated, recombinant, or synthetic polynucleotides that encode the polypeptides or variant polypeptides provided herein.

[0417] In one embodiment, the nucleic acid sequences encoding several polypeptides are co-expressed in a single host, particularly under the control of different promoters.In another embodiment, the nucleic acid sequences encoding several polypeptides can be present on a single transformation vector, or can be co-transformed at the same time, using separate vectors, and selecting transformants containing both chimeric genes.Similarly, genes encoding one or more polypeptides can be expressed in a single plant, cell, microorganism, or organism together with other chimeric genes. 3. Host Applicable to the Present Invention

[0418] Depending on the context, the term "host" can refer to a wild-type host or a genetically altered recombinant host, or both.

[0419] In principle, all prokaryotic or eukaryotic organisms can be considered as hosts or recombinant host organisms for the nucleic acids or nucleic acid constructs according to the invention.

[0420] The vectors of the invention can be used to produce prokaryotic or eukaryotic recombinant hosts, which can be, for example, transformed with at least one vector of the invention and used to produce the polypeptides of the invention. Advantageously, the above-described recombinant constructs of the invention are introduced into a suitable host system and expressed. In particular, the general cloning and transfection methods known to those skilled in the art, such as coprecipitation, protoplast fusion, electroporation, retroviral transfection, etc., are used to express the described nucleic acids in the respective expression systems. Suitable systems are described, for example, in Current Protocols in Molecular Biology, F. Ausubel et al., Ed., Wiley Interscience, New York 1997, or in Sambrook et al. Molecular Cloning: A Laboratory Manual. 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0421] Microorganisms, such as bacteria, are used as host organisms. For example, gram-positive bacteria or gram-positive bacteria are used, in particular bacteria of the Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Streptomycetaceae, Streptococcaceae or Nocardiaceae families, in particular bacteria of the Escherichia, Pseudomonas, Streptomyces, Lactococcus, Nocardia, Burkholderia, Salmonella, Agrobacterium, Clostridium or Rhodococcus genera. Very particular preference is given to the genus and species Escherichia coli. Advantageously, yeasts of the Saccharomyces or Pichia families are also suitable hosts.

[0422] Alternatively, eukaryotic cells can be used as hosts. The eukaryotic cells of the present invention can be selected from, but are not limited to, mammalian cells, insect cells, yeast cells and plant cells. The eukaryotic cells of the present invention can be present as individual cells or can be part of a tissue (e.g., cells in a (cultured) tissue, organ or whole organism).

[0423] Plants or plant cells can function as natural or recombinant hosts. Non-limiting examples include the following plants or cells derived therefrom: Nicotiana, particularly Nicotiana benthamiana and Nicotiana tabacum (tobacco), and Arabidopsis, particularly Arabidopsis thaliana.

[0424] Specific non-limiting examples of insect cells are Sf21, Sf9 and High Five cells.Specific non-limiting examples of mammalian cells are HEK293, HEK293T, HEK293F, CHO, CHO-S, COS and HeLa cells.

[0425] Depending on the host organism, the organisms used in the process of the invention are grown or cultured in a manner known to those skilled in the art. Cultivation can be batch, semi-batch or continuous. Nutrients can be present at the start of the fermentation or can be fed later, semi-continuously or continuously. This is also explained in more detail below.

[0426] 4. POIs Containing One or More than One ncAA and Their Preparation 4.1 POI The POI of the present invention generally relates to any form of polypeptide or protein molecule, which is expressed as a polypeptide or protein molecule, as described above, in any suitable host cell system or cell-free expression system, and which expresses at least one ncAA and an aminoacyl-tRNA synthetase / tRNA as described above. アミノアシル Pairs such as pyrrolysyl-tRNA synthetase and tRNA Pyl It can be recombinantly produced as described above in the presence of

[0427] In certain embodiments, the POI is utilized to form a "targeting agent."

[0428] The primary objective of such targeting agents is the formation of a covalent or non-covalent linkage with a specific "target". The secondary objective of the targeting agent is the targeted transport of a "payload molecule" to said target. To achieve said secondary objective, the POI must be combined (reversibly or irreversibly) with at least one payload molecule. For this, the POI must be functionalized by introducing said at least one ncAA. The functionalized POI carrying said at least one ncAA can then be linked to said at least one payload molecule by bioconjugation via said ncAA residue. The ncAA is in turn reactive with a payload molecule carrying a corresponding moiety reactive with said at least one ncAA residue of the POI. The bioconjugate thus obtained, i.e. the targeting agent, allows the transport of the payload molecule to the intended target.

[0429] For example, a "target" can be any molecule present in and / or on an organism, tissue, or cell. Such targets can be non-specific or specific to a particular organism, tissue, or cell. Targets include cell surface targets, such as receptors, glycoproteins, glycans, carbohydrates, structural proteins, abundant extracellular targets such as amyloid plaques, stroma, etc., extracellular matrix targets, such as growth factors, and proteases, intracellular targets, such as the surface of the Golgi apparatus, the surface of mitochondria, RNA, DNA, enzymes, components of cell signaling pathways, and / or foreign entities, such as pathogens, such as viruses, bacteria, fungi, yeast, or parts thereof.

[0430] Examples of targets include compounds such as proteins whose presence or expression levels correlate with a particular tissue or cell type, or whose expression levels are up- or down-regulated in a particular disease.

[0431] In particular, such targets are proteins such as receptors (internalizing or non-internalizing).

[0432] The target may be selected from any suitable target in the human or animal body or on a pathogen or parasite.

[0433] In the context of the present invention, preferred targets include cellular components, more specifically HER2.

[0434] More specifically, to enable (specific) targeting of said targets, the targeting agent may comprise a compound comprising an ncAA-functionalized peptide sequence, including but not limited to the antibodies, antibody derivatives, antibody fragments, antibody (fragment) fusions (e.g. bispecific and trispecific mAb fragments or derivatives) described herein above.

[0435] Particular examples of peptide molecules, such as antibodies, used as targeting agents include HER2 targeting peptides.

[0436] Human epidermal growth factor receptor 2 (HER2) is a member of the epidermal growth factor receptor family with tyrosine kinase activity. Receptor dimerization results in autophosphorylation of tyrosine residues within the cytoplasmic domain of the receptor, initiating various signaling pathways that lead to cell proliferation and tumorigenesis. Amplification or overexpression of HER2 occurs in approximately 15-30% of breast cancers and approximately 10-30% of gastric / gastroesophageal cancers, and serves as a prognostic and predictive biomarker. HER2 overexpression has also been found in other cancers, such as ovarian, endometrial, bladder, lung, colon, and head and neck. The introduction of HER2-directed therapies has had a major impact on the outcomes of patients with HER2-positive breast and gastric / gastroesophageal cancers. However, results have been shown to be disappointing in other HER2-overexpressing cancers. This review discusses the role of HER2 in various cancers and the available therapeutic modalities targeting HER2 (Iqbal, N. et al Human Epidermal Growth Factor Receptor 2 (HER2) in Cancers: Overexpression and Therapeutic Implications, Mol Biol Int.2014; 2014: 852748) https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC4170925 / .

[0437] In one particular embodiment, Affibodies™ and multimers and derivatives are used.

[0438] In one particular embodiment, an antibody is used to form the targeting agent. Antibodies or immunoglobulins from IgG antibodies are particularly suitable for use in the present invention, but immunoglobulins from any class or subclass may be selected, such as IgG, IgA, IgM, IgD and IgE. Suitably, the immunoglobulin is of the IgG class, including but not limited to the IgG subclasses (IgG1, 2, 3 and 4), or the IgM class, which can specifically bind to a specific epitope on an antigen. The antibody may be an intact immunoglobulin from natural or recombinant sources, or may be an immunoreactive portion of an intact immunoglobulin. The antibody may exist in a variety of forms, including: For example, polyclonal antibodies, monoclonal antibodies, camelized single domain antibodies, recombinant antibodies, anti-idiotypic antibodies, multispecific antibodies, antibody fragments such as Fv, VHH, Fab, F(ab)2, Fab', Fab'-SH, F(ab')2, single chain variable fragment antibodies (scFv), tandem / bis-scFv, Fc, pFc', scFv-Fc, disulfide Fv (dsFv), bispecific antibodies (bc-scFv), such as BiTE antibodies, trispecific antibody derivatives, such as tribodies, camelid antibodies, minibodies, nanobodies, resurf Examples of antibodies include immunizing antibodies, humanized antibodies, fully human antibodies, single domain antibodies (sdAbs, also known as Nanobodies™), chimeric antibodies, chimeric antibodies comprising at least one human constant region, dual affinity antibodies, such as dual affinity retargeting proteins (DART™), and multimers and derivatives thereof, such as bivalent or multivalent single chain variable fragments (e.g., di-scFv, tri-scFv), including but not limited to minibodies, diabodies, triabodies, tribodies, tetrabodies, etc., and multivalent antibodies. See Trends in Biotechnology 2015, 33, 2, 65, Trends Biotechnol. 2012, 30, 575-582, and Cane. Gen. Prot. 2013 10, 1-18, and BioDrugs 2014, 28, 331-343, the contents of which are incorporated herein by reference.

[0439] An "antibody fragment" refers to at least a portion of the variable region of an immunoglobulin that binds to its target, ie, the antigen binding region.

[0440] In other embodiments, antibody mimetics are used as targeting agents, such as, but not limited to, Affimers, Anticalins, Avimers, Alphabodies, Affibodies, DARPins, and their multimers and derivatives, the contents of which are incorporated herein by reference (see Trends in Biotechnology 2015, 33, 2, 65).

[0441] For the avoidance of doubt, in the context of the present invention, the term "antibody" is intended to encompass all of the antibody variations, fragments, derivatives, fusions, analogues and mimetics outlined in this paragraph, unless otherwise specified.

[0442] In preferred embodiments, the targeting agent is selected from agents derived from antibodies and antibody derivatives, such as antibody fragments, fragment fusions, proteins, peptides, and peptidomimetics.

[0443] In another preferred embodiment, the targeting agent is selected from agents derived from antibody fragments, fragment fusions, and other antibody derivatives that do not contain an Fc domain.

[0444] Typical, non-limiting examples of antibody molecules to be further modified to form the ncAA-modified POI of the present invention are selected from biologically, and in particular pharmacologically, active antibody molecules. Non-limiting examples are selected from the group of trastuzumab and pertuzumab.

[0445] According to further particular embodiments of the invention, targets and targeting agents are selected to provide specific or increased targeting of a tissue or disease, such as cancer, particularly breast cancer, which can be achieved by selecting targets with tissue, cell or disease specific expression.

[0446] By way of example, a targeting agent specifically binds to or complexes with a cell surface molecule, such as a cell surface receptor or antigen, for a given cell population. Once the targeting agent specifically binds to or complexes with the receptor, the drug enters the cell.

[0447] As used herein, a targeting agent that "specifically binds to or complexes with" or "targets" a cell surface molecule, an extracellular matrix target, or another target preferentially associates with the target via intermolecular forces. For example, a ligand can preferentially associate with a target with a dissociation constant (Kd or KD) of less than about 50 nM, less than about 5 nM, or less than about 500 pM.

[0448] 4.2 Preparation of site-specifically modified POIs, in particular site-specifically modified immunoglobulins The POI as defined above, in particular the POI comprising a polypeptide moiety comprising one or more ncAA residues, can be prepared according to the present invention using a suitable translation system, in particular an in vivo translation system. The in vivo translation system can be a cell, for example a prokaryotic or eukaryotic cell. The cell can be a bacterial cell, for example an E. coli cell, a fungal cell, for example a yeast cell, for example an S. cerevisiae or a methylotrophic yeast, a plant cell, or an animal cell, for example an insect cell or a mammalian cell, for example an HEK cell or a HeLa cell. The eukaryotic cell used for polypeptide expression can be a single cell or part of a multicellular organism.

[0449] The site-specifically modified antibodies of the present invention can be produced by any of a number of techniques known in the art, such as expression from a host cell, where expression vectors encoding the heavy and light chains are transfected into the host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide range of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. The antibodies of the present invention can be expressed in either prokaryotic or eukaryotic host cells. In certain aspects of the present invention, expression of the antibodies is carried out using eukaryotic cells, such as mammalian host cells, since such eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete properly folded and immunologically active antibodies.

[0450] In one embodiment, mammalian host cells for expressing the recombinant antibodies of the invention include Chinese Hamster Ovary (CHO cells) (including, for example, dhfr- CHO cells as described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220 used with the DHFR selectable marker as described in RJ Kaufman and PA Sharp (1982) Mol. Biol. 159:601-621, or CHO-S cells), NS0 myeloma cells, COS cells, and SP2 cells. Once a recombinant expression vector encoding an antibody gene has been introduced into the mammalian host cells, the antibody is produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or secretion of the antibody into the medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

[0451] Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It will be understood that variations on the above procedures can be made within the scope of the invention. For example, it may be desirable to transfect host cells with DNA encoding functional fragments of either the light and / or heavy chains of the antibody of the invention. Recombinant DNA technology can also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to the antigen of interest. Molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the invention. In addition, bifunctional antibodies, in which one heavy and one light chain is an antibody of the invention and the other heavy and light chains are specific for an antigen other than the antigen of interest, can be produced by crosslinking an antibody of the invention to a second antibody by standard chemical crosslinking methods.

[0452] In an exemplary system for recombinant expression of the antibody of the invention or an antigen-binding portion thereof, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr- CHO cells by calcium phosphate-mediated transfection. On the recombinant expression vector, each of the antibody heavy and light chain genes is operably linked to a CMV enhancer / AdMLP promoter control element to drive high level transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells transfected with the vector using methotrexate selection / amplification. The host cells of the selected transformants are cultured to allow expression of the antibody heavy and light chains, and the intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. Additionally, the invention provides a method of synthesizing a recombinant antibody of the invention by culturing the host cells of the invention in a suitable culture medium until the recombinant antibody of the invention is synthesized. The method may further include isolating the recombinant antibody from the culture medium.

[0453] The preparation of site-specifically modified POIs, especially immunoglobulins, applies the technique of genetic code expansion (GCE), which has been used for several decades and is well established in Escherichia coli, as well as in eukaryotic systems such as mammals (Chatterjee et al, PNAS 2013, 110, 29: 11803-11808), yeast (Chin, JW, Cropp, TA, Anderson, JC, Mukherji, M., Zhang, Z., and Schultz, PG (2003). Science 301, 964-967) or Drosophila (Mukai, T. et al Protein Science 2010, 19: 440-448). See also Lemke, EA The exploding genetic code. ChemBioChem 15, 1691-1694 (2014); de la Torre, D. & Chin, JW Reprogramming the genetic code. Nat. Rev. Genet. 22, 169-184 (2021). This system is used to site-specifically incorporate non-standard amino acids (ncAAs) into proteins. The introduction of non-standard amino acids with various functional groups applies, for example, to label proteins for single molecule studies or super-resolution microscopy, to crosslink proteins, or to attach selected post-translational modifications. For this, a synthetase / tRNA pair (orthogonal to the expression host) must be co-transfected with the protein of interest. The synthetase can recognize the non-standard amino acid, which will be inserted into the elongating protein chain in response to an amber stop codon. Several systems already exist, such as Methanococcus jannaschii TyrRS / tRNATyr or Methanosarcina mazei PylRS / tRNAPyl (Liu, CC, and Schultz, PG (2010). Annual review of biochemistry 79, 413-444). Several other PylRS / tRNA PylPairs are known from the archaeal organisms Methanosarcina barkeri or Methanomethylophilus alvus. PylRS synthase naturally contains a binding site that recognizes pyrrolysine. By changing the specific amino acids of this binding site, various ncAAs can be recognized by this synthase. European patent applications EP-A-2 192 185, EP-A-2 221 370 and EP-A-2 804 872 describe improved variants of the PylRS synthase from M. mazei, which can also be used. Another improved PylRS is described in the applicant's European application No. 21195008.4, filed on September 6, 2021.

[0454] WO2018 / 069481 describes improved archaeal PylRSs, which are modified by introducing a nuclear export signal (NES) or lack a nuclear localization signal (NLS), which can also be applied to GCE.

[0455] The archaeal PylRS can be further modified, optionally by removing an NLS present in said naturally occurring PylRS from which the mutant is derived and / or by introducing at least one NES. NLSs in naturally occurring PylRSs can be identified using known NLS detection tools, such as, for example, cNLS Mapper.

[0456] Removal of an NLS from an archaeal PylRS or a mutant thereof and / or introduction of an NES can alter the localization of the thus modified polypeptide when expressed in a eukaryotic cell, and in particular can prevent or reduce accumulation of the polypeptide in the nucleus of the eukaryotic cell. Thus, the localization of a PylRS mutant of the invention when expressed in a eukaryotic cell can be altered compared to a PylRS or PylRS mutant that differs from the PylRS mutant of the invention in that it (still) contains an NLS and lacks an NES.

[0457] When an archaeal PylRS of the invention comprises an NES but (still) comprises an NLS, the NES is preferably selected such that the strength of the NES exceeds the strength of the NLS and prevents accumulation of the PylRS in the nucleus of a eukaryotic cell.

[0458] Removal of an NLS from a wild-type or mutant PylRS and / or introduction of an NES into a wild-type or mutant PylRS to obtain a PylRS of the present invention does not abolish the enzymatic activity of PylRS. Preferably, the enzymatic activity of PylRS is maintained at essentially the same level. That is, the PylRS of the present invention has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 91, 92, 93, 94, 95, 96, 97, 98, or 99% of the enzymatic activity of the corresponding wild-type or mutant PylRS.

[0459] The NES is conveniently located within the PylRS or mutant PylRS of the present invention such that the NES is functional. For example, the NES can be attached to the C-terminus (e.g., C-terminus of the last amino acid residue) or N-terminus (e.g., between the N-terminal methionine of amino acid residue 1 and amino acid residue 2) of a wild-type or mutant archaeal PylRS.

[0460] The disclosure of WO2018 / 06948 disclosing mutant PylRS modified by incorporation of an NES and / or deletion of an NLS sequence is expressly incorporated herein by reference.

[0461] Expression of modified POI by application of GCE using Methanosarcina mazei PylRS / tRNAPyl in insect cells is described in WO2017 / 093254.

[0462] Specific methods for preparing POIs, such as immunoglobulins, modified by GCE in eukaryotic, particularly mammalian cell systems are described in WO2020 / 165408 and WO2021 / 165410, each of which is incorporated herein by reference. These systems are particularly applicable to the preparation of glycosylated forms of POIs, such as immunoglobulins, modified by GCE. For cytoplasmic expression in mammalian cells, the same constructs described therein can be cloned lacking a secretion signal, such as HAS, as exemplified therein.

[0463] Suitable tRNAs for producing the POI of the present invention Pyl The tRNA / PylRS pair can be selected from a library of mutant tRNAs and PylRSs, for example, based on the results of library screening. Such selection can be performed in a manner similar to known methods for evolving tRNA / RS pairs, for example, as described in WO02 / 085923 and WO02 / 06075. The tRNA of the present invention Pyl To generate a / PylRS pair, one starts with a wild-type or mutant archaeal PylRS that (still) contains a nuclear localization signal and lacks an NES, and ligates the appropriate tRNA Pyl The nuclear localization signal can be removed and / or the NES can be introduced before or after the / PylRS pair is identified.

[0464] The preparation of site-specifically modified POI was carried out using PylRS / tRNA pyl This will be explained in more detail with reference to pairs.

[0465] The above applied cell line comprises (e.g., is fed) at least one non-standard amino acid (ncAA) or a salt thereof corresponding to the ncAA residue of the POI to be prepared. The cell line further comprises: (i) PylRS and tRNA of the present invention Pyl wherein PylRS is a tRNA Pyl can be (preferably selectively) acylated with an ncAA or a salt thereof, and (ii) a polynucleotide encoding a POI, wherein any position in the POI that is occupied by an ncAA residue is Pyl The sequence is encoded by a codon (e.g., a selector codon) that is the reverse complement of the anticodon of the sequence.

[0466] The cell line is cultured to allow translation of the polynucleotide (ii) encoding the POI, thereby producing the POI.

[0467] To produce a POI according to the methods of the invention, the translation in step (b) can be achieved by culturing the cell line under suitable conditions, preferably in the presence of an ncAA or a salt thereof (e.g., in a culture medium containing an ncAA or a salt thereof), for a suitable time to allow translation on the ribosomes of the cells. Pyl Depending on the polynucleotide encoding the POI (and tRNA), it may be necessary to induce expression by adding a compound that induces transcription, such as arabinose, isopropyl β-D-thiogalactoside (IPTG) or tetracycline. Pyl The mRNA (containing one or more codons that are the reverse complement of the anticodon contained in the mRNA) is bound by the ribosome. A polypeptide is then formed by the stepwise attachment of amino acids and ncAAs at the positions encoded by the codons recognized (bound) by each aminoacyl-tRNA. Thus, the tRNA Pyl The ncAA is incorporated into the POI at a position encoded by a codon that is the reverse complement of the anticodon contained in

[0468] The cell line may contain a polynucleotide sequence encoding the PylRS of the present invention that allows the cell to express the PylRS. Pyl is a tRNA contained in cells. Pyl The polynucleotide sequence encoding PylRS and tRNA can be produced by a cell line based on the polynucleotide sequence encoding PylRS. PylThe polynucleotide sequences encoding the may be present on the same polynucleotide or on separate polynucleotides.

[0469] Thus, in one embodiment, the present invention provides a method for producing a POI comprising one or more ncAA residues, wherein the method comprises the steps of: (a) providing a cell line comprising a polynucleotide sequence encoding: - at least one PylRS of the invention, - at least one tRNA that can be acylated by PylRS (tRNA Pyl ), and - at least one POI, where any position of the POI occupied by an ncAA residue is not Pyl and (b) enabling translation of the polynucleotide sequence by the cell line in the presence of the ncAA or a salt thereof, thereby translating the PylRS, tRNA Pyl and producing the POI.

[0470] The cell lines used to prepare POIs containing one or more non-canonical amino acid residues as described herein include those that express PylRS, tRNA Pyl and a polynucleotide sequence encoding the POI into a (host) cell, said polynucleotide sequences may be present on the same or separate polynucleotides and may be introduced into the cell by methods known in the art (e.g., viral-mediated gene delivery, electroporation, microinjection, lipofection, etc.).

[0471] After translation, the POI prepared according to the present invention can optionally be recovered and purified to either partial or substantial homogeneity according to procedures generally known in the art. Unless the POI is secreted into the culture medium, recovery usually requires cell disruption. Methods of cell disruption are well known in the art and include physical disruption, such as by (ultrasonic) sonication, liquid shear disruption (e.g., by French press), mechanical methods (such as those using blenders or grinders) or freeze-thaw cycles, as well as chemical lysis using agents that disrupt lipid-lipid, protein-protein and / or protein-lipid interactions (such as detergents), as well as combinations of physical and chemical disruption techniques. Standard procedures for purifying polypeptides from cell lysates or culture medium are also well known in the art and include, for example, ammonium sulfate or ethanol precipitation, acid or base extraction, column chromatography, affinity column chromatography, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, lectin chromatography, gel electrophoresis, and the like. Protein refolding steps can be used, if necessary, to produce correctly folded mature proteins. If high purity is desired, high performance liquid chromatography (HPLC), affinity chromatography or other suitable methods can be used in the final purification step. Antibodies raised against the polypeptides of the invention can be used as purification reagents, i.e., affinity-based purification of the polypeptides. Various purification / protein folding methods are well known in the art, including those described in Scopes, Protein Purification, Springer, Berlin (1993), and Deutscher, Methods in Enzymology Vol. 182: Guide to Protein Purification, Academic Press (1990), and references cited therein.

[0472] As mentioned above, those skilled in the art will recognize that after synthesis, expression and / or purification, a polypeptide may have a conformation different from the desired conformation of the polypeptide. For example, polypeptides produced by prokaryotic systems are often optimized to achieve proper folding by exposure to chaotropic agents. During purification, for example, from lysates from E. coli, the expressed polypeptide is optionally denatured and then renatured. This is accomplished, for example, by solubilizing the protein in a chaotropic agent such as guanidine HCl. Generally, in some cases, it is desirable to denature and reduce the expressed polypeptide and then refold the polypeptide into a preferred conformation. For example, guanidine, urea, DTT, DTE, and / or chaperonins can be added to the translation product of interest. Methods for reducing, denaturing and renaturing proteins are well known to those skilled in the art. The polypeptide can be refolded in a redox buffer containing, for example, oxidized glutathione and L-arginine.

[0473] The POIs thus prepared can then be converted into their respective bioconjugates by reaction with tetrazine compounds as further described below. 5. Payload molecules

[0474] Typically used payload molecules can be selected from biologically active compounds, in particular drugs, labelling agents and chelating agents, non-limiting examples of which are given in the following paragraphs. 5.1 Biologically active compounds

[0475] The following biologically active compounds are included, but are not limited to:

[0476] Biologically active compounds applicable to the present invention include, but are not limited to, small organic molecule drugs, steroids, lipids, proteins, aptamers, oligopeptides, oligonucleotides, oligosaccharides, as well as peptides, peptoids, amino acids, nucleotides, oligonucleotides or polynucleotides, nucleosides, DNA, RNA, toxins, glycans, and immunoglobulins.

[0477] Exemplary classes of biologically active compounds that can be used in the practice of the present invention include, but are not limited to, hormones, cytotoxins, anti-proliferative / anti-tumor agents, anti-viral agents, antibiotics, cytokines, anti-inflammatory agents, antihypertensives, chemosensitizers, photosensitizers, radiosensitizers, anti-AIDS agents, anti-viral agents, immunosuppressants, immunostimulants, enzyme inhibitors, anti-Parkinson's agents, neurotoxins, channel blockers, modulators of cell-extracellular matrix interactions including cell growth inhibitors and anti-adhesion molecules, DNA, RNA or protein synthesis inhibitors, steroidal and non-steroidal anti-inflammatory agents, anti-angiogenic factors, anti-Alzheimer's agents.

[0478] In some embodiments, the biologically active compound is a low to medium molecular weight compound (eg, about 200 to 5000 Da, about 200 to about 1500 Da, preferably about 300 to about 1000 Da).

[0479] Exemplary cytotoxic drugs are those that are used in particular for cancer treatment.Such drugs generally include DNA damaging agents, antimetabolites, natural products and their analogs, enzyme inhibitors, such as dihydrofolate reductase inhibitors and thymidylate synthase inhibitors, DNA binders, DNA alkylating agents, radiosensitizers, DNA intercalators, DNA cleavage agents, microtubule stabilizers and destabilizers, topoisomerase inhibitors.Examples include, but are not limited to, platinum-based drugs, anthracycline family drugs, vinca drugs, mitomycin, bleomycin, cytotoxic nucleosides, taxanes, lexitropsins, pteridine family drugs, diynenes, podophyllotoxins, dolastatins, maytansinoids, differentiation inducers, and taxol. Particularly useful members of these classes include, for example, auristatins, maytansines, maytansinoids, calicheamicins, dactinomycins, duocarmycins, CC1065 and analogs thereof, camptothecin and analogs thereof, SN-38 and analogs thereof, DXd, tubulysin M, cryptophycins, pyrrolobenzodiazepines and pyrrolobenzodiazepine dimers (PBDs), pyridinobenzodiazepines (PDDs), indolinobenzodiazepines (IBDs) (see US20210206763A1), methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, DNA minor groove binding agents, 6-mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin, anthracyclines (doxorubicin, epirubicin, idarubicin, daunorubicin, PNU-159682 (see US 10,288,745 B2) and analogs thereof, mitomycin C, mitomycin A, caminomycin, aminopterin, tallysomycin, podophyllotoxin and podophyllotoxin derivatives such as etoposide, etoposide phosphate, vinblastine, vincristine, vindesine, taxol, taxotere, retinoic acid, butyric acid, N8-acetylspermidine, staurosporine, colchicine, camptothecin, esperamicin, enediynes and analogs thereof, hemiasterin and analogs thereof.

[0480] Other exemplary drug classes are angiogenesis inhibitors, cell cycle progression inhibitors, PI3K / m-TOR / AKT pathway inhibitors, MAPK signaling pathway inhibitors, kinase inhibitors, protein chaperone inhibitors, HDAC inhibitors, PARP inhibitors, Wnt / Hedgehog signaling pathway inhibitors, RNA polymerase inhibitors, protein degradation inducers (see https: / / pubs.acs.org / doi / 10.1021 / acschembio.0c00285).

[0481] Examples of auristatins include dolastatin 10, monomethylauristatin E (MMAE), auristatin F, monomethylauristatin F (MMAF), auristatin F hydroxypropylamide (AF HPA), auristatin F phenylenediamine (AFP), monomethylauristatin D (MMAD), auristatin PE, auristatin EB, auristatin EFP, auristatin TP, and auristatin AQ. Suitable auristatins are described in U.S. Publication Nos. 2003 / 0083263, 2011 / 0020343, and 2011 / 0070248, PCT Publication Nos. WO09 / 117531, WO2005 / 081711, WO04 / 010957, WO02 / 088172, and WO01 / 24763, and in U.S. Patent Nos. 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,124,431, 6,034,065, 5,780,588, 5,767,237, and the like. Nos. 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,879,278, 4,816,444, and 4,486,414, the disclosures of which are incorporated herein by reference in their entireties.

[0482] Exemplary drugs include dolastatins and their analogs, such as dolastatin A (U.S. Pat. No. 4,486,414), dolastatin B (U.S. Pat. No. 4,486,414), dolastatin 10 (U.S. Pat. Nos. 4,486,444, 5,410,024, 5,504,191, 5,521,284, 5,530,097, 5,599,902, 5,635,483, 5,663,149, 5,665,861, 5,665,902, 5,665,102, 5,665,122, 5,665,162, 5,665,186, 5,665,190, 5,665,200, 5,665,210, 5,665,300, 5,665,420, 5,665,440, 5,665,500, 5,665,510, 5,665,620, 5,665,700, 5,665,861, 5,665,900, 5,665,102, 5,665,122, 5,665,162, 5,665,186, 5,665,200, 5,665,300, 5,665,420, 5,665,510, 5,665,520, 5,665,530, 5,665,540, 5,665,620, 5,665,64 Nos. 5,780,588, 6,034,065, 6,323,315), dolastatin 13 (U.S. Pat. No. 4,986,988), dolastatin 14 (U.S. Pat. No. 5,138,036), dolastatin 15 (U.S. Pat. No. 4,879,278), dolastatin 16 (U.S. Pat. No. 6,239,104), dolastatin 17 (U.S. Pat. No. 6,239,104), and dolastatin 18 (U.S. Pat. No. 6,239,104). Each of these patents is incorporated herein by reference in its entirety.

[0483] Exemplary maytansines, maytansinoids, such as DM-1 and DM-4, ​​or maytansinoid analogs, including maytansinol and maytansinol analogs, are described in U.S. Pat. Nos. 4,424,219, 4,256,746, 4,294,757, 4,307,016, 4,313,946, 4,315,929, 4,331,598, 4,361,650, Nos. 4,362,663, 4,364,866, 4,450,254, 4,322,348, 4,371,533, 5,208,020, 5,416,064, 5,475,092, 5,585,499, 5,846,545, 6,333,410, 6,441,163, 6,716,821, and 7,276,497.

[0484] Other examples include mertansine and ansamitocin. Pyrrolobenzodiazepines (PBDs), explicitly including dimers and analogs, include, but are not limited to, those described in Denny, Exp.Opin.Ther.Patents, 10(4):459-474 (2000); Hartley et al., Expert Opin Investig Drugs. 2011, 20(6):733-44; Antonow et al., Chem Rev. 2011, 111(4), 2815-64.

[0485] Calicheamicins include, for example, enediynes, esperamicins, and those described in US Pat. Nos. 5,714,586 and 5,739,116.

[0486] Examples of duocarmycins and analogs include CC1065, duocarmycin SA, duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, DU-86, KW-2189, adozelesin, bizeresin, carzelesin, seco-adozelesin. Other examples include those described in, for example, U.S. Patent Nos. 5,070,092, 5,101,092, 5,187,186, 5,475,092, 5,595,499, 5,846,545, 6,534,660, 6,548,530, 6,586,618, 6,660,742, 6,756,397, 7,049,316, 7,553,816, 8,815,226, US20150104407, 61 / 988,011 filed May 2, 2014, and 62 / 010,972 filed June 11, 2014. The disclosures of each of these are incorporated herein in their entirety.

[0487] Exemplary vinca alkaloids include vincristine, vinblastine, vindesine, and navelbine, as well as those described in U.S. Publication Nos. 2002 / 0103136 and 2010 / 0305149 and U.S. Patent No. 7,303,749, the disclosures of which are incorporated herein by reference in their entireties.

[0488] Exemplary epothilone compounds include epothilones A, B, C, D, E, and F, and derivatives thereof. Suitable epothilone compounds and their derivatives are described, for example, in U.S. Pat. Nos. 6,956,036, 6,989,450, 6,121,029, 6,117,659, 6,096,757, 6,043,372, 5,969,145, and 5,886,026, as well as WO 97 / 19086, WO 98 / 08849, WO 98 / 22461, WO 98 / 25929, WO 98 / 38192, WO 99 / 01124, WO 99 / 02514, WO 99 / 03848, WO 99 / 07692, WO 99 / 27890, and WO 99 / 28324, the disclosures of which are incorporated herein by reference in their entireties.

[0489] Exemplary cryptophycin compounds are described in US Pat. Nos. 6,680,311 and 6,747,021, the disclosures of which are incorporated herein by reference in their entireties.

[0490] Exemplary platinum compounds include cisplatin, carboplatin, oxaliplatin, iproplatin, ormaplatin, and tetraplatin.

[0491] Exemplary DNA binding or alkylating drugs include CC-1065 and its analogs, anthracyclines, calicheamicins, dactinomycins, mithromycins, pyrrolobenzodiazepines, and the like.

[0492] Exemplary microtubule stabilizing and destabilizing agents include taxane compounds, such as paclitaxel, docetaxel, tesetaxel, and carbazitaxel, maytansinoids, auristatins and their analogs, vinca alkaloid derivatives, epothilones, and cryptophycins.

[0493] Exemplary topoisomerase inhibitors include camptothecin and camptothecin derivatives, camptothecin analogs, and non-natural camptothecins, such as CPT-11, SN-38, topotecan, 9-aminocamptothecin, rubitecan, gimatecan, karenitecin, ciratecan, lurtotecan, exatecan, DXd, diflometotecan, belotecan, lurtotecan, and S39625. Other camptothecin compounds that can be used in the present invention include, for example, those described in J. Med. Chem., 29:2358-2363 (1986), J. Med. Chem., 23:554 (1980), J. Med Chem., 30:1774 (1987).

[0494] Angiogenesis inhibitors include, but are not limited to, MetAP2 inhibitors, VEGF inhibitors, PlGF inhibitors, VGFR inhibitors, PDGFR inhibitors, MetAP2 inhibitors.Exemplary VGFR and PDGFR inhibitors include sorafenib, sunitinib, and vatalanib.Exemplary MetAP2 inhibitors include fumagillol analogues, which means compounds that contain fumagillin core structure.

[0495] Exemplary cell cycle progression inhibitors include CDK inhibitors such as, for example, BMS-387032 and PD0332991, Rho kinase inhibitors such as, for example, AZD7762, Aurora kinase inhibitors such as, for example, AZD1152, MLN8054, and MLN8237, PLK inhibitors such as, for example, BI2536, BI6727, GSK461364, ON-01910, KSP inhibitors such as, for example, SB743921, SB715992, MK-0731, AZD8477, AZ3146, and ARRY-520.

[0496] Exemplary PI3K / m-TOR / AKT signaling pathway inhibitors include phosphoinositide 3-kinase (PI3K) inhibitors, GSK-3 inhibitors, ATM inhibitors, DNA-PK inhibitors, and PDK-1 inhibitors.

[0497] Exemplary PI3 kinases are disclosed in U.S. Pat. No. 6,608,053 and include BEZ235, BGT226, BKM120, CAL263, demethoxyviridin, GDC-0941, GSK615, IC87114, LY294002, Palomid 529, Perifosine, PF-04691502, PX-866, SAR245408, SAR245409, SF1126, wortmannin, XL147, and XL765.

[0498] Exemplary AKT inhibitors include, but are not limited to, AT7867.

[0499] Exemplary MAPK signaling pathway inhibitors include MEK, Ras, JNK, B-Raf, p38 MAPK inhibitors, and the like.

[0500] Exemplary MEK inhibitors are disclosed in U.S. Pat. No. 7,517,944 and include GDC-0973, GSK1120212, MSC1936369B, AS703026, R05126766 and R04987655, PD0325901, AZD6244, AZD8330, and GDC-0973.

[0501] Exemplary B-raf inhibitors include CDC-0879, PLX-4032, and SB590885.

[0502] Exemplary B p38 MAPK inhibitors include BIRB796, LY2228820, and SB202190. Exemplary receptor tyrosine kinase inhibitors include, but are not limited to, AEE788 (NVP-AEE788), BIBW2992 (afatinib), lapatinib, erlotinib (Tarceva), gefitinib (Iressa), AP24534 (ponatinib), ABT-869 (linifanib), AZD2171, CHR-258 (dovitinib), sunitinib (sutent), sorafenib (nexavar), and vatalinib.

[0503] Exemplary protein chaperone inhibitors include HSP90 inhibitors. Exemplary inhibitors include 17AAG derivatives, BIIB021, BIIB028, SNX-5422, NVP-AUY-922, and KW-2478.

[0504] Exemplary HDAC inhibitors include belinostat (PXD101), CUDC-101, droxinostat, ITF2357 (gibinostat, gabinostat), JNJ-26481585, LAQ824 (NVP-LAQ824, dacinostat), LBH-589 (panobinostat), MC1568, MGCD0103 (mosetinostat), MS-275 (entinostat), PCI-24781, pyroxamide (NSC696085), SB939, trichostatin A, and vorinostat (SAHA). Exemplary PARP inhibitors include iniparib (BSI201), olaparib (AZD-2281), ABT-888 (veliparib), AG014699, CEP9722, MK4827, KU-0059436 (AZD2281), LT-673, 3-aminobenzamide, A-966492, and AZD2461.

[0505] Exemplary Wnt / hedgehog signaling pathway inhibitors include vismodegib, cyclopamine, and XAV-939.

[0506] Exemplary RNA polymerase inhibitors include amatoxins. Exemplary amatoxins include alpha-amanitin, beta-amanitin, gamma-amanitin, eta-amanitin, amanulin, amanuric acid, amanisamide, amanone, and proamanitin.

[0507] Exemplary cytokines include IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, TNF.

[0508] Non-limiting examples of specific drugs include auristatins, maytansinoids, PBDs, topoisomerase inhibitors, and anthracyclines.

[0509] In another embodiment, a combination of two or more different drugs, such as those described above, is used.

[0510] According to another embodiment, the biologically active compound may be selected from any synthetic or naturally occurring compound, such as in particular an oligo- or polypeptide or a protein, comprising one or more natural and / or non-natural proteinogenic and / or non-proteinogenic amino acid residues.

[0511] A particular group of such compounds includes immunoglobulin molecules, such as antibodies, antibody derivatives, antibody fragments, antibody (fragment) fusions (e.g. bispecific and trispecific mAb fragments or derivatives), polyclonal or monoclonal antibodies, such as human antibodies, humanized antibodies, murine antibodies or chimeric antibodies.

[0512] Typical non-limiting examples of antibodies for use in the present invention are selected from biologically, particularly pharmacologically active antibody molecules.Non-limiting examples are selected from the following group: trastuzumab, bevacizumab, cetuximab, panitumumab, ipilimumab, rituximab, alemtuzumab, ofatumumab, gemtuzumab, brentuximab, ibritumomab, tositumomab, pertuzumab, adecatumumab, IGN101, INA01, labetuzumab, hua33, pemtumomab, oregovomab, minretumomab (CC49), cG250, J591, MOv-18, farletuzumab (MORAb-003), 3F8, ch14.18, KW-2871, hu3S193, lgN311, IM-2C6, CDP-791, etaracizumab, volociximab, nimotuzumab, MM-121, AMG102, METMAB, SCH900105, AVE1642, IMC-A12, MK-0646, R1507, CP751871, KB004, IIIA4, mapatumumab, HGS-ETR2, CS-1008, denosumab, sibrotuzumab, F19, 81C6, pinatuzumab, rifastuzumab, glembatumumab, cortuximab, lorvotuzumab, indatuximab, anti-PSMA , MLN-0264, ABT-414, milatuzumab, ramucirumab, abagovomab, avituzumab, adecatumumab, afutuzumab, altumomab pentetate, amatuximab, anatumomab, anetumab, apolizumab, arcitumomab, asclinbacumab, atezolizumab, bavituximab, bectumomab, belimumab, bivatuzumab, brontixutuzumab, cantuzumab, capromab, catumaxomab, sitatuzumab, cixutumumab, clivatuzumab, codrituzumab, conatumumab, dacetuzumab, darotuzumab Mab, Daratumumab, Demcizumab, Denintuzumab, Depatuxizumab, Dellotuximab, Detumomab, Dinutuximab, Drozitumab, Durigotumab, Durvalumab, Ducizitumab, Eclomeximab, Edrecolomab, Ergemtumab, Emactuzumab, Enavatuzumab, Emivetuzumab, Enfortumab, Enoblituzumab, Ensituximab, Epratuzumab, Ertumaxomab, Etaracizumab, Farletuzumab, Ficlatuzumab, Figitumumab, Franvotumab, Futuximab, Galiximab,Ganitumab, Icrucumab, Igovomab, Imalumab, Imgatuzumab, Indusatumab, Inebilizumab, Intetumumab, Iratumumab, Isatuximab, Lexatuzumab, Rilotomab, Lintuzumab, Lirilumab, Lucatumumab, Lumuletuzumab, Margetuximab, Matuzumab, Mirvetuximab, Mitsumomab, Mogamulizumab, Moxetumomab, Nacolomab, Naptumomab, Narunatumumab, Necitumumab, Nesvacumab, Nimotuzumab, Nivolumab, Nofetumomab, Obinutuzumab, Okaratuzumab , ofatumumab, olaratumab, onartuzumab, ontuximab, oportuzumab, oregovomab, otlertuzumab, pancomab, palsatuzumab, pasotuximab, patritumab, pembrolizumab, pemtumomab, pidilizumab, pintumomab, polatuzumab, pritumumab, kirisumab, racotumomab, ramucirumab, rilotumumab, lobatumumab, sacituzumab, samalizumab, satumomab, seribantumab, siltuximab, sofituzumab, tacatuzumab, tapritumomab, talextumab , tenatumomab, teprotumumab, tetulomab, ticilimumab, tigatuzumab, tositumomab, tobetumab, tremelimumab, tucotuzumab, ublituximab, urocupulumab, urelumab, utomilumab, vadastuximab, bundletuzumab, vanticutuzumab, vanucizumab, varlilumab, veltuzumab, besencumab, volociximab, borsetuzumab, votumumab, zalutumumab, zatuximab, combinations and derivatives thereof, and CA125, CA15-3, CA19-9, L6, Lewis Y, Lewis and other monoclonal antibodies targeting sucrose X, alpha-fetoprotein, CA242, placental alkaline phosphatase, prostate-specific antigen, prostate-specific membrane antigen, prostatic acid phosphatase, epidermal growth factor, MAGE-1, MAGE-2, MAGE-3, MAGE-4, transferrin receptor, p97, MUC1, CEA, gp100, MART1, IL-2 receptor, CD20, CD52, CD33, CD22, human chorionic gonadotropin, CD38, CD40, mucin, P21, MPG, and the Neu oncogene product.

[0513] 5.2 Labelling agents / radionuclides Labeling agents that can be used in accordance with the present invention can include any type of labeling substance known in the art.

[0514] Labels of the invention include dyes (e.g., fluorescent dyes, luminescent dyes, phosphorescent dyes, such as dansyl, coumarin, fluorescein, acridine, rhodamine, silicon rhodamine, BODIPY, or cyanine dyes), molecules that can fluoresce upon contact with a reagent, chromophores (e.g., phytochromes, phycobilins, bilirubin, etc.), radioactive labels (e.g., radioactive forms of hydrogen, fluorine, carbon, phosphorus, sulfur, or iodine, such as tritium, fluorine-18, carbon-11, carbon-14, phosphorus-32, phosphorus-33, sulfur-33, sulfur- Examples of suitable probes include, but are not limited to, MRI-sensitive spin labels, affinity tags (e.g., biotin, His tags, Flag tags, strep tags, sugars, lipids, sterols, PEG linkers, benzylguanine, benzylcytosine, or coenzymes), polyethylene glycol groups (e.g., branched PEGs, linear PEGs, PEGs of different molecular weights, etc.), photocrosslinkers (e.g., p-azidoiodoacetanilide), NMR probes, X-ray probes, pH probes, IR probes, resins, solid supports, and the biologically active compounds described above.

[0515] In some embodiments, exemplary dyes may include NIR contrast agents that fluoresce in the near infrared region of the spectrum. Exemplary near infrared fluorophores may include dyes and other fluorophores having an emission wavelength (e.g., peak emission wavelength) of about 630-1000 nm, e.g., about 630-800 nm, about 800-900 nm, about 900-1000 nm, about 680-750 nm, about 750-800 nm, about 800-850 nm, about 850-900 nm, about 900-950 nm, or about 950-1000 nm. Fluorescers having an emission wavelength (e.g., peak emission wavelength) greater than 1000 nm may also be used in the methods described herein.

[0516] In some embodiments, exemplary fluorophores include 7-amino-4-methylcoumarin-3-acetic acid (AMCA), Texas Red™ (Molecular (Molecular Probes, Eugene, OR), 5-(and-6)-carboxy-X-rhodamine, Lissamine rhodamine B, 5-(and-6)-carboxyfluorescein, fluorescein-5-isothiocyanate (FITC), 7-diethylaminocoumarin-3-carboxylic acid, tetramethylrhodamine-5-(and-6)-isothiocyanate, 5-(and-6)-carboxytetramethylrhodamine, 7-hydroxycoumarin-3-carboxylic acid, 6-[fluorescein-5-(and-6)-carboxamido]hexanoic acid, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3-indacenepropionic acid, eosin-5-isothiocyanate, erythrosine-5-isothiocyanate, and CASCADE™ blue acetyl azide (Molecular Probes, Eugene, Oreg.) and ATTO dyes.

[0517] Further labelling agents are indium-111, copper-64, copper-67, iodine-124, thorium-227, rhenium-188, lutetium-177, zirconium-89, iodine-131, gallium-68, technetium-99m, actinium-225, bismuth-213, yttrium-90 and lead-212.

[0518] 5.3 Chelating agents Below is a list of typically applicable chelating agents and their abbreviations, the corresponding salts of which are also applicable:

[0519] Acetylacetone (ACAC), ethylenediamine (EN), 2-(2-aminoethylamino)ethanol (AEEA), diethylenetriamine (DIEN), iminodiacetic acid (IDA), triethylenetetramine (TRIEN), triaminotriethylamine, nitrilotriacetic acid (NTA) and its salts, such as Na 3NTA or FeNTA, ethylenediaminotriacetic acid (TED), ethylenediaminetetraacetic acid (EDTA) and its salts, such as Na 2 EDTA and CaNa 2 EDTA, diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), oxalic acid (OX), tartaric acid (TART), citric acid (CIT), dimethylglyoxime (DMG), 8-hydroxyquinoline, 2,2'-bipyridine (BPY), 1,10-phenanthroline (PHEN), dimercaptosuccinic acid (DMSA), 1,2-bis(diphenylphosphino)ethane (DPPE), sodium salicylate, methoxysalicylic acid, British antilewisite or 2,3-dimercaprol (BAL), meso-2,3-dimercaptosuccinic acid (DMSA), siderophores secreted by microorganisms, e.g., Streptomyces desferrioxamine or deferoxamine B, also known as Deferral (Novartis), produced by Streptomyces spp., deferoxamine (DFO), a trihydroxamic acid secreted by Streptomyces pilosus, phytochemicals such as curcuminoids and mugineic acid derivatives, such as 3-hydroxymugineic acid and 2'-deoxymugineic acid, synthetically produced chelating agents such as ibuprofen, catechol derivatives, hydroxamic acid derivatives, and hydroxypyridinone derivatives, such as the hydroxamic acid desferal and the hydroxypyridinone deferiprone, deferiprone (L1 or 1,2-dimethyl-3-hydroxypyrid-4-one), β-β-dimethylcysteine ​​or 3-mercapto-D-valine, D-penicillamine (DPA or D-PEN), tetraethylenetetramine (TETA) or trientine and its two main metabolites N 1 -Acetyltriethylenetetramine (MAT) and N 1 ,N 10-Diacetyltriethylenetetramine (DAT), hydroxyquinoline, the halogenated derivative of 8-hydroxyquinoline, clioquinol, and 5,7-dichloro-2-[(dimethylamino)methyl]quinolin-8-ol (PBT2).

[0520] 6. ncAA ncAA useful in the methods and kits of the present invention have been described in the prior art (for reviews, see, e.g., Liu et al., Annu Rev Biochem 83:379-408, 2010; Lemke, ChemBioChem 15:1691-1694, 2014).

[0521] The ncAA may contain a group (herein referred to as a "labeling group") that facilitates reaction with a suitable group (herein referred to as a "docking group") of another molecule (herein referred to as a "binding partner molecule"), thereby covalently attaching the binding partner molecule to the ncAA. When an ncAA containing a labeling group is translationally incorporated into a POI, the labeling group becomes part of the POI. Thus, the POI prepared by the method of the present invention can react with one or more binding partner molecules, thereby covalently binding the binding partner molecule to (the labeling group of) a non-standard amino acid residue of the POI. Such conjugation reactions can be used for in situ coupling of the POI within cells or tissues expressing the POI, or for site-specific conjugation of an isolated or partially isolated POI.

[0522] Particularly useful options for combinations of labeling and docking groups (of binding partner molecules) are those that can react by metal-free click reactions, including strain-promoted inverse electron demand Diels-Alder cycloaddition (SPIEDAC, see e.g. Devaraj et al., Angew Chem Int Ed Engl 2009, 48:7013) and cycloadditions of strained cycloalkynyl groups or strained cycloalkynyl analogues in which one or more of the ring atoms not connected by a triple bond are substituted with an amino group with azides, nitrile oxides, nitrones and diazocarbonyl reagents (see e.g. Sanders et al., J Am Chem Soc 2010, 133:949; Agard et al., J Am Chem Soc 2004, 126:15046), such as strain-promoted alkyne azide cycloaddition (SPAAC). Such a click reaction allows for ultrafast and bioorthogonal covalent site-specific coupling of the ncAA labeling group of a POI with a suitable group of a coupling partner molecule.

[0523] The above-mentioned click-reactive docking group and labeling group pairs are known in the art. Examples of suitable ncAAs that contain docking groups include, but are not limited to, the ncAAs described in WO2012 / 104422 and WO2015 / 107064, the disclosures of which are expressly incorporated herein by reference.

[0524] Examples of particularly suitable pairs of docking groups (contained in the binding partner molecule) and labeling groups (contained in the ncAA residue of the POI) include, but are not limited to, the following: (a) a docking group comprising (or consisting essentially of) a group selected from an azide group, a nitrile oxide functionality (i.e., a radical of the formula: ##STR00002##), a nitrone functionality, or a diazocarbonyl group, in combination with a labeling group comprising (or consisting essentially of) an optionally substituted strained alkynyl group (such groups are covalently reactable by copper-free strain-promoted alkyne azide cycloaddition (SPAAC)). (b) a docking group comprising (or consisting essentially of) an optionally substituted strained alkynyl group, in combination with a labeling group comprising (or consisting essentially of) a group selected from an azide group, a nitrile oxide functionality (i.e., a radical of the formula:), a nitrone functionality, or a diazocarbonyl group (such groups are covalently reactable by copper-free strain-promoted alkyne azide cycloaddition (SPAAC)). (c) a docking group comprising (or consisting essentially of) a group selected from an optionally substituted strained alkynyl group, an optionally substituted strained alkenyl group, and a norbornenyl group, in combination with a labeling group comprising (or consisting essentially of) an optionally substituted tetrazinyl group (such groups are covalently reactable by copper-free strain-promoted inverse electron demand Diels-Alder cycloaddition (SPIEDAC)). (d) a docking group comprising (or consisting essentially of) an optionally substituted tetrazinyl group, in combination with a labeling group comprising (or consisting essentially of) a group selected from an optionally substituted strained alkynyl group, an optionally substituted strained alkenyl group, and a norbornenyl group (such groups are covalently reactable by copper-free strain-promoted inverse electron demand Diels-Alder cycloaddition (SPIEDAC)).

[0525] Optionally substituted strained alkynyl groups include, but are not limited to, optionally substituted trans cyclooctenyl groups, such as those described in WO2012 / 104422 and WO2015 / 107064. Optionally substituted strained alkenyl groups include, but are not limited to, optionally substituted cyclooctynyl groups, such as those described in WO2012 / 104422 and WO2015 / 107064. Optionally substituted tetrazinyl groups include, but are not limited to, those described in WO2012 / 104422 and WO2015 / 107064.

[0526] The azide group has the formula -N 3 is a radical.

[0527] The nitrone functional group has the formula -C(R x )=N+ (R y )-O - is a radical of, where R x and R y is an organic residue, e.g., C 1 -C 6 alkyl.

[0528] The diazocarbonyl group has the formula -C(O)-CH=N 2 is a radical.

[0529] The nitrile oxide functional group has the formula -C≡N + -O - or preferably a radical of the formula -C=N + (R x )-O - is a radical of, where R x is an organic residue, e.g., C 1 -C 6 alkyl.

[0530] "Cyclooctynyl" is an unsaturated alicyclic radical having eight carbon atoms and one triple bond in the ring structure.

[0531] "Transcyclooctenyl" is an unsaturated alicyclic radical having eight carbon atoms and one double bond in the ring structure in a trans configuration.

[0532] "Tetradyl" is a six-membered monocyclic aromatic radical having four ring nitrogen atoms and two ring carbon atoms.

[0533] Unless otherwise specified, the term "substituted" means that the radical is substituted with one, two or three, particularly one or two, substituents. In certain embodiments, these substituents are hydrogen, halogen, C 1 -C 4 Alkyl, (R a O) 2 P(O)OC 1 -C 4 Alkyl, (Rb O) 2 P(O)-C 1 -C 4 Alkyl, CF 3 , CN, hydroxyl, C 1 -C 4 Alkoxy, -O-CF 3 , C 2 -C 5 Alkenoxy, C 2 -C 5 Alkanoyloxy, C 1 -C 4 Alkylaminocarbonyloxy or C 1 -C 4 Alkylthio, C 1 -C 4 Alkylamino, di(C 1 -C 4 Alkyl)amino, C 2 -C 5 Alkenylamino, NC 2 -C 5 Alkenyl-NC 1 -C 4 Alkylamino and di(C 2 -C 5 alkenyl)amino, where R a and R b , R a , R b are independently hydrogen or C 2 -C 5 It is alkanoyloxymethyl.

[0534] The term halogen denotes in each case a fluorine, bromine, chlorine or iodine radical, in particular a fluorine radical.

[0535] C 1 -C 4 Alkyl is a straight or branched alkyl group having 1 to 4, especially 1 to 3, carbon atoms. Examples include methyl and C 2 -C 4 Alkyl includes, for example, ethyl, n-propyl, isopropyl, n-butyl, 2-butyl, isobutyl, and tert-butyl.

[0536] C 2 -C 5 Alkenyl is a monovalent unsaturated hydrocarbon radical having 2, 3, 4 or 5 carbon atoms. Examples include vinyl, allyl (2-propen-1-yl), 1-propen-1-yl, 2-propen-2-yl, methallyl (2-methylprop-2-en-1-yl), 1-methylprop-2-en-1-yl, 2-buten-1-yl, 3-buten-1-yl, 2-penten-1-yl, 3-penten-1-yl, 4-penten-1-yl, 1-methylbut-2-en-1-yl and 2-ethylprop-2-en-1-yl.

[0537] C 1 -C 4 Alkoxy is a radical of the formula RO-, where R is C as defined herein. 1 -C 4 It is an alkyl group.

[0538] C 2 -C 5 Alkenoxy is a radical of the formula RO-, where R is C as defined herein. 2 -C 5 It is alkenyl.

[0539] C 2 -C 5 Alkanoyloxy is a radical of the formula RC(O)-O-, where R is C as defined herein. 1 -C 4 It is an alkyl.

[0540] C 1 -C 4 Alkylaminocarbonyloxy is a radical of the formula R-NH-C(O)-O-, where R is C as defined herein. 1 -C 4 It is an alkyl.

[0541] C 1 -C 4 Alkylthio is a radical of the formula RS-, where R is C as defined herein. 1 -C4 It is an alkyl.

[0542] C 1 -C 4 Alkylamino is a radical of the formula R-NH-, where R is C as defined herein. 1 -C 4 It is an alkyl.

[0543] Ji (C 1 -C 4 Alkyl)amino is a group of the formula R x -N(R y )-radical, where R x and R y are independently C as defined herein. 1 -C 4 It is an alkyl.

[0544] C 2 -C 5 Alkenylamino is a radical of the formula R-NH-, where R is C as defined herein. 2 -C 5 It is alkenyl.

[0545] NC 2 -C 5 Alkenyl-NC 1 -C 4 Alkylamino is a group of the formula R x -N(R y )-radical, where R x is defined herein as C 2 -C 5 alkenyl, R y is defined herein as C 1 -C 4 It is an alkyl.

[0546] Ji (C 2 -C 5 alkenyl)amino has the formula R x -N(R y )-radical, where R x and R y are independently C as defined herein. 2 -C 5It is alkenyl.

[0547] C 2 -C 5 Alkanoyloxymethyl is a compound of the formula R x -C(O)-O-CH 2 - is a radical of, where R x is defined herein as C 1 -C 4 It is an alkyl.

[0548] The ncAAs used in the context of the present invention can be used in the form of their salts. The salts of the ncAAs described herein refer to acid or base addition salts, particularly to physiologically tolerated acid or base addition salts. Physiologically tolerated acid addition salts can be formed by treating the base form of the ncAA with a suitable organic or inorganic acid. The ncAAs containing acidic protons can be converted into their non-toxic metal or amine addition salt forms by treating with a suitable organic and inorganic base. The ncAAs and their salts described in the context of the present invention also include their hydrates and solvent addition forms, such as hydrates, alcoholates, etc.

[0549] In particular, a physiologically tolerated acid or base is one that is tolerated by the translation system used to prepare the POI having ncAA residues, e.g., is substantially non-toxic to living eukaryotic cells.

[0550] The ncAAs and their salts useful in the context of the present invention can be prepared in analogy with methods well known in the art, for example those published in the various documents cited herein.

[0551] The nature of the coupling partner molecule depends on the intended application: for example, the POI can be coupled to a molecule suitable for imaging methods or can be functionalized by coupling to a biologically active molecule as already described in more detail above.

[0552] Examples of useful coupling partner molecules include, but are not limited to, members of receptor / ligand pairs, members of antibody / antigen pairs, members of lectin / carbohydrate pairs, members of enzyme / substrate pairs, biotin / avidin, biotin / streptavidin, and digoxin / antidigoxin.

[0553] The ability of certain ncAA residues (labeling groups) to be covalently coupled in situ to (docking groups of) binding partner molecules, particularly by click reactions as described herein, can be used to detect POIs bearing such ncAA residues in eukaryotic cells or tissues expressing the POI, as well as to study the distribution and fate of the POI. In particular, the method of the invention for preparing a POI by expression in eukaryotic cells can be combined with super-resolution microscopy (SRM) for detecting the POI within cells or tissues of such cells. Several SRM methods are known in the art and can be adapted to utilize click chemistry for the detection of POIs expressed by eukaryotic cells of the invention. Specific examples of such SRM methods include DNA-PAINT (DNA point accumulation for imaging in nanoscale topography, e.g. as described in Jungmann et al., Nat Methods 11:313-318, 2014), dSTORM (direct stochastic optical reconstruction microscopy), and STED (stimulated emission depletion) microscopy.

[0554] 7. Complex Preparation The conjugates of the invention, in particular APCs and more particularly ADCs, are essentially active agents of known class, which may have diagnostic and / or therapeutic applications.

[0555] Different strategies are available for selectively and reversibly binding at least one corresponding payload compound to immunoglobulin molecules without destroying their ability to recognize and bind to a specific medical target of interest, such as a tumor marker molecule. Walsh, SJ et al., Chem. Soc. Rev. 2021, 50, 1305, review currently available strategies for site-selective modification in antibody-drug conjugates. Their respective preparation methods can be taken from the prior art cross-referenced therein.

[0556] A particular strategy of conjugation is based on coupling drug molecules to ncAA residues of correspondingly modified antibody molecules via Diels-Alder cycloaddition. A more specific approach has been reported by the inventors in Koehler, C. et al Nat. Methods, 2016, 13, 997. In this approach, highly reactive ncAAs such as cyclooctene and cyclooctyne-lysine (TCO-K and SCO-K) are incorporated into trastuzumab (i.e. Herceptin) by genetic code expansion. Such antibodies are then reacted with 1,2,4,5-tetrazine functionalized fluorophores to detect human cancer cells. Following the same principle, Oller-Salvia, B. et al. reported in Angew. Chem. Int. Ed. 2018, 57, In 2831, they report the conjugation of tetrazine-functionalized drugs to cyclopropene-lysine-functionalized trastuzumab. The tetrazine-functionalized drugs contain a linker moiety between the benzyltetrazine end group and the MMAE moiety. The linker contains a protease-labile valine-citrulline dipeptide linker, which is cleaved by cathepsin B in the lysosome to release the toxin. The tetrazine-functionalized drugs are conjugated to the functionalized antibody molecules in greater than 95% yield at 25° C. for 3 hours in PBS containing 10% MeCN.

[0557] Bioorthogonal conjugation with the above-mentioned types of highly reactive ncAAs can be carried out with payload molecules functionalized with different types of dienophilic tetrazine groups. See, for example, Kozma, E,. et al., ChemBioChem,2017,18 486, which discloses tetrazine derivatives in which the tetrazine moiety is substituted with one or two optionally further functionalized aromatic moieties. Further examples of suitable tetrazine groups are disclosed, for example, in Audebert, P. et al., New. J. Chem., 2004, 28, 387, or Yang, J. et al., Angew. Chem. Int. Ed. 2012, 51, 5222, or Knall, A.-C., et al. Chem. Soc. Rev., 2012,42,5131.

[0558] As a non-limiting example, (1,2,4,5-tetrazine-3-yl)benzoic acid is included as a starting material for the preparation of tetrazine-functionalized payload molecules of the invention.

[0559] European Patent Application No. EP21213081.9, filed December 8, 2021 (herein incorporated by reference), discloses a new class of substituted tetrazinyl-functionalized payloads containing tetrazinyl residues bearing specific polar substituents, which are applicable for the preparation of APCs and ADCs of the invention.

[0560] In the conjugates of the present invention, any type of non-cleavable, cleavable or pH-sensitive linker can be placed between the tetrazine moiety and the payload molecule.

[0561] Non-limiting examples of suitable peptide linkers that can be used to prepare the conjugates of the present invention can be selected from di-, tri- and tetrapeptides. Particular examples are: valine-citrulline, valine-glycine, valine-alanine, glycine-glycine, alanine-alanine, valine-lysine-glycine, alanine-alanine-asparagine, asparagine-proline-valine or aspartic acid-glutamic acid-valine-aspartic acid, glycine-glycine-phenylalanine-glycine. Suitable functionalized peptide linkers adapted to be incorporated into APC or ADC molecules are commercially available, for example from BroadPharm, San Diego, CA, USA. Similarly, functionalized peptide linkers covalently attached to payload molecules are commercially available, for example from the same suppliers.

[0562] Instead of cleavable peptide linkers, pH-sensitive or cleavable non-peptide linkers can also be used, for example hydrazone, disulfide or glucuronide groups can be used to prepare the cleavable conjugates of the invention.

[0563] More particularly, said linker group is an enzymatically or chemically cleavable linker group selected from: a) peptidyl groups, in particular di-, tri- or tetrapeptidyl groups, b) Formula - (CR 7 R 8 ) n -SS-(CR 7 R 8 ) n -X 5 - disulfide group, where residue R 7 and R 8 are independently selected from H or lower alkyl, in particular methyl, or two residues R 7 and R 8 together with the carbon atom to which they are attached form a ring C 4 -C 8 Forming an alkyl group, Part X 5 is selected from -C(O)- and -O-; c)>C=NN(R9 )- and -N(R 9 )-N=C<, where R 9 is H or lower alkyl, and d) beta-glucuronidase sensitive cleavable linker groups, particularly those having a beta-glucuronic acid derived trigger residue.

[0564] Non-limiting examples are residues of formula 8, 9 or 10 below. [ka]

[0565] Non-limiting examples illustrating the types of linkages between the tetrazine moiety and the -LD moiety include residues of formulas 5, 6, 7, 11, 12 and 13 below. [ka]

[0566] The residues of formulae 5, 6, 7, 11, 12 and 13 may be linked directly to L or, in particular, to the linear moiety -((CH 2 ) x1 -O) y1 - or -(O-(CH 2 ) x1 ) y1 - and their branched analogues, wherein x1 independently of one another represent an integer selected from 1, 2, 3 or 4, in particular 1 or 2, and y1 independently of one another represent an integer from 1 to 20, in particular 1 to 4.

[0567] If desired, well-known self-immolating groups can also be incorporated into the linker moiety, particularly adjacent to the payload molecule. Non-limiting examples of suitable self-immolating moieties include p-aminobenzyl groups, carbonate groups, dicarbamate groups, and methylenealkoxycarbamate groups.

[0568] The APCs and ADCs of the invention can be prepared in a similar manner as described above. Non-limiting examples include reaction sequences starting from tetrazine reactants. Payload molecules already carrying linker moieties such as those exemplified above can be dissolved in a suitable solvent and reacted with functionalized tetrazine moieties. The tetrazine moieties can have suitable reactive groups, such as carboxylic acid groups or succinimidyl ester groups that can react with the terminal amino groups of the peptide linkers. The reaction can be carried out at low temperatures, preferably around ambient temperature, optionally in the presence of a suitable coupling agent such as EDC. During the reaction, organic or inorganic acids or bases can be added to adjust the required pH. Non-limiting examples of suitable solvents are polar protic or aprotic organic solvents such as DMF, DMSO, pyridine, etc.

[0569] Of course, other synthetic routes are available or can be readily developed by one skilled in the art.

[0570] 8. General Description of the Synthesis of H-Tet-Glucuronide-D Type Payloads The H-Tet-glucuronide-D type payload molecules of formula 20, more specifically formula 23, and especially formula 34, can be prepared by applying standard techniques of organic synthesis. In particular, the synthesis can be carried out according to the following reaction scheme: [ka]

[0571] Step 1: Synthesis of intermediate (2) To a solution of a drug D of formula 30 containing a reactive secondary amino group and a glucuronide of formula 31 (i.e., (2S,3S,4S,5R,6R)-6-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamide)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid) (preferably in approximately equimolar amounts) in a suitable solvent, especially in admixture with a basic solvent such as pyridine, is added an organic base, especially N,N-diisopropylethylamine (DIPEA) in approximately equimolar amounts and hydroxybenzotriazole (HOBt) (catalytic amount). Suitable solvents include, for example, polar aprotic solvents selected from ethers, esters, ketones, acid anhydrides, tertiary amines, asymmetric halogenated hydrocarbons such as furan, thiophene, 1,1,1-trichloroethane, anisole, nitromethane, or especially DMF.

[0572] The resulting mixture is stirred at a suitable temperature, for example 0-100° C., for example at room temperature for a sufficient period of time, for example overnight. 1M NaOH (for example in excess, for example 4-20 fold or 5-10 fold molar excess) is then added and the mixture is stirred at an appropriate temperature for a sufficient period of time, for example 1 hour at room temperature. The resulting reaction product is subjected to purification to obtain the desired product of formula 32. Step 2: Synthesis of H-Tet-glucuronide-D (34) [ka]

[0573] To a solution of a compound of formula 32 and H-tetrazine of formula 33 in a suitable ratio (e.g., an excess, particularly a 2-fold excess, of the tetrazine of formula 33) in a suitable solvent (e.g., a polar aprotic solvent as described above, e.g., DMF), is added NaHCO 3 is added (e.g., in excess, particularly a 10-fold excess relative to the tetrazine of formula 33). The mixture is stirred for a sufficient time (e.g., 4 hours) at elevated temperature (e.g., 50° C.). After the conversion is complete, an excess of 1 M HCl is added to quench the reaction, and the reaction mixture is directly purified to provide the compound of formula 34.

[0574] A specific example of this general process is given in the Experimental Section below (see Synthesis Example 2).

[0575] 8. Pharmaceutical Compositions The APCs, particularly ADCs, of the invention (i.e., active agents or components) are generally administered as "pharmaceutical compositions" comprised of a therapeutically and / or prophylactically effective amount or a diagnostically effective amount of at least one such active component, or a pharma- ceutical acceptable salt thereof, and, optionally, at least one pharma- ceutical acceptable excipient.

[0576] The pharmaceutical compositions may be delivered by any suitable route of administration, such as oral, rectal, transmucosal, topical, ophthalmic, otic, or intestinal administration, parenteral delivery, such as intramuscular, subcutaneous, intraosseous injection, and intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injection, depending on the circumstances.

[0577] Depending on the nature of the composition or the mode and form of administration, the at least one additional pharmaceutical excipient may vary.

[0578] "Excipients" are substances that are formulated with an active ingredient and are included for a variety of purposes, such as long-term stabilization, bulking up solid formulations containing small amounts of potent active ingredients (and thus often also referred to as "bulking agents," "fillers," or "diluents"), or imparting therapeutic enhancements to the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, or enhancing solubility. Excipients can also be useful in the manufacturing process of pharmaceutical compositions to aid in active handling issues, such as by promoting powder flowability or non-stickiness, in addition to aiding in in vitro stability, such as preventing denaturation or aggregation during the expected shelf life. Selection of appropriate excipients depends on the specific active ingredient and other factors, as well as the route of administration and dosage form.

[0579] Excipients can be selected from the following classes: immunological adjuvants, anti-adherents, binders, coatings, colorants, disintegrants, flavors, flow agents, lubricants, preservatives, adsorbents, sweeteners, and carriers.

[0580] Non-limiting examples of excipients include diluents, preservatives, stabilizers, emulsifiers such as polysorbates and emulsifying polymers such as poloxamers, antioxidants, mitigating agents, chelating and stabilizing salts such as chlorides, sulfates, phosphates, diphosphates, hydrobromides and nitrates, suspending agents, antibacterial or antifungal agents. In addition, buffers can be used, for example buffer systems of low molecular weight organic acids with their respective salts, or inorganic buffer substances, for example phosphate buffers. Further suitable ingredients are also known from standard texts of relevant pharmacology. The proportions of the various components also depend on the nature of the particular components used and are generally known to those skilled in the art (Remington's Pharmaceutical science ("Handbook of Pharmaceutical Excipients" 2nd Edition (1994), Edited by A Wade and PJ Weller or Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985).

[0581] A pharmaceutical composition as used herein may be expressed in the form of a "dosage form" or "unit dose" and may contain one or more APCs, particularly ADCs, as described herein. Thus, a pharmaceutical composition as used herein may provide, for example, two active agents mixed together in a unit dose, or may provide two active agents combined in a dosage form in which the active agents are physically separated.

[0582] Furthermore, the pharmaceutical composition can be administered in a targeted drug delivery system, for example, in a liposome coated with endothelial cell-specific antibody.

[0583] The pharmaceutical compositions of the present invention can be manufactured in a manner known per se, for example by conventional mixing, dissolving, emulsifying, encapsulating, entrapping or combinations thereof. Proper formulation depends on the chosen route of administration.

[0584] The phrase "pharmacologically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of a patient, within the scope of sound medical judgment, without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable risk / benefit ratio.

[0585] The present invention encompasses all "pharmaceutical acceptable salt forms" of the active ingredient. Pharmaceutically acceptable salts are those in which the counter ion does not contribute significantly to the biological activity or toxicity of the compound and therefore function as pharmacological equivalents. These salts can be made by common organic techniques using commercially available reagents. Some anionic salt forms include acetate, acetonitrate, besylate, bromide, chloride, citrate, fumarate, glucuronate, hydrobromide, hydrochloride, hydroiodide, iodide, lactate, maleate, mesylate, nitrate, pamoate, phosphate, succinate, sulfate, tartrate, tosylate, and xinofoate. Some cationic salt forms include ammonium, aluminum, benzathine, bismuth, calcium, choline, diethylamine, diethanolamine, lithium, magnesium, meglumine, 4-phenylcyclohexylamine, piperazine, potassium, sodium, tromethamine, and zinc.

[0586] A "therapeutically effective amount" and / or a "prophylactically effective amount" means an amount effective to provide some therapeutic and / or prophylactic benefit when administered to a human or non-human patient. More specifically, a "therapeutically effective amount" is an amount of an active ingredient disclosed herein, or a combination of two or more such active ingredients, that inhibits in whole or in part the progression of a condition or at least partially alleviates one or more symptoms of a condition.

[0587] By "diagnostically effective amount" is meant an amount effective to enable diagnostically valuable information regarding the status or progression of a disease state to be obtained from a patient.

[0588] Therapeutic benefit can be an amount effective to improve symptoms in a patient with a disease, for example, to reduce symptoms in a patient with a disease. Under certain circumstances, the patient may not show symptoms of the condition being treated. Thus, a prophylactically effective amount of a compound is also an amount sufficient to provide a significant positive effect on any symptoms of a disease, disorder or condition, for example, an amount sufficient to significantly reduce the frequency and severity of disease symptoms.

[0589] A therapeutically effective amount can also be a prophylactically effective amount.

[0590] As used herein, a "patient" refers to a human or non-human animal, especially a human animal.

[0591] A "dosage form" is any unit administration ("unit dose") of one or more active agents described herein.

[0592] The term "treat" or "treatment" refers to (i) preventing the occurrence of a disease, disorder, or condition in a patient who may be predisposed to, but has not yet been diagnosed as having, the disease, disorder, and / or condition, (ii) inhibiting the disease, disorder, or condition, i.e., arresting its progression, and (iii) palliating the disease, disorder, or condition, i.e., causing the regression of the disease, disorder, and / or condition. In particular, it encompasses prophylactic or therapeutic treatment, or a combination thereof.

[0593] The "frequency" of administration may vary depending on the compound used and the specific type of infection being treated. A once-daily administration regimen is possible. A regimen in which the active ingredient is administered several times a day, for example 2-10 times, for example 2, 3, 4, 5, 6, 7, 8, 9 or 10 times, may be more beneficial in some cases.

[0594] It will be understood, however, that the specific dosage and frequency for any particular patient will depend on a variety of factors, including the activity of the particular compound used, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disease of the patient being treated. Patients can generally be monitored for therapeutic or prophylactic effectiveness using assays suitable for the condition being treated or prevented, as will be familiar to those of skill in the art.

[0595] Particular examples of pharmaceutical compositions of the present invention are preparations in liquid form, such as solutions, suspensions and emulsions, which contain a therapeutically effective amount of at least one APC, in particular an ADC component, as defined above, optionally together with at least one further pharma- ceutically acceptable excipient, as defined above, and can be administered by any suitable route.

[0596] Further examples of pharmaceutical compositions of the present invention are solid form preparations such as powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.

[0597] Numerous possible variations that will become readily apparent to those of ordinary skill in the art after considering the disclosure provide...

Claims

1. A site-selectively modified immunoglobulin molecule comprising at least one immunoglobulin heavy chain (IgH) and at least one immunoglobulin light chain (IgL), The IgH CDR-H1 selected from SEQ ID NOs: 9 and 10, CDR-H2 selected from SEQ ID NOs: 11 and 12, and Variable region V comprising CDR-H3 selected from SEQ ID NOs: 13 and 14 H ,and Constant region C H Including, The IgL CDR-L1 selected from SEQ ID NOs: 15 and 16, CDR-L2 selected from SEQ ID NOs: 17 and 18, and Variable region V comprising CDR-L3 selected from SEQ ID NOs: 19 and 20 L ,and Constant region C L wherein the site-selectively modified immunoglobulin molecule has the ability to bind to human epidermal growth factor receptor 2 (ERBB2 or HER2 / neu), the site-selectively modified immunoglobulin molecule exists in a non-glycosylated, glycosylated, or deglycosylated form; The site-selectively modified immunoglobulin molecule comprises: SEQ ID NO: 2 VH position: S25, P41, G42, K43, R50, D62, K65, E89, D102; C H1 positions: E155, P156, S194, E219; and C H2 positions: K249, K251, E275, K277, D283, H288, K291, K293, E296, R304, K320, K323, and K343 a site-selectively modified IgH comprising a non-canonical amino acid (ncAA) residue at one amino acid sequence position corresponding to a position selected from and / or SEQ ID NO: 4 VL positions: G41, K42, K45, A51, P59, R61, D70, E81; and CL position: A111, E143, D151, G157, K169, G200 a site-selectively modified IgL comprising a non-canonical amino acid (ncAA) residue at one amino acid sequence position corresponding to a position selected from have or SEQ ID NO: 2 C H1 position: E155, P156, S194, E219; C H2 positions: K249, K251, E275, K277, D283, H288, K291, K293, E296, R304, K320, K323, and K343 or a double modification at two amino acid sequence positions of at least one IgH corresponding to positions selected from SEQ ID NO: 4 CL position: A111, E143, D151, G157, K169, G200 Dual modifications at two amino acid sequence positions of at least one IgL corresponding to positions selected from having Site-selectively modified immunoglobulin molecules.

2. a) IgH single variants P41, G42, K249, K251, K291, K320, and K343 of SEQ ID NO: 2 in each IgH; b) IgL single variants G41, A51, P59, A111, and K169 of SEQ ID NO: 4 in each IgL; c) the double mutants K249 / K320 and K249 / K343 of the IgH of SEQ ID NO: 2, more specifically CH 2, in each IgH; d) the IgH / IgL mixed double mutants K249 / K169, K249 / G41, K320 / K169, K320 / G41, and P41 / G41 of SEQ ID NO: 2 and SEQ ID NO: 4 in each IgH / IgL pair; or e) IgH single mutants P41 and G42 of SEQ ID NO: 2; IgL single mutants G41, A51 and P59 of SEQ ID NO: 4; or An antigen-binding fragment of any of a) to d) selected from the IgH / IgL mixed double mutants K249 / G41, K320 / G41 and P41 / G41 of SEQ ID NO: 2 and SEQ ID NO: 4; and selected from trastuzumab variants selected from: In particular, in non-glycosylated, glycosylated or deglycosylated form, The site-selectively modified immunoglobulin molecule of claim 1.

3. a) IgH single mutants P41, G42, K248, K250, K290, K319 and K342 of SEQ ID NO: 6 in each IgH; b) IgL single variants G41, A51, P59, A111 and K169 of SEQ ID NO: 8 in each IgL; c) the IgH of SEQ ID NO: 6, more specifically the double mutants K248 / K319 and K248 / K342 of CH 2, in each IgH; d) the IgH / IgL mixed double mutants K248 / K169, K248 / G41, K319 / K169, K319 / G41, and P41 / G41 of SEQ ID NO: 6 and SEQ ID NO: 8 in each IgH / IgL pair; or e) IgH single mutants P41 and G42 of SEQ ID NO: 6; IgL single mutants G41, A51 and P59 of SEQ ID NO:8; IgH / IgL mixed double mutants K248 / G41, K319 / G41, and P41 / G41 of SEQ ID NO: 6 and SEQ ID NO: 8 an antigen-binding fragment of any of a) to d) selected from: and a trastuzumab variant selected from:

2. The site-selectively modified immunoglobulin molecule of claim 1, in particular in non-glycosylated, glycosylated or deglycosylated form.

4. the ncAA carries a functional side chain, wherein the functional side chain is capable of reacting via a Diels-Alder type cycloaddition reaction; (i) a trans-cyclooctenyl dienophile group of the formula: 【Chemistry 1】 where: R 1 are hydrogen, halogen, C 1 -C 4 Alkyl, (R a O) 2 P(O)OC 1 -C 4 Alkyl, (R b O) 2 P(O)-C 1 -C 4 Alkyl, CF 3 , CN, hydroxyl, C 1 -C 4 Alkoxy, -O-CF 3 , C 2 -C 5 Alkenoxy, C 2 -C 5 Alkanoyloxy, C 1 -C 4 Alkylaminocarbonyloxy or C 1 -C 4 Alkylthio, C 1 -C 4 Alkylamino, di(C 1 -C 4 Alkyl)amino, C 2 -C 5 Alkenylamino, C 2 -C 5 Alkenyl-C 1 -C 4 Alkyl-amino or di(C 2 -C 5 alkenyl)amino, R a , R b are independently hydrogen or C 2 -C 5 alkanoyloxymethyl or (ii) a cyclooctynyl dienophile group of the formula: 【Chemistry 2】 where: R 2 are hydrogen, halogen, C 1 -C 4 Alkyl, (R c O) 2 P(O)OC 1 -C 4 Alkyl, (R d O) 2 P(O)-C 1 -C 4 Alkyl, CF 3 , CN, hydroxyl, C 1 -C 4 Alkoxy, -O-CF 3 , C 2 -C 5 Alkenoxy, C 2 -C 5 Alkanoyloxy, C 1 -C 4 Alkylaminocarbonyloxy or C 1 -C 4 Alkylthio, C 1 -C 4 Alkylamino, di(C 1 -C 4 Alkyl)amino, C 2 -C 5 Alkenylamino, C 2 -C 5 Alkenyl-C 1 -C 4 Alkyl-amino or di(C 2 -C 5 alkenyl)amino, R c , R d are independently hydrogen or C 2 -C 5 alkanoyloxymethyl selected from, in particular: The ncAA may be SCO (2-amino-6-(cyclooct-2-yn-1-yloxycarbonylamino)hexanoic acid), TCO-Lys (N-ε-((trans-cyclooct-4-en-1-yloxy)carbonyl)-L-lysine), TCO*-Lys (N-ε-((trans-cyclooct-2-en-1-yloxy)carbonyl)-L-lysine), TCO # 4. The site-selectively modified immunoglobulin molecule of claim 3, wherein the TCO*A-Lys is selected from TCO*A-Lys (N-ε-((trans-cyclooct-3-en-1-yloxy)carbonyl)-L-lysine), TCO*B-Lys (N-ε-((trans-cyclooct-3-en-1-yloxy)carbonyl)-L-lysine), TCO*C-E-Lys (N6-((((R,E)-cyclooct-4-en-1-yl)oxy)carbonyl)-L-lysine) and TCO*A-Lys (N6-((((S,E)-cyclooct-2-en-1-yl)oxy)carbonyl)-L-lysine).

5. An antibody payload conjugate (APC), in particular an antibody drug conjugate (ADC), comprising at least one site-selectively modified immunoglobulin molecule according to any one of claims 1 to 4.

6. 5. A nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide chain of at least one site-selectively modified immunoglobulin molecule of any one of claims 1 to 4, wherein the nucleic acid molecule comprises at least one codon that allows incorporation of said ncAA into the encoded polypeptide sequence upon protein expression.

7. 5. A method for preparing a site-selectively modified immunoglobulin molecule according to any one of claims 1 to 4, comprising one or more non-canonical amino acid (ncAA) residues, the method comprising the steps of: (a) providing a eukaryotic cell comprising: (i) pyrrolysyl-tRNA synthetase, (ii) tRNA (tRNA) Pyl ) (iii) an ncAA or a salt thereof, and (iv) a polynucleotide encoding the site-selectively modified immunoglobulin molecule; wherein any position of the site-selectively modified immunoglobulin molecule occupied by an ncAA residue is Pyl is encoded by the reverse complement of the anticodon contained in wherein the pyrrolysyl-tRNA synthetase (i) is Pyl (ii) can be acylated with the non-standard amino acid or its salt (iii); and (b) allowing said polynucleotide (iv) to be translated by said eukaryotic cell, thereby producing said site-selectively modified immunoglobulin molecule.

8. 1. A method for preparing a polypeptide complex, comprising the steps of: (a) preparing a site-selectively modified immunoglobulin molecule containing one or more ncAA residues using the method of claim 7; and (b) reacting the site-selectively modified immunoglobulin molecule of step a) with one or more binding partner molecules such that the binding partner molecules covalently bind to the ncAA residues of the site-selectively modified immunoglobulin molecule.

9. A pharmaceutical composition comprising at least one ADC of claim 5 in a pharmaceutically acceptable carrier, or a diagnostic composition comprising at least one ADC of claim 5 in a diagnostically applicable carrier.

10. An antibody payload conjugate (APC), particularly an ADC, as described in claim 5, for medical use, particularly in diagnosis and therapy.

11. 6. An antibody-payload conjugate (APC), particularly an ADC, according to claim 5 for use in the diagnosis or treatment of breast cancer, gastric cancer or other Her2-overexpressing tumors, particularly tumors of the ovary, endometrium, bladder, lung, colon and head and neck.

12. 1. An ADC comprising at least one site-selectively modified immunoglobulin molecule comprising at least one immunoglobulin heavy chain (IgH) and at least one immunoglobulin light chain (IgL), The IgH CDR-H1 of SEQ ID NO: 9, CDR-H2 of SEQ ID NO: 11, and CDR-H3 of SEQ ID NO: 13 The variable region V encompasses H ,and Constant region C H Including, The IgL CDR-L1 of SEQ ID NO: 15, CDR-L2 of SEQ ID NO: 17, and CDR-L3 of SEQ ID NO: 19 The variable region V encompasses L ,and Constant region C L Including, where: at least one IgH is site-selectively modified by incorporating one or two SCO residues into its amino acid sequence at sequence positions corresponding to positions selected from K249 and K320 of SEQ ID NO: 2, respectively; the site-selectively modified immunoglobulin molecule has the ability to bind to human epidermal growth factor receptor 2 (ERBB2 or HER2 / neu); each SCO is linked to an H-tetrazine-functionalized payload moiety P comprising a drug moiety D selected from an auristatin and a maytansinoid; The ADC of claim 5.

13. The ADC of claim 5, which has the ability to bind to human epidermal growth factor receptor 2 (ERBB2 or HER2 / neu) and has the following general formula (1): 【Transformation 3】 where: n represents the number of attached side chains, each chain containing a payload moiety -LD; where: D is selected from auristatins and maytansinoids; L is an optionally cleavable linker moiety; A represents a site-selectively modified immunoglobulin molecule comprising at least one immunoglobulin heavy chain (IgH) and at least one immunoglobulin light chain (IgL); where: The IgH is CDR-H1 of SEQ ID NO: 9, CDR-H2 of SEQ ID NO: 11, and CDR-H3 of SEQ ID NO: 13 The variable region V encompasses H ,and containing the constant region CH, The IgL is CDR-L1 of SEQ ID NO: 15, CDR-L2 of SEQ ID NO: 17, and CDR-L3 of SEQ ID NO: 19 The variable region V encompasses L ,and comprising a constant region CL, at least one IgH is site-selectively linked to said payload moiety-LD at one or two sequence positions corresponding to positions selected from K249 and K320 of SEQ ID NO: 2, respectively; Either in the form of any stereoisomer and / or positional isomer, or as a mixture of at least two different stereoisomers and positional isomers thereof, as well as in non-glycosylated, glycosylated, or deglycosylated form.

14. A method for preparing an ADC of the following general formula (1): 【Chemistry 4】 [where: n, L, D and A are as defined above; in any stereoisomeric and / or positional isomeric form, or as a mixture of at least two different stereoisomeric and positional isomeric forms thereof, and either in non-glycosylated, glycosylated or deglycosylated form; The method comprises the steps of: 【Transformation 5】 where n and A are as defined above. and an H-tetrazine-functionalized payload molecule of the following general formula (3): 【Transformation 6】 where L and D are as defined above. to obtain an ADC of general formula (1), and optionally isolating the product.

15. 14. A pharmaceutical composition comprising at least the ADC of claim 13 in a pharmaceutically acceptable carrier.

16. 14. The ADC of claim 13 for use in the diagnosis or treatment of breast cancer.

17. 6. An APC, particularly an ADC, according to claim 5, site-selectively linked to at least one payload moiety comprising the moiety -LP of formula 4.1 below. 【Transformation 7】 where: m is an integer from 1 to 8; M is a radioactive metal isotope selected from 111-indium, 64-copper, 67-copper, 227-thorium, 188-rhenium, 177-lutetium, 89-zirconium, 68-gallium, 99m-technetium, 225-actinium, 213-bismuth, 90-yttrium and 212-lead, preferably 177-lutetium.

18. 18. The APC of claim 17, wherein at least one IgH is site-selectively linked to the payload moiety-LP at one sequence position corresponding to position A121 of SEQ ID NO:

2.

19. at least one IgH is site-selectively modified by incorporating a TCO*A, e.g., TCO*A-Lys, residue into its amino acid sequence at a sequence position corresponding to position A121 of SEQ ID NO:2; the site-selectively modified immunoglobulin molecule has the ability to bind to human epidermal growth factor receptor 2 (ERBB2 or HER2 / neu); At least one, in particular each TCO*A, for example TCO*A-Lys, is bound to an H-tetrazine functionalized payload moiety-LP; 19. The APC of claim 18.

20. 20. The APC of claim 19, wherein the H-tetrazine functionalized payload moiety-LP is of formula (5): 【Transformation 8】 where: m is an integer from 1 to 8; M is a radioactive metal isotope selected from 111-indium, 64-copper, 67-copper, 227-thorium, 188-rhenium, 177-lutetium, 89-zirconium, 68-gallium, 99m-technetium, 225-actinium, 213-bismuth, 90-yttrium and 212-lead, preferably 177-lutetium.

21. An antibody-payload conjugate (APC), particularly an ADC, as described in claim 17, particularly for medical use in diagnosis and therapy, more particularly in the diagnosis or treatment of breast cancer.

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